JP2004525607A - Oligonucleotide-attached nanoparticles and methods of use - Google Patents

Abstract

The present invention provides a method for detecting a nucleic acid. The method comprises contacting a nucleic acid with one or more types of particles having oligonucleotides attached thereto. In one embodiment of the method, the oligonucleotide is attached to the nanoparticle and has a sequence that is complementary to a portion of the sequence of the nucleic acid. As a result of the hybridization of the oligonucleotide on the nanoparticle to the nucleic acid, a detectable change (preferably a color change) occurs. The invention also provides compositions and kits that include the particles. The present invention further provides a method for synthesizing a unique nanoparticle-oligonucleotide conjugate, a conjugate obtained by the method, and a method for using the conjugate. Moreover, the present invention provides nanomaterials including nanoparticles and methods of nanofabrication using nanostructures and nanoparticles. Finally, the present invention provides a method for separating a selected nucleic acid from other nucleic acids.

Description

表１から分るように、６０℃のハイブリダイゼーションでは、完全対合標的３の場合のみ青色スポットが得られた。５０℃のハイブリダイゼーションでは、標的３と６の両方で青色スポットを生成した。４５℃のハイブリダイゼーションでは、標的３、５および６で青色スポットが得られた。
【０３０２】
関連シリーズにおいて、１誤対合Ｔヌクレオチドを含む標的は、５８℃で陽性テスト（青色）を生成し、共役体１および２は６４℃で陰性テスト（赤色）を生成した。同一条件下で、完全対合標的（３）は、どちらの温度下にも陽性テストを生成したが、これは、このテストが完全対合標的と、１誤対合塩基を含む標的とを識別し得ることを示している。
【０３０３】
異なるハイブリダイゼーション法を用いても同様な結果が得られた。特に、凍結、解凍し、次いで急速にストリンジェントな温度に温めることにより、選択的ハイブリダイゼーションが達成された。例えば、１５ｎＭの各オリゴヌクレオチド−コロイド共役体１および２と、１０ｐｍｏｌの標的オリゴヌクレオチド３、４、５、６または７を含有する１００μｌの０．１Ｍ ＮａＣｌを、ドライアイス−イソプロピルアルコール浴中で５分間凍結し、室温下に解凍し、次いで、急速に以下の表２に示されている温度に冷却し、混合物をこの温度で１０分間インキュベートしてハイブリダイゼーションを実施した。次いで、各反応混合物の３μｌ試料をＣ−１８ＴＬＣシリカプレート上にスポットした。結果を表２に示す。
【０３０４】
【表２】

これらの系の重要な特徴は、温度変化に関連する変色が極めて急激であり、かつ約１℃の温度範囲にわたって発生することである。これは、コロイド共役体が関与する融解および会合プロセスにおける高い協同性を示しており、完全対合配列と１塩基対誤対合を含むオリゴヌクレオチド標的を容易に識別し得ることを示している。
【０３０５】
高い識別度は２つの特徴によるものと考えられる。第１に、陽性シグナルを得るには、標的上の２つの比較的短いプローブオリゴヌクレオチドセグメント（１５ヌクレオチド）の整列が必要である。同等な２成分検出系において、どちらかのセグメント中に誤対合があると、もっと長いプローブ（例えば、３０塩基長のオリゴヌクレオチド）中の誤対合より不安定になる。第２に、溶液中の標的オリゴヌクレオチドとナノ粒子共役体とのハイブリダイゼーションで得られた２６０ｎｍでのシグナルは、ＤＮＡに基づくものではなく、ナノ粒子に基づいている。このシグナルは、多重オリゴヌクレオチド二本鎖によるポリマー網目構造として組織化されたナノ粒子集合体の解離に依存する。これによって、凝集体の解離に関して観察される温度範囲が、標準ＤＮＡ熱変性と比べて狭められる。簡単に説明すると、架橋凝集体中の一部の二本鎖は、ナノ粒子を溶液中に分散させることなく解離し得る。したがって、凝集体を融解させる温度範囲は、ナノ粒子を含まない同等な系の融解に関わる温度範囲（１２℃）に比べて極めて狭い（４℃）。この検出法のさらに顕著かつ有利な点は、Ｃ−１８シリカプレート上で観察される比色応答（＜１℃）の温度範囲である。原則的に、この３成分ナノ粒子をベースとする方法は、標的核酸とハイブリダイズしている一本鎖プローブをベースとする任意の２成分検出系よりも選択的であろう。
【０３０６】
１００μｌのハイブリダイゼーション緩衝液（０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸、ｐＨ７）中で、１ｎｍｏｌの標的３を含有するマスター溶液を調製した。この溶液１μｌは、標的オリゴヌクレオチド１０ｐｍｏｌに相当する。マスター溶液からアリコートを取り、ハイブリダイゼーション緩衝液で所望の濃度に希釈して、連続希釈を実施した。表３は、プローブ１および２と異なる量の標的３との混合物３μｌを用いて得た感度を示している。凍結−解凍条件を用いてハイブリダイゼーションを実施した後、これらの溶液の３μｌアリコートをＣ−１８ ＴＬＣプレート上にスポットして、色を定量した。以下の表３において、ピンク色は陰性テストを示し、青色は陽性テストを示す。
【０３０７】
【表３】

この実験は、１０ｆｍｏｌがこの特定の系の検出下限であることを示している。
【０３０８】
実施例６： ナノ粒子−オリゴヌクレオチド共役体を用いたアッセイ
図１３Ｂに示されているように、ＤＮＡ修飾ナノ粒子を修飾透明基板上に吸着させた。この方法は、ＤＮＡハイブリダイゼーション相互作用を用いて、ＤＮＡ修飾ナノ粒子をガラス製基板に付着しているナノ粒子に結合させるステップから成る。
【０３０９】
ガラス製顕微鏡スライドはフィッシャー・サイエンティフック社（Ｆｉｓｈｅｒ Ｓｃｉｅｎｆｉｔｉｃ）から購入した。先端にダイヤモンドが付いたけがきペンを用いて、スライドを約５×１５ｍｍの部片に切断した。スライドを、５０℃の４：１Ｈ_２ＳＯ_４：Ｈ_２Ｏ溶液中に２０分間浸漬して清浄した。次いで、スライドを、多量の水、次いでエタノールでリンスし、乾燥窒素流下に乾燥させた。スライド表面をチオール末端シランで官能化するために、スライドを脱気エタノール１％（質量）メルカプトプロピル−トリメトキシシラン溶液に１２時間浸漬した。スライドをエタノール溶液から取り出し、エタノール、次いで水でリンスした。直径１３ｎｍの金ナノ粒子を含有する溶液（実施例１に記載の配合物）にナノ粒子を浸漬して、スライドのチオール末端表面上に吸着させた。コロイド溶液中で１２時間後、スライドを取り出し、水でリンスした。得られたスライドは、吸着されたナノ粒子に帰するピンク色を有し、水性金ナノ粒子コロイド溶液と同様なＵＶ−可視吸光度プロファイル（５２０ｎｍでの表面プラズモン吸光度ピーク）を示す。図１４Ａ参照。
【０３１０】
ガラス製スライドを、新たに精製された３′チオールオリゴヌクレオチド（３′チオール ＡＴＧＣＴＣＡＡＣＴＣＴ［配列番号３３］（実施例１および３に記載のように合成）を含有する０．２ＯＤ（１．７μＭ）溶液中に浸漬して、ＤＮＡをナノ粒子修飾表面に付着させた。１２時間浸漬させた後、スライドを取り出し、水でリンスした。
【０３１１】
検体ＤＮＡ鎖がナノ粒子を修飾表面に結合させる能力を証明するために、連結オリゴヌクレオチドを調製した。（実施例２に記載のように調製した）連結オリゴヌクレオチドは、既に基板表面上に吸着されているＤＮＡ（配列番号３３）と相補的な１２ｂｐ末端を有する配列を含む２４ｂｐ長（５′ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴＧＡＡＴＧＣＧ）［配列番号３４］であった。次いで、基板を、連結オリゴヌクレオチド（０．４ＯＤ、１．７μＭ）を含有するハイブリダイゼーション緩衝液（０．５Ｍ ＮａＣｌ，１０ｍＭ リン酸緩衝液ｐＨ７）溶液中に１２時間浸漬した。基板を取り出し、類似緩衝液でリンスした後、基板を、基板に付着している連結オリゴヌクレオチドのハイブリダイズしていない部分と相補的なオリゴヌクレオチド（ＴＡＧＧＡＣＴＴＡＣＧＣ５′チオール［配列番号３５］）（実施例３に記載のように調製）で修飾された直径１３ｎｍの金ナノ粒子を含有する溶液中に浸漬した。１２時間浸漬した後、基板を取り出し、ハイブリダイゼーション緩衝液でリンスした。基板の色は紫色に変わっており、５２０ｎｍでのＵＶ−可視吸光度はほぼ二倍になった（図１４Ａ）。
【０３１２】
オリゴヌクレオチド修飾金ナノ粒子が、連結オリゴヌクレオチドとのＤＮＡハイブリダイゼーション相互作用によりオリゴヌクレオチド／ナノ粒子修飾表面に付着したことを確認するために、融解曲線を作成した。融解実験のために、基板を１ｍｌのハイブリダイゼーション緩衝液を含有するキュベットに入れ、実施例２のパートＢで用いたものと同じ装置を用いた。基板の温度を０．５℃／分の速度で増大させながら、ナノ粒子に帰する吸光度シグナル（５２０ｎｍ）をモニターした。温度が６０℃を越えたときに、ナノ粒子のシグナルは劇的に低下した。図１４Ｂ参照。シグナルの一次導関数は６２℃の融解温度を示したが、これは、ナノ粒子を含まない溶液中でハイブリダイズした３つのＤＮＡ配列に関して見られた温度と一致する。図１４Ｂ参照。
【０３１３】
実施例７： ナノ粒子−オリゴヌクレオチド共役体を用いたアッセイ
図１５Ａ−Ｇに示されている検出系は、２種のプローブ１および２が相補的標的４上に末端同士で整列するように設計した。これは、２つのプローブが標的鎖上に近接して整列している実施例５に記載の系とは異なる（図１２Ａ−Ｆ参照）。
【０３１４】
図１５Ａ−Ｇに示されているオリゴヌクレオチド−金ナノ粒子共役体は、実施例３に記載のように調製したが、但し、ナノ粒子は、ハイブリダイゼーション緩衝液（０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸、ｐＨ７）に再分散させた。最終ナノ粒子−オリゴヌクレオチド共役体濃度は、赤色のナノ粒子を生成する５２２ｎｍでの表面プラズモンバンド強度の低下を測定して、１３ｎＭであると予測した。図１５Ａ−Ｇに示されているオリゴヌクレオチド標的は、イリノイ州エバンストン所在のノースウエスタン大学バイオテクノロジー施設（Ｎｏｒｔｈｗｅｓｔｅｒｎ Ｕｎｉｖｅｒｓｉｔｙ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｆａｃｉｌｉｔｙ，Ｅｖａｎｓｔｏｎ，ＩＬ）から購入した。
【０３１５】
１３ｎＭ オリゴヌクレオチド−ナノ粒子共役体１および２を含有するハイブリダイゼーション緩衝液１５０μｌと、６０ｐｍｏｌ（６０μｌ）の標的４とを混合すると、溶液の色は直ぐに赤色から紫色に変化した。この変色は、金ナノ粒子の大きなオリゴヌクレオチド結合ポリマー網目構造の形成の結果として起こり、これは、ナノ粒子の表面プラズモン共鳴の赤色シフトをもたらす。溶液を２時間にわたって放置すると、大きな巨視的性質の凝集体の沈殿が観察された。懸濁凝集体を含む溶液の「融解分析」を実施した。「融解分析」を実施するために、溶液をハイブリダイゼーション緩衝液で１ｍｌに希釈し、温度を２５℃から７５℃まで上昇させながら、１分／１℃の保持時間で、２６０ｎｍでの凝集体の光学シグナチャーを１分間隔で記録した。５３．５℃の「融解温度」（Ｔ_ｍ）で、オリゴヌクレオチド−ナノ粒子ポリマーとしての凝集体の特性決定と一致する、特徴的な急激な転移（半値全幅、一次導関数のＦＷ_１／２＝３．５℃）が観察された。これは、ナノ粒子を含まないオリゴヌクレオチドに関して観察されたもっと広幅の転移（Ｔ_ｍ＝５４℃、ＦＷ_１／２＝〜１３．５℃）に関連するＴ_ｍに十分匹敵する。ナノ粒子を含む分析と類似の条件下に、ナノ粒子を含まないオリゴヌクレオチド溶液の「融解分析」を実施したが、但し、温度は１０℃から８０℃まで増大させた。また、溶液の各オリゴヌクレオチド成分は１．０４μＭであった。
【０３１６】
系の選択性をテストするために、プローブ１と２の完全相補体４から形成された凝集体のＴ_ｍを、１単塩基誤対合、欠失または挿入を含む標的から形成された凝集体のＴ_ｍ′と比較した（図１５Ａ−Ｇ参照）。不完全標的を含むすべての金ナノ粒子−オリゴヌクレオチド凝集体は、種々の凝集体のＴ_ｍ値によって証明されているように、完全相補体から形成された凝集体と比べて、有意な測定可能不安定化を示した（図１５Ａ−Ｇ参照）。不完全標的を含有する溶液は、５２．５℃に保持された水浴に入れたときのそれらの色によって完全相補体を含有する溶液から容易に区別し得る。この温度は、誤対合ポリヌクレオチドのＴ_ｍより高く、したがって、完全標的を含有する溶液のみがこの温度下に紫色を呈した。半相補的標的を含むプローブ溶液に関しても「融解分析」を実施した。２６０ｎｍでの吸光度は極くわずかに増大したことが観察された。
【０３１７】
次いで、ハイブリダイゼーション緩衝液中に５０μｌの各プローブ（１３ｎＭ）を含有する溶液に、２μｌ（２０ｐｍｏｌ）の各オリゴヌクレオチド標的（図１５Ａ−Ｇ参照）を加えた。室温下に１５分間放置した後、溶液を、温度制御水浴に移し、以下の表４に示されている温度下に５分間インキュベートした。次いで、各反応混合物の３μｌ試料をＣ−１８シリカプレート上にスポットした。凝集、したがって変色を誘発させるためには両プローブを標的上で整列させる必要があることを証明するために、２回の対照実験を行った。１回目の対照実験は、標的が存在しないプローブ１とプローブ２とから構成した。２回目の対照実験は、一方のプローブ配列のみと相補的な標的３を含むプローブ１とプローブ２とから構成した（図１５Ｂ参照）。結果は以下の表４に示されている。ピンク色のスポットは陰性テストを示し、青色のスポットは陽性テストを示している。
【０３１８】
注目すべきは、肉眼で検出し得る比色変化は、１℃未満にわたって発生し、それによって、完全標的４は、誤対合（５および６）、末端欠失（７）および２種のオリゴヌクレオチドプローブが出会う標的個所での１単塩基の挿入（８）を有する標的から容易に区別し得る（表４参照）ことである。比色変化Ｔ_ｃは、Ｔ_ｍと温度では近いが、同一ではない。どちらの対照の場合も、すべての温度下に観察されたピンクがかった赤色によって証明されるように、溶液中の粒子の凝集または不安定性の徴候はなく、すべての温度下のプレートテストで陰性スポット（ピンク色）を示した（表４）。
【０３１９】
１単塩基挿入標的８が完全相補性標的４から区別し得るという観察結果は、２種のプローブ配列を有する挿入鎖の完全相補性を考えれば、本当に注目すべきことである。８およびナノ粒子プローブから形成された凝集体の不安定化は、２つの短いプローブの使用と、完全相補性標的にハイブリダイズしたときにプローブの末端が出会う２つのチミジン塩基間の塩基の積み重ねの喪失とによるようである。同等条件（Ｔ_ｍ＝５１℃）下に３塩基対挿入（ＣＣＣ）を含む標的をプローブとハイブリダイズさせると、類似の結果が観察された。実施例５で上述した系において、塩基挿入を有する標的は、完全相補性標的から区別できなかった。したがって、この実施例に記載されている系は選択性の点で極めて好ましい。また、この系は、増幅技術を用いずにおよそ１０ｆｍｏｌの実施例５に記載の系と同じ感度を示した。
【０３２０】
これらの結果は、標的鎖に沿った任意の１単塩基誤対合と共に、標的鎖への任意の挿入をも検出し得ることを示している。変色を検出し得る温度範囲は極めて急勾配であり、変化は極めて狭い温度範囲にわたって生じる。この急激な変化は、標的オリゴヌクレオチド鎖を介して結合された大きなコロイド網目構造が関与する融解プロセスに大きな協同度が存在することを示している。これは、データで示されているように顕著な選択性をもたらす。
【０３２１】
【表４】

実施例８： ナノ粒子−オリゴヌクレオチド共役体を用いたアッセイ
「充填」二本鎖オリゴヌクレオチドとのハイブリダイゼーションを含む１式の実験を行った。図１６Ａに示されているナノ粒子−オリゴヌクレオチド共役体１および２を、図１６Ａ−Ｃに示されているような種々の長さの標的（２４、４８および７２塩基長）および相補的フィラーオリゴヌクレオチドと共にインキュベートした。条件は、その他の点では、実施例７に記載の通りであった。また、オリゴヌクレオチドとナノ粒子−オリゴヌクレオチド共役体は実施例７に記載のように調製した。
【０３２２】
予測したように、異なる反応溶液は、ハイブリダイゼーション後に、金ナノ粒子の間隔依存性光学特性による顕著に異なる光学特性を有していた。以下の表５参照。しかし、これらの溶液をＣ−１８ ＴＬＣプレート上にスポットして、室温または８０℃で乾燥させると、標的オリゴヌクレオチドの長さや金ナノ粒子間の間隔とは無関係に、青色を呈色した。表５参照。これは、固相支持体がハイブリダイズしたオリゴヌクレオチド−ナノ粒子共役体の凝集を促進するために起こると考えられる。これは、溶液をＴＬＣプレート上にスポットすることにより、金ナノ粒子間の間隔がかなり（少なくとも７２塩基）になるが、それでも比色検出が可能であることを証明している。
【０３２３】
【表５】

この実施例や他の実施例で観察された変色は、金ナノ粒子間の間隔（粒子間間隔）がナノ粒子の直径とほぼ同じかそれより小さいときに起こる。したがって、ナノ粒子のサイズ、ナノ粒子に付着しているオリゴヌクレオチドのサイズ、およびナノ粒子を標的核酸にハイブリダイズさせるときのナノ粒子の間隔は、オリゴヌクレオチド−ナノ粒子共役体が核酸標的とハイブリダイズして凝集体を形成するときに変色が観察可能であるかどうかに影響を与える。例えば、１３ｎｍの直径を有する金ナノ粒子は、長さが１０〜３５ヌクレオチドの標的配列とハイブリダイズするように設計されたナノ粒子に付着しているオリゴヌクレオチドを用いて凝集させると、変色を生じるであろう。ナノ粒子を標的核酸にハイブリダイズさせたときに変色を生じさせるのに適したナノ粒子間間隔は、結果が証明しているように、凝集の度合いに応じて異なるであろう。これらの結果はさらに、固体表面が既に凝集している試料の凝集をさらに促進し、金ナノ粒子をさらに近接させることを示している。
【０３２４】
金ナノ粒子に関して観察された変色は、金の表面プラズモン共鳴のシフトや広がりによるものであり得る。この変色が直径が約４ｎｍ未満の金ナノ粒子に関して起こる可能性はありそうにない。というのは、核酸の特異的検出に必要なオリゴヌクレオチドの長さがナノ粒子の直径を超えるからである。
【０３２５】
実施例９： ナノ粒子−オリゴヌクレオチド共役体を用いたアッセイ
それぞれプローブ１と２（図１２Ａ）から５μｌを緩衝液（１０ｍＭ リン酸、ｐＨ７）と合わせて０．１Ｍ ＮａＣｌの最終濃度とし、この溶液にヒト尿１μｌを添加した。この溶液を凍結、解凍し、次いでＣ−１８ ＴＬＣプレート上にスポットしたが、青色は発生しなかった。１２．５μｌの各プローブと、２．５μｌのヒト尿を含有する類似溶液に、０．２５μｌ（１０ｐｍｏｌ）の標的３（図１２Ａ）を加えた。溶液を凍結、解凍し、次いでＣ−１８ ＴＬＣプレート上にスポットすると、青色スポットが得られた。
【０３２６】
ヒト唾液の存在下で類似実験を行った。１２．５μｌの各プローブ１および２と、２および２．５μｌの標的３を含有する溶液を７０℃に加熱した。室温に冷ました後、２．５μｌの唾液溶液（水で１：１０希釈したヒト唾液）を加えた。得られた溶液を凍結、解凍し、次いで、Ｃ−１８ ＴＬＣプレート上にスポットした後、プローブと標的のハイブリダイゼーションを示す青色スポットが得られた。標的を加えなかった対照実験では、青色スポットは観察されなかった。
【０３２７】
実施例１０： ナノ粒子−オリゴヌクレオチド共役体を用いたアッセイ
図１３Ａに示されているようにアッセイを実施した。先ず、フィッシャー・サイエンティフィック社から購入したガラス製顕微鏡スライドを先端にダイヤモンドが付いたけがきペンで約５×１５ｍｍの部片に切断した。スライドを５０℃の４：１のＨ_２ＳＯ_４：Ｈ_２Ｏ_２溶液中に２０分間浸漬して清浄した。次いで、スライドを多量の水、次いでエタノールでリンスし、乾燥窒素流下に乾燥させた。改変した文献記載手順（Ｃｈｒｉｓｅｙら，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．，第２４巻，３０３１−３０３９ページ（１９９６年））を用いてチオール修飾ＤＮＡをスライド上に吸着させた。先ず、スライドを、室温下に、Ｎａｎｏｐｕｒｅ水中の１ｍＭ酢酸中のトリメトキシシリルプロピルジエチルトリアミン１％溶液（ＤＥＴＡ、ペンシルベニア州ブリストル所在のユナイテッド・ケミカル・テクノロジー社（Ｕｎｉｔｅｄ Ｃｈｅｍｉｃａｌ Ｔｅｃｈｎｏｌｏｇｙ，Ｂｒｉｓｔｏｌ，ＰＡ）から購入）中に２０分間浸漬した。スライドを水、次いでエタノールでリンスした。乾燥窒素流で乾燥させた後、温度制御加熱ブロックを用いて１２０℃で５分間焼成した。スライドを冷まし、次いで、室温下に２時間、８０：２０メタノール：ジメトキシスルホキシド中１ｍＭのスクシンイミジル−４−（マレイミドフェニル）−ブチレート（ＳＭＰＢ、シグマ・ケミカルズ社（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌｓ）から購入）中に浸漬した。ＳＭＰＢ溶液から取り出して、エタノールでリンスした後、ＳＭＰＢ架橋剤と結合しなかったアミン部位に以下のようにキャップ形成した。先ず、スライドを、１０％ １−メチルイミダゾールを含有する８：１ ＴＨＦ：ピリジン溶液中に５分間浸漬した。次いで、スライドを、９：１ ＴＨＦ：無水酢酸溶液中に５分間浸漬した。これらのキャッピング溶液は、ヴァージニア州スターリング所在のグレン・リサーチ社から購入した。スライドをＴＨＦ、次いでエタノール、最後に水でリンスした。
【０３２８】
修飾ガラス製スライドを、新たに精製されたオリゴヌクレオチド（３′チオールＡＴＧＣＴＣＡＡＣＴＣＴ［配列番号３３］を含有する０．２ＯＤ（１．７μＭ）溶液中に浸漬して、ＤＮＡを表面に付着させた。１２時間浸漬した後、スライドを取り出し、水でリンスした。
【０３２９】
検体ＤＮＡ鎖がナノ粒子を修飾基板表面に結合させる能力を証明するために、連結オリゴヌクレオチドを調製した。連結オリゴヌクレオチドは、既に基板表面上に吸着されているＤＮＡと相補的な１２ｂｐ末端を含む配列を有する２４ｂｐ長（５′ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴＧＡＡＴＧＣＧ）［配列番号３４］であった。次いで、基板を、連結オリゴヌクレオチド（０．４ＯＤ、１．７μＭ）を含有するハイブリダイゼーション緩衝液（０．５Ｍ ＮａＣｌ，１０ｍＭ リン酸緩衝液ｐＨ７）溶液中に１２時間浸漬した。基板を取り出し、類似緩衝液でリンスした後、基板を、基板に付着している連結オリゴヌクレオチドのハイブリダイズしていない部分と相補的なオリゴヌクレオチド（ＴＡＧＧＡＣＴＴＡＣＧＣ５′チオール［配列番号３５］）で修飾されている直径１３ｎｍの金ナノ粒子を含有する溶液中に浸漬した。１２時間浸漬した後、基板を取り出し、ハイブリダイゼーション緩衝液でリンスした。ガラス製基板の色は透明な無色から透明なピンク色に変わっていた。図１９Ａ参照。
【０３３０】
スライドを上記のような連結オリゴヌクレオチド溶液に浸漬し、次いで、オリゴヌクレオチド（３′チオールＡＴＧＣＴＣＡＡＣＴＣＴ［配列番号３３］）が付着している直径１３ｎｍの金ナノ粒子を含有する溶液に浸漬して、スライドに追加のナノ粒子層を加えた。１２時間浸漬した後、上記のように、スライドをナノ粒子溶液から取り出し、リンスして、ハイブリダイゼーション溶液中に浸漬した。スライドの色は、はっきり分るほど赤色が濃くなっていた。図１９Ａ参照。最終ナノ粒子層としてオリゴヌクレオチド（ＴＡＧＧＡＣＴＴＡＣＧＣ５′チオール［配列番号３５］）で修飾した１３ｎｍの金ナノ粒子を用いて連結オリゴヌクレオチドおよびナノ粒子浸漬手順を繰り返して最終ナノ粒子層を加えた。この場合も、色は濃くなり、５２０ｎｍのＵＶ−可視吸光度は増大した。図１９Ａ参照。
【０３３１】
オリゴヌクレオチド修飾金ナノ粒子が連結オリゴヌクレオチドとのＤＮＡハイブリダイゼーション相互作用によりオリゴヌクレオチド修飾表面に付着したことを確認するために、融解曲線を作成した。融解実験のために、スライドを、１．５ｍｌのハイブリダイゼーション緩衝液を含有するキュベットに入れ、実施例２のパートＢで用いたものと類似の装置を用いた。基板の温度を２０℃から８０℃まで増大させながら、１℃／１分の保持時間で、ナノ粒子に帰する吸光度シグナル（５２０ｎｍ）をモニターした。温度が５２℃を越えたとき、ナノ粒子のシグナルは劇的に低下した。図１９Ｂ参照。シグナルの一次導関数は、５５℃の融解温度を示したが、これは、溶液中でハイブリダイズしたオリゴヌクレオチド−ナノ粒子共役体と連結オリゴヌクレオチドに関して見られた温度と一致する。図１９Ｂ参照。
【０３３２】
実施例１１： プローブとしてナノ粒子−オリゴヌクレオチド共役体を用いたポリリボヌクレオチドアッセイ
これまでの実施例は、アッセイにおける標的としてオリゴ−デオキシリボヌクレオチドを利用した。本実施例は、ナノ粒子−オリゴヌクレオチド共役体をポリリボヌクレオチドアッセイにおけるプローブとして用い得ることを証明する。ポリ（ｒＡ）（０．００４_２６０単位）の溶液１μｌを、５′末端のメルカプトアルキルリンカーを介してｄＴ_２０（チミジレート残基を含有する２０ｍｅｒオリゴヌクレオチド）に結合している１００μＬの金ナノ粒子（粒子中〜１０ｎＭ）に加えて、実験を行った。結合手順は、実施例３に記載のものであった。実施例４に記載のように、ドライアイス／イソプロピルアルコール浴中で凍結し、室温下に解凍し、Ｃ１８ ＴＬＣプレート上にスポットした後、ハイブリダイゼーションによるナノ粒子の凝集の特徴を示す青色スポットが観察された。標的の不在下に実施した対照実験では、青色スポットではなく、ピンク色のスポットを得た。
【０３３３】
実施例１２： ナノ粒子−オリゴヌクレオチド共役体を用いた炭疽菌保護抗原セグメントのアッセイ
多くの場合、ＰＣＲによる二本鎖ＤＮＡ標的の増幅にはアッセイに十分な材料を提供することが必要である。本実施例は、ナノ粒子−オリゴヌクレオチド共役体が、ＤＮＡ鎖をその相補体の存在下にアッセイする（すなわち、二本鎖標的の熱デハイブリダイゼーション後に一本鎖に関してアッセイする）ために使用し得、かつＰＣＲ反応から得られたアンプリコンを認識して、特異的に結合し得ることを証明する。
【０３３４】
炭疽菌の保護抗原セグメントの１４１塩基対二本鎖アンプリコンを含有するＰＣＲ溶液は海軍省（Ｎａｖｙ）から提供された（図１２に示されている配列）。このアンプリコンのアッセイは、Ｑｉａｑｕｉｃｋ Ｎｕｃｌｅｏｔｉｄｅ Ｒｅｍｏｖａｌ Ｋｉｔ（カリフォルニア州サンタ・クラリタ所在のキアゲン社（Ｑｉａｇｅｎ，Ｉｎｃ．，Ｓａｎｔａ Ｃｌａｒｉｔａ，ＣＡ））およびこのキット用の標準プロトコルを用いて、１００μｌのＰＣＲ溶液からＤＮＡを単離して実施したが、但し、ＤＮＡの溶離は、キットで提供された緩衝液を用いるのではなく、ｐＨ８．５の１０ｍＭ リン酸緩衝液を用いて実施した。次いで、Ｓｐｅｅｄ Ｖａｃ（サバント社（Ｓａｖａｎｔ））上で溶離液を蒸発乾固した。この残留物に、２種の異なるオリゴヌクレオチド−ナノ粒子プローブ（図２３参照）からなる２種の各溶液を等量混合して調製した５μｌのマスター混合物を添加した。各オリゴヌクレオチド−ナノ粒子プローブは、実施例３に記載のように調製した。マスター混合物を形成するために合わせたプローブ溶液は、１０μｌの２Ｍ ＮａＣｌおよび５μｌのオリゴヌクレオチドブロッカー溶液（０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸、ｐＨ７．０溶液中５０ｐｍｏｌの各ブロッカーオリゴヌクレオチド（図２３および以下参照））を、５μｌの全強度（約１０ｎＭ）のナノ粒子−オリゴヌクレオチド溶液に加えて調製した。アンプリコン−プローブ混合物を３分間で１００℃に加熱し、次いで、ドライアイス／エタノール浴中で凍結し、室温に解凍した。小アリコート（２μｌ）をＣ１８ ＴＬＣプレート上にスポットし、乾燥させた。ハイブリダイゼーションを示す濃い青色のスポットを得た。
【０３３５】
アンプリコン標的の不在下、プローブ１の不在下、プローブ２の不在下、または塩化ナトリウムの不在下に、同様な方法で対照テストを実施したが、すべて陰性、すなわち、ピンク色のスポットを得た。同様に、保護抗原セグメントの代わりに炭疽菌の致死因子セグメント由来のＰＣＲアンプリコンを含むプローブ１および２を用いて実施したテストは陰性（ピンク色のスポット）。これらの対照により、どちらのプローブも必要不可欠であり、ハイブリダイゼーションに適した塩条件が必要であり、かつテストは特定の標的配列特異的であることが確認された。
【０３３６】
初期二本鎖標的の第２鎖（すなわち、標的鎖と相補的な鎖）がプローブに結合するセグメントの外側の標的核酸領域に結合するのを阻止するためにオリゴヌクレオチドブロッカーを付加した（配列に関しては図２３参照）。というのは、そのような結合はナノ粒子オリゴヌクレオチドプローブと標的鎖との結合に干渉するからである。この実施例では、ブロッカーオリゴヌクレオチドは、プローブによって被覆されない領域の一本鎖標的と相補的であった。代替計画は、プローブオリゴヌクレオチドと競合する領域外のＰＣＲ相補鎖（標的鎖と相補的な鎖）と相補的なブロッカーオリゴヌクレオチドを用いるものである。
【０３３７】
実施例１３： ＰＣＲ溶液からアンプリコンを単離しないＰＣＲアンプリコンの直接アッセイ
実施例１２に記載の手順は、ナノ粒子−オリゴヌクレオチドプローブを付加する前にＰＣＲ溶液からＰＣＲアンプリコンを分離するステップを含むものであった。多くの目的のためには、前以てポリヌクレオチド産物を単離することなく、ＰＣＲ溶液中で直接アッセイを実施し得ることが望ましいであろう。そのようなアッセイ用のプロトコルが開発されており、それを以下に説明する。このプロトコルは、Ａｍｐｌｉｔａｑ ＤＮＡポリメラーゼを含むＧｅｎｅＡｍｐ ＰＣＲ Ｒｅａｇｅｎｔ Ｋｉｔを用い、標準条件下に誘導されたいくつかのＰＣＲ産物を使って首尾良く実施されている。
【０３３８】
５０μｌのＰＣＲ試料溶液に、２種の金ナノ粒子−オリゴヌクレオチドプローブ（それぞれ０．００８Ａ_５２０単位）の混合物５μｌを添加し、次いで、１μｌのブロッカーオリゴヌクレオチド（各１０ｐｍｏｌ）と、５μｌの５Ｍ ＮａＣｌと、２μｌの１５０ｍＭ ＭｇＣｌ_２を加えた。この混合物を１００℃下に２分間加熱して二本鎖標的の鎖を分離し、管を直接冷浴（例えば、ドライアイス／エタノール）に２分間浸漬し、次いで、管を取り出して、溶液を室温下に解凍した（凍結−解凍サイクルにより、プローブと標的オリゴヌクレオチドとのハイブリダイゼーションが容易になる）。最後に、数μｌの溶液をプレート（例えば、Ｃ１８ ＲＰ ＴＬＣプレート、シリカプレート、ナイロン膜など）上にスポットした。例の通り、青色はＰＣＲ溶液中の標的核酸の存在を示し、ピンク色はこの標的に関して陰性である。
【０３３９】
実施例１４： ナノ粒子−オリゴヌクレオチド共役体の集合体を用いた、デハイブリダイゼーションを行わない二本鎖オリゴヌクレオチドの直接認識
これ以前の実施例では、ナノ粒子に結合している一本鎖オリゴヌクレオチドプローブと相互作用させる一本鎖を生成するために、二本鎖標的を加熱してデハイブリダイズさせた。本実施例は、三本鎖複合体を形成し得る場合、前以て標的をデハイブリダイズさせなくても、二本鎖オリゴヌクレオチド配列がナノ粒子によって認識され得ることを証明する。
【０３４０】
２種の異なる系、ポリＡ：ポリＵおよびｄＡ_４０：ｄＴ_４０を用い、１００μｌの緩衝液（０．１Ｍ ＮａＣｌ、１０ｍＭ リン酸、ｐＨ７．０）中に０．８Ａ_２６０単位の標的二本鎖を含有する溶液１μｌを、０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液（ｐＨ７．０）中のＡｕ−ｓｄＴ_２０ナノ粒子−オリゴヌクレオチド共役体（粒子中〜１０ｎＭ；実施例１１参照）のコロイド溶液１００μｌに加えて、テストを実施した。その後、管をドライアイス／イソプロピルアルコール浴中に浸漬して急速凍結し、管を浴から取り出して解凍して、室温（２２℃）下に放置し、次いで、３μｌの溶液をＣ１８ ＴＬＣプレート上にスポットして、ナノ粒子のハイブリダイゼーションおよび凝集の特徴を示す青色のスポットを得た。
【０３４１】
このテストの原理は、（この実施例ではピリミジンオリゴヌクレオチドを担持する）ナノ粒子プローブが、配列特異的に、二本鎖標的に沿ったプリンオリゴヌクレオチド／ピリミジンオリゴヌクレオチド部位で結合することである。各二本鎖体上の多くの結合部位が利用可能なので、結合によって、ナノ粒子凝集体が形成される。これらの結果は、ナノ粒子プローブを含む三本鎖複合体をベースとするこのアッセイが、オリゴリボヌクレオチド二本鎖標的にも、オリゴデオキシリボヌクレオチド二本鎖標的にも機能することを示している。
【０３４２】
実施例１５： 蛍光検出と比色検出とを用いるアッセイ
すべてのハイブリダイゼーション実験は、０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸、ｐＨ７．０、緩衝液中で実施した。ＡｃｅｔａｔｅＰｌｕｓ^ＴＭ濾過メンブレン（０．４５μｍ）は、マサチューセッツ州ウエストボロ所在のミクロン・セパレ−ションズ社（Ｍｉｃｒｏｎ Ｓｅｐａｒａｔｉｏｎｓ Ｉｎｃ．，Ｗｅｓｔｂｏｒｏ，ＭＡ）から購入した。アルキルアミン官能化ラテックスミクロスフェア（３．１μｍ）は、インディアナ州フィッシャーズ所在のバングス・ラボラトリーズ社（Ｂａｎｇｓ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｆｉｓｈｅｒｓ，ＩＮ）から購入した。Ａｍｉｎｏ−Ｍｏｄｉｆｉｅｒ Ｃ７ ＣＰＧ固相支持体（グレン・リサーチ社）およびＥｘｐｅｄｉｔｅ ８９０９合成機上の５′−フルオロセインホスホロアミダイト（６−ＦＡＭ，グレン・リサーチ社）を使う標準ホスホロアミダイト化学（Ｅｃｋｓｔｅｉｎ編，Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ ａｎｄ Ａｎａｌｏｇｕｅｓ，第１版，Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ，Ｎｅｗ Ｙｏｒｋ，Ｎ．Ｙ．，１９９１年）を用いて、３′末端をアルキルアミノ基で官能化した発蛍光団標識オリゴヌクレオチドを合成し、逆相ＨＰＬＣで精製した。これらのオリゴヌクレオチドを、Ｃｈａｒｒｅｙｒｅら，Ｌａｎｇｍｕｉｒ，第１３巻，３１０３−３１１０ページ（１９９７年）に記載のようにジチオ尿素結合を生成するジイソチオシアネートカップリングを用いて、アミン官能化ラテックスミクロスフェアに付着させた。簡単に説明すると、１０００倍過剰の１，４−フェニレンジチオシアネートのＤＭＦ溶液を、アミノ修飾オリゴヌクレオチドの水性ホウ酸緩衝液（０．１Ｍ、ｐＨ９．３）に加えた。数時間後、過剰な１，４−フェニレンジイソチオシアネートをブタノールで抽出し、水溶液を凍結乾燥した。活性化オリゴヌクレオチドをホウ酸緩衝液に再溶解させて、炭酸緩衝液（０．１Ｍ、ｐＨ９．３、１Ｍ ＮａＣｌ）中でアミノ官能化ラテックスミクロスフェアと反応させた。１２時間後、粒子を遠心分離し、緩衝塩類溶液（０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸、ｐＨ７．０）で３回洗浄した。５′−オリゴヌクレオチド修飾金ナノ粒子を実施例３に記載のように調製した。
【０３４３】
標的オリゴヌクレオチド（１〜５μｌ、３ｎＭ）を、３μｌの発蛍光団で標識したオリゴヌクレオチド修飾ラテックスミクロスフェアプローブ溶液（３．１μｍ；１００ｆＭ）に添加した。５分後、３μｌの５′オリゴヌクレオチド修飾金ナノ粒子プローブ溶液（１３ｎｍ；８ｎＭ）を、標的およびラテックスミクロスフェアプローブを含有する溶液に加えた。さらに１０分間放置した後、プローブと標的とを含有する溶液をＡｃｅｔａｔｅＰｌｕｓメンブレンに通じて真空濾過した。メンブレンは、比較的大きいラテックス粒子を保持し、ハイブリダイズしなかった金ナノ粒子プローブはすべて通過させた。十分な濃度の標的と、標的にハイブリダイズしたラテックスミクロスフェアおよび金ナノ粒子との存在下、メンブレン上に赤色スポットが観察された（陽性結果）。標的オリゴヌクレオチドを含有する溶液のアリコートを等量の水に代えた対照実験を常に行った。この場合、メンブレン上には白色スポットが残された（陰性結果）。２４塩基対モデル系の場合、比色計で３ｆｍｏｌの標的オリゴヌクレオチドを肉眼検出できた。
【０３４４】
二本鎖標的オリゴヌクレオチド（１〜５μｌ、２０ｎＭ）、３μｌの発蛍光団標識オリゴヌクレオチド−ラテックスミクロスフェア（３．１μｍ；１００ｆＭ）および３μｌの５′−オリゴヌクレオチド−金ナノ粒子（１３ｎｍ；８ｎＭ）を合わせ、３分間で１００℃に加熱した。次いで、直ぐに溶液を含有する反応容器を液体Ｎ_２浴に３分間に浸漬して凍結した。次いで、この溶液を室温下に解凍し、上記のように濾過した。２４塩基対のモデル系に関して、比色計で、２０ｆｍｏｌの二本鎖オリゴヌクレオチドが肉眼検出できた。
【０３４５】
蛍光によってモニターする場合、上記検出法はメンブレンからのバックグラウンド蛍光のために困難であることが証明された。この問題は、アリコートを逆相ＴＬＣプレート上にスポットする前に過剰な金ナノ粒子プローブを除去するためにラテックスミクロスフェアを遠心して「洗浄」することによって克服された。ハイブリダイゼーション実験を上述のように実施した。プローブと標的とのハイブリダイゼーションを実施した後、溶液に１０μｌの緩衝液を添加し、次いで、１０，０００×ｇで２分間遠心した。上清を除去し、沈殿物の再懸濁を支援するために５μｌの緩衝液を添加した。次いで、３μｌアリコートを逆相ＴＬＣプレート上にスポットした。一本鎖オリゴヌクレオチドと二本鎖標的オリゴヌクレオチドのどちらに関しても、比色計により２５ｆｍｏｌが肉眼検出できた。スポットの形成に用いた３μｌアリコート中の標的の量が５０ｆｍｏｌの低さになるまで、ハンドヘルドＵＶランプを用いて肉眼で蛍光スポットを視ることができた。この系を最適化することにより、もっと低量の標的核酸の検出も可能になるであろう。
【０３４６】
実施例１６： 銀染色法を用いたアッセイ
溶液中の特定のＤＮＡ配列の存在を検出するためには、一般に、オリゴヌクレオチド修飾基板上のＤＮＡハイブリダイゼーションテストが用いられる。遺伝子情報を精査するための組合せＤＮＡ配列の有望性が増してきていることは、未来の科学に対するこれらの異種配列アッセイの重要性を物語っている。ほとんどのアッセイにおいて、発蛍光団標識標的と表面結合プローブとのハイブリダイゼーションは、蛍光顕微鏡検査またはデンシトメーター検査によりモニターされている。蛍光検出は極めて感度が良いが、その使用は、実験装置費用とほとんどの慣用基板からのバックグラウンド放射とにより制限される。さらに、１単塩基誤対合を有するものに比べ完全相補性プローブに対する標識オリゴヌクレオチド標的への選択性の方が低いために、一ヌクレオチド多形を検出するための表面ハイブリダイゼーションテストが使用できない。蛍光法の簡易性、感度および選択性が改良された検出計画によって、組合せ配列の全ての可能性を具現化することができる。本発明はそのような改良型検出計画を提供する。
【０３４７】
例えば、３成分サンドイッチアッセイにおいて、オリゴヌクレオチド修飾金ナノ粒子と非修飾ＤＮＡ標的を、ガラス基板に付着しているオリゴヌクレオチドプローブとハイブリダイズさせることができた（図２５Ａ−Ｂ参照）。ナノ粒子は、個別のもの（図２５Ａ参照）であっても、複数のナノ粒子の「ツリー」（ｔｒｅｅ）（図２５Ｂ参照）であってもよい。「ツリー」は、個別のナノ粒子に比べてシグナル感度を増大させ、ハイブリダイズした金ナノ粒子の「ツリー」は、ガラス基板上の暗色領域として肉眼で観察できることが多い。「ツリー」を用いない場合、または「ツリー」によって生成されたシグナルを増幅させるためには、ハイブリダイズした金ナノ粒子を銀染色溶液で処理し得る。「ツリー」は、染色プロセスを促進し、個別ナノ粒子に比べて標的核酸の検出をより高速にする。
【０３４８】
以下は、（図２５Ａに例示されている）１つの特定の系の説明である。捕獲オリゴヌクレオチド（３′−ＨＳ（ＣＨ_２）_３−Ａ_１０ＡＴＧＣＴＣＡＡＣＴＣＴ；配列番号４３）を、実施例１０に記載のように、ガラス基板上に固定した。標的オリゴヌクレオチド（５′−ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴＧＡＡＴＧＣＧ−３′、配列番号４４、各実験用に以下の表６に示されている濃度）と捕獲オリゴヌクレオチドとを、実施例１０に記載のように、０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液中でハイブリダイズさせた。基板を同一緩衝液で２回リンスし、標的相補性ＤＮＡ（５′−ＨＳ（ＣＨ_２）_６Ａ_１０ＣＧＣＡＴＴＣＡＧＧＡＴ、配列番号４５）（実施例３に記載の配合物）で官能化した金ナノ粒子プローブを含有する溶液中に１２時間浸漬した。次いで、基板を０．３Ｍ ＮａＮＯ_３で何回もリンスしてＣｌ⁻を除去した。次いで、基板を銀染色溶液（Ｓｉｌｖｅｒ Ｅｎｈａｎｃｅｒ Ｓｏｌｕｔｉｏｎ ＡとＢの１：１混合物、シグマ・ケミカル社、＃Ｓ−５０２０および＃Ｓ−５１４５）で３分間現像した。グレースケール測定値を計算し得るソフトウエア（例えば、Ａｄｏｂｅ Ｐｈｏｔｏｓｈｏｐ）をロードしたコンピュータに連結した（通常、文書をコンピュータにスキャンするのに用いられる）平台型スキャナで基板をスキャンしてグレースケール測定を行った。結果を以下の表６に示す。
【０３４９】
【表６】

実施例１７： 量子ドットを含む集合体
この実施例は、半導体ナノ粒子量子ドット（ＱＤ）上の合成一本鎖ＤＮＡの固定化を説明する。天然ＣｄＳｅ／ＺｅＳコア／シェルＱＤ（〜４ｎｍ）は有機媒質にのみ可溶であるために、アルキルチオールを末端基とする一本鎖ＤＮＡと直接反応しにくい。この問題は、先ず、ＱＤに３−メルカプトプロピオン酸でキャップ形成することにより回避された。次いで、カルボン酸基を４−（ジメチルアミノ）ピリジンで脱保護して、粒子を水溶性とし、ＱＤと、３′−プロピルチオールまたは５′−ヘキシルチオール修飾オリゴヌクレオチド配列とを反応し易くした。ＤＮＡ修飾後、透析により、反応しなかったＤＮＡから粒子を分離した。次いで、「リンカー」ＤＮＡ鎖を表面結合配列とハイブリダイズさせて、長いナノ粒子集合体を生成した。ＴＥＭ、ＵＶ／可視分光法および蛍光顕微鏡検査により特性決定したＱＤ集合体は、溶液温度を制御することにより可逆的に集合させ得る。新規ＱＤ集合体およびＱＤと金ナノ粒子（〜１３ｎｍ）間に形成された複合凝集体の温度依存性ＵＶ−可視スペクトルを得た。
【０３５０】
Ａ． 一般法
ＮＡＮＯｐｕｒｅ超純水精製系を用いて調製したナノピュア水（１８．１ＭΩ）を一貫して用いた。Ｐｅｒｋｉｎ Ｅｌｍｅｒ ＬＳ ５０Ｂ蛍光分光計を用いて蛍光スペクトル得た。Ｈｐ ９０９０ａ Ｐｅｌｔｉｅｒ Ｔｅｍｐｅｒａｔｕｒｅ Ｃｏｎｔｒｏｌｌｅｒを具備したＨＰ ８４５３ダイオードアレイ分光光度計を用いて融解分析を実施した。Ｅｐｐｅｎｄｏｒｆ ５４１５遠心機またはＢｅｃｋｍａｎ Ａｖａｎｔｉ ３０遠心機を用いて遠心を行った。２００ｋＶで操作する日立 ＨＦ−２０００電界放出ＴＥＭを用いてＴＥＭ画像を得た。
【０３５１】
Ｂ． オリゴヌクレオチド−ＱＤ共役体の調製
半導体量子ドット（ＱＤ）の合成法は最近の数年間に大きく改良されており、ある種の材料、最も注目すべきことにはＣｄＳｅに関して、今や予備決定サイズの単分散試料を比較的容易に調製し得る。Ｍｕｒｒａｙら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１１５巻，８７０６ページ，１９９３年；Ｈｉｎｅｓら，Ｊ．Ｐｈｙｓ．Ｃｈｅｍ．，第１００巻，４６８ページ，１９９６年。その結果、ＱＤを発光ダイオード（Ｓｃｈｌａｍｐら，Ｊ．Ａｐｐｌ．Ｐｈｙｓ．，第８２巻，５８３７ページ，１９９７年；Ｄａｂｂｏｕｓｉら，Ａｐｐｌ．Ｐｈｙｓ．Ｌｅｔｔ．，第６６巻，１３１６ページ、１９９５年）および非放射性生物学的標識（Ｂｒｕｃｈｅｚら，Ｓｃｉｅｎｃｅ，第２８１巻，２０１３ページ，１９９８年；Ｃｈａｎら，Ｓｃｉｅｎｃｅ，第２８１巻，２０１６ページ，１９９８年）などの多様なテクノロジーに用いるための道を開く可能性があるこれらの粒子の固有の電子特性および蛍光特性が広範囲に研究されている（Ａｌｉｖｉｓａｔｏｓ，Ｊ．，Ｐｈｙｓ．Ｃｈｅｍ．，第１００巻，１３２２６ページ，１９９６年、およびその中の参考文献；Ｋｌｅｉｎら，Ｎａｔｕｒｅ，６９９，１９９７年；Ｋｕｎｏら，Ｊ．Ｃｈｅｍ．Ｐｈｙｓ．，第１０６巻，９８６９ページ，１９９７年；Ｎｉｒｍａｌら，Ｎａｔｕｒｅ，第３８３巻，８０２ページ，１９９６年参照）。しかし、多くの用途では、これらの粒子は表面上に空間的に配列されるか、または三次元物質中に組織化されることが要求されるであろう（Ｖｏｓｓｍｅｙｅｒら，Ｊ．Ａｐｐｌ．Ｐｈｙｓ．，第８４巻，３６６４ページ，１９９８年）。さらに、１種以上のナノ粒子を超格子構造（Ｍｕｒｒａｙら，Ｓｃｉｅｎｃｅ，第２７０巻，１３３５ページ，１９９５年）中に組織化する能力があれば、新規かつ潜在的に興味深く有用な特性を有する完全に新しい種類のハイブリッド材料の構築が可能になるであろう。
【０３５２】
ＤＮＡは、ナノスケールの構築ブロック集合体を周期的な二次元および三次元拡張構造中にプログラムするための理想的なシントンである。合成し易さ、非常に高い結合特異性およびヌクレオチド配列による実質的に無制限のプログラム可能性を含むＤＮＡの多くの特性をＱＤ集合体の使用に利用し得る。
【０３５３】
ＤＮＡによるＱＤの修飾は、金ナノ粒子の場合よりも困難であることが証明された。高い蛍光ＣｄＳｅ ＱＤを調製するための一般法によって、トリオクチルホスフィンオキシド（ＴＯＰＯ）とトリオクチルホスフィン（ＴＯＰ）の混合物でコートされた材料が得られる。その結果、これらのＱＤは、非極性溶媒にのみ可溶になり、そのため、ＱＤを高荷電ＤＮＡ鎖で直接反応により官能化するのは難しい。この難点は、初めて半導体ナノ粒子を一本鎖ＤＮＡで修飾することに成功した以下に記載の方法により克服された。他の人々は、精密な研究において、ＱＤと二本鎖ＤＮＡとの相互作用を考えていたが、これらの研究は、拡張ＱＤ構造の集合体を誘導するＤＮＡの配列特異的結合特性を利用しなかったことに留意されたい。Ｃｏｆｆｅｒら，Ａｐｐｌ．Ｐｈｙｓ．Ｌｅｔｔ．，第６９巻，３８５１ページ，１９９６年；Ｍａｈｔａｂら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１４８巻，７０２８ページ，１９９６年。
【０３５４】
ＣｄＳｅ／ＺｎＳコア／シェルＱＤの表面は有機チオールと結合するので、これらの半導体粒子を、置換反応により、アルキルチオールを末端基とするＤＮＡ鎖で修飾することが望ましかった。しかし、これらのＱＤは水溶性ではないので、そのような方法は妨げられた。最近、ＱＤを水溶性にして、ＱＤ表面上にタンパク質構造を固定化させるための２種の異なる方法が報告された。一方は、シリカ層でコア／シェル構造を封入するステップを含み（Ｂｒｕｃｈｅｚら，Ｓｃｉｅｎｃｅ，第２８１巻，２０１３ページ，１９９８年）、他方は、粒子を安定させ、かつ水溶性とするためにメルカプト酢酸を利用する（Ｃｈａｎら，Ｓｃｉｅｎｃｅ，第２８１巻，２０１６ページ，１９９８年）。ＤＮＡハイブリダイゼーション条件下に著しく安定したコロイドを生成する、この実施例に記載されている手順は、ＱＤ表面を不動態化するために３−メルカプトプロピオン酸を利用する。
【０３５５】
１．０ｍｌのＮ，Ｎ−ジメチルホルムアミド（ＤＭＦ；オールドリッチ社）中の（Ｈｉｎｅｓら，Ｊ．Ｐｈｙｓ．Ｃｈｅｍ．，第１００巻，４６８ページ，１９９６年に記載のように調製した）〜２０ｍｇのＴＯＰ／ＴＯＰＯ安定化ＣｄＳｅ／ＺｎＳ ＱＤの懸濁液に、シリンジを用いて、過剰な３−メルカプトプロピオン酸（０．１０ｍｌ、１．１５ｍｍｏｌ；オールドリッチ社）を添加して、３−メルカプトプロピオン酸官能化ＱＤを含有する透明な黒みがかったオレンジ色の溶液を生成した。反応は急速に起こった。後続反応のために、過剰な３−メルカプトプロピオン酸を除去せず、粒子を室温下にＤＭＦ中に保存した。
【０３５６】
しかし、ＱＤの特性決定のために、試料の一部から反応しなかった３−メルカプトプロピオン酸を以下のように除去して精製した。０．５０ｍｌ試料を（３０，０００ｒｐｍで４時間）遠心し、上清を除去した。残りの溶液を〜０．３ｍｌのＤＭＦで洗浄し、再遠心した。このステップをさらに２回繰り返してから、ＦＴＩＲスペクトルを記録した。ＦＴＩＲ（ポリエチレンカード、３Ｍ社）：１７１０ｃｍ^−１（ｓ）、１４７２ｃｍ^−１（ｍ）、１２７８ｃｍ^−１（ｗ）、１１８９ｃｍ^−１（ｍ）、１０４５ｃｍ^−１（ｗ）、９９３ｃｍ^−１（ｍ）、９４６ｃｍ^−１（ｗ）、７７６ｃｍ^−１（ｍ）、６７１ｃｍ^−１（ｍ）。ＴＯＰ／ＴＯＰＯ安定化天然ＱＤとは異なり、３−メルカプトプロピオン酸修飾ＱＤは、表面結合プロピオン酸に特徴的な１７１０ｃｍ^−１のｖ_ｃｏバンドを示した。
【０３５７】
３−メルカプトプロピオン酸修飾ＱＤは水に実質的に不溶であるが、それらの溶解度は、以下のパラグラフに記載のように、表面結合メルカプトプロピオン酸部位を４−（ジメチルアミノ）ピリジン（ＤＭＡＰ；オールドリッチ社）で脱保護して有意に高めることができた。そうすると、ＱＤは水に容易に分散し、オレンジ色の溶液となり、この溶液は室温下に１週間まで安定であった。
【０３５８】
オリゴヌクレオチドをＱＤに付着させるために、ＤＭＦ中の３−メルカプトプロピオン酸官能化粒子溶液１５０μｌ（５３０ｎｍでの光学密度＝２１．４）を、０．４ｍｌのＤＭＦ中のＤＭＡＰ（８．０ｍｇ、０．０６５ｍｍｏｌ）の溶液に添加した。オレンジ色の沈殿物が形成された。これを、遠心（３，０００ｒｐｍで〜３０秒）により分離し、次いで、３′プロピルチオール−または５′ヘキシルチオール−を末端基とするオリゴヌクレオチド（１．０〜２．０ＯＤ／ｍｌ；実施例１に記載のように調製；配列は以下に記載）の溶液１．０ｍｌに溶解させた。（水に溶解した）沈殿物をＩＲスペクトロスコピー（ポリエチレンカード、３Ｍ社）によって特性決定した。ＩＲ（ｃｍ^−１）： １６４７（ｍ）、１５５９（ｓ）、１４６２（ｍ）、１２１４（ｗ）、７１９（ｗ）、４７８（ｓ）。１２時間放置した後、オリゴヌクレオチド含有溶液を０．１５Ｍ ＮａＣｌに加え、粒子をさらに１２時間熟成させた。次いで、ＮａＣｌの濃度を０．３Ｍに高め、混合物をさらに２４〜４０時間放置してから、１００ｋＤａメンブレン（Ｓｐｅｃｔｒａ／Ｐｏｒ Ｃｅｌｌｕｌｏｓｅ Ｅｓｔｅｒ Ｍｅｍｂｒａｎｅ）を用いてＰＢＳ（０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液、ｐＨ７、０．０１％アジ化ナトリウム）で透析した。透析は４８時間にわたって実施し、その間に、透析浴を３回新しくした。
【０３５９】
このようにして調製したオリゴヌクレオチド−ＱＤ共役体は、不定な水安定性を示した。さらに、コロイドは、（〜３．２ｎｍ ＣｄＳｅコアを示す；Ｍｕｒｒａｙら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１１５巻，８７０６ページ，１９９３年）５４６ｎｍでの急激な［半値全幅（ＦＷＨＭ）＝３３ｎｍ］対称発光を有し、強力に蛍光性のままであった。
【０３６０】
このプロトコルにより、２種の異なるオリゴヌクレオチド−ＱＤ共役体を調製し、ＰＢＳ中に保存した。一方は、３′末端のプロピルチオール官能基と、１２ｍｅｒ捕獲配列と、介在１０塩基（すべてＡ）スペーサー：５′−ＴＣＴＣＡＡＣＴＣＧＴＡＡ_１０−（ＣＨ_２）_３−ＳＨ［配列番号４６］とからなる２２ｍｅｒで修飾した。他方は、５′−ヘキシルチオールを末端基とする配列と、さらに１０塩基（すべてＡ）スペーサー、および３′−プロピルチオール配列とは非相補的な１２ｍｅｒ捕獲配列：５′−ＳＨ−（ＣＨ_２）_６−Ａ_１０ＣＧＣＡＴＴＣＡＧＧＡＴ−３′［配列番号４７］を用いた。
【０３６１】
Ｃ． ＱＤ集合体の調製
ほぼ等量のこれら２種のオリゴヌクレオチド（それぞれ、２００μｌ、ＯＤ_５３０＝０．２２４および０．２０６）を混合し、次いで、相補的な連結２４ｍｅｒ配列（５′−ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴ−ＧＡＡＴＧＣＧ−３′、配列番号４８）の溶液６μｌ（６０ｐｍｏｌ）と合わせて、室温下に２０〜３０分以内でＱＤ集合体を形成した（図２６）。混合物を凍結（−７８℃）し、ゆっくり室温まで温めると、連結が速く起こった。
【０３６２】
生成したクラスターは、溶液から沈殿するほど大きくはなかった。しかし、クラスターは、連結しなかった粒子（３０，０００ｒｐｍで２〜３時間）と比べて、比較的低速で遠心（１０，０００ｒｐｍで１０分間）することにより分離することができた。
【０３６３】
ハイブリダイゼーション直後の蛍光の減少を、４対の試料の４７５ｎｍから６２５ｎｍまでの蛍光シグナル（励起波長３２０ｎｍ）の積分により定量した。各対は以下の方法で調製した。Ｅｐｐｅｎｄｏｒｆ遠心管中で、３′プロピルチオールを末端基とするＤＮＡで修飾した粒子（３０μｌ、５３０ｎｍでの光学密度＝０．２２４）の溶液を、５′ヘキシルチオールを末端基とするＤＮＡで修飾したＱＤ（３０μｌ、５３０ｎｍでの光学密度＝０．２０６）と合わせ、次いで１４０μｌのＰＢＳで希釈した。次いで、混合物を２つの等量部分に分け、一方には相補的「リンカー」ＤＮＡ（３μｌ、３０ｐｍｏｌ）を、他方には、非相補的「リンカー」ＤＮＡ（５′−ＣＴＡＣＴＴＡＧＡＴＣＣＧＡＧＴＧＣＣＣＡＣＡＴ−３′、配列番号４９）（３μｌ、３０ｐｍｏｌ）を加えた。次いで、全８種の試料をドライアイス／アセトン浴（−７８℃）中で凍結し、その後、試料を浴から取り出し、ゆっくり室温まで温めた。ハイブリダイゼーション時の蛍光効率の変化を予測するために、「標的」（相補的「リンカー」）試料の蛍光強度を、非相補的「リンカー」を含む対応する対照試料由来の３２０ｎｍでの吸光度における差を明らかにするように調整した。
【０３６４】
結果は、ＱＤ／ＱＤ集合体が、平均して２６．４±６．１％の積分蛍光強度の減少と、ＱＤ間の協同事象によると考えられる発光最大の〜２ｎｍのレッドシフトを伴うことを示した。興味深いことには、Ｂａｗｅｎｄｉらは、密集ＱＤと凍結マトリックス中で広く分離している隔離ドットの蛍光を比較したときに、類似ではあるが、わずかに大きなレッドシフトに気づいていた（Ｍｕｒｒａｙら，Ｓｃｉｅｎｃｅ，第２７０巻，１３３５ページ，１９９５年）。蛍光スペクトルにおけるこれらの変化は、ＱＤ間にエキシマが形成されたことを示しているのかもしれないが、そのような複合体の正確な性質は、大部分まだ類推の域を出ないものである。予測したように、「リンカー」が無いか、もしくは相補的でない場合、または２種の粒子のどちらかが不在の場合に、凝集は観察されなかった。
【０３６５】
温度の関数として凝集体のＵＶ−可視スペクトルを観察して、ＤＮＡの「融解」挙動をモニターした。この「融解」分析のために、ＱＤ／ＱＤ集合体を含む沈殿物を１０，０００ｒｐｍで１０分間遠心し、７μｌのＰＢＳで洗浄、再遠心して、０．７ｍｌのＰＢＳに懸濁させた。温度を２５℃から７５℃まで上昇させながら、各測定の前に１分間の保持時間をとって、集合体のＵＶ／可視分光シグナチャーを２℃間隔で記録した。実験を通じて均一性を確実にするために、混合物を５００ｒｐｍの速度で攪拌した。次いで、６００ｎｍでの消光から温度対消光プロファイルを作製した。これらのプロファイルの一次導関数を用いて「融解」温度を決定した。
【０３６６】
結果、図２７Ｂ（Ｔ_ｍ＝５７℃）は、ＤＮＡがＱＤ表面上に固定化され、ハイブリダイゼーションが集合体プロセスに関与することを明確に証明した。ＤＮＡだけと比較したときの偏移は極めて急激（それぞれの一次導関数のＦＷＨＭ：４℃対９℃）であり、これは、１粒子当たり多重ＤＮＡ結合を有する凝集体構造の形成と一致する。変性により消光の増大が観察されたが、これは、集合体内の粒子がその周囲のＱＤによる光の吸収から防止されるスクリーニング効果のためである可能性が高い。
【０３６７】
Ｄ． ＱＤ／金集合体の調製
ＤＮＡ官能化ＱＤが得られたので、多重種類のナノ粒子構築ブロックから作製したハイブリッド集合体の構成が実現可能になった。これらのハイブリッド集合体を作製するために、Ｅｐｐｅｎｄｏｒｆ遠心管中で、〜１７ｎＭの３′−ヘキシルチオールで修飾した１３ｎｍの金ナノ粒子（３０μｌ、〜５ｆｍｏｌ；実施例３に記載のように調製）の溶液を、５′−ヘキシルチオールを末端基とするＤＮＡ修飾ＱＤ（１５μｌ、５３０ｎｍでの光学密度は０．２０６）と混合した。「リンカー」ＤＮＡ（５μｌ、５０ｐｍｏｌ）を加え、混合物を−７８℃に冷却し、次いで、ゆっくり室温まで温めると、赤みを帯びた紫色の沈殿物が生成した。両種類の粒子と相補的標的の不在下には、凝集挙動は観察されなかった。遠心（３，０００ｒｐｍで１分間）し、上清を除去した後、沈殿物を１００μｌのＰＢＳで洗浄し、再遠心した。
【０３６８】
「融解」分析のために、洗浄した沈殿物を０．７ｍｌのＰＢＳに懸濁した。ＵＶ−可視分光法を用いて、金ナノ粒子の表面プラズモン共鳴の変化を追跡して、５２５ｎｍでの温度対消光プロファイルを作製した。金ナノ粒子の表面プラズモン共鳴を用いると、ハイブリダイゼーションをモニターするために、ＱＤだけのＵＶ−可視分光シグナチャーを用いるよりも、はるかに高感度のプローブが得られる。したがって、「融解」実験は、はるかに小さい試料（ＱＤ溶液の〜１０％が必要）に関して実施し得るが、プラズモンバンドの強さによって、ＱＤ由来のＵＶ／可視シグナルが不鮮明になる。上記の純粋ＱＤ系と同様に、急激な（一次導関数のＦＷＨＭ＝４．５℃）融解転移は、５８℃で起こった（図２７Ｄ参照）。
【０３６９】
これらの集合体の高解像度ＴＥＭ画像は、多重ＱＤにより相互連絡された金ナノ粒子網目構造を示した（図２７Ｃ）。ＴＥＭ画像では金ナノ粒子よりはるかにコントラストが弱いＱＤは、それらの格子干渉縞によって識別し得る。ＱＤは、高解像度ＴＥＭでかろうじて解像できるが、これらの複合体集合体の周期的構造やそれらの形成にＤＮＡが果たす役割を明瞭に示している。
【０３７０】
Ｅ． 要約
この実施例に記載されている結果は、ＱＤ表面へのＤＮＡの固定化が達成され、かつ今やこれらの粒子をハイブリダイゼーション条件下にＤＮＡと組合せて使用し得ることを最も確実に立証している。ＤＮＡ官能化ＱＤを用いて、最初のＤＮＡ誘導ＱＤ形成および混合金／ＱＤナノ粒子構造が実証された。半導体ＱＤをＤＮＡで首尾良く修飾することは、材料研究にとって重要な意味を有しており、これらの固有の構築ブロックは新規かつ機能的な多成分ナノ構造およびナノスケール材料に組み込まれるので、今や、これらのブロックの蛍光、電子、および化学特性の広範囲な研究への扉が開かれている。
【０３７１】
実施例１８： オリゴヌクレオチド−ナノ粒子共役体の合成法およびそれらの方法により生成された共役体
Ａ． 一般法
ＨＡｕＣｌ_４・３Ｈ_２Ｏおよびクエン酸三ナトリウムは、ウィスコンシン州ミルウォーキー所在のオールドリッチ・ケミカル社（Ａｌｄｒｉｃｈ Ｃｈｅｍｉｃａｌ Ｃｏｍｐａｎｙ、Ｍｉｌｗａｕｋｅｅ，ＷＩ）から購入した。純度９９．９９９％の金ワイヤ、およびチタンワイヤは、イリノイ州エバンストン所在のゴールドスミス社（Ｇｏｌｄｓｍｉｔｈ Ｉｎｃ．，Ｅｖａｎｓｔｏｎ，ＩＬ）から購入した。厚さ１ミクロンの酸化物層を有するシリコンウェーハ（１００）は、カリフォルニア州サンタクララ所在のシリコン・クエスト・インターナショナル社（Ｓｉｌｉｃｏｎ Ｑｕｅｓｔ Ｉｎｔｅｒｎａｔｉｏｎａｌ，Ｓａｎｔａ Ｃｌａｒａ，ＣＡ）から購入した。５′−チオール修飾剤Ｃ６−ホスホロアミダイト試薬、３′−プロピルチオール修飾剤ＣＰＧ、フルオレセインホスホロアミダイト、およびオリゴヌクレオチド合成に必要な他の試薬は、ヴァージニア州スターリング所在のグレン・リサーチ社から購入した。すべてのオリゴヌクレオチドは、標準ホスホロアミダイト化学（Ｅｃｋｓｔｅｉｎ，Ｆ．，Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ ａｎｄ Ａｎａｌｏｇｕｅｓ；第１版；Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ，Ｎｅｗ Ｙｏｒｋ，１９９１年）を用いるＤＮＡ自動合成機（エキスペダイト社（Ｅｘｐｅｄｉｔｅ））を使って調製した。５′ヘキシルチオール修飾体のみを含むオリゴヌクレオチドを実施例１に記載のように調製した。５−（および６−）−カルボキシフルオレセイン、スクシンイミジルエステルは、オレゴン州ユージーン所在のモレキュラー・プローブス社（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ，Ｅｕｇｅｎｅ，ＯＲ）から購入した。ＮＡＰ−５カラム（Ｓｅｐｈａｄｅｘ Ｇ−２５Ｍｅｄｉｕｍ、ＤＮＡグレード）は、ファルマシア・バイオテク社（Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）から購入した。すべての実験に、Ｂａｒｎｓｔｅａｄ ＮＡＮＯｐｕｒｅ超純水系を用いて精製したナノピュアＨ_２Ｏ（＞１８．０ＭΩ）を用いた。Ａｕナノ粒子溶液の遠心には、Ｅｐｐｅｎｄｏｒｆ ５４１５ＣまたはＢｅｃｋｍａｎ Ａｖａｎｔｉ ３０遠心機を用いた。高速液体クロマトグラフィーは、ＨＰシリーズ１１００ＨＰＬＣを用いて実施した。
【０３７２】
Ｂ． 物理測定
オリゴヌクレオチドおよびナノ粒子溶液の電子吸収スペクトルは、Ｈｅｗｌｅｔｔ−Ｐａｃｋａｒｄ（ＨＰ）８４５２ａダイオードアレイ分光光度計を用いて記録した。蛍光分光法は、Ｐｅｒｋｉｎ−Ｅｌｍｅｒ ＬＳ５０蛍光光度計を用いて実施した。透過型電子顕微鏡検査（ＴＥＭ）は、２００ｋＶで操作する日立８１００透過型電子顕微鏡で実施した。ナノ粒子溶液中の金の原子濃度の定量には、誘導結合プラズマ（ＩＣＰ）源を備えたＡｔｏｍＳｃａｎ２５原子分光計を用いた（金の発光は２４２．７９５ｎｍでモニターした）。
【０３７３】
Ｃ． 蛍光標識したアルカンチオール修飾オリゴヌクレオチドの合成および精製
５′ヘキシルチオール部分および３′フルオレセイン部分を用いて、１２塩基を含むチオール修飾オリゴヌクレオチド鎖を調製した。１２ｍｅｒ（Ｓ１２Ｆ）の配列は、ＨＳ（ＣＨ_２）_６５−ＣＧＣ−ＡＴＴ−ＣＡＧ−ＧＡＴ−３′−（ＣＨ_２）_６−Ｆ［配列番号５０］であり、３２ｍｅｒ（ＳＡ_２０１２Ｆ）は同じ１２ｍｅｒ配列とさらなる２０ｄＡスペーサー配列を５′端に有していた［配列番号５１］。チオール修飾オリゴヌクレオチドは、Ｓｔｏｒｈｏｆｆら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１２０巻：１９５９−１９６４ページ（１９９８年）に記載のように調製した。自動合成にはアミノ修飾剤Ｃ７ ＣＰＧ固相支持体を用い、５′末端は、先に記載のように、ヘキシルチオールホスホロアミダイトで手で修飾した。３′アミノ、５′トリチルで保護したチオール修飾オリゴヌクレオチドを、２５４ｎｍでＤＮＡのＵＶシグナルをモニターしながら、０．０３Ｍ トリエチルアンモニウムアセテート（ＴＥＡＡ）、ｐＨ７および１％／分勾配の９５％ＣＨ_３ＣＮ／５％０．０３Ｍ ＴＥＡＡを含むＨＰ ＯＤＳ Ｈｙｐｅｒｓｉｌカラム（５ｍｍ、２５０×４ｍｍ）を用いて、１ｍｌ／分の流速で逆相ＨＰＬＣにより精製した。５′−Ｓ−トリチル、３′アミノ修飾１２塩基および３２塩基オリゴヌクレオチドの保持時間はそれぞれ３６分、３２分であった。
【０３７４】
凍結乾燥産物を１ｍｌの０．１Ｍ Ｎａ_２ＣＯ_３に再分散させ、調製業者の指示（モレキュラー・プローブ社の文献）に従って、暗所で攪拌しながら、無水ＤＭＦ中１０ｍｇ／ｍｌのフルオレセインスクシンイミジルエステル（５，６ＦＡＭ−ＳＥ、モレキュラー・プローブス社）１００μｌを１．５時間かけて加えた。溶液を室温下にさらに１５時間攪拌し、次いで、−２０℃下に１００％エタノールから沈殿させた。沈殿物を遠心して回収し、Ｈ_２Ｏに溶解させ、イオン交換ＨＰＬＣにより、結合産物を反応しなかったアミノを末端基とするオリゴヌクレオチドから分離した。１０ｍＭ ＮａＯＨ水性溶離液および１％／分勾配の１Ｍ ＮａＣｌ／１０ｍＭ ＮａＯＨを用いて、０．８ｍｌ／分の流速で、Ｄｉｏｎｅｘ Ｎｕｃｌｅｏｐａｃ ＰＡ−１００カラム（２５０×４ｍｍ）を操作した。５′−Ｓ−トリチル、３′フルオレセイン修飾１２ｍｅｒおよび３２ｍｅｒの保持時間はそれぞれ５０分、４９分であった。オリゴヌクレオチド産物を逆相ＨＰＬＣで脱塩した。フルオレセインを末端基とするトリチルオリゴヌクレオチドのトリチル保護基の除去は、先に記載のように、（Ｓｔｏｒｈｏｆｆら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１２０巻，１９５９−１９６４（１９９８年））、硝酸銀およびジチオトレイトール（ＤＴＴ）を用いて行った。オリゴヌクレオチドの収率および純度は、アルキルチオールオリゴヌクレオチドに関して先に記載した技術（Ｓｔｏｒｈｏｆｆら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１２０巻：１９５９−１９６４ページ（１９９８年））を用いて評価した。オリゴヌクレオチドは、チオール基のトリチル除去処理直後に使用した。
【０３７５】
３′がプロピルチオールで５′フルオレセイン部分（ＨＳ（ＣＨ_２）_３−３′− （Ｗ）_２０−ＴＡＧ−ＧＡＣ−ＴＴＡ−ＣＧＣ−５′−（ＣＨ_２）_６−Ｆ、Ｗ＝ＡまたはＴ）である３２塩基を含むチオール修飾オリゴヌクレオチド［配列番号５２］を、３′チオール修飾剤ＣＰＧを使用して自動合成装置で合成した。各オリゴヌクレオチドの５′末端を、手作業で、フルオレセインホスホロアミダイト（６−ＦＡＭ（６−ＦＡＭ、Ｇｌｅｎ Ｒｅｓｅａｒｃｈ）につないた。修飾オリゴヌクレオチドを、イオン交換ＨＰＬＣで精製した（１Ｍ ＮａＣｌの１％／分勾配、１０ｍＭ ＮａＯＨ；保持時間（Ｒｔ）〜４８分（Ｗ＝Ｔ）、Ｒｔ〜２９分（Ｗ＝Ａ））。精製後、オリゴヌクレオチド溶液を逆相ＨＰＬＣで脱塩した。３′チオール部分を、以前に記述された方法（Ｓｔｏｒｈｏｆｆら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２０：１９５９−１９６４（１９９８））により、ジチオスレイトールで脱保護した。
【０３７６】
Ｄ．フルオレセイン標識オリゴヌクレオチドの合成および精製
発蛍光団標識相補体（１２′Ｆ）は、Ｓ１２ＦおよびＳＡ_２０１２Ｆの１２ｍｅｒ配列と相補的な１２塩基３′−ＧＣＧ−ＴＡＡ−ＧＴＣ−ＣＴＡ−５′−（ＣＨ_２）_６−Ｆ［配列番号５３］から構成した。標準法を用いてオリゴヌクレオチドを合成し、５′アルキルチオール修飾に関して上述した手順に類似のシリンジベース手順を用いて、フルオレセインホスホロアミダイト（６−ＦＡＭ、グレン・リサーチ社）とＣＰＧ連結オリゴヌクレオチドの５′末端とをカップリングした。上記のような逆相ＨＰＬＣを用いて精製を実施した。フルオレセイン標識オリゴヌクレオチドの保持時間は１８分であった。自動合成機用のアミノ修飾剤Ｃ７ ＣＰＧ固相支持体を用いて発蛍光団標識相補体、３′１２Ｆ（５′−ＡＴＣ−ＣＴＧ−ＡＡＴ−ＧＣＧ−Ｆ；［配列番号５４］）を調製し、次いで、上記手順を用いて、５−（６）−カルボキシフルオレセインスクシンイミジルエステルを３′アミンにカップリングした。
【０３７７】
Ｅ．金ナノ粒子の調製および特性決定
金ナノ粒子は、実施例１に記載のように、ＨＡｕＣｌ_４をクエン酸塩で還元して調製した。得られたナノ粒子のサイズ分布の定量には、日立８１００ＴＥＭを用いて実施する透過型電子顕微鏡検査（ＴＥＭ）を用いた。グラフィックソフトウエア（ＩａｍｇｅＴｏｏｌ）を用いてＴＥＭネガから少なくとも２５０粒子を分粒した。典型的な粒子配合物の平均直径は１５．７±１．２ｎｍであった。球状ナノ粒子と密度がバルク金の密度（１９．３０ｇ／ｃｍ^２）と等しいと仮定して、粒子当たりの平均分子量を計算した（２．４×１０^７ｇ／ｍｏｌ）。金ナノ粒子溶液中の金原子の濃度を、ＩＣＰ−ＡＥＳ（誘導結合プラズモン原子発光分光法）によって決定した。較正には、金原子吸着標準溶液（オールドリッチ社）を用いた。粒子溶液中の金原子濃度とＴＥＭ分析によって得た平均粒子質量とを比較して、所与の配合物中の金粒子のモル濃度、典型的には〜１０ｎＭを得た。表面プラズモン周波数（５２０ｎｍ）でのナノ粒子溶液のＵＶ−可視吸光度を測定して、粒子のモル消光係数（５２０ｎｍでのε）を計算すると、典型的には、直径１５．７±１．２ｎｍの粒子では４．２×１０^８Ｍ^−１ｃｍ^−１であった。
【０３７８】
Ｆ．金薄膜の調製
シリコンウェーハを〜１０ｍｍ×６ｍｍ部片に切断し、５０℃下にピラニア腐蝕溶液（４：１の濃Ｈ_２ＳＯ_４：３０％Ｈ_２Ｏ_２）で３０分清浄し、次いで、多量の水、次いでエタノールでリンスした。（警告：ピラニア腐蝕溶液は有機物質と激しく反応するので、取り扱いには厳重な注意が必要である。）Ｅｄｗａｒｄｓ ＦＴＭ６石英結晶微量天秤を具備したＥｄｗａｒｄｓ Ａｕｔｏ ３０６エバポレータ（３×１０−７ミリバールの基準圧力）を用いて０．２ｎｍ／秒の速度で金属を蒸着させた。シリコンの酸化された側面を、５ｎｍのＴｉ接着層、次いで２００ｎｍの金でコートした。
【０３７９】
Ｇ．５′アルキルチオールオリゴヌクレオチド修飾金ナノ粒子の調製
新たに脱保護したオリゴヌクレオチドをナノ粒子水溶液（粒子濃度〜１０ｎＭ）に３μＭの最終オリゴヌクレオチド濃度まで加えて、金ナノ粒子をフルオレセイン−アルキルチオールオリゴヌクレオチドで修飾した。２４時間後、溶液をｐＨ７（０．０１Ｍ リン酸）で緩衝し、ＮａＣｌ溶液を（０．１Ｍの最終濃度まで）加えた。これらの条件下にさらに４０時間、溶液を「熟成」させた。次いで、１４，０００ｒｐｍで３０分間遠心して、過剰な試薬を除去した。上清を除去した後、赤色の油性沈殿物を、０．３Ｍ ＮａＣｌ、１０ｍＭリン酸緩衝液、ｐＨ７（ＰＢＳ）で２回洗浄し、連続遠心して、再分散させ、最後に新鮮な緩衝液中に再分散させた。洗浄手順中、常に、少量（ＵＶ−可視分光法により測定して〜１０％）のナノ粒子が上清と共に廃棄される。したがって、最終ナノ粒子濃度を、ＴＥＭ、ＩＣＰ−ＡＥＳ、およびＵＶ−可視分光法（上記参照）で測定した。消光係数および粒度分布は、オリゴヌクレオチド修飾の結果として有意には変化しなかった。
【０３８０】
Ｈ．５′アルキルチオールオリゴヌクレオチド修飾金薄膜の調製
シリコン支持金薄膜を、金ナノ粒子の場合と同じ回数および同じ緩衝条件下に、脱保護したアルキルチオール修飾オリゴヌクレオチドの蒸着溶液中に浸漬した。オリゴヌクレオチド蒸着の後、膜を０．３Ｍ ＰＢＳでよくリンスし、緩衝液中に保存した。不動態化されていないケイ素／酸化ケイ素面を残して、片面の金のみを蒸発させた。しかし、アルキルチオール修飾ＤＮＡは、ＰＢＳに浸漬した何も結合していない酸化ケイ素表面には有意に吸着しなかった。
【０３８１】
Ｉ．ナノ粒子上に充填したアルキルチオールオリゴヌクレオチドの定量
オリゴヌクレオチドを置換するために、０．３Ｍ ＰＢＳ中の発蛍光団標識オリゴヌクレオチドで修飾したナノ粒子または薄膜に、メルカプトエタノール（ＭＥ）を（１２ｍＭの最終濃度まで）添加した。間欠的に振とうしながら、室温下に１８時間後、金ナノ粒子を遠心するか、または金薄膜を除去して、置換オリゴヌクレオチドを含有する溶液を金から分離した。０．３Ｍ ＰＢＳ、ｐＨ７を添加して、上清のアリコートを２倍希釈した。試料および較正標準溶液のｐＨとイオン強度を、これらの条件に対するフルオレセインの光学的性質の感度によるすべての測定値に関して、同じに維持するように注意した（Ｚｈａｏら，Ｓｐｅｃｔｒｏｃｈｉｍｉｃａ Ａｃｔａ，４５Ａ：１１１３−１１１６ページ（１９８９年））。（５２０ｎｍで測定した）最大蛍光を、標準線形較正曲線からの補間によりフルオレセイン−アルキルチオール修飾オリゴヌクレオチドのモル濃度に変換した。標準曲線は、同一の緩衝液および塩濃度を用いて発蛍光団標識オリゴヌクレオチドの既知濃度を用いて作製した。最後に、測定したオリゴヌクレオチドのモル濃度を元の金ナノ粒子濃度で割って、粒子当たりの平均オリゴヌクレオチド数を得た。次いで、ナノ粒子溶液中の（球状粒子を想定して）予測した粒子表面積で割って、正規表面被覆面積値を計算した。真円度の仮定条件は、計算した０．９３の平均真円度に基づく。真円度因数は、Ｂａｘｅｓ，Ｇｒｅｇｏｒｙ，Ｄｉｇｉｔａｌ Ｉｍａｇｅ Ｐｒｏｃｅｓｓｉｎｇ，１５７ページ（１９９４年）から引用した（４×ｐｉ×面積）（周長×２）として計算する。
【０３８２】
Ｊ．ハイブリダイズされた標的表面密度の定量
付着したオリゴヌクレオチドのハイブリダイゼーション活性を測定するために、蛍光発色団で標識したオリゴヌクレオチド（表面結合オリゴヌクレオチド（１２′Ｆ）に相補的）を、ハイブリダイゼーション条件（３μＭ 相補的オリゴヌクレオチド、０．３Ｍ ＰＢＳ、ｐＨ７、２４時間）下で、オリゴヌクレオチド修飾表面（金のナノ粒子または薄膜）と反応させた。ハイブリダイズしなかったオリゴヌクレオチドは、上述したように緩衝塩溶液で２度リンスすることにより金から取り外した。その後、蛍光発色団で標識したオリゴヌクレオチドを、ＮａＯＨの添加（最終濃度〜５０ｍＭ、ｐＨ１１〜１２、４時間）により、デハイブリダイズした。遠心によってナノ粒子溶液から１２′Ｆ含有溶液を分離した後、１Ｍ ＨＣｌの追加によって溶液を中和し、ハイブリダイズしたオリゴヌクレオチドの濃度とそれに対応するハイブリダイズされた標的表面密度を、蛍光分光法により測定した。
【０３８３】
Ｋ．表面被覆面積の定量とハイブリダイゼーション
クエン酸塩安定化金ナノ粒子を１２ｍｅｒフルオレセイン修飾アルキルチオールＤＮＡ（ＨＳ−（ＣＨ_２）_６５′−ＣＧＣ−ＡＴＴ−ＣＡＧ−ＧＡＴ−（ＣＨ_２）_４−Ｆ［配列番号５０］）で官能化した。次いで、化学吸着していないオリゴヌクレオチドを完全に洗い流した後で、発蛍光団標識オリゴヌクレオチドを金表面から除去して表面被覆面積研究を実施し、（上記のような）蛍光分光法を用いてオリゴヌクレオチド濃度を定量した。
【０３８４】
金表面からすべてのオリゴヌクレオチドを除去し、次いで溶液から金ナノ粒子を除去することは、いくつかの理由で、蛍光による正確な被覆面積データを得るために重要である。第１に、標識した表面結合ＤＮＡの蛍光シグナルは、金ナノ粒子への蛍光共鳴エネルギー移動（ＦＲＥＴ）の結果として効率的に消光される。実際、フルオレセイン修飾オリゴヌクレオチド（１２〜３２ヌクレオチド鎖、配列は上記に記載）が１５．７±１．２ｎｍの金ナノ粒子上に固定化され、溶液中の残留オリゴヌクレオチドが洗い流された後では、フルオレセイン修飾オリゴヌクレオチドに関して測定可能なシグナルはほとんど存在しない。第２に、金ナノ粒子は、２００〜５３０ｎｍの間に有意な量の光を吸収し、そのために、蛍光測定時の溶液中の金ナノ粒子の存在は、フィルターとしての役割を果たし、利用可能な励起エネルギーだけでなく放出される放射線の強度をも低下させる。フルオレセインの最大放出時には、５２０ｎｍでの金表面プラズモンバンドが減少する。
【０３８５】
メルカプトエタノール（ＭＥ）を用いて、交換反応により表面結合オリゴヌクレオチドを急速に置換させた。置換動力学を調べるために、遠心および蛍光測定の前に、オリゴヌクレオチド修飾ナノ粒子を、増加時間、ＭＥ（１２ｍＭ）に暴露した。ナノ粒子を含まない溶液に関連する蛍光の強さを用いて、ナノ粒子からどの位多くのオリゴヌクレオチドが放出されるかを測定することができる。ＭＥと交換に放出されたオリゴヌクレオチドの量は、約１０時間の暴露まで増大した（図２９）が、これは、完全なオリゴヌクレオチドの置換を示している。置換反応は高速であり、これは、オリゴヌクレオチド膜がＭＥの金表面へのアクセスをブロックし得ないためであると考えられる（Ｂｉｅｂｕｙｃｋら，Ｌａｎｇｍｕｉｒ，第９巻，１７６６ページ（１９９３年））。
【０３８６】
金ナノ粒子上のアルキルチオール修飾１２ｍｅｒオリゴヌクレオチド（Ｓ１２Ｆ）の平均オリゴヌクレオチド表面被覆面積は、３４±１ｐｍｏｌ／ｃｍ^２（試料の１０回の個別測定値の平均）であった。直径１５．７±１．２ｎｍの粒子の場合、これは、金粒子１個当たりおよそ１５９チオール結合１２ｍｅｒ鎖に相当する。バッチ毎に粒径はわずかに異なるものの、面積が正規化された表面被覆面積は、異なるナノ粒子配合物に関して類似していた。
【０３８７】
この方法が正確なオリゴヌクレオチド表面被覆面積を得るために有用であることを確認するために、金薄膜から発蛍光団標識オリゴヌクレオチドを置換する方法を用い、表面被覆面積データと類似の情報を得ることを目的とするが異なる技術を用いた実験と比較した。これらの実験では、金薄膜に対し、クエン酸塩安定化金ナノ粒子（上記参照）と類似のオリゴヌクレオチド修飾およびＭＥ置換手順を課した。金薄膜のオリゴヌクレオチド置換対時間曲線は、金ナノ粒子に関して測定したものと極めて類似している。これは、これらの薄膜に関して測定した典型的な表面被覆面積値が金ナノ粒子上のオリゴヌクレオチド被覆面積よりいくらか低かったにも拘わらず、薄膜の置換速度は類似していることを示唆している。本発明者らの技術によって測定した金薄膜上のオリゴヌクレオチド表面被覆面積（１８±３ｐｍｏｌ／ｃｍ^２）が、既に報告されたオリゴヌクレオチド薄膜上の被覆面積の範囲内（電気化学または表面プラズモン共鳴分光法（ＳＰＲＳ）を用いて測定された金電極上の２５塩基オリゴヌクレオチドに関しては１０ｐｍｏｌ／ｃｍ^２（Ｓｔｅｅｌら，Ａｎａｌ．Ｃｈｅｍ．，第７０巻，４６７０−４６７７ページ（１９９８年））に含まれることは重要である。表面被覆面積の相違は、オリゴヌクレオチドの配列と長さの違いおよび薄膜の調整方法の違いによるものと考えられる。
【０３８８】
表面結合ｌ２ｍｅｒオリゴヌクレオチドを備えたナノ粒子への相補的な蛍光発色団標識されたオリゴヌクレオチド（１２′Ｆ））のハイブリダイゼーションの範囲を、上述のように測定した。簡潔に説明すると、Ｓ１２Ｆ修飾ナノ粒子を、１２Ｆ′に３μＭの濃度でハイブリダイゼーション条件（０．３Ｍ ＰＢＳ、ｐＨ７）下で２４時間暴露し、次に、緩衝液で入念にリンスした。やはり、蛍光測定前に金からハイブリダイズした鎖を取り外すことが必要であった。これは、高ｐＨ溶液（ＮａＯＨ、ｐＨ１１）中で二重鎖ＤＮＡを変性させてから遠心にかけることにより遂行した。ハイブリダイズした１２′Ｆは、総計１．３±０．２ｐｍｏｌ／ｃｍ^２（１５．７ｎｍ粒子当たり約６つの二本鎖；１粒子当たりの二本鎖の平均数は、正規化したハイブリダイズされた表面被覆範囲（ｐｍｏｌ／ｃｍ^２）に、ＴＥＭによって測定した粒度分布から求められる平均粒子表面積を掛けることにより算出した。）に達した。非特異性吸着の程度を測定するために、Ｓ１２Ｆ修飾金ナノ粒子を、０．３Ｍ ＰＢＳ中で、蛍光発色団標識した非相補的な１２塩基オリゴヌクレオチド（１２Ｆ′）に暴露した。入念なリンス（連続する遠心／再分散ステップ）とそれに続く高ｐＨ処理後、ナノ粒子上の非特異的に吸着したオリゴヌクレオチドの被覆範囲は約０．１ｐｍｏｌ／ｃｍ^２であるとわかった。金電極に関する報告値とハイブリダイゼーション結果を比較するために、類似の方法を使用して、Ｓ１２Ｆ修飾金薄膜へのハイブリダイゼーションを測定した。ハイブリダイゼーション６＋２ｐｍｏｌ／ｃｍ^２の程度は、金電極上の混合塩基２５ｍｅｒに対して報告されているハイブリダイゼーション（２−６ｐｍｏｌ／ｃｍ^２）（Ｓｔｅｅｌら、Ａｎａｌ．Ｃｈｅｍ．７０：４６７０−４６７７（１９９８）と一致していた。
【０３８９】
ナノ粒子用システムおよび薄膜の両方に対するＳ１２Ｆ／１２Ｆ′系の表面被覆範囲およびハイブリダイゼーション値を、表７に要約する。最も顕著な結果は、ハイブリダイゼーション効率の低さである（ナノ粒子上の表面結合鎖の４％未満、薄膜上の鎖の３３％がハイブリダイズ）。以前の研究では、十分に密に充填されたオリゴヌクレオチド単分子層に対して、同様に低いハイブリダイゼーションが示されていた。これは、塩基の立体的密集性の組合せ（特に金表面付近の）と静電気反発相互作用とにより、入って来るハイブリダイズ鎖への接近容易性が低いことを反映している可能性がある。
【０３９０】
Ｌ．表面被覆範囲とハイブリダイゼーションに対するオリゴヌクレオチドスペーサーの影響
Ｓ１２Ｆオリゴヌクレオチドの適用範囲が高いことは、ナノ粒子安定化の点から有利であるが、その低いハイブリダイゼーション効率により、本願発明者らは、ハイブリダイズする配列の周囲の立体的混雑を減少させる手段を考案するように促された。アルキルチオール基と最初の１２塩基認識配列との間に２０ｄＡスペーサー配列が挿入された、オリゴヌクレオチド（３２ｍｅｒ）を合成した。このストラテジーを、以下の２つの仮定に基づき選択した。すなわち、１）ナノ粒子表面付近の塩基は、含窒素塩基と金表面間の相互作用の弱さと、鎖間の立体密集との故に、立体的に接近不能であること、および、２）直径１５．７ｎｍの概ね球状の粒子では、概ねその表面に対して垂直な２０ｍｅｒスペーサーユニットの端部に付着された１２ｍｅｒ配列（Ｌｅｖｉｃｋｙら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２０：９７８７−９７９２（１９９８））は、表面に直接結び付けられた同じ１２ｍｅｒから形成されたフィルムに比べてより大きな自由体積を有するフィルムにつながるだろう。
【０３９１】
一本鎖ＳＡ_２０１２Ｆ鎖の表面密度（１５±４ｐｍｏｌ／ｃｍ^２）はＳｌ２Ｆ（３４の±１ｐｍｏｌ／ｃｍ^２）よりも低いが、同一の表面修飾を使用して３２ｍｅｒで修飾した粒子は、１２ｍｅｒで修飾した粒子と比較して、かなりの安定性ｗｐ示した。予想されたように、ＳＡ_２０１２Ｆ／１２Ｆ′系のハイブリダイゼーション効率」（６．６±０．２ｐｍｏｌ／ｃｍ^２、４４％）は、元のＳ１２Ｆ／１２Ｆ′系のハイブリダイゼーション効率の約１０倍に増大した（表７）。
【０３９２】
Ｍ．オリゴヌクレオチド付着時の電解質濃度の効果
Ｓ１２Ｆ配列を用いた研究において、塩熟成ステップが、安定したオリゴヌクレオチド修飾ナノ粒子を得るのに重要であることが判明した（実施例３参照）。純水中Ｓ１２Ｆで修飾した金ナノ粒子を不可逆的に融解し、遠心すると、黒色沈殿物が形成されたが、塩中で熟成させたものは、高イオン強度溶液中で遠心しても凝集しなかった。安定性の増大は、立体保護作用および静電保護作用をもたらす高いオリゴヌクレオチド表面被覆面積によるものと考えられる。ＳＡ_２０１２Ｆ修飾粒子を用いて、オリゴヌクレオチド表面添加に及ぼす電解質条件の効果を調べた。図８に示すように、５′ヘキシルチオールおよび３′フルオレセイン部分を有するチオール修飾オリゴヌクレオチドを調製し、上記のように金ナノ粒子に付着させ、ナノ粒子上のこれらのオリゴヌクレオチドの表面被覆面積を上記のように定量した。結果は以下の表７に示されている。水中のオリゴヌクレオチドに４８時間暴露した金ナノ粒子の最終表面被覆面積は、塩で「熟成」させるか、または実験の最後の２４時間にわたって漸進的に塩濃度を増大させて（最終濃度は１．０Ｍ ＮａＣｌ）調製したものと比べると、はるかに低かった（７．９±０．２ｐｍｏｌ／ｃｍ^２）（上記参照）。
【０３９３】
合成された金ナノ粒子は、極めて低いイオン強度媒質中でさえ不可逆的に凝集することは注目に値する。確かに、金ナノ粒子は本来、塩、特にオリゴヌクレオチドなどのポリアニオンには不相溶である。安定したオリゴヌクレオチド粒子を調製するためには、この熟成処理が必須である。したがって、粒子は、イオン強度を徐々に増大させる前に、先ず、水中のアルキルチオールオリゴヌクレオチドで修飾しなければならない。オリゴヌクレオチドは、初期には、窒素含有塩基と金との弱い相互作用を介して結合した状態で平らに横たわっていると思われる。類似の相互作用モードが薄膜に付けたオリゴヌクレオチドに関して提案されている（Ｈｅｒｎｅら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１１９巻，８９１６−８９２０ページ（１９９７年））。しかし、オリゴヌクレオチドと正に帯電したナノ粒子表面との相互作用は、さらに強力であると予想される（Ｗｅｉｔｚら，Ｓｕｒｆ．Ｓｃｉ．，第１５８巻，１４７−１６４ページ（１９８５年））。熟成ステップにおいて、高イオン強度媒質は、隣接するオリゴヌクレオチド間の電荷反発だけでなくポリアニオンオリゴヌクレオチドと正に帯電した金表面との間の引力をも効率的に遮蔽する。これにより、より多くのオリゴヌクレオチドがナノ粒子表面に結合し、それによってオリゴヌクレオチド表面被覆面積が増大する。
【０３９４】
Ｎ．表面被覆範囲に対するオリゴヌクレオチドスペーサー配列の影響
スペーサーの配列がどのようにＡｕナノ粒子上のオリゴヌクレオチドの被覆範囲に影響するかを調べるために、３′プロピルチオールとフルオレセイン標識１２ｍｅｒ配列の間に２０ｄＡスペーサーおよび２０ｄＴスペーサーが挿入されたフルオレセイン修飾３２ｍｅｒ鎖を、調製した。Ｓ３′Ｔ_２０１２ＦとＳ３′Ａ_２０１２Ｆで修飾したナノ粒子に関する表面被覆範囲とハイブリダイゼーションの研究で最も顕著な結果は、２０ｄＡスペーサー（２４±１ｐｍｏｌ／ｃｍ^２）と比較して、２０ｄＴスペーサー（３５±１ｐｍｏｌ／ｃｍ^２）でより大きな表面被覆範囲が達成されたことである。ハイブリダイズした表面結合鎖のパーセンテージはＳＴ_２０ｌ２ｍｅｒナノ粒子（７９％）ではＳＡ_２０１２ナノ粒子（「〜９４％」より低かったが、ハイブリダイズした鎖の数は匹敵していた。これらの結果は、ｄＴ豊富なオリゴヌクレオチド鎖が、ｄＡ豊富なオリゴヌクレオチドよりも低い程度でナノ粒子表面と非特異的に相互作用することを示唆している。従って、２０ｄＡスペーサーセグメントが粒子表面上で平坦に横渡ることにより金の部位をブロックする一方、２０ｄＴスペーサーセグメントは、金の表面から垂直に延びることができ、これはより高い表面被覆範囲を促す。
【０３９５】
Ｏ．同時吸着された希釈剤オリゴヌクレオチドの影響
効率的なハイブリダイゼーションに加えて、オリゴヌクレオチド修飾ナノ粒子の別の重要な特性は、ハイブリダイゼーション事象の総数を調節する可能性である。これは、認識鎖の表面密度の調節により最も容易に遂行される。他の研究者は、ハイブリダイゼーションを制御するために、金電極上の修飾オリゴヌクレオチドと共に、メルカプトヘキサノールのよう同時吸着される希釈剤アルキルチオールを使用した（Ｓｔｅｅｌら、Ａｎａｌ．Ｃｈｅｍ．７０：４６７０−４６７７（１９９８）；Ｈｅｒｎｅら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１１９：８９１６−８９２０（１９９７））。しかしながら、保護されていない金ナノ粒子の固有の低い安定性は、希釈剤分子の選択に重大な制約を提起した。チオール修飾２０ｄＡ配列（ＳＡ_２０）［配列番号５５］は、ハイブリダイゼーションに必要な高いイオン強度の緩衝液中での粒子安定性を維持し、かつ非特異的吸着から表面を保護する点で、適切していることがわかった。
【０３９６】
ナノ粒子を、種々の認識鎖（ＳＡ_２０１２Ｆ）対希釈剤（ＳＡ_２０）鎖の分子比を有する溶液を用いて修飾した。得られた粒子を、ＳＡ_２０１２Ｆ表面密度の測定のために上述の蛍光方法で分析し、次に、１２′Ｆとのハイブリダイゼーション効率に関してテストした。
【０３９７】
ＳＡ_２０１２Ｆ表面密度は、堆積溶液中のＳＡ_２０１２Ｆ対ＳＡ_２０の比率に関して直線的に増加した（図３０）。これは、ナノ粒子に付けられたＳＡ_２０１２ＦとＳＡ_２０の比が溶液の比を反映することを示唆しているので、面白い結果である。この結果は、溶解度と鎖長が吸着動力学に重大な役割を果たす、短鎖アルキルまたはＴ官能化チオールの混合物に通常見られる結果と対照的である（Ｂａｉｎら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１１１：７１５５−７１６４（１９８９）；Ｂａｉｎら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１１１：７１６４−７１７５（１９８９）。
【０３９８】
個々の異なるサンプルにハイブリダイズした相補的な１２Ｆオリゴヌクレオチドの量も、ＳＡ_２０１２Ｆ表面被覆範囲の増加につれて直線的に増加した（図３１）。この関係が良好に定義されるという事実は、ナノ粒子−オリゴヌクレオチド共役体のハイブリダイゼーションの程度を予測および制御できることを示す。これは１２Ｆ′のハイブリダイゼーションがより高いＳＡ_２０１２Ｆ被覆範囲ではより困難になり、オリゴヌクレオチド間に立体的密集と静電気反発が起こる可能性が高いことを示唆している。
【０３９９】
Ｐ．要約
本研究は、オリゴヌクレオチドを付着させたナノ粒子を安定化させる程度に高いが、高い割合の鎖が溶液中のオリゴヌクレオチドとのハイブリダイゼーションのために接近できる程度に低く、オリゴヌクレオチド被覆範囲間のバランスを達成することが重要であることを示している。それは、オリゴヌクレオチドを、高いオリゴヌクレオチド表面被覆範囲を得るためにナノ粒子に付着させたり、静電相互作用を減らすためにオリゴヌクレオチドスペーサーセグメントに付着させたり、各ナノ粒子に対するハイブリダイゼーション事象の平均数を再現可能に制御するために同時吸着鎖を付着したりしている間に、塩条件を調節させることにより達成される。鎖（スペーサー）の配列の性質が、金ナノ粒子に装填されたオリゴヌクレオチド鎖の数に影響を及ぼすことも示された。この研究は、オリゴヌクレオチドとナノ粒子の間の相互作用の理解ならびにナノ粒子−オリゴヌクレオチド検出方法の感度の最良化に関して重要な意味合いを持つ。
【０４００】
【表７】

【化１】

【表８】

【表９】

【０４０１】
実施例１９： 遺伝子チップアッセイ
この実施例では、オリゴヌクレオチド官能化金ナノ粒子を用いたコンビナトリアルＤＮＡアレイの選択的が非常に高くかつ非常に高感度な分析法を説明する。非常に狭いナノ粒子−標的複合体熱解離温度範囲により、ヌクレオチド誤対合を１つ有する標的から所与のオリゴヌクレオチド配列を、非常に高い選択性で識別することができる。さらに、銀（Ｉ）のナノ粒子触媒還元に基づくシグナル増幅法と組合せると、このナノ粒子配列検出系の感度は、慣用の類似発蛍光団系の感度よりも２桁の大きさだけ優れている。
【０４０２】
科学者が病気の遺伝的根拠を明らかにし、この新規な情報を医学診断や治療の改良に用いるにつれ、配列選択的ＤＮＡ検出はますますその重要度を増している。サザンブロットやコンビナトリアルＤＮＡチップなどの一般に用いられている異種ＤＮＡ配列検出系は、標的ＤＮＡと相補的な表面結合一本鎖オリゴヌクレオチドの特異的ハイブリダイゼーションに基づいている。これらのアッセイの特異性も感度も、全対合配列および誤対合配列にハイブリダイズした捕獲鎖の解離特性に依存する。以下に説明するように、驚くべきことには、基板にハイブリダイズした単１種類のナノ粒子が類似の発蛍光団ベース系や非標識ＤＮＡよりも実質的に急激な融解プロファイルを示すことが発見された。さらに、ナノ粒子二本鎖の融解温度は、同一配列を有する類似の発蛍光団系の場合より１１℃も高い。これら２つの観察結果を、銀（Ｉ）のナノ粒子触媒還元に基づく定量的シグナル増幅法の開発と合わせると、１単塩基誤対合選択性および類似の慣用発蛍光団ベースアッセイよりも２桁も高い感度を有する新規なチップベースＤＮＡ検出系の開発が可能になる。
【０４０３】
３成分サンドイッチアッセイ形式（図３２参照）において、透明な基板にハイブリダイズした特定のＤＮＡ配列の存在を示すために、実施例３に記載のように調製したオリゴヌクレオチドが付着している金ナノ粒子（直径１３ｎｍ）を用いた。典型的な実験では、基板は、実施例１０に記載のようなアミン修飾プローブオリゴヌクレオチドを用いて、フロートガラス製顕微鏡スライド（フィッシャー・サイエンティフック社）を官能化して作製した。この方法を用いて、スライドの表面全体に１種類のオリゴヌクレオチドかまたは市販のマイクロアレイヤーを用いてスポットした複数種類のオリゴヌクレオチドアレイで官能化したスライドを作製した。次いで、指示オリゴヌクレオチドが付着しているナノ粒子および（炭疽菌保護抗原配列をベースとする）合成３０ｍｅｒオリゴヌクレオチド標的をこれらの基板に同時ハイブリダイズさせた（図３２参照）。したがって、表面のナノ粒子の存在によって、特定の３０塩基配列の検出が示された。標的濃度が高い（≧１ｎＭ）と、表面上のハイブリダイズした高密度のナノ粒子が、表面を明るいピンク色にした（図３３参照）。標的濃度が低い場合、付着しているナノ粒子は、（電界放出走査電子顕微鏡検査では画像が見られたが）肉眼で見ることはできなかった。基板表面にハイブリダイズしたナノ粒子の視覚化を容易にするために、銀イオンをヒドロキノンによって触媒的に還元してスライド表面上に銀金属を形成するシグナル増幅法を用いた。この方法は、組織化学顕微鏡検査研究において、タンパク質−および抗体−結合金ナノ粒子を拡大するために用いられている（Ｈａｃｋｅｒ，Ｃｏｌｌｏｉｄａｌ Ｇｏｌｄ：Ｐｒｉｎｃｉｐｌｅｓ，Ｍｅｔｈｏｄｓ，ａｎｄ Ａｐｐｌｉｃａｔｉｏｎｓ，Ｍ．Ａ．Ｈａｙａｔ編（Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｓａｎ Ｄｉｅｇｏ，１９８９年），第１巻，第１０章；Ｚｅｈｂｅら，Ａｍ．Ｊ．Ｐａｔｈｏｌ．，第１５０巻，１５５３ページ（１９９７年））が、その定量的ＤＮＡハイブリダイゼーションアッセイにおける使用は新規である（Ｔｏｍｌｉｎｓｏｎら，Ａｎａｌ．Ｂｉｏｃｈｅｍ．，第１７１巻，２１７ページ（１９８８年））。この方法は、ナノ粒子プローブの極めて低い表面被覆面積を簡単な平台型スキャナや肉眼で視覚化し得た（図３３）だけでなく、染色面積の光学密度に基づく標的ハイブリダイゼーションの定量も可能になった（図３４）。重要なことには、標的の不在下、または非相補的標的の存在下には、表面の染色は観察されず、これは、ナノ粒子の表面への非特異的結合も、非特異的銀染色も起こらないことを証明している。この結果は、核酸の非常に高い感度かつ非常に選択的な検出を可能にするこれらのナノ粒子−オリゴヌクレオチド共役体の優れた特徴である。
【０４０４】
さらに、本発明のオリゴヌクレオチド官能化ナノ粒子の固有のハイブリダイゼーション特性を、さらにコンビナトリアルオリゴヌクレオチドアレイ（または「遺伝子チップ」）の選択性を改良するために用い得ることが確認された（Ｆｏｄｏｒ，Ｓｃｉｅｎｃｅ，第２７巻，３９３ページ（１９９７年））。オリゴヌクレオチドアレイの異なる素子にハイブリダイズした標的の相対比は、標的配列決定時のアレイの正確性を決定するであろう。この比率は、異なる捕獲鎖とＤＮＡ標的との間で形成された二本鎖のハイブリダイゼーション特性に依存する。驚くべきことには、これらのハイブリダイゼーション特性は、発蛍光団標識の代わりにナノ粒子標的を用いることにより劇的に改良される。図３５に示されているように、ナノ粒子標識標的の表面結合捕獲鎖からのデハイブリダイゼーションは、同一配列を有する発蛍光団標識標的のものよりはるかに温度感受性である。発蛍光団標識標的は、極めて広範な温度範囲（一次導関数ＦＷＨＭ＝１６℃）にわたって表面捕獲鎖からデハイブリダイズしたが、同一ナノ粒子標識標的ははるかに急激に融解した（一次導関数ＦＷＨＭ＝３℃）。これらの急勾配の解離プロファイルは、通常、ハイブリダイゼーションストリンジェンシー洗浄によって影響を受ける、チップベースの配列分析のストリンジェンシーを改善すると予想された。確かに、相補的表面プローブにハイブリダイズした標的と、（図３５の水平線によって表される）特定温度下のストリンジェンシー洗浄後に、誤対合プローブにハイブリダイズした標的との比率は、発蛍光団標識よりナノ粒子標識の方がはるかに高い。これは、チップ検出形式における高選択性を意味する筈である。さらに、ナノ粒子標識は、それによって、それより低いと、二本鎖が室温下に自発融解する臨界濃度を低下させる表面二本鎖の融解温度（Ｔ_ｍ）を上昇させることによりアレイ感度を向上させるであろう。
【０４０５】
オリゴヌクレオチドアレイの比色指示剤としてのナノ粒子の有効性を評価するために、テストチップを合成標的でプローブし、発蛍光団指示剤とナノ粒子指示剤とで標識した。テストアレイとオリゴヌクレオチド標的は、公開されているプロトコル（Ｇｕｏら，Ｎｕｃｌ．Ａｃｉｄｓ Ｒｅｓ．，第２２巻，５４５６ページ（１９９４年）；Ｇｅｎｅｔｉｃ Ｍｉｃｒｏｓｙｓｔｅｍｓ ４１７ Ｍｉｃｒｏａｒｒａｙｅｒを用いて、３７５μｍで分離された直径１７５μｍのスポットアレイをパターン化した）に従って作製した。アレイは、標的の８位の４つの可能なヌクレオチド（Ｎ）それぞれに対応する４つの素子を含んでいた（図３２参照）。ハイブリダイゼーション緩衝液中で、合成標的と、蛍光標識またはナノ粒子標識プローブとを段階的にアレイにハイブリダイズさせたが、各ステップの後で、３５℃のストリンジェンシー緩衝液洗浄を行った。先ず、２×ＰＢＳ（０．３Ｍ ＮａＣｌ、１０ｍＭ Ｎａ_２Ｈ_２ＰＯ_４／Ｎａ_２ＨＰＯ_４緩衝液、ｐＨ７）中の１ｎＭ 合成標的溶液２０μｌとアレイとを、室温下に４時間、ハイブリダイゼーションチャンバ（Ｇｒａｃｅ Ｂｉｏ−Ｌａｂｓ Ｃｏｖｅｒ Ｗｅｌｌ ＰＣ２０）中でハイブリダイズさせ、次いで、３５℃下に清浄な２×ＰＢＳ緩衝液で洗浄した。次いで、２×ＰＢＳ中１００ｐＭ オリゴヌクレオチド官能化金ナノ粒子溶液２０μｌとアレイとを、室温下に４時間、新鮮なハイブリダイゼーションチャンバ中でハイブリダイズさせた。アレイを３５℃下に清浄な２×ＰＢＳで洗浄し、次いで、２×ＰＢＳ（０．３Ｍ ＮａＮＯ_３、１０ｍＭ ＮａＨ_２ＰＯ_４／Ｎａ_２ＨＰＯ_４緩衝液、ｐＨ７）で２回洗浄した。次いで、ナノ粒子アレイを銀増幅溶液（シグマ・ケミカル社、Ｓｉｌｖｅｒ Ｅｎｈａｎｃｅｅｒ Ｓｏｌｕｔｉｏｎ）中に５分間浸漬し、水で洗浄した。銀増幅により、アレイ素子はかなり暗色化し、直径２００μｍの素子は平台型スキャナまたは肉眼でさえ容易に見ることができた。
【０４０６】
モデル標的およびナノ粒子標識プローブで攻撃し、銀溶液で染色したアレイは、相補的アレイ素子への選択性の高いハイブリダイゼーションを明瞭に示した（図３６Ａ）。同じ捕獲配列の重複スポットは、再現性があってばらつきのないハイブリダイゼーションシグナルを示した。ナノ粒子または銀染料によるバックグラウンド吸着は全く観察されなかった。平台型スキャナによって記録された画像グレースケール値は、清浄な顕微鏡スライドに関して観察されたものと同じであった。８位のアデニン（Ｎ＝Ａ）に対応する暗いスポットは、オリゴヌクレオチド標的が、３：１を超える比率で、誤対合鎖よりも完全相補性捕獲鎖に選択的にハイブリダイズしたことを示している。さらに、各スポットセットの総合グレースケール値は、ワトソン・クリック型塩基対、Ａ：Ｔ＞Ｇ：Ｔ＞Ｃ：Ｔ＞Ｔ：Ｔ（Ａｌｌａｗｉら，Ｂｉｏｃｈｅｍｉｓｔｒｙ，第３６巻，１０５８１ページ（１９８８年））の予測安定性に従う。通常、Ｇ：Ｔ誤対合をＡ：Ｔ相補体から識別するのは特に難しく（Ｓａｉｋｉら，Ｍｕｔａｔｉｏｎ Ｄｅｔｅｃｔｉｏｎ，Ｃｏｔｔｏｎら編（Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ，Ｏｘｆｏｒｄ，１９９８年），第７章；Ｓ．Ｉｋｕｔａら，Ｎｕｃｌ．Ａｃｉｄｓ Ｒｅｓ．，第１５巻，７９７ページ（１９８７年））、これら２種のアレイ素子の識別は、１つのヌクレオチド誤対合の検出におけるナノ粒子標識の驚異的な分解能を証明している。ナノ粒子ベースアレイの選択性は、発蛍光団指示アレイの選択性より高く（図３６Ｂ）、発蛍光団標識は８位のアデニンに対して２：１の選択性を提供したに過ぎない。
【０４０７】
ナノ粒子標識プローブを利用するアッセイは、発蛍光団標識プローブを利用するものより感度が有意に高かった。ハイブリダイゼーションシグナルを、５０ｆＭ程度の低さ（または、２０μｌの溶液を含有するハイブリダイゼーションチャンバの場合は、１×１０^６総コピー数）の標的濃度のＮ＝Ａ要素で分解することができた。これは、通常、１ｐＭ以上の標的濃度が要求される慣用のＣｙ３／Ｃ７５発蛍光団標識アレイに比べて劇的な感度の向上を示している。表面上に固定化されたナノ粒子−標的複合体に関して観察された高融解温度がアレイの感度に寄与していることは疑いもない。ナノ粒子系の場合に発蛍光団系に比べてプローブ／標的／表面−オリゴヌクレオチド複合体の安定性が増大すると、洗浄ステップ中に失われる標的およびプローブが少なくなると考えられる。
【０４０８】
コンビナトリアルオリゴヌクレオチドアレイの比色性ナノ粒子標識は、誤対合１つの解像、感度、コスト、使用し易さが重要な要素である一ヌクレオチド多型分析などの用途に有用であろう。さらに、全体的に最適化する必要があるこの系の感度は、ポリメラーゼ連鎖反応などの標的増幅計画を必要とせずに、オリゴヌクレオチド標的を検出するための潜在的な方法を示している。
【０４０９】
実施例２０： ナノ粒子構造
ハイブリダイズしたＤＮＡリンカーを媒介としたガラス支持体上への超分子重層金ナノ粒子構造の可逆的集合体を説明する。オリゴヌクレオチド官能化ナノ粒子を、相補的ＤＮＡリンカーの存在下、オリゴヌクレオチド官能化ガラス支持体に連続付着させる。ＤＮＡの独特な認識特性により、相補的リンカーの存在下にナノ粒子構造が選択的に集合する。さらに、これらの構造は、溶液の温度、ｐＨ、およびイオン強度を含めた連結二本鎖ＤＮＡのハイブリダイゼーションを媒介する外部刺激に応答して集合したり、離散したりし得る。この系は、固相支持体上にナノ粒子ベース構造を構築する極めて選択的かつ制御された方法を提供することに加えて、ＤＮＡによって連結されたナノ粒子網目構造の光学特性と融解特性とに影響を与える要素の研究を可能にする。
【０４１０】
別の者は、いかにして、二官能価有機分子（Ｇｉｔｔｉｎｓら，Ａｄｖ．Ｍａｔｅｒ．，第１１巻，７３７ページ（１９９９年）；Ｂｒｕｓｔら，Ｌａｎｇｍｕｉｒ，第１４巻：５４２５ページ（１９９８年）；Ｂｒｉｇｈｔら，Ｌａｎｇｍｕｉｒ，第１４巻：５６９６ページ（１９９８年）；Ｇｒａｂａｒら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１１８巻：１１４８ページ（１９９６年）；Ｆｒｅｅｍａｎら，Ｓｃｉｅｎｃｅ，第２６７巻：１６２９ページ（１９９５年）；Ｓｃｈｍｉｄら，Ａｎｇｅｗ．Ｃｈｅｍ．Ｉｎｔ．Ｅｄ．Ｅｎｇｅｌ．，第３９巻：１８１ページ（２０００年）；Ｍａｒｉｎａｋｏｓら，Ｃｈｅｍ．Ｍａｔｅｒ．，第１０巻：１２１４ページ（１９９８年））または高分子電解質（Ｓｔｏｒｈｏｆｆら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１２０巻，１９５９ページ（１９９８年）；Ｓｔｏｒｈｏｆｆら，Ｊ．Ｃｌｕｓｔｅｒ Ｓｃｉ．，第８巻：１７９ページ（１９９７年）；Ｅｌｇｈａｎｉａｎら，Ｓｃｉｅｎｃｅ，第２７７巻：１０７８ページ（１９９７年）；Ｍｉｒｋｉｎら，Ｎａｔｕｒｅ 第３８２巻，６０７ページ（１９９６年））を使用して平面構造から単層および多層ナノ粒子材料を制御可能に構築し得るかを証明した。ナノ粒子の相互連結体としてＤＮＡを用いることの魅力的な特徴は、ＤＮＡ配列を選択することにより、粒子間間隔、粒子周期性および粒子組成を合成的にプログラムし得ることである。さらに、オリゴヌクレオチドの可逆結合特性を利用して、動構造ではなく熱力学的構造を確実に形成し得る。この方法は、固相支持体からのナノ粒子ベース構造の成長を制御する新規かつ強力な方法を提供するだけでなく、ナノ粒子凝集体サイズと、凝集体ＤＮＡ相互連結構造の融解特性および光学特性との関係を評価することもできる。これら２つの物理的パラメータとそれらの材料構築物との関係は、特にバイオ検出分野におけるナノ粒子網目構造材料の利用に必須である。
【０４１１】
多層集合体の構築に用いられる直径１３ｎｍのオリゴヌクレオチド官能化金ナノ粒子を実施例１および３に記載のように調製した。これらのナノ粒子には、それぞれナノ粒子ａおよびｂを生成する、５′−ヘキシルチオール−キャップオリゴヌクレオチド１（５′−ＨＳ（ＣＨ_２）_６Ｏ（ＰＯ_２ ⁻）Ｏ−ＣＧＣＡＴＴＣＡＧＧＡＴ−３′［配列番号５０］と、３′−プロパンチオール−キャップオリゴヌクレオチド２（３′−ＨＳ（ＣＨ_２）_３Ｏ（ＰＯ_２ ⁻）Ｏ−ＡＴＧＣＴＣＡＡＣＴＣＴ−５′［配列番号５９］が付着していた（図３７参照）。実施例１０に記載のように、ガラス製スライドを１２ｍｅｒオリゴヌクレオチド２で官能化した。ナノ粒子層を構築するために、先ず、基板を２４ｍｅｒリンカー３（５′−ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴＧＡＡＴＧＣＧ−３′［配列番号６０］）の１０ｎＭ溶液に浸漬し、室温下に４時間ハイブリダイズさせた（図３７参照）。支持体を清浄な緩衝液で洗浄し、次いで、第１ナノ粒子層を付着させるために室温下に４時間、粒子ａの２ｎＭ溶液とハイブリダイズさせた。同様に基板表面をリンカー３とナノ粒子ｂの溶液に暴露するだけで第２ナノ粒子層を第１ナノ粒子層に付着させることができた。これらのハイブリダイゼーションステップを繰り返して、各層がリンカー３を介して前の層に連結したナノ粒子ａとｂの多重交互層を付着させることができた。リンカーの不在下、または非相補的オリゴヌクレオチドの存在下では、表面へのナノ粒子のハイブリダイゼーションは観察されなかった。さらに、ＤＮＡリンカーのハイブリダイゼーションを促進させる条件：中性ｐＨ、穏和な塩濃度（＞０．０５Ｍ ＮａＣｌ）および二本鎖融解温度（Ｔ_ｍ）を下回る温度下にのみ多層集合体が観察された。
【０４１２】
ハイブリダイズした各ナノ粒子層は、基板をより暗紅色とし、１０層がハイブリダイズした後の支持ガラス製スライドには、反射による金色が出現した。表面へのナノ粒子の連続ハイブリダイゼーションのモニターには、基板の透過ＵＶ−可視分光法を用いた（図３８Ａ）。初期ナノ粒子層の吸光度の低さは、この層がさらなる層の形成を促進するもととなったことを示唆しており、層を追加するに従って、プラズモンバンドの強度がほぼ線形に増大することが示された（各連続ナノ粒子層の形成に関して、リンカー３またはナノ粒子溶液の暴露時間を長くしたり、濃度を増大させたりしても、さらなる吸光度の増大は観察されなかった）。初期ナノ粒子層生成後に吸光度が線形に増大したことは、連続的に層を加えるに従って、表面がハイブリダイズしたナノ粒子で飽和されたことを示している（図３８Ｂ）。これは、１層の場合には低いナノ粒子被覆を示すが、２層の場合にはほぼ完全な被覆を示す表面上の１つ（図３９Ａ）および２つ（図３９Ｂ）のナノ粒子層の電界放出走査型電子顕微鏡（ＦＥ−ＳＥＭ）画像によって支持される。多層集合体のプラズモンバンドλ_ｍａｘは、５層になった後でさえ、１０ｎｍ以下しかシフトしない。このシフトの検出は、金ナノ粒子凝集体の他の実験的処理（Ｇｒａｂａｒら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１１８巻，１１４８ページ（１９９６年））および理論的処理（Ｑｕｉｎｔｅｎら，Ｓｕｒｆ．Ｓｃｉ．，第１７２巻，５５７ページ（１９９６年）；Ｙａｎｇら，Ｊ．Ｃｈｅｍ．Ｐｈｙｓ．，第１０３巻：８６９ページ（１９９５年））と一致する。しかし、シフトの大きさは、λ_ｍａｘ＞５７０ｎｍを示す、オリゴヌクレオチドに連結した金ナノ粒子網目構造の懸濁液に関して先に観察されたものと比較すると小さい（先の実施例参照）。これは、金ナノ粒子ベースのオリゴヌクレオチドプローブに関して観察された赤色から青色への劇的な変色を生成するには、恐らく何百または何千ものもっと多くのナノ粒子が連結する必要があることを示唆している。（Ｓｔｏｒｈｏｆｆら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１２０巻，１９５９ページ（１９９８年）；Ｓｔｏｒｈｏｆｆら，Ｊ．Ｃｌｕｓｔｅｒ Ｓｃｉ．，第８巻，１７９ページ（１９９７年）；Ｅｌｇｈａｎｉａｎら，Ｓｃｉｅｎｃｅ，第２７７巻，１０７８ページ（１９９７年）；Ｍｉｒｋｉｎら，Ｎａｔｕｒｅ，第３８２巻，６０７ページ（１９９６年））。凝集した金ナノ粒子の表面プラズモンシフトは、粒子間間隔に大きく依存することが明らかにされた（Ｑｕｉｎｔｅｎら，Ｓｕｒｆ．Ｓｃｉ．，第１７２巻：５５７ページ（１９８６年）；Ｓｔｏｒｈｏｆｆら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，前掲）。オリゴヌクレオチドリンカーにより提供される大きな間隔（この系の場合８．２ｎｍ）によって、金表面プラズモンバンドに及ぼすナノ粒子凝集の累積的効果が有意に低下する。
【０４１３】
集合したナノ粒子多層の解離特性は、層の数に大きく依存していた。多層でコートされた基板を緩衝液に懸濁し、温度を連結オリゴヌクレオチドのＴ_ｍより高い温度（５３℃）に上げると、ナノ粒子は、無色のガラス表面を残して、溶液中に解離した。緩衝懸濁液のｐＨを上下させたり（＞１１または＜３）、塩濃度を低下させたり（０．０１Ｍ ＮａＣｌ未満）しても、連結したＤＮＡをデハイブリダイズすることによりナノ粒子は解離した。多層集合体は完全に可逆性であり、ナノ粒子は、ガラス基板にハイブリダイズしたり、ガラス基板からデハイブリダイズしたりし得る（例えば、３つのサイクルは、検出可能な不可逆性ナノ粒子結合がないことが示された）。
【０４１４】
重要なことには、表面結合ナノ粒子集合体はすべて連結オリゴヌクレオチドのＴ_ｍを超える温度で解離したが、これらの転移の急激さは、支持されている凝集体のサイズに依存していた（図３９Ｄ−Ｆ）。驚くべきことには、支持体からの第１ナノ粒子層の解離は、溶液中にナノ粒子を含まない同じオリゴヌクレオチド（図３９Ｃ）のものより急激な転移（図３９Ｄ、一次導関数のＦＷＨＭ＝５℃）を示した。基板にさらにナノ粒子層がハイブリダイズするにつれ、オリゴヌクレオチド連結ナノ粒子の融解転移は、溶液中に見出された大きなナノ粒子網目構造集合体の融解転移と同等になるまで連続的に急激さを増した（図３９Ｅ−Ｆ、一次導関数のＦＷＨＭ＝３℃）。（Ｇｉｔｔｉｎｓら，Ａｄｖ．Ｍａｔｅｒ．，第１１巻：７３７ページ（１９９９年）；Ｂｒｕｓｔら，Ｌａｎｇｍｕｉｒ，第１４巻：５４２５ページ（１９９８年）。）これらの実験は、最適な急激融解曲線を得るためには、３つ以上のナノ粒子と多重ＤＮＡの相互連結体が必要であることを立証している。これらの実験はさらに、この系における光学的変化が融解特性から完全に切り離されている（すなわち、凝集が小さいと、急激な転移は生じ得るが、それでも変色は生じない）ことを示している。
【０４１５】
実施例２１： 金ナノ粒子集合体の電気的性質
ＤＮＡを介した電子的移動は、過去数年にわたり化学の分野で最も広範囲に討議されている問題の１つである。（Ｋｅｌｌｅｙら，Ｓｃｉｅｎｃｅ，第２８３巻：３７５−３８１ページ（１９９９年）；Ｔｕｒｒｏら，ＪＢＩＣ、第３巻：２０１−２０９ページ（１９９８年）；Ｌｅｗｉｓら，ＪＢＩＣ，第３巻：２１５−２２１ページ（１９９８年）；Ｒａｔｎｅｒ，Ｍ．，Ｎａｔｕｒｅ，第３９７巻，４８０−４８１ページ（１９９９年）；Ｏｋａｈａｔａら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１２０巻，６１６５−６１６６ページ（１９９８年））。ＤＮＡは効率的に電子を移動させ得ると主張するものもいるが、ＤＮＡは絶縁体であると考えるものもいる。
【０４１６】
表面上類似点の無い研究分野において、ナノ粒子をベースとした材料の電気的性質の研究に多大な努力が傾けられてきた（Ｔｅｒｒｉｌｌら，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，第１１７巻，１２５３７−１２５４８ページ（１９９５年）；Ｂｒｕｓｔら，Ａｄｖ．Ｍａｔｅｒ．，第７巻，７９５−７９７ページ（１９９５年）；Ｂｅｔｈｅｌｌら，Ｊ．Ｅｌｅｃｔｒｏａｎａｌ．Ｃｈｅｍ．，第４０９巻：１３７−１４３ページ（１９９６年）；Ｍｕｓｉｃｋら，Ｃｈｅｍ．Ｍａｔｅｒ．，第９巻：１４９９−１５０１ページ（１９９７年）；Ｂｒｕｓｔら，Ｌａｎｇｍｕｉｒ，第１４巻，５４２５−５４２９ページ（１９９８年）；Ｃｏｌｌｉｅｒら，Ｓｃｉｅｎｃｅ，第２７７巻：１９７８−１９８１ページ（１９９７年））。確かに、多くのグループがナノ粒子を二次元および三次元網目構造に集合させる方法を探究し、そのような構造の電子特性を調査してきた。しかし、ＤＮＡと連結させたナノ粒子ベース材料の電気的性質については実質的に何も分っていない。
【０４１７】
この研究において初めて、異なる長さのＤＮＡ相互連結体によって形成された金ナノ粒子集合体の電気的性質が調査された。以下に示されているように、これらのハイブリッド無機集合体は、２４〜７２ヌクレオチド範囲にわたるオリゴヌクレオチド粒子相互連結体長にも拘わらず、半導体として挙動する。本明細書に記載されている結果は、ＤＮＡ相互共役体が、金属ナノ粒子間の絶縁バリヤを形成不要で、このため粒子の電気的性質を破壊することのない、金属ナノ粒子用の化学的に特異的な足場材料として用い得ることを示している。これらの結果は、そのようなハイブリッド集合体を電子材料として利用し得る新規な方法を示している。
【０４１８】
この主題の中心には以下の問題がある：ＤＮＡを介して集合したナノ粒子は、それでも電気を伝導し得るか、または各粒子上に重度に添加されたＤＮＡ相互共役体は絶縁シェルとして作用し得るか、ということである（Ｍｕｃｉｃ，Ｒ．Ｃ．，Ｓｙｎｔｈｅｔｉｃａｌｌｙ Ｐｒｏｇｒａｍｍａｂｌｅ Ｎａｎｏｐａｒｔｉｃｌｅ Ａｓｓｅｍｂｌｙ Ｕｉｓｎｇ ＤＮＡ，Ｔｈｅｓｉｓ Ｐｈ．Ｄ．，Ｎｏｒｔｈｗｅｓｔｅｒｎ Ｕｎｉｖｅｒｓｉｔｙ（１９９９年））。これらの材料の導電率を、温度、オリゴヌクレオチド長および相対湿度の関数として調べた。ＤＮＡによって連結されたナノ粒子構造を、電界放出走査型電子顕微鏡検査（ＦＥ−ＳＥＭ）、シンクロトロン小角Ｘ線散乱（ＳＡＸＳ、ｓｙｎｃｈｒｏｔｒｏｎ ｓｍａｌｌ ａｎｇｌｅ ｘ−ｒａｙ ｓｃａｔｔｅｒｉｎｇ）実験、熱変性プロファイル、およびＵＶ−可視分光法によって特性決定した。
【０４１９】
典型的な実験（図４０参照）では、クエン酸塩で安定化した１３ｎｍ金ナノ粒子を、実施例１および３に記載のように、３′および５′アルカンチオールでキャップした１２ｍｅｒオリゴヌクレオチド１（３′ＳＨ（ＣＨ_２）_２Ｏ（ＰＯ_２ ⁻）Ｏ−ＡＴＧＣＴＣＡＡＣＴＣＴ５′［配列番号５９］）および２（５′ＳＨ（ＣＨ_２）_６Ｏ（ＰＯ_２ ⁻）Ｏ−ＣＧＣＡＴＴＣＡＧＧＡＴ３′［配列番号５０］）で修飾した。２４、４８または７２塩基長のＤＮＡ鎖３（５′ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴＧＡＡＴＧＣＧ３′［配列番号６０］）、４（５′ＴＡＣＧＡＧＴＴＧＡＧＡＣＣＧＴＴＡＡＧＡＣＧＡＧＧＣＡＡＴＣ−ＡＴＧＣＡＡＴＣＣＴＧＡＡＴＧＣＧ３′［配列番号６１］）、および５（５′ＴＡＣＧＡＧＴＴＧＡＧＡＣＣＧＴＴＡＡＧＡＣＧＡＧＧＣＡＡＴＣＡＴＧＣＡＴＡＴＡＴＴＧＧＡＣＧＣＴＴＴＡＣＧＧＡＣＡＡＣＡＴＣＣＴＧＡＡＴＧＣＧ３′［配列番号６２］）をリンカーとして用いた。充填剤６（３′ＧＧＣＡＡＴＴＣＴＧＣＴＣＣＧＴＴＡＧＴＡＣＧＴ５′［配列番号６３］）および７（３′ＧＧＣＡＡＴＴＣＴＧＣＴＣＣＧＴＴＡＧＴＡＣＧＴＡＴＡＴＡＡＣＣＴＧＣＧＡＡＡＴＧＣＣＴＧＴＴＧ５′［配列番号６４］）を４８塩基リンカーおよび７２塩基リンカーと共に用いた。ＤＮＡ修飾ナノ粒子とＤＮＡリンカーおよび充填剤は、使用前に、０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸（ｐＨ７）緩衝液（０．３Ｍ ＰＢＳと称される）中に貯蔵した。ナノ粒子集合体を構築するために、１で修飾した金ナノ粒子（６５２μｌ、９．７ｎＭ）と２で修飾した金ナノ粒子（６５２μｌ、９．７μＭ）をリンカーＤＮＡ３、４、または５（３０μｌ、１０ｎＭ）に加えた。完全に沈殿させた後、凝集体を０．３Ｍ ＣＨ_３ＣＯＯＮＨ_４溶液で洗浄して過剰なＤＮＡおよびＮａＣｌを除去した。
【０４２０】
凝集体を凍結乾燥（１０^−３〜１０^−２トル）乾固してペレットを得、揮発性塩ＣＨ_３ＣＯＯＮＨ_４を除去した。Ｆｒｅｎｓ法（Ｆｒｅｎｓ，Ｎａｔｕｒｅ Ｐｈｙｓ．Ｓｃｉ．，第２４１巻：２０−２２ページ（１９７３年））により調製した官能化されていないクエン酸塩安定化粒子を膜として乾燥させ、比較用に用いた。得られた乾燥凝集体は曇った黄銅に似た色を有しており、非常に脆かった。ＦＥ−ＳＥＭ画像は、オリゴヌクレオチド修飾ナノ粒子は乾燥しても無傷のままであるが、クエン酸塩安定化ナノ粒子は互いに融合したことを示した。乾燥したＤＮＡを介して連結された凝集体が０．３Ｍ ＰＢＳ緩衝液（１ｍｌ）に再分散し得、かつ優れた融解特性を示したことは重要である。そのような分散液を６０℃に加熱すると、ＤＮＡ相互共役体のハイブリダイゼーションが起こり、分散したナノ粒子の赤色溶液が得られた。これをＦＥ−ＳＥＭデータと合わせると、ＤＮＡ修飾金ナノ粒子は、乾燥させると、不可逆的には凝集しないことが最終的に証明された。
【０４２１】
コンピュータ制御４プローブ技術を用いて、３種の試料（それぞれ３、４および５を介して連結された乾燥凝集体）の導電率を測定した。電気接点は、金ペーストでペレットに付着させた細い金ワイヤ（直径２５および６０μｍ）から構成した。試料を中程度の真空（１０^−３〜１０^−２トル）下に冷却し、低圧のドライヘリウムガス下に温度を増大させながら導電率を測定した。起こり得る光電子事象を排除するために、試料チャンバを光から隔離した。励起電流を１００ｎＡ以下に維持し、試料全体にわたる圧力を最大２０Ｖに制限した。驚くべきことには、３種すべてのリンカーから形成された凝集体の導電率は、室温下に１０^−５〜１０^−４Ｓ／ｃｍの範囲であり、これらの凝集体は、類似した温度依存性挙動を示した。ＤＮＡ連結凝集体の導電率は、半導体材料の特徴を示す、約１９０°Ｋまでのアレニウス挙動を示した。これは、不連続金属島状膜に認められる活性化電子ホッピング挙動に似ている（Ｂａｒｗｉｎｓｋｉ，Ｔｈｉｎ Ｓｏｌｉｄ Ｆｉｌｍｓ，第１２８巻：１−９ページ（１９８５年））。アルカンジチオールを介して連結された金ナノ粒子の網目構造は類似の温度依存性を示した（Ｂｒｕｓｔら，Ａｄｖ．Ｍａｔｅｒ．，第７巻：７９５−７９７ページ（１９９５年）；Ｂｅｔｈｅｌｌら．Ｊ．Ｅｌｅｃｔｒｏａｎａｌ．Ｃｈｅｍ．，第４０９巻：１３７−１４３ページ（１９９６年））。電荷移動の活性化エネルギーは、方程式（１）
【０４２２】
σ＝σ_ｏｅｘｐ［−Ｅ_α／ｋＴ］］ （１）
を用いる１ｎσ対１／Ｔのプロットから得ることができる。３つの測定値から計算した平均活性化エネルギーは、それぞれ２４、４８および７２ｍｅｒリンカーに関して、７．４±０．２ｍｅＶ、７．５±０．３ｍｅＶ、７．６±０．４ｍｅＶであった。これらの計算には、５０°Ｋから１５０°Ｋまでの導電率を用いた。
【０４２３】
これらの種類の材料の電気的性質は粒子間間隔に依存する筈なので、シンクロトロンＳＡＸＳ実験を用いて、分散した乾燥凝集体の粒子間間隔を測定した。ＳＡＸＳ実験は、Ａｄｖａｎｃｅｄ Ｐｒｏｔｏｎ Ｓｏｕｒｃｅ、Ａｒｇｏｎｎｅ Ｎａｔｉｏｎａｌ ＬａｂｏｒａｔｏｒｙのＤｕｐｏｎｔ−Ｎｏｒｔｈｗｅｓｔｅｒｎ−Ｄｏｗ Ｃｏｌｌａｂｏｒａｔｉｖｅ Ａｃｃｅｓｓ Ｔｅａｍ（ＤＮＤ−ＣＡＴ）Ｓｅｃｔｏｒ５で実施した。ＤＮＡ連結凝集体およびＤＮＡ修飾コロイドの希釈物試料を０．３ミクロンビームの１．５４Å放射線で照射し、散乱放射線をＣＣＤ検出器で回収した。２Ｄデータを循環的に平均し、散乱ベクトル量、ｓ＝２ｓｉｎ（θ）／λ（ここで、２θは散乱角、λは入射放射線の波長）の関数、Ｉ（ｓ）に変換した。すべてのデータをバックグラウンド散乱および試料吸収に対して補正した。３種すべての凝集体構造を乾燥させると、粒子間間隔感受性の第１ピーク位置は、それぞれ、２４ｍｅｒ、４８ｍｅｒおよび７２ｍｅｒ連結凝集体の０．０６３ｎｍ^−１、０．０４８ｎｍ^−１、および０．０３７ｎｍ^−１のｓ値から０．０８７ｎｍ^−１のｓ値へ大きく変化した。これは、乾燥させると、粒子が殆ど接触するポイントまで粒子間間隔が有意に減少したこと、およびそのような間隔は実質的にはリンカー長とは無関係であるが、溶液中での粒子間隔はリンカー長に大きく依存していたことを示している。これによって、乾燥ペレットの導電率実験における３種の異なる系に関して何故類似の活性化エネルギーが観察されたかが分る。さらに、これによって、何故、ＤＮＡの電子特性の見方とは無関係に比較的高い導電率が観察されたかも明らかになる。ＤＮＡ連結材料とは異なり、乾燥させたクエン酸塩安定化金ナノ粒子膜は、金属様挙動を示した。これは、そのような粒子が互いに融合することを示したＳＥＭデータと一致する。
【０４２４】
１９０°Ｋを超えると、測定したＤＮＡ連結試料の導電率は非常に急な低下挙動を示した。すべての試料について、導電率は約１９０°Ｋで急激に低下し始め、およそ２５０°Ｋまで低下し続け、そこから再度増大した。この挙動を詳細に調査するために、試料を繰返し冷却、加熱しながら導電率を測定した。興味深いことには、導電率の低下は、温度が増大方向にあるときにのみ起こった。ＤＮＡは親水性であり、かつ水は潜在的にハイブリッド構造の電気的性質に影響を与え得るので、金凝集体の導電率に及ぼす相対湿度の影響を調べた。湿度を１％から１０％に増大させると、抵抗が１０因数増大した。導電率測定前に、試料を真空（１０^−６トル）下に４８時間維持したときには、特有な急低下が非常に弱かったことに留意されたい。これらの観察結果から、１９０°Ｋを超えたときの導電率の非常に急な低下およびその後の増大は、粒子間間隔を（蒸発が起こるまで）一時的に増大させた水の融解およびＤＮＡの吸湿性に関連すると推断した。この仮説と一致して、０．３Ｍ ＰＢＳ緩衝液で湿らせた乾燥凝集体に関するＳＡＸＳ測定値は、粒子間間隔の２００％の増大（〜２ｎｍ）を示した。
【０４２５】
これらの研究は以下の理由から重要である。先ず、これらの研究は、ＤＮＡの分子認識特性を用いれば、ナノ粒子ベース材料を、不動態化したり、それらの離散構造または電気的性質を破壊せずに集合させ得ることを証明している。これらのＤＮＡ官能化粒子を用いて三次元巨視的集合体または、さらにリトグラフィーパターン化構造（Ｐｉｎｅｒら，Ｓｃｉｅｎｃｅ，第２８３巻：６６１−６６３（１９９９年））における電気的移動を研究しようとする場合、それらの電気的移動特性を正確に叙述することが絶対に必要である。第２に、これらの研究は、かなり長いリンカー間隔（８〜２４ｎｍ）にわたって、乾燥集合体の導電率がＤＮＡリンカー長には実質的に無関係であることを示している。これは、これらの実験において、水を除去したり、揮発性塩を用いたりした結果であると考えられる。確かに、溶媒および塩の除去によって創出された自由体積によって、ＤＮＡが表面に圧縮され、凝集体内の粒子が近接する。第３に、ＤＮＡ保護ナノ粒子を有する凝集体は半導体として挙動するが、クエン酸塩安定化粒子から形成された膜は、不可逆的粒子融合および金属様挙動を示す。最後に、これらの結果は、オリゴヌクレオチドで官能化されたナノ粒子と標的ＤＮＡとの間の配列特異的結合事象が回路の閉鎖および導電率の急激な増大（すなわち、絶縁体から半導体へ）を起こすＤＮＡ診断用途におけるこれらの材料の使用を示している（次ぎの実施例参照）。
【０４２６】
実施例２２： 金電極を用いた核酸の検出
金電極を用いた核酸検出法が図３９に略図で示されている。Ｇｕｏら，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．，第２２巻，５４５６−５４６５ページ（１９９４年）の方法に従って、２つの金電極の間のガラス表面を、標的ＤＮＡ３（５′ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴＧＡＡＴＧＣＧ［配列番号６０］）と相補的な１２ｍｅｒオリゴヌクレオチド１（３′ＮＨ_２（ＣＨ_２）_７Ｏ（ＰＯ_２ ⁻）Ｏ−ＡＴＧ−ＣＴＣ−ＡＡＣ−ＴＣＴ［配列番号５９］）で修飾した。オリゴヌクレオチド２（５′ＳＨ（ＣＨ_２）_６Ｏ（ＰＯ_２ ⁻）Ｏ−ＣＧＣ−ＡＴＴ−ＣＡＧ−ＧＡＴ［配列番号５０］）を作製し、実施例１および１８に記載のように１３ｎｍ金ナノ粒子に付着させて、ナノ粒子ａを生成した。標的ＤＮＡ３とナノ粒子ａをデバイスに付加した。ガラス表面の色はピンク色に変わったが、これは、標的ＤＮＡ−金ナノ粒子集合体がガラス基板上に形成されたことを示している。次いで、デバイスを０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液中に浸漬し、４０℃で１時間加熱して、非特異的に結合したＤＮＡを除去し、次いで、実施例１９に記載のような銀染色溶液で５分間処理した。電極の抵抗は６７ｋΩであった。
【０４２７】
比較のために、電極間に、オリゴヌクレオチド１の代わりにオリゴヌクレオチド４を付着させて対照デバイスを修飾した。オリゴヌクレオチド４は、ナノ粒子上のオリゴヌクレオチド２と同じ配列（５′ＮＨ_２（ＣＨ_２）_６Ｏ（ＰＯ_２ ⁻）Ｏ−ＣＧＣ−ＡＴＴ−ＣＡＧ−ＧＡＴ［配列番号５０］）を有しており、ナノ粒子の結合を阻止するように標的ＤＮＡ３に結合するであろう。その他の点では上記のようにテストを実施した。抵抗は、使用したマルチメーターの検出限度である４０ＭΩより高かった。
【０４２８】
この実験は、相補的標的ＤＮＡ鎖のみがデバイスの２つの電極間にナノ粒子集合体を形成し、回路は、ナノ粒子のハイブリダイゼーションおよびその後の銀染色によって完成し得ることを示している。したがって、相補的ＤＮＡおよび非相補的ＤＮＡは、導電率を測定することにより識別し得る。この形式は、何千もの異なる核酸を同時にテストし得る何千対もの電極を用いた基板アレイ（チップ）にも適用範囲を広げ得る。
【０４２９】
実施例２３：環式ジスルフィドリンカーを用いたオリゴヌクレオチド修飾金ナノ粒子の調製
この実施例では、調製が簡単で、広く有用で、メルカプトヘキシルリンカーを使用して調製した金−オリゴヌクレオチド共役体よりもＤＴＴに対して大きな安定性を示す金−オリゴヌクレオチド共役体を与える、ステロイドジスルフィド１ａ（図４２）に基づく金表面にオリゴヌクレオチドを結合させる新しい環式ジスルフィドリンカーについて説明する。１，２−ジチアン−４，５−ジオールのエステル誘導体は金表面上で単分子層を形成することが知られているため（Ｎｕｚｚｏら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１０５，４４８１−４４８３）、環式ジスルフィドを、アンカーユニットの反応部位として選択した。環式ジスルフィドは、両方の硫黄原子を介して立とうに表面に結合し（Ｕｌｍａｎ，Ａ．、ＭＲＳ Ｂｕｌｌｅｔｉｎ、６月４６−５１）、向上した安定性を示し得るキレート構造を与えるだろう。リンク要素としては、入手が容易で、容易に誘導退化されるケトアルコールであり、大きな疎水性表面を備えた置換体として、金の表面への水溶性分子の接近を選別するのに役立つよう期待されるため、エピアンドロステロンを選択した（Ｒｅｔｓｉｎｇｅｒら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１１５、７５３５−７５３６―Ｂｉｏｃｏｎｊｕｇａｔｅ Ｃｈｅｍ．９、８２６−８３０）。
【０４３０】
先の研究に使用していたオリゴヌクレオチド−金プローブを、水性緩衝液中で末端メルカプトヘキシル基を有するオリゴヌクレオチドを金ナノ粒子と反応させることによって調製した。該プローブは、驚くほど丈夫で、１００℃まで加熱した後や５℃で３年間保存した後でさえ良好に機能することが分かった。しかしながら、本願発明者らは、この共役体が、金の表面から誘導体化したオリゴヌクレオチドを取り外すことで作用するチオールを含む溶液に浸漬させた時に、ハイブリダイゼーションプローブとしての活性を失うことを見出した。この特徴は、ナノ粒子プローブが、チオールを含む溶液（例えばポリメラーゼ酵素の安定化剤としてジチオスレイトール（ＤＴＴ）を含むＰＣＲ溶液）中で使用される場合に、問題を提起する。
【０４３１】
（ａ）一般法
ＮＭＲスペクトルを、溶媒としてＣＤＣｌ_３、内部（Ｈ）標準としてＴＭＳ、外部（^３１Ｐ）標準としてＨ_３ＰＯ_４を使用して、５００ＭＨｚ（^１Ｈ）および４００ＭＨｚ（^３１Ｐ；１６１．９ＭＨｚの獲得）Ｖａｒｉａｎ分光計により記録した。化学シフトはδユニットで表される。ＭＳデータをＱｕａｔｔｒｏ ＩＩ三重四極子質量分析計により得た。自動オリゴヌクレオチド合成を、ミリジーンＥｘｐｅｄｉｔｅ ＤＮＡＤＮＡ合成装置で行った。Ｓの分析は、Ｏｎｅｉｄａ研究サービスによって行われた。
【０４３２】
（ｂ）ステロイド−ジスルフィドケタール（１ａ）の調製
合成スキームは図４３に示される。エピアンドロステロン（０．５ｇ）、１，２−ジチアン−４，５ジオール（０．２８ｇ）およびｐ−トルエンスルホン酸（１５ｍｇ）をトルエン（３０ｍＬ）に溶かした溶液を、脱水条件下で７時間環流した（Ｄｅａｎ Ｓｔａｒｋ装置）。その後、トルエンを、減圧下で除去し、残留物を酢酸エチルに溶解した。この溶液を水で洗い、硫酸ナトリウムで乾燥し、シロップ条の残留物に濃縮した。これをペンタン／エーテル中に一晩静置すると、白色固体（４００ｍｇ）として化合物ｌａが得られた。Ｒｆ値（ＴＬＣ、シリカプレート、溶離液としてエーテル）は０．５であった。比較として、同じ条件下でのエピアンドロステロンおよびの１，２−ジチアン−４、５−ジオールのＲｆ値は、それぞれ０．４および０．３である。ペンタン／エーテルからの再結晶により、白い粉末（融点１１０〜１１２℃）を生じた。^１Ｈ ＮＭＲ、δ３．６、（１Ｈ，Ｃ^３ＯＨ）３．５４−３．３９（２Ｈ，ｍ ジチアン環の２ＯＣＨ）、３．２−３．０（４Ｈ，ｍ ２ＣＨ_２Ｓ）、２．１−０．７（２９Ｈ，ｍ ステロイドのＨ）；質量スペクトル（ＥＳ^＋） Ｃ_２３Ｈ_３６Ｏ_３Ｓ_２（Ｍ＋Ｈ）に対する計算値４２５．２１７９、実測値４２５．２１５１ （Ｃ_２３Ｈ_３７Ｏ_３Ｓ_２）分析 計算値１５．１２、実測値１５．２６。
【０４３３】
（ｃ）ステロイド−ジスルフィドケタールホスホロアミダイト誘導体（１ｂ）の調製
化合物１ａ（１００ｍｇ）をＴＨＦ（３ｍＬ）に溶解し、ドライアイスアルコール浴で冷却した。Ｎ，Ｎ−ジイソプロピルエチルアミン（８０μＬ）およびβ−シアノエチルクロロジイソプロピルホスホルアミダイト（８０μＬ）を連続して加えた。その後、混合物を室温まで温め、２時間撹拌し、酢酸エチル（１００ｍＬ）と混ぜ、５％ＮａＨＣＯ_３水溶液で洗い、硫酸ナトリウムで乾燥させ、干し上がるまで濃縮した。残留物を最小量のジクロロメタンに溶かし、ヘキサンを加えて−７０℃で沈降させ、真空下で乾燥させ、１００ｍｇを生成した。；^３１Ｐ ＮＭＲ １４６．０２。
【０４３４】
（ｄ）５′修飾オリゴヌクレオチド−ナノ粒子共役体の調製
最後の亜リン酸化工程で化合物１ｂ（図４２）を使用した以外には、従来のホスホロアミダイト化学機構を使用して５′修飾オリゴヌクレオチドＩｃ１および１ｃ２をＣＰＧ支持体上に構築した。生成物を、濃縮ＮＨ_４ＯＨで１６時間、５５℃で処理することにより支持体から外した。オリゴヌクレオチドを、Ｈｅｗｌｅｔｔ Ｐａｃｋａｒｄ ＯＤＳ Ｈｙｐｅｒｓｉｌカラム（４．６×２００ｎｍ、５μｍ粒径）を備えたＤｉｏｎｅｘ ＤＸ５００システムで、ＴＥＡＡ緩衝液（ｐＨ７．０）と１％／分勾配の９５％ＣＨ_３ＣＮ／５％０．０３ＴＥＡＡを１ｍＬ／分の流量で使用して、逆相ＨＰＬＣにより精製した。疎水性ステロイド基の優れた特徴は、キャップされた誘導体が、キャップが外されたオリゴマーから楽に分離されることである。硫黄で誘導体化したオリゴヌクレオチドＩＩａ、ＩＩｃ１，およびＩＩｃ２（図４３）を、市販の「５′−チオール−修飾試薬」（Ｃ６Ｇｌｅｎ Ｒｅｓｅａｒｃｈ）と、以前に説明されたような（Ｓｔｏｒｈｏｆｆ、Ｊ．Ｊ．ら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２０、１９５９−１９６４）硝酸銀によるトリチル保護基の解離とを使用して調製した。ジスルフィドＩＩｃ１の調製に関しては、「チオール−修飾Ｃ６ Ｓ−Ｓ」（Ｇｌｅｎ Ｒｅｓｅａｒｃｈ）を最後の亜リン酸化に使用し、末端のジメトキシトリチル基を８０％酢酸水溶液中で解離させた。
【０４３５】
各修飾オリゴヌクレオチドを、固定されたメルカプトヘキシル基を介したオリゴヌクレオチドの固定に使用された方法（Ｓｔｏｒｈｏｆｆら（１９９８）Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２０、１９５９−１９６４）により、〜１３ｎｍの金ナノ粒子に固定化した。これは、クエン酸塩で安定化ナノ粒子（直径〜１３ｎｍ）を５６時間末端硫黄置換基（ＨＳ−または非環式または環式ジスルフィド）を有するオリゴヌクレオチドを含む緩衝塩溶液に浸漬してから、０．１ＭまでのＮａＣＬを添加し、２４時間静置することを含んでいた。ナノ粒子を遠心によってペレットにし、上澄溶液を除去した。ナノ粒子を洗浄し、緩衝液に再懸濁し、再び遠心し、０．１Ｍ ＮａＣｌ、１０ｍＭリン酸塩に懸濁した。この方法により、装填プロセスに使用される過剰な硫化オリゴヌクレオチドのない、ナノ粒子−オリゴヌクレオチド共役体が得られた。
【０４３６】
（ｅ）ハイブリダイゼーション
ジスルフィド−ステロイドアンカーを含むナノ粒子−オリゴヌクレオチドプローブの一体性を評価するために、金ナノ粒子上の修飾オリゴヌクレオチドの固定により、プローブＩｃ１、Ｉｃ２、ＩＩｃ１、ＩＩｃ２、およびＩＩＩｃ１を作製した。与えたシリーズ中のオリゴマーは同じヌクレオチド配列を有するが、５′先端基Ｙの構造が異なっている。Ｉｃ１とＩｃ２はステロイド−ジスルフィド先端基を有し；ＩＩｃ１、ＩＩｃ２はメルカプトヘキシル先端基を有し、ＩＩＩｃ１は非環式ジスルフィド先端基を有する。（ｄＡ）_２０鎖はハイブリダイゼーションを促進するための、金とオリゴヌクレオチド認識部分との間のスペーサーとして機能する。硫黄で誘導体化されたオリゴマーの多くは各ナノ粒子に結合する。標的オリゴヌクレオチドとのナノ粒子プローブ対のハイブリダイゼーションは３次元のネットワークの生成と、および赤から青灰色への色の変化につながる（Ｍｕｃｉｃ，Ｒ．Ｃ．ら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２０、１２６７４−１２６７５）。
【０４３７】
プローブのハイブリダイゼーションを、プローブに相補的な配列を含む７９ｍｅｒオリゴヌクレオチド標的を用いて調べた。（図４３）。その反応は、０．５ＭのＮａＣｌ、１０ｍＭリン酸塩（ｐＨ７．０）で、プローブ対Ｉｃ１、Ｉｃ２、およびＩＩｃ１、ＩＩｃ２、およびＩＩＩｃ１、ＩＩＩｃ２のコロイド溶液（各ナノ粒子プローブに関して５０μＬおよび１．０Ａ_５２０ユニット）に１μＬの標的溶液（１０ｐｍｏｌのＩＶ）を加えることにより、室温で行った。１０秒、５分、および１０分目に、アリコート（３μＬ）を取り、Ｃ−１８逆相ＴＬＣプレート上にスポットした。様々なプローブ対はすべて同じに作用した。すなわち、１０秒間の反応のスポットは赤くなり（ナノ粒子が自由であること示す）；１０分間の反応のスポットは紺青灰色となり（ナノ粒子の凝集物の特徴）；５分間の反応は、赤茶けた青色のスポットを与えた（非結合ナノ粒子と結合ナノ粒子の混合物を示す）。オリゴヌクレオチドのハイブリダイゼーションによって達成されるナノ粒子の凝集に関する以前の観察と一致して（Ｓｔｏｒｈｏｆｆ，Ｊ．Ｊ．ら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２０、１９５９−１９６４；Ｅｌｇｈａｎｉａｎ、Ｓｃｉｅｎｃｅ、２７７、１０７８−１０８１；Ｍｕｃｉｃ Ｒ．Ｃ．ら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２０、１２６７４−１２６７５；Ｍｉｔｃｈｅｌｌ，Ｇ．Ｐ．、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２１、８１２２−８１２３）、反応は可逆的だった。凝集物を９０℃（ナノ粒子を一緒にリンクするオリゴマーの解離温度を超える温度）に温め、熱いうちにスポットすると、赤いスポットが得られた。オリゴヌクレオチド標的を省略したかオリゴヌクレオチド標的がプローブに相補的でない対照実験では、すべての条件下で色が赤かった。
【０４３８】
本願発明者が結論づけるのは、ステロイド−ジスルフィドアンカーによって生成されたナノ粒子共役体が、ハイブリダイゼーションプローブとして有効に機能することである。さらに、スポット試験によって判断されるように、様々なアンカーユニットを備えた共役体が、かなりの割合で標的オリゴヌクレオチドと反応する。この特徴は、プローブが、ナノ粒子の表面にかなりの密度のオリゴヌクレオチドを有し、５′先端基から比較的遠くに離れているヌクレオチド認識部分を有するという予想と一致している。
【０４３９】
（ｆ）ナノ粒子プローブとジチオスレイトールの反応
金ナノ粒子かメルカプトヘキシル−オリゴヌクレオチドを装填した金ナノ粒子のコロイド溶液へのチオールの追加は、ナノ粒子の凝集につながる。色は赤から紺青まで変化し、静置すると、暗色沈澱物が沈着する。蛍光標識したオリゴヌクレオチドを有するナノ粒子の実験で実証されるように、チオールは、金表面に結び付けられたメルカプトアルキル−オリゴヌクレオチドを置換する（Ｍｕｃｉｃ、Ｒ．Ｃ．（１９９９） Ｓｙｎｔｈｅｔｉｃ Ｐｒｏｇｒａｍｍａｂｌｅ Ｎａｎｏｐａｒｔｉｃｌｅ Ａｓｓｅｍｂｌｙ Ｕｓｉｎｇ ＤＮＡ ＰｈＤ論文、ノースウェスタン大学）。オリゴヌクレオチド−ナノ粒子共役体のハイブリダイゼーションによって起こった凝集とは対照的に、そのような反応は不可逆であり、加熱しても、ＮａＯＨを加えても、凝集物を分解することはできない。
【０４４０】
本願発明者らは、この色を、ステロイド環式ジスルフィド先端基、メルカプトヘキシル先端基、および非環式ジスルフィド先端基を用いて作製したプローブとＤＴＴとの反応をモニタするために使用した。実験は、１μＬの１Ｍ ＤＴＴ溶液を、０．５Ｍ ＮａＣｌおよび１０ｍＭリン酸塩（ｐＨ７．０）の１００μＬのナノ粒子−オリゴヌクレオチドプローブ溶液（ナノ粒子の２Ａ_５２０ユニット）に加え、次に、様々な時間に３μＬのアリコートをＴＬＣプレート上にスポットし、色を観察するにより行った。表１に示されるように、メルカプトヘキシル（ＩＩｃ１とＩＩｃ２）と非環式オリゴヌクレオチド先端基（ＩＩＩｃ１）を備えたオリゴヌクレオチドに由来するコロイドプローブは、急速に反応した。赤青色のスポットが２０秒で得られ、強い青色のスポットが５分以内に得られた。１００分までに、ほとんどの金は沈殿した。対照的に、ステロイド環式ジスルフィド先端基（Ｉｃ１、Ｉｃ２）を用いて作製したプローブの反応では、４０分以内に、色の変化が観察されなかった。ＩＩｃ１、ＩＩｃ２を用いて作製したプローブで得られたのと同じ色に達するには１００分かかった。ＩＩＩｃ１では２０秒かかった。これに基づいて、本願発明者らは、ステロイドジスルフィドプローブのＤＴＴとの反応速度は、他のプローブの反応速度の約３００分の１であると推測する、非環式ジスルフィドアンカー３ｃから作製したプローブは、メルカプトヘキシルアンカーから作製したプローブとほぼ同じ速度で反応する。この後者の結果は、非環式ジスルフィドの金との反応がＳ−Ｓ結合の開裂に関与しているという証拠を考慮すれば、驚くことではない（Ｚｈｏｎｇ、Ｃ．Ｊ．Ｌａｎｇｍｕｉｒ、１５、５１８−５２５）。従って、非環式先端基を備えたオリゴヌクレオチドは、メルカプトヘキシル−オリゴヌクレオチド誘導体の場合のように、１つの硫黄原子によって恐らく金にリンクされるだろう。
【０４４１】
ＤＴＴの存在下で静置した後に、ＩｃｌおよびＩｃ２から作製したプローブが実際にハイブリダイゼーションプローブとして依然として機能するかどうか確かめるために、本願発明者らは、表１の反応に使用した条件下でプローブ混合物の２つのサンプルをＤＴＴで処理した。３０分後に、１μＬの７９ｍｅｒ標的オリゴヌクレオチド溶液（１０ｐｍｏｌ）を加えた。サンプルを急速凍結し、解凍し、スポット試験によって測定した。標的を含むサンプルのスポットは青く、標的を欠く対照のスポットは赤かった。これは、ナノ粒子共役体が安定しているだけでなく、メルカプトヘキシルか線形ジスルフィドアンカー基に由来するプローブの凝集を生じさせる条件下でＤＴＴに暴露した後でプローブとして有効であることを実証した。
【０４４２】
【表１０】

（ｇ）結論
この環式ジスルフィドリンカーを用いて作られた金ナノ粒子−オリゴヌクレオチド共役体は、特定のオリゴヌクレオチド配列を検出するのに有効なプローブとして機能し、従来のメルカプトヘキシル基または非環式ジスルフィドユニットを用いて作製された対応する共役体よりも、ジチオスレイトールへのはるかに大きな安定性を示す。チオール不活性化に対する高い安定性は、少なくとも一部では、２つの硫黄原子によって各オリゴヌクレオチドを金に固定することに因ると考えられる。
【０４４３】
実施例２４：単純な環式ジスルフィドリンカーを使用したオリゴヌクレオチド修飾金ナノ粒子の調製
この実施例では、本願発明者らは非ステロイド環式ジスルフィドリンカーと、このリンカーからオリゴヌクレオチド−ナノ粒子プローブとを調製し、ステロイド環式ジスルフィドとアルキルチオールリンカーにより調製されたプローブに対する、チオール含有溶液の存在下でのプローブ安定性を評価した。アルキルチオールアンカー基Ｉ（図４４）［Ｃ．Ａ．Ｍｉｒｋｉｎら、Ｎａｔｕｒｅ、３８２、６０７（１９９６）；Ｓｔｏｒｈｏｆｆら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１２０、１９５９年（１９９８）］またはステロイド環式ジスルフィドアンカー基ＩＩ（図４４）［Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒら、Ｂｉｏｃｏｎｊｕｇａｔｅ Ｃｈｅｍｉｓｔｒｙ、１１、２８９（２０００）］を使用して金ナノ粒子にオリゴヌクレオチドを結合することにより、ＤＮＡまたはＲＮＡの配列を検出するためのプローブの調製方法が記載されている。プローブとしてステロイド環式ジスルフィドリンカーを使用して調製した共役体は、メルカプトエタノールやジチオスレイトール（ＤＴＴ）のようなチオール化合物の存在下ではるかにより安定している点でアルキルチオールアンカーを使用して調製した共役体よりも遊離であることがわかった。検出するＤＮＡサンプルを増幅するのに使用されるＰＣＲ溶液は、酵素を保護するために少量のＤＴＴを含んでいるため、この特徴は重要である。ＰＣＲ産物の単純かつ迅速な検出の場合、最初に増幅されたＤＮＡを分離せずにＰＣＲ溶液中で直接試験を行えるように、ＤＴＴへの高い安定性を有するプローブを使用することが望ましい。
【０４４４】
２つの特徴がステロイド環式アンカー（化合物１、図４２）を際だたせている。すなわち、（１）環式ジスルフィド（金表面で所与オリゴヌクレオチドを保持するのに共同的に作用する２つの結合部位を原則的に与えることができる）と、（２）疎水的相互作用により金上の隣接鎖を安定させることが可能なステロイドユニット（例えばＲ．Ｌ．Ｌｅｔｓｉｎｇｅｒら、Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１１５，７５３５（１９９３）参照）である。それらの寄与の重要性を評価するために、本願発明者らは、ステロイド基を欠く環式ジスルフィド化合物ＩＩＩｃ（図４４）によって固定された金共役体を調製し、調べた。
【０４４５】
トランス−１，２−ジチアン−４，５−ジチオールを、トルエン中でアセトールと加熱することにより調製した化合物２ａを、シアノエチルＮ，Ｎ−ジ−ｉ−プロピルホスホロアミダイト試薬２ｂに変換した。該試薬２ｂは、修飾オリゴヌクレオチド２ｃ１および２ｃ２の合成の最終結合ステップに使用した。１つの金共役体プローブを、２ｃ１および等モル量の２ｄで金コロイド溶液を処理することにより調製した。２ｄは金表面上の希釈剤として機能する。コンパニオンプローブを、２ｃ２および２ｄから同様に作製した。これらのナノ粒子共役体は、静置と、凍結・融解との両方に関して、塩化ナトリウム溶液の０．１、０．３、０．５、０．７Ｍの範囲で安定していた。
【０４４６】
（ａ）化合物２ａ、２ｂ、２ｃ１、２ｃ２の調製
実施例２３に記述したように化合物２ａを調製した。２ａの亜リン酸化とオリゴヌクレオチド２ｃ１および２ｃ２の合成を、実施例２３や他の箇所でステロイド環式ジスルフィド誘導体に関して先に述べたように行った［Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒら、Ｂｉｏｃｏｎｊｕｇａｔｅ Ｃｈｅｍｉｓｔｒｙ、１１、２８９（２０００）、この文献の開示は引用によりその全体が組み込まれる］。ＣＰＧ支持体上のオリゴマーにＩＩＩｂを縮合することに関与するステップの反応時間は、１０分であった。
【０４４７】
（ｂ）金−オリゴヌクレオチド共役体の調製
１．７μモル／ｍＬの各オリゴヌクレオチドを含む溶液を提供するために、等モル量のオリゴヌクレオチド２ｃ１および２ｄまたは２ｃ２および２ｄを１３ｎｍの金コロイド（〜１０ｎＭ）に加えた。該溶液を暗所で２４時間保存し、その後、塩類を加えて、該溶液を０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸塩（ｐＨ７．０）、０．０１％アジ化ナトリウムとなるようにした。２４時間後に、ＮａＣｌ濃度を０．８Ｍに増大し、溶液をさらに２４時間静置した。その後、凝集物を除去するためにコロイドをろ過し、溶液を遠心してナノ粒子を集める。ペレットを、ナノピュア水で洗浄し、再び遠心し、０．１Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液（ｐＨ７．０）、０．０１％アジ化ナトリウムに再分散した。
【０４４８】
（ｃ）ナノ粒子プローブとジチオスレイトールの反応
置換の研究は、２０μＬの２ｃ１と２ｃ２から得たコロイド共役体の等量混合物に、２μＬの０．１Ｍ ＤＴＴを加えることにより、室温（２２℃）で行った。アリコート（３μＬ）を定期的に取り、白いナイロン膜上にスポットした。最初、スポットは赤かった。ＤＴＴによる金からのオリゴヌクレオチド硫黄誘導体の置換により、スポット試験で青灰色のスポットを与える混合物が生じた。ＤＴＴによる置換の時間を、混合物がスポット試験で強い青灰色を与える時間として採用した。２ｃ１および２ｃ２に由来する共役体の混合物の場合、この時は１０時間だった。
【０４４９】
比較のために、オリゴヌクレオチド共役体を、同様にメルカプトヘキシルアンカー（化合物２、図４２）およびステロイド環式ジスルフィドアンカー（化合物１、図４２）を使用して、オリゴヌクレオチド配列ｃ１およびｃ２（図４４）から調製した。青灰色のスポットを与える時間によって測定される、単チオール誘導体（２および図４２）およびステロイド環式ジスルフィドから調製した共役体の反応時間は、それぞれ５分と５３時間であった。これらの値は、メルカプトアルキル誘導体、非ステロイド環式ジスルフィドおよびステロイド環式ジスルフィドに由来するナノ粒子−オリゴヌクレオチド共役体に対する相対安定性の１、〜６０、〜３００に対応する。結果は、環式ジスルフィドアンカーユニット自体が、これらの系でメルカプトアルキル基に対して高い安定性を与えるのに十分であることを示す。化合物１に由来するナノ粒子共役体中の大きな疎水基も、オリゴヌクレオチドのチオール置換に対して安定性を増強するのに役割を果たしているように思われる。
【０４５０】
実施例２５：オリゴヌクレオチド修飾金ナノ粒子の調製
この実施例では、本願発明者らは、チオール含有溶液の存在下での、アルキルチオールおよびステロイド環式ジスルフィドリンカーと比較した、新しい非環式ジスルフィドリンカーであるトリチオールの安定性を評価した。比較のために、メルカプトヘキシルアンカー（化合物５、図４５）およびステロイド環式ジスルフィドアンカー（化合物６、図４５）を備えたオリゴヌクレオチドを調製した。
【０４５１】
（ａ）５′−トリメルカプトアルキルオリゴヌクレオチドの合成および特徴付け
図４７に示したように、５′−トリメルカプトアルキルチオールオリゴヌクレオチドを合成した。Ｔｒｅｍｂｌｅｒホスホロアミダイト７（Ｇｌｅｎ Ｒｅｓｅａｒｃｈ社、バージニア州スターリング）（図４５）およびチオール修飾剤Ｃ６ Ｓ−Ｓホスホロアミダイトを、ＣＰＧ支持体に結合された保護オリゴマーの５′端に逐次結合した。生成物をＣＰＧから解離し、上述のように精製した。３つのＤＭＴ基を備えたトリチオールオリゴヌクレオチドに対する保持時間は、約６４分である。続いて、５′−ＤＭＴ基を、８０％酢酸に３０分間オリゴヌクレオチドを溶解し、その後蒸発により取り外した。オリゴヌクレオチドを５００μＬのナノピュア水に再溶解した、溶液を酢酸エチル（３×３００μＬ）で抽出した。溶媒の蒸発後、オリゴヌクレオチドを白色固体として得られた。ＤＭＴ基がないこの５′−トリ−ジスルフィドオリゴヌクレオチドの保持時間は、逆相カラムで約３５分であり、イオン交換カラムでは２４分であった。これらのピークはいずれもスペクトル面積の９７％以上であり、オリゴヌクレオチドが高純度であることを示している。オリゴヌクレオチド８（図４５）の式量を、エレクトロスプレーＭＳ（計算値１２２４２．８５、実測値１２２４４．１）により得た。ＤＮＡ鎖上の３つのジスルフィド基を５′モノチオールＤＮＡに対して上述したようにトリチオール基に還元し、その後、ＮＡＰ−５カラムでオリゴヌクレオチドを精製した。
【０４５２】
（ｂ）５′−チオールまたはジスルフィドＤＮＡ修飾金ナノ粒子の調製
ベクター研究所（カリフォルニア州Ｂｕｒｌｉｎｇａｍｅ）より購入した金のナノ粒子を使用した。１０ｍＬの３０ｎｍ金コロイドに、５ＯＤのチオール修飾ＤＮＡを添加した。溶液を、徐々に０．３Ｍ ＮａＣｌ／１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７）（ＰＢＳ）にした。その後、ナノ粒子を遠心によって沈降させた。無色の上清を除去した後、赤色の油状沈殿物を１０ｍＬの新しいＰＢＳ緩衝液に再分散した。このプロセスを繰り返すことにより、コロイドを１０ｍＬの新しいＰＢＳ緩衝液を用いて２回洗浄した。
【０４５３】
（ｃ）チオール−ＤＮＡ修飾金ナノ粒子の安定性試験
固体のＤＴＴを、ＤＴＴ濃度が０．０１７Ｍとなるまで、様々な種類のチオールまたはジスルフィドＤＮＡで修飾した３０ｎｍ金ナノ粒子コロイドの６００μＬ溶液に加えた。ＤＴＴがオリゴヌクレオチドを置換するにつれて、コロイドの色は赤から青に代わる。ＵＶ／ＶＩＳスペクトルを時間の関数として取った。分散した３０ｎｍ金粒子に関連する〜５２８ｎｍの吸光度は減少し始め、７００ｎｍの広幅のバンドが伸び始めた。７００ｎｍのバンドはコロイドの凝集に関連している。図４８に示されるように、１つのチオールオリゴヌクレオチド（１）で修飾した３０ｎｍ金の粒子は、０．０１７Ｍ ＤＴＴで迅速に凝集物を形成し、１．５時間後にはコロイドが完全に青くなる。ジスルフィドオリゴヌクレオチド（４）で修飾したナノ粒子を含む溶液は、同一条件下では２０時間後に青くなる。トリチオール−オリゴヌクレオチド（開裂した６）で修飾したナノ粒子では、溶液が青くなるのに４０時間かかった。
【０４５４】
実施例２６：ナノ粒子−核酸−ｓｆｐメンバー共役体を用いた検体の検出
この実施例では、ナノ粒子−ストレプトアビジンプローブを用いて検体（ビオチン）の検出を実証する（図５４，５６）。
【０４５５】
（ａ）ナノ粒子−核酸−タンパク質共役体の調製
ナノ粒子−核酸共役体を、実施例３に記載したように調製する。これらの共役体を調製するために用いたオリゴヌクレオチド修飾因子は、以下の配列を有する：５’ＳＨ（ＣＨ_２ ）_６ −Ａ_１０−ＣＧＣＡＴＴＣＡＧＧＡＴ３’（２）。図５６に示すように、ビオチン標識オリゴヌクレオチド（１）［３’−ビオチン−ＴＥＧ−Ａ_１０−ＡＴＧＣＴＣＡＡＣＴＣＴ５’］を、ビオチン ＴＥＧ ＣＰＧ支持体（グレン・リサーチ社（Ｇｌｅｎ Ｒｅｓｅａｒｃｈ），Ｓｔｅｒｌｉｎｇ，Ｖｉｒｇｉｎｉａ；カタログ番号２０−２９５５−０１）を用いて文献の手順により調製する。ストレプトアビジン／ＤＮＡ共役体を作製するために、ストレプトアビジンを２０ｍＭ Ｔｒｉｓ（ｐＨ７．２）、０．２ｍＭ ＥＤＴＡ緩衝液中の１当量のビオチン修飾オリゴヌクレオチドと、攪拌器にて室温で２時間反応させた。異なる数のオリゴヌクレオチドと複合化したストレプトアビジンを、２０ｍＭ Ｔｒｉｓ（ｐＨ７．２）及び０．５％／分 勾配の２０ｍＭ Ｔｒｉｓ、１Ｍ ＮａＣｌによる流速１ｍＬ／分のイオン交換ＨＰＬＣで分離しながら、ＤＮＡのＵＶシグナルを２６０ｎＭ及び２８０ｎＭでモニタリングした。ストレプトアビジン／ビオチン修飾オリゴヌクレオチド混合物は、４５分、５６分、６７分、及び７１分に、それぞれ１：１、２：１、３：１、及び４：１オリゴヌクレオチド−ストレプトアビジン複合体に対応する４つのピークを示した。ストレプトアビジン／オリゴヌクレオチドの１：１複合体（１０μＭ、５μＬ）を単離して、０．３Ｍ ＰＢＳ（０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液、ｐＨ７）中のリンカーＤＮＡ［５’ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴＧＡＴＴＧＣＧ３’］（３）（１０μＭ、５μＬ）と予備混合し、その後その混合物を０．３Ｍ ＰＢＳ中のナノ粒子−オリゴヌクレオチド共役体（１０ｎＭ、１３０μＬ）に添加して、ナノ粒子−オリゴヌクレオチド−ストレプトアビジン共役体を調製した。ナノ粒子−オリゴヌクレオチド共役体、ストレプトアビジン／オリゴヌクレオチド、及びリンカーＤＮＡを含有する溶液を、ドライアイス中で１０分間凍結させ、解凍してＤＮＡハイブリダイゼーションを促進させた。１２時間後に、その溶液を１０００ｒｐｍで２０分間遠心分離して、上清を除去した。ナノ粒子−オリゴヌクレオチド−ストレプトアビジン共役体の赤色油状沈殿物を、０．３Ｍ ＰＢＳ緩衝液に再分散させた。
【０４５６】
その後、ナノ粒子−オリゴヌクレオチド−ストレプトアビジン共役体を使用して、ターゲット検体、つまりガラス表面に固定化したビオチンの存在を検出した。
【０４５７】
（ｂ）ビオチンの検出
ビオチンは、以下の手順に従ってガラススライドに固定化した：オリゴヌクレオチド修飾ガラススライドを、過去に報告された方法（Ｓｃｉｅｎｃｅ，２０００年，第２８９巻，１７５７−１７６０ページ）により調製し、その後ビオチン修飾オリゴヌクレオチドを表面ＤＮＡにハイブリダイズさせた。（ビオチン修飾ガラススライドは、様々な方法により調製することができる。１つの例は、アミン修飾ガラススライドを調製し、その後表面のアミンを、アミンと反応する様々な市販のビオチン化試薬と反応させることである。）図５４に示すように、固定化ビオチンガラススライドは、その後ナノ粒子−オリゴヌクレオチド−ストレプトアビジンを含有する媒体（０．３Ｍ ＰＢＳ中に２ｎＭ）で処理した。洗浄し銀強化溶液で処理した後、灰色又は光沢のある銀色のスポットの発現により、捕獲したプローブの存在が実証された［銀強化溶液Ａ（カタログ番号Ｓ５０２０，シグマ社（Ｓｉｇｍａ Ｃｏｍｐａｎｙ），米国ミズーリ州セントルイス）、及び銀強化溶液Ｂ（カタログ番号Ｓ５１４５，シグマ社（Ｓｉｇｍａ Ｃｏｍｐａｎｙ））、Ｓｃｉｅｎｃｅ，２０００年，第２８９巻、１７５７ページも参照］。所望なら、オリゴヌクレオチドに連結する蛍光標識した特異的結合対メンバーを用いて、ナノ粒子−オリゴヌクレオチド−ｓｐｆプローブを調製してもよい。その後、この発蛍光団標識プローブを用いて、検体の存在を検出してもよい。
【０４５８】
実施例２７：金コロイドストレプトアビジン共役体の特異的結合の比較
この実施例において、実施例２６に従って調製したナノ粒子ストレプトアビジンプローブ、及び市販のＡｕコロイド（２０ｎｍ）／ストレプトアビジン共役体（フルネーム：金標識ストレプトアビジン標識）（シグマ社（Ｓｉｇｍａ Ｃｏｍｐａｎｙ），米国ミズーリ州セントルイスから購入，カタログ番号Ｓ６５１４）の非特異的結合を評価した。共役体を含有する市販の溶液及び本発明のプローブ（０．３Ｍ ＰＢＳ中に２ｎＭ）を含有する溶液各５μｌを、ガラススライド上にスポットした。２０時間後に、スライドを水で洗浄して、銀強化溶液［実施例２６参照］で処理した。市販の共役体は、発色の５分後に灰色のスポットを示しており、ガラス表面にＡｕコロイドが存在することが示されたが、実施例３のナノ粒子ストレプトアビジンプローブでは、認識できる銀色染色は示されなかった（図５３）。シグナルの差は、銀色染色したスライドに第２のＤＮＡ修飾ナノ粒子を載せてそれを再染色することにより高めることができた。これにより、本発明のナノ粒子−オリゴヌクレオチド−ｓｂｐ共役体が、市販のＡｕコロイド／ストレプトアビジン共役体よりも非特異的結合に対して抵抗性があることが実証される。
【０４５９】
実施例２８：ナノ粒子アセンブリの調製
本明細書に論じたように、特異的結合対メンバー、例えば受容体又はリガンドは、オリゴヌクレオチドにより機能化され、オリゴヌクレオチド修飾ナノ粒子に固定化されて、ＤＮＡよりむしろ特異的結合対メンバーが指向する分子認識特性を備える以外に、オリゴヌクレオチド修飾粒子の高度安定性を呈する新しい分類のハイブリッド粒子を生成することができる。或いは、受容体修飾オリゴヌクレオチドを備えた多重受容体結合部位を有するタンパク質を機能化し、それにより我々の本来の材料の組立スキームにおいて、タンパク質受容体複合体を無機ナノ粒子の１つの代わりに部分構造の１つとして用いることができる。この実施例において、我々は、１３ｎｍ金ナノ粒子、ストレプトアビジン、及びビオチン化ＤＮＡを用いて、新しいナノ粒子アセンブリ（図５２）を調製して、得られた新しい生物無機材料の物理的及び化学的特性の幾つかを研究する。
【０４６０】
（ａ）実験
オリゴヌクレオチド修飾１３ｎｍ Ａｕ粒子（２−Ａｕ）及びリンカーＤＮＡ（３）を調製するための方法は、他の文献で報告されている。これらの共役体を調製するために用いるオリゴヌクレオチド修飾因子は、以下の配列を有する：５’ＳＨ（ＣＨ_２ ）_６ −Ａ_１０−ＣＧＣＡＴＴＣＡＧＧＡＴ３’。リンカーＤＮＡ（３）は、以下の配列を有する：［５’ＴＡＣＧＡＧＴＴＧＡＧＡＡＴＣＣＴＧＡＴＴＧＣＧ３’］。Ｊ．Ｊ．Ｓｔｏｒｈｏｆｆ，Ｒ．Ｅｌｇｈａｎｉａｎ，Ｒ．Ｃ．Ｍｕｃｉｃ，Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１９９８年，第１２０巻，１９５９ページを参照されたい。Ａｕ粒子（２−Ａｕ）及びリンカーＤＮＡ（３）は、使用の前に０．３Ｍ ＮａＣｌ、１０ｍＭリン酸緩衝液（ｐＨ７、０．３Ｍ ＰＢＳ）に保存した。ビオチン修飾ＤＮＡ（２）は、ビオチン ＴＥＧ ＣＰＧ支持体（グレン・リサーチ社（Ｇｌｅｎ Ｒｅｓｅａｒｃｈ））を用いて合成し、文献の方法により精製した。Ｊ．Ｊ．Ｓｔｏｒｈｏｆｆ，Ｒ．Ｅｌｇｈａｎｉａｎ，Ｒ．Ｃ．Ｍｕｃｉｃ，Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．１９９８年，第１２０巻，１９５９ページ；Ｔ．Ｂｒｏｗｎ，Ｄ．Ｊ．Ｓ．Ｂｒｏｗｎ，ｉｎ Ｏｌｉｇｏｎｕｃｌｉｅｏｔｉｄｅｓ ａｎｄ Ａｎａｌｏｇｕｅｓ（Ｆ．Ｅｃｋｓｔｅｉｎ編集），Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ，Ｎｅｗ Ｙｏｒｋ，１９９１年を参照されたい。ストレプトアビジンは、シグマ社（Ｓｉｇｍａ）から購入し、２０ｍＭ Ｔｒｉｓ緩衝液（３０Ｍ、ｐＨ７．２）に溶解した。ストレプトアビジン／ＤＮＡ共役体（１−ＳＴＶ）を作製するために、ストレプトアビジンを２０ｍＭ Ｔｒｉｓ（ｐＨ７．２）、０．２ｍＭ ＥＤＴＡ緩衝液中の８当量のビオチン−オリゴヌクレオチド共役体１と攪拌器にて室温で２時間反応させた。ビオチン−オリゴヌクレオチド共役体は、配列［３−ビオチン−ＴＥＧ−Ａ_１０−ＡＴＧＣＴＣＡＡＣＴＣＴ５’］を有している。ＤＮＡに対するストレプトアビジンの割合が異なる混合物（１：０．４、１：１、１：４）も、ＨＰＬＣ分析のために調製した。ストレプトアビジン／ＤＮＡ共役体は、２０ｍＭ Ｔｒｉｓ（ｐＨ７．２）及び０．５％／分 勾配の２０ｍＭ Ｔｒｉｓ、１Ｍ ＮａＣｌによる流速１ｍＬ／分のイオン交換ＨＰＬＣで過剰のＤＮＡから分離しながら、２６０ｎｍ及び２８０ｎｍでＤＮＡのＵＶシグナルをモニタリングした。１：０．４ストレプトアビジン／ＤＮＡ混合物は、４５分，５６分に２つのピークを、１：１混合物は４５分、５６分、６７分、及び７１分に、それぞれ１：１、２：１、３：１、及び４：１オリゴヌクレオチド−ストレプトアビジン複合体に対応する４つのピークを示した。１：４混合物は、３番目のピーク（６７分）及び４番目のピーク（７１分）の強度が増大することを除けば、同様の位置に４つのピークを示した。１：８ストレプトアビジン／ＤＮＡ混合物のＨＰＬＣスペクトルは、２つの主なピーク、つまり１つは５９分の未反応のＤＮＡのもの、もう一方は７１分の４：１オリゴヌクレオチド−ストレプトアビジン複合体のものを示した。加えて、３：１複合体にあたる６７分のショルダーも存在した。精製したストレプトアビジン−ビオチン化ＤＮＡ共役体は、濃縮して、限外濾過（セントリコン３０）により０．３Ｍ ＰＢＳに分散させた。ＴＥＭ、熱変性実験、及びＳＡＸＳ測定用の凝集体は、ストレプトアビジン−オリゴヌクレオチド共役体（１−ＳＴＶ）、金ナノ粒子ＤＮＡ共役体（２−Ａｕ）、及びリンカーＤＮＡ（３）を含有する溶液をドライアイス中で１０分間凍結することにより調製し、測定する前に解凍して、ハイブリダイゼーションを促進させた。
【０４６１】
（ｂ）結果
本明細書に報告したナノ粒子／タンパク質アセンブリ（図５２）は、３つの部分構造：４つのビオチン化オリゴヌクレオチドに複合化したストレプトアビジン（１−ＳＴＶ）と、オリゴヌクレオチド修飾金ナノ粒子（２−Ａｕ）と、１に相補的な配列の半分及び２に相補的なもう半分を有するリンカーオリゴヌクレオチド（３）とに依存する（図５２参照）。典型的な実験において、リンカーＤＮＡ３（１０μＭ、２１μＬ）を、２−Ａｕ（９．７ｎＭ、２６０μＬ）と１−ＳＴＶ（１．８μＭ、２７μＬ）の混合物に導入するか、或いはリンカーＤＮＡ３（１０μＭ、２１μＬ）を、１−ＳＴＶ（１．８μＭ、２７μＬ）と予備混合し、その後混合物を０．３Ｍ ＰＢＳ中の２−Ａｕ（９．７ｎＭ、２６０μＬ）に添加した。同等の特性を備えた凝集体が、両方の方法から形成し得るが、３及び１−ＳＴＶの予備混合は、凝集体の形成を促進する。１３ｎｍ金ナノ粒子は、ストレプトアビジン（４ｎｍｘ４ｎｍｘ５ｎｍ）よりも実質的に大きいため［Ｐ．Ｃ．Ｗｅｂｅｒ，Ｄ．Ｈ．Ｏｈｌｅｎｄｏｒｆ，Ｊ．Ｊ．Ｗｅｎｄｏｌｏｓｋｉ，Ｆ．Ｒ．Ｓａｌｅｍｍｅ，Ｓｃｉｅｎｃｅ，１９８９年，第２４３巻，８５ページ；Ｎ．Ｍ．Ｇｒｅｅｎ，Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ．１９９０年，第１８４巻，５１ページ］、ストレプトアビジンに対するＡｕナノ粒子のモル比１：２０を利用して、数個のナノ粒子を含む小さな凝集体より、又はストレプトアビジンのハイブリダイズされた層に機能化された単一の金ナノ粒子からなる構造よりどちらかといえば伸長した高分子構造の形成を好適化した。
【０４６２】
興味深いことに、室温では、１−ＳＴＶ、２−Ａｕ、及び３を混合した場合、１−ＳＴＶ、２−Ａｕ、及び３を含有する溶液のＵＶ−ｖｉｓスペクトルが安定していることが示すように、３日後でも観察可能な粒子の凝集は起こらない。しかし、溶液の温度（５３℃）をＤＮＡ相互連結の融点（Ｔｍ）未満で２、３℃上昇させると、ミリサイズの凝集体の成長（図５７Ａ）と、粒子アセンブリに関連するＡｕプラズモン共鳴の特徴的な赤色のシフト及び低下とがもたらされた。Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ｒ．Ｃ．Ｍｕｃｉｃ，Ｊ．Ｊ．Ｓｔｏｒｈｏｆｆ，Ｎａｔｕｒｅ １９９６年，第３８２巻，６０７ページを参照されたい。透過電子顕微鏡（ＴＥＭ）像（図５７Ｂ）は、凝集体内の粒子がアニーリングの前後にその物理的形状を保持していることを示していることから、粒子の融合がないことが示され、更に表面オリゴヌクレオチド層の安定した影響が実証される。粒子アセンブリを開始するための熱活性化の要件は、過去に研究されたシステム（図５２ｂ）とは相反しており、過去のシステムは、非常に類似した条件下でオリゴヌクレオチド修飾金粒子が室温でリンカーＤＮＡを加えると数分以内に凝集体内に組込まれる２つの金ナノ粒子共役体を含んでいる。Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ｒ．Ｃ．Ｍｕｃｉｃ，Ｊ．Ｊ．Ｓｔｏｒｈｏｆｆ，Ｎａｔｕｒｅ １９９６年，第３８２巻，６０７ページを参照されたい。この特徴は、：（１）Ａｕナノ粒子上のＤＮＡ被覆（ＤＮＡ：粒子＝〜１１０：１）に比べストレプトアビジン上のもの（ＤＮＡ：ストレプトアビジン＝４：１）が比較的少ないことによる、Ａｕ−ＳＴＶアセンブリシステム内の低い衝突確率［Ｌ．Ｍ．Ｄｅｍｅｒ，Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｒ．Ｃ．Ｍｕｃｉｃ，Ｒ．Ａ．Ｒｅｙｎｏｌｄｓ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ｒ．Ｅｌｇｈａｎｉａｎ，Ｇ．Ｖｉｓｗａｎａｄｈａｍ，Ａｎａｌ．Ｃｈｅｍ．２０００年，第７２巻，５５３５ページを参照］、又は（２）単一ストレプトアビジン上のＤＮＡのほとんど又は全てが単一粒子上のＤＮＡに結合する動力学的構造の初期形成、を原因とする可能性がある。凝集体形成は、溶液の凍結だけでなく加熱により促進することができる。Ｒ．Ｅｌｇｈａｎｉａｎ，Ｊ．Ｊ．Ｓｔｏｒｈｏｆｆ，Ｒ．Ｃ．Ｍｕｃｉｃ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｓｃｉｅｎｃｅ １９９７年，第２７７巻，１０７８ページを参照されたい。
【０４６３】
ＵＶ−ｖｉｓ分光学に基づいた融解実験を、ストレプトアビジン／ナノ粒子凝集体で実施し、それらがＤＮＡハイブリダイゼーションにより形成されることが確認された（図５８Ａ）。加熱すると、５２０ｎｍでの吸光度が最初に低下し、その結果融解曲線が一時的に下降し、その後ＤＮＡ融解が起こり粒子が分散すると、その吸光度が急激に上昇する。この一時的下降という挙動は、純粋な金ナノ粒子システムで観察され、融解の前の凝集体成長、つまりある種の凝集体の「熟成」に起因させた。Ｊ．Ｊ．Ｓｔｏｒｈｏｆｆ，Ａ．Ａ．Ｌａｚａｒｉｄｅｓ，Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ｒ．Ｃ．Ｍｕｃｉｃ，Ｇ．Ｃ．Ｓｃｈａｔｚ，Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．２０００年，第１２２巻，４６４０ページを参照されたい。融解の後、Ａｕプラズモン共鳴が５２０ｎｍに集中し、それは分散した１３ｎｍナノ粒子の特徴である。これらの実験により、結果的に、非特異的相互作用よりむしろ配列特異性ハイブリダイゼーションが、粒子／タンパク質アセンブリを実行すること、及びその工程が完全に可逆的であることが実証される。
【０４６４】
Ａｕナノ粒子／タンパク質アセンブリは、ハイブリダイゼーション誘導性アセンブリとは逆に、ストレプトアビジン／ビオチン相互作用によっても形成することができる。例えば、典型的な実験において、１（０．２４ｍＭ、１．３μＬ）、３（１０μＭ、３２μＬ）、及び２−修飾ナノ粒子（Ａｕナノ粒子濃度：９．７ｎＭ、２６０μＬ）を、０．３Ｍ ＰＢＳ緩衝液中で混合して、ビオチン修飾Ａｕ粒子を生成した。この混合物を６０℃に１０分間加熱し、その後室温に冷却して、ハイブリダイゼーションを促進させた。２４時間後に、１、３、及び２−修飾Ａｕナノ粒子を含有する溶液を、０．３Ｍ ＰＢＳ中のストレプトアビジン（１０μＭ、４．２μＬ）に添加し、５０℃に加熱して、ナノ粒子凝集体を形成した。溶液の色は、凝集体の形成を示す紫色に変化し、溶液温度をＴｍ（６５℃）を超えて上昇させることにより、凝集体を２−修飾Ａｕ粒子、１−修飾ストレプトアビジン、及び３に解体することができた。小角Ｘ線散乱（ＳＡＸＳ）［Ｓ．Ｊ．Ｐａｒｋ，Ａ．Ａ．Ｌａｚａｒｉｄｅｓ，Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｐ．Ｗ．Ｂｒａｚｉｓ，Ｃ．Ｒ．Ｋａｎｎｅｗｕｒｆ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ａｎｇｅｗ．Ｃｈｅｍ．Ｉｎｔ．Ｅｄ．２０００年，第３９巻，３８４５ページ；Ａ．Ｇｕｉｎｉｅｒ、Ｆ．Ｇ．，Ｓｍａｌｌ Ａｎｇｌｅ Ｓｃａｔｔｅｒｉｎｇ ｏｆ Ｘ−ｒａｙｓ，Ｗｉｌｅｙ，Ｎｅｗ Ｙｏｒｋ，１９９５年；Ｂ．Ａ．Ｋｏｒｇｅｌ，Ｄ．Ｆｉｔｚｍａｕｒｉｃｅ，Ｐｈｙｓ．Ｒｅｖ．Ｂ １９９９年，第５９巻，１４１９１ページ］のデータを、Ａｕ及びストレプトアビジン部分構造（１−ＳＴＶ、２−Ａｕ）と２つの異なる長さのＤＮＡリンカー（２４量体（３）及び４８量体）とから形成した凝集体について回収し、Ａｕ−Ａｕアセンブリ及び同様の連結オリゴヌクレオチド（２−Ａｕ、４−Ａｕ、２４量体リンカー（３）、４８量体リンカー）を基にした凝集体のものと比較した（図５９）。４８量体リンカーは、：１及び２に相補的な１２量体の付着末端を含有する５’ＴＡＣＧＡＧＴＴＧＡＧＡＣＣＧＴＴＡＡＧＡＣＧＡＧＧＣＡＡＴＣＡＴＧＣＡＡＴＣＣＴＧＡＡＴＧＣＧ及び３’ＧＧＣＡＡＴＴＣＴＧＣＴＣＣＧＴＴＡＧＴＡＣＧＴからなる２本鎖であった（２本鎖領域は下線で示す）。ＳＡＸＳ実験を設計するために、上記実験で用いられ、ＤＮＡハイブリダイゼーションでより高い接近容易性を提供した１，２，及び４のＡ_１０スペーサ［Ｌ．Ｍ．Ｄｅｍｅｒ，Ｃ．Ａ．Ｍｉｒｋｉｎ，Ｒ．Ｃ．Ｍｕｃｉｃ，Ｒ．Ａ．Ｒｅｙｎｏｌｄｓ，Ｒ．Ｌ．Ｌｅｔｓｉｎｇｅｒ，Ｒ．Ｅｌｇｈａｎｉａｎ，Ｇ．Ｖｉｓｗａｎａｄｈａｍ，Ａｎａｌ．Ｃｈｅｍ．２０００年，第７２巻，５５３５ページを参照］を除去して、より強固なシステムを生成した。ＤＮＡにより連結された凝集体は、比較的明確な回折ピークを示し、Ａｕ−ＳＴＶ凝集体は、同じリンカーから形成されたＡｕ−Ａｕ凝集体よりも小さなｓ値で回折ピークを呈した。これは、Ａｕ−ＳＴＶシステムでのＡｕ粒子間距離がより大きいことを示唆している。その上、Ａｕ−Ａｕ及びＡｕ−ＳＴＶアセンブリの両者でリンカーとして２４量体 ＤＮＡの代わりに４８量体 ＤＮＡを用いた場合、回折ピークはより小さなｓ値にシフトする（図５９）。
【０４６５】
対距離分布関数（ｐａｉｒ ｄｉｓｔａｎｃｅ ｄｉｓｔｒｉｂｕｔｉｏｎ ｆｕｎｃｔｉｏｎ ）（ＰＤＤＦ）であるｇ（ｒ）を、方程式（１）（式中、ｑ＝（４π／λ）ｓｉｎ（θ／２）で、ρは粒子数密度である）を用いて、ＳＡＸＳパターンから計算した［Ａ．Ｇｕｉｎｉｅｒ，Ｆ．Ｇ．，Ｓｍａｌｌ Ａｎｇｌｅ Ｓｃａｔｔｅｒｉｎｇ ｏｆ Ｘ−ｒａｙｓ，Ｗｉｌｅｙ，Ｎｅｗ Ｙｏｒｋ，１９５５年；Ｂ．Ａ．Ｋｏｒｇｅｌ，Ｄ．Ｆｉｔｚｍａｕｒｉｃｅ，Ｐｈｙｓ．Ｒｅｖ．Ｂ １９９９年，第５９巻，１４１９１ページ］。
【０４６６】
ｇ（ｒ）＝１＋（１／２Ｂ^２ Δｒ）Ｉｑ（ｓ（ｑ）−１）ｓｉｎ（ｑｒ）ｄｑ 方程式（１）
ここで、ｇ（ｒ）は、第２の粒子を選択された粒子からの距離ｒの関数として見出す確率を示している。ＰＤＤＦから得られた最も近い近接Ａｕ粒子間距離（中心から中心までの距離）は、２４量体連結Ａｕ−Ａｕ、４８量体連結Ａｕ−Ａｕ、２４量体連結Ａｕ−ＳＴＶ、及び４８量体連結Ａｕ−ＳＴＶアセンブリで、それぞれ１９．３ｎｍ、２５．４ｎｍ、２８．７ｎｍ、及び４０．０ｎｍである。Ａｕ−ＳＴＶシステムとＡｕ−Ａｕシステムの粒子間距離を比較すると、Ａｕ−ＳＴＶアセンブリが予測されたＡｕナノ粒子／ストレプトアビジン周期性を有し、２つの成分（Ａｕナノ粒子及びストレプトアビジン）が、強固なＤＮＡ２本鎖リンカーにより十分に分離されることが明瞭に示される。
【０４６７】
（ｃ）結論
この研究は、以下の理由により重要である。１）ＤＮＡ依存性ナノ粒子アセンブリ（組立）法（ＤＮＡ−ｄｉｒｅｃｔｅｄ ｎａｎｏｐａｒｔｉｃｌｅ ａｓｓｅｍｂｌｙ ｍｅｔｈｏｄ ）が、これまで研究された無機ナノ粒子に加えて、タンパク質の構造にまで拡張できることが実証される。実際にこれは、Ａｕナノ粒子の可逆的なＤＮＡ依存性アセンブリと、ＤＮＡにより機能化されたタンパク質性構造とを実証する最初の報告である。２）ストレプトアビジンをはじめとする多くのタンパク質が、コロイド金の表面に吸着し、強力に結合した複合体を形成することは周知である。我々の実験は、ナノ粒子表面の高密度ＤＮＡ層の安定した影響と、ハイブリダイゼーションを行わない場合のストレプトアビジン吸着への抵抗性を実証している。それ故、それらは、タンパク質の活性を実質的に乱さないような方法で、非常に特異的な相互作用によりナノ粒子表面のタンパク質構造を特異的に固定化する方法に向かうものである。表面に吸着されたタンパク質を備えた粒子は、免疫化学において重要な役割を演じ［Ｍ．Ａ．Ｈａｙａｔ，Ｃｏｌｌｏｉｄａｌ Ｇｏｌｄ：Ｐｒｉｎｃｉｐｌｅｓ，Ｍｅｔｈｏｄｓ， ａｎｄ Ａｐｐｌｉｃａｔｉｏｎｓ，Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ， Ｓａｎ Ｄｉｅｇｏ，１９９１年］、タンパク質がナノ粒子表面と直接相互作用する安定したタンパク質／粒子共役体の開発により、組織化学的研究及び免疫検査のための改良ナノ粒子プローブが誘導され得る。
【０４６８】
実施例２９：電場を横切ってのナノ粒子の加速
本発明は、クエン酸で安定化されたナノ粒子や、荷電生体分子で被覆されたナノ粒子を、電場の中で移動させる方法について説明する。ナノ粒子と核酸を検出するためのナノ粒子の使用については、いずれもその全体が本明細書に組み込まれるＰＣＴ出願第ＷＯ９８／０４７４０号（ノースウエスタン大学）や第ＷＯ００／３３０７９号（ナノスフェア エルエルシー）に詳細に説明されている。生体分子で官能化したナノ粒子プローブを利用する検出技術は、プローブ分子が固定化標的を見つける能力やオリゴヌクレオチド鎖を捕獲する能力（チップの場合）に基づいている。このプロセスは、プローブの「捕獲された標的」を備えた電極表面への移動や、捕獲された標的を溶液中で有するプローブのセットのプローブの対象表面への移動を加速することができる。本発明の方法は、ナノ粒子に基づくプローブを使用する重要な生体診断用途を有する。例えば、本発明の方法は、ナノ粒子プローブまたはプローブのセットの、相補的ＤＮＡまたは相補的抗原／抗体で官能化された表面へのハイブリダイゼーションを加速するために使用することができる。
【０４６９】
蛍光ハイブリダイゼーション検出システムにおいて、ＤＮＡなどの生体分子の電場中での標的部位に向かう能動的移動を促進するために、電場を適用するという考え方は、例えば米国特許第５，８４９，４８６号に記されている。しかしながら、そのような特許はいずれも、生体分子に結び付けられたナノ粒子の標的部位への移動を加速するために電場を適用することについては記していない。
【０４７０】
ＤＮＡ修飾ナノ粒子の輸送に対する電場の影響を調べるために、１３ｎｍ金ナノ粒子を３’プロパンチオールでキャップした２２ｍｅｒ ＤＮＡで修飾し、この修飾粒子を水中に分散した。ケースレー（Ｋｅｉｔｈｌｅｙ）４８７ピコアンメータ／電圧源（Ｋｅｉｔｈｌｅｙ Ｉｎｓｔｒｕｍｅｎｔｓ社、アメリカ合衆国オハイオ州クリーヴランドオーロラロード２８７７５所在、カタログ番号４８７ピコアンメータ）を使用して、２つのインジウム−スズ酸化物（ＩＴＯ）電極を横切ってＤＣ電場を適用し、電流を測定した。電極とナノ粒子の移動を例示した図式１を参照されたい。ＩＴＯ被覆ガラス（Ｄｅｌｔａ Ｔｅｃｈｎｏｌｏｇｉｅｓ社、アメリカ合衆国ミネソタ州スチルウォーター所在（番号ＣＤ−５０ＩＮ−ＣＵＶ））を０．８×１ｃｍに切断した。２つのインジウム−スズ酸化物被覆ガラス基板を、カソード（陰極）およびアノード（陽極）として使用した。電極をＤＮＡ修飾ナノ粒子の溶液に浸漬し、３Ｖの直流電場を１５分間適用した（４μＡ）。電場の適用後、電極を水で洗浄した。正バイアスした電極（アノード）上にはピンク色が観察され、カソード上には肉眼で見える変化はなかった。アノード上でのナノ粒子の捕獲が、Ａｇ（Ｉ）とヒドロキノンの溶液による銀を用いたシグナルの増強により確認された。いずれの電極も銀染色溶液に３分間浸漬した。アノードは黒色になり、カソードには実質的な変化はなかった。この観察は、ＤＮＡ修飾粒子が、粒子上の高い負電荷のために電場によって輸送され得ることを示す。またこのことは、ＤＮＡ修飾粒子の電極表面上の相補的オリゴヌクレオチドへのハイブリダイゼーションを促進するために電場を利用することができることを示している。それはさらに、そのことが荷電粒子が関与する他の生体認識プロセスにまで拡張し得ることを示唆している。
【０４７１】
本明細書に引用した特許、特許出願および参考文献はすべて、引用によりその全体が組み込まれる。
【図面の簡単な説明】
【図１】相補的オリゴヌクレオチドが付着しているナノ粒子を結合させることによるナノ粒子凝集体の形成を示す略図。ナノ粒子は、相補的オリゴヌクレオチドのハイブリダイゼーションの結果として凝集体をなして結合している。Ｘは、（−Ｓ（ＣＨ_２）_３ＯＰ（Ｏ）（Ｏ⁻）−（ここで、Ｓは金ナノ粒子に接合している）などの）任意の共有結合アンカーを表す。図１および後続のいくつかの図面では、分かり易くするために、各ナノ粒子には単一のオリゴヌクレオチドしか付着していないように示されているが、実際には、各ナノ粒子には、いくつかのオリゴヌクレオチドが付着している。また、図１および後続図面において、金ナノ粒子とオリゴヌクレオチドとの相対サイズは正確な縮尺率では描かれていない点に留意することが重要である。
【図２】オリゴヌクレオチドが付着しているナノ粒子を用いて核酸を検出する系を示す略図。２つのナノ粒子上のオリゴヌクレオチドは、示されている一本鎖ＤＮＡの２つの異なる部分に対して相補的な配列を有している。その結果、これらのオリゴヌクレオチドがＤＮＡとハイブリダイズすると、（凝集体を形成し、変色を起こす）検出可能な変化が生じる。
【図３】図２に示されている系の変型の略図。２つのナノ粒子上のオリゴヌクレオチドは、示されているナノ粒子上のオリゴヌクレオチドとは相補的でない第３部分によって分離された一本鎖ＤＮＡの２つの異なる部分に対して相補的な配列を有している。一本鎖ＤＮＡの非相補的部分とのハイブリダイズに用い得る任意のフィラーオリゴヌクレオチドも示されている。ＤＮＡ、ナノ粒子およびフィラーオリゴヌクレオチドを合わせると、ナノ粒子は、切れ目が入った（ｎｉｃｋｅｄ）二本鎖オリゴヌクレオチドコネクタを形成して凝集する。
【図４】連結オリゴヌクレオチドとのハイブリダイゼーションおよびデハイブリダイゼーションの結果としての、オリゴヌクレオチドが付着しているナノ粒子の可逆的凝集を示す略図。例示されている連結オリゴヌクレオチドは、ナノ粒子に付着しているオリゴヌクレオチドと相補的なオーバーハング末端（付着末端）を有する二本鎖ＤＮＡである。
【図５】オリゴヌクレオチドが付着しているナノ粒子とナノ粒子に付着しているオリゴヌクレオチドに対して相補的な配列を有する連結オリゴヌクレオチドとを合わせることによるナノ粒子凝集体の形成を示す略図。
【図６Ａ】それぞれ異なるオリゴヌクレオチドが付着している２つの種類の金コロイドと、ナノ粒子に付着しているオリゴヌクレオチドと相補的な付着末端を有する連結二本鎖オリゴヌクレオチドとが入ったキュベット（図４参照）。キュベットＡ−連結ＤＮＡのＴｍを超える８０℃；デハイブリダイズ（熱変性）した。色は暗赤色。
【図６Ｂ】それぞれ異なるオリゴヌクレオチドが付着している２つの種類の金コロイドと、ナノ粒子に付着しているオリゴヌクレオチドと相補的な付着末端を有する連結二本鎖オリゴヌクレオチドとが入ったキュベット（図４参照）。キュベットＢ−リンカーＤＮＡのＴｍより低い室温に冷却後。ハイブリダイゼーションが起って、ナノ粒子は凝集したが、凝集体は沈殿しなかった。色は紫色。
【図６Ｃ】それぞれ異なるオリゴヌクレオチドが付着している２つの種類の金コロイドと、ナノ粒子に付着しているオリゴヌクレオチドと相補的な付着末端を有する連結二本鎖オリゴヌクレオチドとが入ったキュベット（図４参照）。キュベットＣ−室温下に数時間後、凝集したナノ粒子はキュベットの底に沈殿した。溶液は清澄で、沈殿物はピンクがかった灰色。ＢまたはＣを加熱するとＡが生じる。
【図７】図４に示されているように、オリゴヌクレオチドが付着している金ナノ粒子が温度を下げると連結オリゴヌクレオチドとハイブリダイズしたために凝集するときの吸光度の変化を示す吸光度対波長（ｎｍ）のグラフ。
【図８Ａ】図４に示されている系に関する吸光度対温度／時間の変化のグラフである。低温下、オリゴヌクレオチドが付着している金ナノ粒子は、連結オリゴヌクレオチドとのハイブリダイゼーションにより凝集する（図４参照）。高温（８０℃）下で、ナノ粒子はデハイブリダイズする。経時的な温度の変化は、これが可逆的プロセスであることを示している。
【図８Ｂ】修飾していない金ナノ粒子の水溶液を用いて図８Ａと同じ方法で実施した吸光度対温度／時間における変化のグラフである。図８Ａに見られた可逆的変化は観測されない。
【図９Ａ】透過型電子顕微鏡（ＴＥＭ）画像。金ナノ粒子上のオリゴヌクレオチドと連結オリゴヌクレオチドとのハイブリダイゼーションにより結合した凝集金ナノ粒子のＴＥＭ画像である。
【図９Ｂ】透過型電子顕微鏡（ＴＥＭ）画像。結合したナノ粒子の順序づけ（ｏｒｄｅｒｉｎｇ）を示す二次元凝集体のＴＥＭ画像である。
【図１０】ピリミジン：プリン：ピリミジンモチーフを有するナノ粒子間の熱安定性三本鎖オリゴヌクレオチドコネクタの形成を示す略図。このような三本鎖コネクタは、二本鎖コネクタより剛性が大きい。図１０において、一方のナノ粒子には、全てがプリンからなるオリゴヌクレオチドが付着しており、他方のナノ粒子には、全てがピリミジンからなるオリゴヌクレオチドが付着している。（ナノ粒子には付着していない）三本鎖コネクタを形成するための第３オリゴヌクレオチドはピリミジンからなる。
【図１１】相補的オリゴヌクレオチドが付着しているナノ粒子を合わせることによるナノ粒子凝集体の形成を示す略図であり、ナノ粒子は、相補的オリゴヌクレオチドのハイブリダイゼーションの結果として凝集体をなして結合している。図１１において、丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、ｓはチオ−アルキルリンカーである。２種類のナノ粒子上の多数のオリゴヌクレオチドは、互いにハイブリダイズして凝集体構造を形成し得る。
【図１２Ａ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的３が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、点線および破線は、ヌクレオチドを連結するつなぎ目を表す。
【図１２Ｂ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、点線および破線は、ヌクレオチドを連結するつなぎ目を表す。
【図１２Ｃ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的４が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、点線および破線は、ヌクレオチドを連結するつなぎ目を表す。
【図１２Ｄ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的５が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、点線および破線は、ヌクレオチドを連結するつなぎ目を表す。
【図１２Ｅ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的６が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、点線および破線は、ヌクレオチドを連結するつなぎ目を表す。
【図１２Ｆ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的７が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、点線および破線は、ヌクレオチドを連結するつなぎ目を表す。
【図１３Ａ】ナノ粒子および透明基板を用いたＤＮＡ（検体ＤＮＡ）検出系を示す略図である。
【図１３Ｂ】ナノ粒子および透明基板を用いたＤＮＡ（検体ＤＮＡ）検出系を示す略図である。
【図１４Ａ】オリゴヌクレオチドが付着している金ナノ粒子（その１つの集団は溶液中にあり、１つの集団は図１３Ｂに示されているような透明基板に付着している）が連結オリゴヌクレオチドとのハイブリダイゼーションにより凝集したときの吸光度の変化を示す吸光度対波長（ｎｍ）のグラフである。
【図１４Ｂ】温度の増大（融解する）に従った、図１４Ａに示されているハイブリダイズした系に関する吸光度の変化のグラフである。
【図１５Ａ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１５Ｂ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的３が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１５Ｃ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的４が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１５Ｄ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的５が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１５Ｅ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的６が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１５Ｆ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的７が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１５Ｇ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を示す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、一本鎖オリゴヌクレオチド標的８が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１６Ａ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を表す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、長さが異なる複数の一本鎖オリゴヌクレオチド標的が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１６Ｂ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を表す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、長さが異なる複数の一本鎖オリゴヌクレオチド標的が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１６Ｃ】オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系を表す略図。オリゴヌクレオチド−ナノ粒子共役体１および２と、長さが異なる複数の一本鎖オリゴヌクレオチド標的が示されている。丸はナノ粒子を表し、式はオリゴヌクレオチド配列であり、Ｓはチオ−アルキルリンカーを表す。
【図１７Ａ】ナノ粒子−オリゴヌクレオチド共役体と、オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系とを示す略図。丸はナノ粒子を表し、直線はオリゴヌクレオチド鎖（塩基は示さず）を表し、間隔の狭い２本の平行線は二本鎖分子セグメントを表し、小文字は特異的ヌクレオチド配列（ａはａ′と相補的、ｂはｂ′の相補的である、など）を示す。
【図１７Ｂ】ナノ粒子−オリゴヌクレオチド共役体と、オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系とを示す略図。丸はナノ粒子を表し、直線はオリゴヌクレオチド鎖（塩基は示さず）を表し、間隔の狭い２本の平行線は二本鎖分子セグメントを表し、小文字は特異的ヌクレオチド配列（ａはａ′と相補的、ｂはｂ′の相補的である、など）を示す。
【図１７Ｃ】ナノ粒子−オリゴヌクレオチド共役体と、オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系とを示す略図。丸はナノ粒子を表し、直線はオリゴヌクレオチド鎖（塩基は示さず）を表し、間隔の狭い２本の平行線は二本鎖分子セグメントを表し、小文字は特異的ヌクレオチド配列（ａはａ′と相補的、ｂはｂ′の相補的である、など）を示す。
【図１７Ｄ】ナノ粒子−オリゴヌクレオチド共役体と、オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系とを示す略図。丸はナノ粒子を表し、直線はオリゴヌクレオチド鎖（塩基は示さず）を表し、間隔の狭い２本の平行線は二本鎖分子セグメントを表し、小文字は特異的ヌクレオチド配列（ａはａ′と相補的、ｂはｂ′の相補的である、など）を示す。
【図１７Ｅ】ナノ粒子−オリゴヌクレオチド共役体と、オリゴヌクレオチドが付着しているナノ粒子を用いた核酸検出系とを示す略図。丸はナノ粒子を表し、直線はオリゴヌクレオチド鎖（塩基は示さず）を表し、間隔の狭い２本の平行線は二本鎖分子セグメントを表し、小文字は特異的ヌクレオチド配列（ａはａ′と相補的、ｂはｂ′の相補的である、など）を示す。
【図１８】リポソーム（大きい二重丸）、ナノ粒子（小さい白丸）および透明基板を用いた核酸検出系を示す略図。黒い四角はコレステリル基を表し、ねじれ線はオリゴヌクレオチドを表し、梯子は二本鎖（ハイブリダイズした）オリゴヌクレオチドを表す。
【図１９Ａ】金ナノ粒子−オリゴヌクレオチド共役体が図１３Ａに示されているような透明基板上の多重層として集合したときの吸光度の変化を示す吸光度対波長（ｎｍ）のグラフである。
【図１９Ｂ】温度の増大（融解）に従った図１９Ａに示されているハイブリダイズした系に関する吸光度の変化のグラフである。
【図２０Ａ】金属もしくは半導体消光ナノ粒子（図２０Ａ）に付着している蛍光標識オリゴヌクレオチドを用いた計画の略図。
【図２０Ｂ】非金属、非半導体粒子（図２０Ｂ）に付着している蛍光標識オリゴヌクレオチドを用いた計画の略図。
【図２１】オリゴヌクレオチドが付着している金ナノ粒子および蛍光標識オリゴヌクレオチドが付着しているラテックスミクロスフェアを用いた標的核酸検出系を示す略図。小さい黒丸はナノ粒子を表し、大きい白丸はラテックスミクロスフェアを表し、大きい楕円は微孔質膜を表す。
【図２２】２つの種類の蛍光標識オリゴヌクレオチド−ナノ粒子共役体を用いた標的核酸検出系を示す略図。黒丸はナノ粒子を表し、大きな楕円は微孔質膜を表す。
【図２３】炭疽菌保護抗原（実施例１２参照）用アッセイに利用される材料の配列。
【図２４】オリゴヌクレオチド（直線）が付着している磁気ナノ粒子（黒球）、ナノ粒子に付着しているオリゴヌクレオチドにハイブリダイズしたプローブオリゴヌクレオチド（直線）を含む「サテライトプローブ」を用いた標的核酸検出系を示す略図であり、プローブオリゴヌクレオチドはレポーター基（白四角）で標識されている。Ａ、Ｂ、Ｃ、Ａ′、Ｂ′、およびＣ′は、特異的ヌクレオチド配列を表し、Ａ、ＢおよびＣはそれぞれ、Ａ′、Ｂ′およびＣ′と相補的である。
【図２５Ａ】ナノ粒子と透明基板を用いたＤＮＡ検出系を示す略図。これらの図面において、ａ、ｂおよびｃは、異なるオリゴヌクレオチド配列を表し、ａ′、ｂ′およびｃ′は、それぞれａ、ｂおよびｃと相補的なオリゴヌクレオチド配列を表す。
【図２５Ｂ】ナノ粒子と透明基板を用いたＤＮＡ検出系を示す略図。これらの図面において、ａ、ｂおよびｃは、異なるオリゴヌクレオチド配列を表し、ａ′、ｂ′およびｃ′は、それぞれａ、ｂおよびｃと相補的なオリゴヌクレオチド配列を表す。
【図２６】ＣｄＳｅ／ＺｎＳコア／シェル量子ドット（ＱＤ）の集合体を形成する系を示す略図。
【図２７Ａ】４００ｎｍでの励起で、分散ＱＤと凝集ＱＤを比較する蛍光スペクトルを示している。試料は同じように調製したが、但し、一方には相補的「リンカー」ＤＮＡを付加し、他方には等量、等濃度の非相補的ＤＮＡを付加した。
【図２７Ｂ】「融解」の前、間および後の異なる温度下のＱＤ／ＱＤ集合体のＵＶ−可視スペクトルを示している。図中の挿入図は、集合体の熱変性に関する温度対消光プロファイルを示している。変性実験は、０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液（ｐＨ７）、０．０１％アジ化ナトリウム中、１３ｎｍの金ナノ粒子および／または〜４ｎｍのＣｄＳｅ／ＺｎＳコア／シェルＱＤを用いて実施した。
【図２７Ｃ】ハイブリッド金／ＱＤ集合体の一部の高解像度ＴＥＭ画像を示している。指紋に似たＱＤの格子干渉縞が各金ナノ粒子の近くに見える。
【図２７Ｄ】「融解」の前、間および後の異なる温度下のハイブリッド金／ＱＤ集合体のＵＶ−可視スペクトルを示している。図中の挿入図は、集合体の熱変性に関する温度対消光プロファイルを示している。変性実験は、０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液（ｐＨ７）、０．０１％アジ化ナトリウム中、１３ｎｍの金ナノ粒子および／または〜４ｎｍのＣｄＳｅ／ＺｎＳコア／シェルＱＤを用いて実施した。
【図２８Ａ】コアプローブ、凝集体プローブの作製およびこれらのプローブを用いたＤＮＡ検出系を示す略図。これらの図面において、ａ、ｂ、ｃおよびｄは、異なるオリゴヌクレオチド配列を表し、ａ′、ｂ′、ｃ′およびｄ′はそれぞれ、ａ、ｂ、ｃおよびｄと相補的なオリゴヌクレオチド配列を表す。
【図２８Ｂ】コアプローブ、凝集体プローブの作製およびこれらのプローブを用いたＤＮＡ検出系を示す略図。これらの図面において、ａ、ｂ、ｃおよびｄは、異なるオリゴヌクレオチド配列を表し、ａ′、ｂ′、ｃ′およびｄ′はそれぞれ、ａ、ｂ、ｃおよびｄと相補的なオリゴヌクレオチド配列を表す。
【図２８Ｃ】コアプローブ、凝集体プローブの作製およびこれらのプローブを用いたＤＮＡ検出系を示す略図。これらの図面において、ａ、ｂ、ｃおよびｄは、異なるオリゴヌクレオチド配列を表し、ａ′、ｂ′、ｃ′およびｄ′はそれぞれ、ａ、ｂ、ｃおよびｄと相補的なオリゴヌクレオチド配列を表す。
【図２８Ｄ】コアプローブ、凝集体プローブの作製およびこれらのプローブを用いたＤＮＡ検出系を示す略図。これらの図面において、ａ、ｂ、ｃおよびｄは、異なるオリゴヌクレオチド配列を表し、ａ′、ｂ′、ｃ′およびｄ′はそれぞれ、ａ、ｂ、ｃおよびｄと相補的なオリゴヌクレオチド配列を表す。
【図２８Ｅ】コアプローブ、凝集体プローブの作製およびこれらのプローブを用いたＤＮＡ検出系を示す略図。これらの図面において、ａ、ｂ、ｃおよびｄは、異なるオリゴヌクレオチド配列を表し、ａ′、ｂ′、ｃ′およびｄ′はそれぞれ、ａ、ｂ、ｃおよびｄと相補的なオリゴヌクレオチド配列を表す。
【図２９】メルカプトエタノールによる、オリゴヌクレオチドが付着しているナノ粒子（黒丸）または金薄膜（白四角）からのオリゴヌクレオチドの置換分率のグラフ。
【図３０】ナノ粒子−オリゴヌクレオチド共役体の調製に使用される、様々な認識オリゴヌクレオチド：希釈剤オリゴヌクレオチド比に対して得られた、ナノ粒子上の認識オリゴヌクレオチドの表面被覆範囲のグラフ。
【図３１】ナノ粒子上の認識オリゴヌクレオチドの様々な表面被覆範囲に対する、ハイブリダイズした相補的なオリゴヌクレオチドの表面被覆範囲のグラフ。
【図３２】ナノ粒子−オリゴヌクレオチド共役体および銀染色法による増幅を用いた基板上の４素子アレイ中の標的ＤＮＡを検出する系を示す略図。
【図３３】平台型スキャナを用いて得た、７ｍｍ×１３ｍｍのオリゴヌクレオチド官能化フロートガラススライドの画像。（Ａ）ＤＮＡ標的と金ナノ粒子−オリゴヌクレオチド指示共役体とがハイブリダイズする前のスライド。（Ｂ）１０ｎＭの標的ＤＮＡと５ｎＭのナノ粒子−オリゴヌクレオチド指示共役体とがハイブリダイズした後のスライドＡ。ピンク色は赤い直径１３ｎｍ金ナノ粒子が付着したためである。（Ｃ）銀増幅溶液に５分間暴露した後のスライドＢ。（Ｄ）（Ａ）と同じ。（Ｅ）１００ｐＭの標的と５ｎＭのナノ粒子−オリゴヌクレオチド指示共役体とがハイブリダイズした後のスライドＤ。ナノ粒子層の吸光度は低過ぎて肉眼または平台型スキャナで観測できなかった。（Ｆ）銀増幅溶液に５分間暴露した後のスライドＥ。スライドＦはスライドＣよりはるかに明るく、これは、標的濃度が低いことを示している。（Ｇ）５ｎＭのナノ粒子−オリゴヌクレオチド指示共役体に暴露し、かつ銀増幅溶液に５分間暴露した対照スライド。スライドの黒ずみは観測されなかった。
【図３４】種々の濃度の標的ＤＮＡに暴露した後、５ｎＭの金ナノ粒子−オリゴヌクレオチド指示共役体に暴露し、銀増幅溶液に５分間暴露した、オリゴヌクレオチド官能化ガラス表面のグレースケール（光学密度）のグラフ。
【図３５Ａ】発蛍光団で標識した標的のオリゴヌクレオチド官能化ガラス表面からの解離を示すハイブリダイズ標的％対温度のグラフ。
【図３５Ｂ】ナノ粒子で標識した標的（図３５Ｂ）のオリゴヌクレオチド官能化ガラス表面からの解離を示すハイブリダイズ標的％対温度のグラフ。図３５ＡおよびＢにおいて、測定は、ガラス表面上の溶液中の解離した標識の蛍光（図３５Ａ）および吸光度（図３５Ｂ）を測定して実施した。「ｂ」と標示された線は、ガラス上の完全対合オリゴヌクレオチドの解離曲線を示し、「ｒ」と標示されている線は、ガラス上の誤対合オリゴヌクレオチド（１塩基誤対合）の曲線を示している。グラフ中の垂線は、各測定に関して所与の温度（各曲線の融解温度Ｔ_ｍの中間）で解離した標的分画および発蛍光団およびナノ粒子ベース遺伝子チップに関する予測された配列同定選択率を示している。蛍光（図３５Ａ）：相補体（６９％）／誤対合（３８％）＝１．８：１。吸光度（図３５Ｂ）：相補体（８５％）／誤対合（１４％）＝６：１。発蛍光団標識曲線（図３５Ａ）の幅は、発蛍光団標識標的の遺伝子チップからの解離に特徴的なものである（Ｆｏｒｍａｎら，Ｍｏｌｅｃｕｌａｒ Ｍｏｄｅｌｉｎｇ ｏｆ Ｎｕｃｌｅｉｃ Ａｃｉｄｓ，Ｌｅｏｎｔｉｓら編（ＡＣＳ Ｓｙｍｐｏｓｉｕｍ Ｓｅｒｉｅｓ ６８２，Ａｍｅｒｉｃａｎ Ｃｈｅｍｉｃａｌ Ｓｏｃｉｅｔｙ，Ｗａｓｈｉｎｇｔｏｎ，Ｄ．Ｃ．，１９９８年），２０６−２２８ページ）。
【図３６Ａ】合成標的および蛍光標識ナノ粒子−オリゴヌクレオチド共役体プローブで攻撃したモデルオリゴヌクレオチド配列の画像。
【図３６Ｂ】ナノ粒子標識ナノ粒子−オリゴヌクレオチド共役体プローブで攻撃したモデルオリゴヌクレオチド配列の画像。図３６ＡおよびＢにおいて、Ｃ、Ａ、ＴおよびＧは、基板に付着しているオリゴヌクレオチドに１単塩基変化が起こって標的との完全対合（塩基Ａ）または１塩基誤対合（塩基Ａとの完全対合の代わりに塩基Ｃ、ＴまたはＧ）をもたらした配列上のスポット（要素）を表している。要素Ｃ：Ａ：Ｔ：Ｇのグレースケール比は、図３６Ａに関しては９：３７：９：１１、図３６Ｂに関しては３：６２：７：３４である。
【図３７】連結核酸（３）を介して結合したナノ粒子（ａおよびｂ）の凝集体（Ａ）または層（Ｂ）を形成する系を示す略図。
【図３８Ａ】相補的リンカー３を介してオリゴヌクレオチド官能化ガラス製顕微鏡スライドにハイブリダイズした金ナノ粒子のａとｂの交互層（図３７参照）のＵＶ−可視スペクトル。これらのスペクトルは、１（ａ、λ_ｍａｘ＝５２４ｎｍ）、２（ｂ、λ_ｍａｘ＝５２９ｎｍ）、３（ｃ、λ_ｍａｘ＝５３２ｎｍ）、４（ｄ、λ_ｍａｘ＝５３４ｎｍ）または５（ｅ、λ_ｍａｘ＝５３４ｎｍ）層を有する集合体に関する。これらのスペクトルは、スライドを介して直接測定した。
【図３８Ｂ】層の数の増大に伴うナノ粒子集合体（図３８Ａ参照）のλ_ｍａｘでの吸光度のグラフ。
【図３９Ａ】ＤＮＡリンカーと共にオリゴヌクレオチド官能化導電性インジウム−スズ−オキシド（ＩＴＯ）スライド（オリゴヌクレオチド官能化ガラススライドと同じ方法で作製）に同時ハイブリダイズしたオリゴヌクレオチド官能化金ナノ粒子の１つの層のＦＥ−ＳＥＭ。このスライドの可視吸光度スペクトルは、図３８Ａと同一であったが、これは、ＩＴＯ上の官能化およびナノ粒子被覆面積がガラス上のものと類似であることを示している。１０枚のそのような画像から計数したナノ粒子の平均密度は約８００ナノ粒子／μｍ^２であった。
【図３９Ｂ】ＩＴＯスライド上の２つのナノ粒子層のＦＥ−ＳＥＭ画像。１０枚のそのような画像から計数したナノ粒子の平均密度は約２８００ナノ粒子／μｍ^２であった。
【図３９Ｃ】０．３Ｍ ＮａＣｌ、１０ｍＭ リン酸緩衝液（ｐＨ７）中の一本鎖とのオリゴヌクレオチド二本鎖分子（１＋２＋３；図３５Ａ参照）の０．５μＭ溶液の解離を示す２６０ｎｍ（Ａ_２６０）の吸光度。
【図３９Ｄ】０．３Ｍ ＮａＣｌ、１０ｍＭ 燐酸緩衝液に浸漬したガラススライドからの１層のオリゴヌクレオチド官能化金ナノ粒子の解離を示す２６０ｎｍ（Ａ_２６０）の吸光度。
【図３９Ｅ】０．３Ｍ ＮａＣｌ、１０ｍＭ 燐酸緩衝液に浸漬したガラススライドからの４層のオリゴヌクレオチド官能化金ナノ粒子の解離を示す２６０ｎｍ（Ａ_２６０）の吸光度。
【図３９Ｆ】０．３Ｍ ＮａＣｌ、１０ｍＭ 燐酸緩衝液に浸漬したガラススライドからの１０層のオリゴヌクレオチド官能化金ナノ粒子の解離を示す２６０ｎｍ（Ａ_２６０）の吸光度。図Ｄ〜Ｆに関して、温度の増大に従って減少するスライドを介した５２０ｎｍ（Ａ_５２０）での吸光度を測定して融解プロファイルを得た。図３９Ｄ−Ｆそれぞれの挿入図は、測定した解離曲線の一次導関数を示している。これらの曲線のＦＷＨＭは、（図３９Ｃの挿入図）１３．２℃、（図３９Ｄの挿入図）５．６℃、（図３９Ｅの挿入図）３．２℃、（図３９Ｆの挿入図）２．９℃であった。
【図４０】ＤＮＡを介して結合している金ナノ粒子集合体の電気的性質の測定に用いられる系を示す略図。分かり易くするために、単一のハイブリダイゼーション事象のみが描かれている。
【図４１】金電極および金ナノ粒子を用いた核酸検出法を示す略図。
【図４２】オリゴヌクレオチドをナノ粒子に連結する際に使用されるステロイド部分を有する好ましい化合物１を含む、環式ジスルフィド１の構造を例証する略図。ステロイドジスルフィド分子は、４，５−ジヒドロキシ−１，２−ジチアンをエピアンドロステロンと縮合することにより得られる。金のナノ粒子−オリゴヌクレオチド共役体は、ステロイドジスルフィドにより修飾したオリゴヌクレオチドを使用して調製され、その調製用にオリゴヌクレオチド−メルカプトヘキシルリンカーを使用したナノ粒子−オリゴヌクレオチド共役体と比べて、ＤＴＴに対するより大きな安定性を示す。
【図４３】ステロイド環式スルフィドアンカー基に対する合成と化学式を示す略図。
【図４４】実施例２４に説明したオリゴヌクレオチド環式ジスルフィドリンカーの調製に使用される化学式２の環式ジスルフィドと、アンカー基として使用される同様な関連環式ジスルフィドを示す略図。
【図４５】実施例２５に記述した構造を示す略図。図４５（ａ）は、５′−モノチオール−修飾オリゴヌクレオチド５、３５塩基５′−ステロイドジスルフィドオリゴマー６、Ｔｒｅｍｂｌｅｒホスホルアミド７、および５−トリ−メルカプトアルキルオリゴヌクレオチド８を示す。
【図４６】新規トリチオールオリゴヌクレオチドを作製する化学的機構を示す略図。
【図４７】高密度オリゴヌクレオチドナノ粒子共役体Ｉ、オリゴヌクレオチドタンパク質共役体１、ナノ粒子−オリゴヌクレオチド−タンパク質共役体ＩＩ、及びプローブ−ターゲット複合体ＩＩＩの略図。オリゴヌクレオチド共役体Ｉは、共役体Ｉに結合したオリゴヌクレオチドに相補的な配列を有している。プローブＩＩは、タンパク質のための代表的プローブである。複合体ＩＩＩは、タンパク質ターゲットとプローブに結合したタンパク質との特異的結合相互作用からもたらされる。
【図４８】特異的タンパク質を、ガラス表面に固定化した異なるタンパク質のアレイ中ので検出する際のプローブＩＩの適用例の略図。金ナノ粒子は、視覚的に、或いは銀染色により認識することができる。蛍光ナノ粒子は、それらの蛍光により認識することができる。この図に示すように、ガラスプレートは、表面に固定化した３種の異なるタンパク質を示しており、そのうち１種は、プローブＩＩでタンパク質に結合する。段階１において、そのプレートをプローブＩＩを含有する溶液に暴露する。段階２において、非結合のプローブＩＩを洗い流す。段階３において、その一連のタンパク質のうち第１のタンパク質が占める部位の結合したナノ粒子（ＩＶ）の存在を観察する。
【図４９】ナノ粒子−オリゴヌクレオチド−受容体プローブ（ＩＩ’）の略図、及び特異的ターゲットを、滑面に固定化した物質のアレイで検出する際の適用例の略図。認識受容体ユニット（ターゲットに相補的な特異的結合）は、Ａと呼称し、ターゲットはＢと称する。
【図５０】（ａ）受容体（Ｒ）に連結したオリゴヌクレオチドが、ナノ粒子に結合したオリゴヌクレオチドに相補的であり、ナノ粒子共役体が、受容体に結合したオリゴヌクレオチドに、ハイブリダイゼーションにより結合することを示すナノ粒子−オリゴヌクレオチド−受容体プローブ（ＩＩ）の略図、及び（ｂ）受容体（Ｒ）に連結するオリゴヌクレオチドが、ハイブリダイゼーションの結果、連結オリゴヌクレオチドの第１部分に結合し、ナノ粒子に結合したオリゴヌクレオチドが、ハイブリダイゼーションの結果、連結オリゴヌクレオチドの第２部分に結合することを示すナノ粒子−オリゴヌクレオチド−受容体プローブ（ＩＩ）の略図。簡略化のために、オリゴヌクレオチド−ナノ粒子共役体に結合したオリゴヌクレオチド−タンパク質共役体を１種のみ示しているが、実際の数はもっと多い可能性がある。
【図５１Ａ】特異的ターゲット、例えば薬物又はタンパク質を、ガラス表面に固定化した異なる薬物又はタンパク質のアレイで検出する際のプローブ（図５０ｂ）の適用例の略図。
【図５１Ｂ】特異的ターゲットを、異なる分子の混合物を有する細胞で検出する際のプローブ図５０ｂの適用例の略図。図５１Ａ，Ｂにおいて、プローブは、検体、例えばタンパク質又は薬物が多重結合部位を有する場合も、スポットテストにおいて用いることができる。
【図５２】結合したオリゴヌクレオチド１、ナノ粒子オリゴヌクレオチド共役体２、及びターゲット核酸３を有するビオチン分子に結合したストレプトアビジンの複合体のハイブリダイゼーション。
【図５２Ｂ】ある種のナノ粒子のオリゴヌクレオチドを第２の種のナノ粒子とハイブリダイゼーションさせることによる金ナノ粒子アセンブリ。
【図５２Ｃ】ナノ粒子に結合したオリゴヌクレオチドに相補的な配列を有するオリゴヌクレオチドに各ビオチン分子が結合するような４種のビオチン分子に特異的に結合したアビジンの複合体のハイブリダイゼーションによる金ナノ粒子アセンブリ、の結果形成した３次元金ナノ粒子−ストレプトアビジンアセンブリ又は凝集体の略図。図５２Ａ〜Ｂの３例全てにおいて、凝集体が形成すると、溶液の色が赤色から紫色又は青灰色に変化した。
【図５３】本発明の金ナノ粒子／オリゴヌクレオチド／ストレプトアビジン共役体（左）及び市販の金コロイド／ストレプトアビジン共役体（右）の非特異的結合テスト（実施例２７）を示す写真。市販の共役体は、灰色のスポットを示し、その共役体がガラス表面に接着していることが示されたが、本発明の共役体は、有意な銀染色を示さなかった。
【図５４】表面に結合したビオチンへの金ナノ粒子−オリゴヌクレオチド−ストレプトアビジン共役体の結合の略図。ビオチンは、支持体表面に結合したオリゴヌクレオチドに相補的な配列を有するオリゴヌクレオチドに連結し、オリゴヌクレオチド−ビオチン共役体は、表面に結合したオリゴヌクレオチドにハイブリダイズされる。
【図５５】リンカーをタンパク質のためのＤＮＡ受容体として用いるタンパク質検出方法の略図。図示するように、結合した受容体ＤＮＡを有するナノ粒子−オリゴヌクレオチド−ビオチン共役体は、ストレプトアビジンを付加すると、３次元ナノ粒子アセンブリを形成する。その凝集体は、その後単離され、デハイブリダイゼーションに付されて、ナノ粒子−オリゴヌクレオチド共役体と、結合するオリゴヌクレオチドを有するビオチン分子に結合するストレプトアビジンの複合体と、その受容体とを遊離する。そのタンパク質は、ＤＮＡチップの使用をはじめとする従来法での受容体ＤＮＡの同定により検出される。
【図５６】（ａ）オリゴヌクレオチドに連結した単一のビオチンに結合するストレプトアビジン（ビオチンに結合したオリゴヌクレオチドは、ハイブリダイゼーションの結果、リンカーオリゴヌクレオチドの第１部分に更に結合する）、及び（ｂ）結合するオリゴヌクレオチドを有する金ナノ粒子（ナノ粒子に結合したそのオリゴヌクレオチドは、リンカーオリゴヌクレオチドの第２部分に相補的な配列を有する）、からの金ナノ粒子−オリゴヌクレオチド−ストレプトアビジン共役体の調製（実施例２６）を示す略図。
【図５７】（ａ）ストレプトアビジン−オリゴヌクレオチド共役体（１−ＳＴＶ）、１３ｎｍの金粒子（２−Ａｕ）、及びリンカーＤＮＡ（３）の溶液を５３℃に加熱すると凝集体を形成すること、及び（ｂ）アニーリングの前後に凝集体内の粒子がそれらの物理的形状を残していること、を示す透過電子顕微鏡（ＴＥＭ）像（実施例２８）は、粒子の融合がないことを示しており、更に表面オリゴヌクレオチド層の安定した影響を実証している。
【図５８Ａ】温度の関数としての融解曲線により、ストレプトアビジン−ナノ粒子凝集体が非特異的相互作用ではなくＤＮＡハイブリダイゼーション相互作用により形成し、その工程が可逆的であることが確認される。実施例２８を参照されたい。
【図５８Ｂ】波長の関数としての融解曲線により、ストレプトアビジン−ナノ粒子凝集体が非特異的相互作用ではなくＤＮＡハイブリダイゼーション相互作用により形成し、その工程が可逆的であることが確認される。実施例２８を参照されたい。
【図５９】実施例２８に記載するような様々な凝集体の様々な粒子間距離を示す小角Ｘ線散乱回折パターン。
【図６０】実施例２９で説明されているＩＴＯ電極装置一式の例示図。[0001]
This invention was made with government support under United States National Institutes of Health (NIH) grant number GM10265 and Army Research Institute (ARO) grant number DAAG55-0967-0133. The government has certain rights in the invention.
[0002]
This application is a continuation-in-part of pending PCT application No. PCT / US97 / 12783, filed July 21, 1997, pending application Ser. No. 09 / 240,755 (Jan. 29, 1999). No. 09 / 603,830 (June 2000), which is a continuation-in-part of Japanese Patent Application No. 09 / 344,667 (filed on June 25, 1999). (Filed on Jan. 12, 2001), a pending application of application number 09 / 760,500 (filed on Jan. 12, 2001). No. (filed on March 28, 2001). Each of the above references is incorporated herein by reference. Provisional Application Nos. 60 / 031,809 (filed on July 29, 1996), 60 / 176,409 (filed on January 13, 2000), and 60 / 200,161 (filed on April 26, 2000) No. 60 / 192,699 (filed on Mar. 28, 2000), No. 60 / 254,392 (filed on Dec. 8, 2000), and No. 60 / 255,235 (dec. 2000) Claims No. 60 / 224,631). The disclosure of each of the above documents is incorporated herein by reference.
[0003]
(Field of the Invention)
The present invention relates to a method for detecting analytes containing nucleic acids and proteins, natural or synthetic, and modified or unmodified. The present invention also relates to materials for detecting nucleic acids and methods for preparing those materials. The invention further relates to a nanofabrication method. Finally, the invention relates to a method for separating selected nucleic acids from other nucleic acids.
[0004]
(Background of the Invention)
The development of methods for detecting and screening nucleic acids is important for the diagnosis of genetic, bacterial and viral diseases. Mansfield, E .; S. See Molecular and Cellular Probes, Vol. 9, pages 145-156 (1995). Currently, there are a variety of methods used to detect a particular nucleic acid sequence. Same as above. However, these methods are complex, time consuming and / or require specialized and expensive equipment. Clearly, a simple and fast nucleic acid detection method that does not require such a device is desirable.
[0005]
Colloidal gold protein probes have found wide application in immunocytochemistry [S. Garzon and M.S. Bendayan, "Immuno-Gold Electron Microscopy", "Colloidal Gold Probes: An Overview of Applications in Virus Cell Chemistry" and "Overview of the Applications of Clinical Applications." D. Hyatt and B.A. T. Eaton, CrC Publishing, Ann Arbor, MI (1993); E. FIG. Beesley, Colloidal Gold: A New Perspective for Cytochemical Marking, "Oxford University Press, Oxford (1989)]. These probes are prepared by adsorbing antibodies on gold surfaces from aqueous solutions under carefully defined conditions. Although conjugates produced in this way are functional, they have several disadvantages: for example, some such proteins desorb on standing and compete with adsorbed antibodies against antigen targets Liberates antibody into solution; low activity due to low adsorption and partial denaturation of antibody upon adsorption; and particles coated with such proteins are self-assembled, especially in solutions with high ionic strength There is fluorescence. Another means for preparing nanoparticle protein probes is described in E. FIG. Hainfeld, R.A. D. Leone, F.C. R. Furuya, and R.A. D. Powell (U.S. Pat. No. 5,521,289 (May 28, 1996), "Small Organometallic Probes"). Typically, this method involves reducing a gold salt in an organic solvent containing a triallyl phosphine or mercaptoalkyl derivative to yield a reactive substituent (X) and attaching the gold salt to the surface via an Au-P or Au-S bond. Involves providing small nanoparticles (50-70 gold atoms) that carry the X substituent on the attached linker. Subsequently, the colloid solution is reacted with a protein having a substituent Y that reacts with the protein X, and the protein is covalently linked to the nanoparticles. The effect on such nanoparticles is limited by the poor solubility of many proteins in water, which limits the range of protein-nanoparticle conjugates that can be used effectively. Furthermore, since there are only a few gold atoms on the surface of these particles, the number of "capture" chains that can be attached to the surface of a given particle is very low.
[0006]
Various methods have been developed for assembling metal and semiconductor colloids into nanomaterials. These methods focus on the use of covalently linked linker molecules with functionalities that have chemical affinity for the colloid of interest at opposite ends. One of the most successful methods to date, Brust et al., Adv. Mater. 7, Vol. 795-797 (1995), which involves the use of colloidal gold and well-established thiol adsorption chemistry. Bain & Whitesides, Angew. Chem. Int. Ed. Engl. 28, 506-512 (1989) and Dubois & Nuzzo, Annu. Rev .. Phys. Chem. 43, pp. 437-64 (1992). In this method, a linear alkanedithiol is used as the particle linker molecule. The thiol groups at each end of the linker molecule covalently bind to the colloid particles to form an aggregate structure. The disadvantage of this method is that the process is difficult to control and the aggregates are formed irreversibly. If one wants to take full advantage of the material properties of these structures, a method is needed to systematically control the assembly process.
[0007]
The potential use of DNA in the preparation of biomaterials for medical use and nanofabrication has been recognized. In this study, researchers focus on using the sequence-specific molecular recognition properties of oligonucleotides to design impressive structures with well-defined geometry and size . Shekhtman et al., New J. Med. Chem. 17, pp. 757-763 (1993); Shaw & Wang, Science, 260, 533-536 (1993); Chen et al., J. Am. Am. Chem. Soc. 111, 6402-6407 (1989); Chen & Seeman, Nature, 350, 631-633 (1991); Smith and Feigon, Nature, 356, 164-168 (1992). Wang et al., Biochem. 32, 1899-1904 (1993); Chen et al., Biochem. 33, 13540-13546 (1994); Marsh et al., Nucleic Acids Res. 23, 696-700 (1995); Mirkin, Annu. Review Biophys. Biomol. Struct. 23, 541-576 (1994); Biol. Chem. 263: 1095-1098 (1988); Wang et al., Biochem. 30, Vol. 5, pp. 5667-567 (1991). However, it is still far from confirming the theory of DNA structure generation experimentally. Seeman et al., New J. Am. Chem. 17, Vol. 739-755 (1993).
[0008]
(Summary of the Invention)
The present invention provides a method for detecting a nucleic acid. In one embodiment, the method comprises the step of contacting certain types of nanoparticles (nanoparticle-oligonucleotide conjugate) with oligonucleotides attached to the nucleic acid. The nucleic acid has at least two portions, and the oligonucleotide on each nanoparticle has a sequence that is complementary to the sequence of at least two portions of the nucleic acid. Contacting occurs under conditions that allow the nucleic acid to effectively hybridize the oligonucleotide on the nanoparticle. Hybridization of the oligonucleotide and nucleic acid on the nanoparticle results in a detectable change.
[0009]
In another embodiment, the method comprises the step of contacting at least two types of nanoparticles having oligonucleotides attached thereto with a nucleic acid. The oligonucleotide on the first type of nanoparticle has a sequence that is complementary to a first portion of the sequence of the nucleic acid. The oligonucleotide on the second type of nanoparticle has a sequence that is complementary to a second portion of the sequence of the nucleic acid. Contacting occurs under conditions that allow the nucleic acid to effectively hybridize the oligonucleotide on the nanoparticle and observes a detectable change caused by this hybridization.
[0010]
In a further embodiment, a method includes providing a substrate having a first type of nanoparticles attached thereto. An oligonucleotide is attached to the first type of nanoparticle, the oligonucleotide having a sequence that is complementary to a first portion of the sequence of the nucleic acid. The substrate is contacted with the nucleic acid under conditions that allow the oligonucleotide on the nanoparticle to hybridize effectively with the nucleic acid. Thereafter, a second type of nanoparticles having the oligonucleotide attached thereto is provided. The oligonucleotide has a sequence that is complementary to one or more other portions of the sequence of the nucleic acid. The nucleic acid bound to the substrate is contacted with a second type of nanoparticle oligonucleotide under conditions that effectively hybridize the oligonucleotide on the second type of nanoparticle to the nucleic acid. A detectable change is now observable. The method further comprises providing a binding oligonucleotide having a selected sequence having at least two portions. The first of the two portions is complementary to at least a portion of the sequence of the oligonucleotide on the second type of nanoparticle. The binding oligonucleotide is contacted with a second type of nanoparticle-oligonucleotide conjugate bound to the substrate under conditions that allow the binding oligonucleotide to effectively hybridize to the oligonucleotide on the nanoparticle. Thereafter, a third type of nanoparticle to which an oligonucleotide having a sequence complementary to the sequence of the second portion of the binding oligonucleotide is attached is subjected to conditions under which the binding oligonucleotide is effectively hybridized to the oligonucleotide on the nanoparticle. To contact with the binding oligonucleotide bound to the substrate. Finally, the detectable change caused by such hybridization is observed.
[0011]
In yet another embodiment, the method comprises contacting the nucleic acid with a substrate having an oligonucleotide having a sequence complementary to the first portion of the sequence of the nucleic acid attached. Contacting occurs under conditions that allow the oligonucleotides on the substrate to effectively hybridize to the nucleic acid. Thereafter, the nucleic acid bound to the substrate is contacted with a first type of nanoparticles having an oligonucleotide having a sequence complementary to the second portion of the nucleic acid sequence attached thereto. Contacting occurs under conditions that effectively hybridize the oligonucleotide on the nanoparticle to the nucleic acid. Next, the first type of nanoparticle-oligonucleotide conjugate bound to the substrate is brought into contact with the second type of nanoparticle to which the oligonucleotide is attached. The oligonucleotide on the second type of nanoparticles has a sequence that is complementary to at least a portion of the sequence of the oligonucleotide on the first type of nanoparticles. Contacting occurs under conditions that allow the oligonucleotides on the first and second types of nanoparticles to effectively hybridize. Finally, the detectable change caused by such hybridization is observed.
[0012]
In another embodiment, the method comprises the step of contacting the nucleic acid with a substrate having an oligonucleotide having a sequence complementary to the first portion of the sequence of the nucleic acid attached. Contacting occurs under conditions that allow the oligonucleotides on the substrate to effectively hybridize to the nucleic acid. Thereafter, the nucleic acid bound to the substrate is contacted with a liposome to which an oligonucleotide having a sequence complementary to a part of the sequence of the nucleic acid is attached. Contacting occurs under conditions that effectively hybridize the oligonucleotide on the liposome to the nucleic acid. Next, the liposome-oligonucleotide conjugate bound to the substrate is contacted with the first type of nanoparticles to which at least the first type of oligonucleotide is attached. The end of the first type of oligonucleotide that is not attached to the nanoparticle has a hydrophobic group. Contacting occurs under conditions where the oligonucleotide on the nanoparticle effectively attaches to the liposome as a result of the hydrophobic interaction. A detectable change is now observable. The method may further comprise the step of contacting the first type of nanoparticle-oligonucleotide conjugate associated with the liposome with a second type of nanoparticle having the oligonucleotide attached thereto. A second type of oligonucleotide having a sequence complementary to at least a portion of the sequence of the oligonucleotide on the second type of nanoparticles is attached to the first type of nanoparticle. The oligonucleotide on the second type of nanoparticles has a sequence that is complementary to at least a portion of the sequence of the second type of oligonucleotide on the first type of nanoparticles. Contacting occurs under conditions that allow the oligonucleotides on the first and second types of nanoparticles to effectively hybridize. Thereafter, a detectable change is observed.
[0013]
In another embodiment, the method comprises the step of contacting the nucleic acid to be detected with a substrate having oligonucleotides attached thereto. The oligonucleotide has a sequence that is complementary to a first portion of the sequence of the nucleic acid. Contacting occurs under conditions that allow the oligonucleotides on the substrate to effectively hybridize to the nucleic acid. Next, the nucleic acid bound to the substrate is contacted with a type of nanoparticle to which an oligonucleotide having a sequence complementary to the second part of the sequence of the nucleic acid is attached. Contacting occurs under conditions that effectively hybridize the oligonucleotide on the nanoparticle to the nucleic acid. Thereafter, the substrate is contacted with a silver dye to produce a detectable change and the detectable change is observed.
[0014]
In yet another embodiment, the method includes providing a substrate having the first type of nanoparticles attached thereto. An oligonucleotide having a sequence complementary to the first portion of the sequence of the nucleic acid to be detected is attached to the nanoparticle. Thereafter, the nucleic acid is contacted with the nanoparticles attached to the substrate under conditions that allow the nucleic acid to effectively hybridize the oligonucleotides on the nanoparticles. Next, an aggregate probe including at least two types of nanoparticles having oligonucleotides attached thereto is provided. The nanoparticles of the aggregate probe are linked together as a result of the hybridization of a portion of the oligonucleotide attached to the nanoparticles. An oligonucleotide having a sequence complementary to the second portion of the sequence of the nucleic acid is attached to at least one of the at least two nanoparticles of the aggregate probe. Finally, the nucleic acid bound to the substrate is contacted with the aggregate probe under conditions that effectively hybridize the nucleic acid to the oligonucleotide on the aggregate probe, and a detectable change is observed.
[0015]
In a further embodiment, the method includes providing a substrate having the oligonucleotide attached thereto. The oligonucleotide has a sequence that is complementary to a first portion of the sequence of the nucleic acid to be detected. An aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto is provided. The nanoparticles of the aggregate probe are linked together as a result of the hybridization of a portion of the oligonucleotide attached to the nanoparticles. At least one of the at least two nanoparticles of the aggregate probe has an oligonucleotide attached thereto having a sequence complementary to a second portion of the sequence of the nucleic acid. The nucleic acid, the substrate, and the aggregate probe are contacted under conditions that allow the nucleic acid to effectively hybridize the oligonucleotide on the aggregate probe and the oligonucleotide on the substrate, and a detectable change is observed.
[0016]
In a further embodiment, the method includes providing a substrate having the oligonucleotide attached thereto. An aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto is provided. The nanoparticles of the aggregate probe are linked together as a result of the hybridization of a portion of the oligonucleotide attached to the nanoparticles. An oligonucleotide having a sequence complementary to the first part of the sequence of the nucleic acid to be detected is attached to at least one of the at least two types of nanoparticles of the aggregate probe. Certain types of nanoparticles having at least two types of oligonucleotides attached thereto are provided. The first type of oligonucleotide has a sequence complementary to a second portion of the sequence of the nucleic acid, and the second type of oligonucleotide has a sequence complementary to at least a portion of the sequence of the oligonucleotide attached to the substrate. . Nucleic acids, aggregate probes, nanoparticles and substrates are effectively hybridized with oligonucleotides on aggregate probes and nanoparticles and nucleic acids, and oligonucleotides on nanoparticles are effectively hybridized with oligonucleotides on substrate Contact under conditions to be observed and observe for detectable changes.
[0017]
In another embodiment, the method comprises contacting the nucleic acid to be detected with a substrate having oligonucleotides attached thereto. The oligonucleotide has a sequence that is complementary to a first portion of the sequence of the nucleic acid. Contacting occurs under conditions that allow the oligonucleotide on the substrate to effectively hybridize to the nucleic acid. The nucleic acid bound to the substrate is contacted with a liposome to which an oligonucleotide having a sequence complementary to a part of the sequence of the nucleic acid is attached. Contacting occurs under conditions that allow the oligonucleotides on the liposome to effectively hybridize to the nucleic acid. An aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto is provided. The nanoparticles of the aggregate probe are linked together as a result of the hybridization of a portion of the oligonucleotide attached to the nanoparticles. At least one of the at least two types of nanoparticles of the aggregate probe has an oligonucleotide having a hydrophobic group attached to the end that does not adhere to the nanoparticles. The liposome bound to the substrate is contacted with the aggregate probe under conditions effective for the oligonucleotide on the aggregate probe to attach to the liposome as a result of the hydrophobic interaction, and a detectable change is observed.
[0018]
In yet another embodiment, the method includes providing a substrate having the oligonucleotide attached thereto. The oligonucleotide has a sequence that is complementary to a first portion of the sequence of the nucleic acid to be detected. A core probe comprising at least two types of nanoparticles is provided. Each type of nanoparticle has an oligonucleotide attached thereto that is complementary to the oligonucleotide on at least one of the other types of nanoparticles. The nanoparticles of the aggregate probe are linked together as a result of the hybridization of the oligonucleotide attached to the nanoparticles. Next, a type of nanoparticle having two types of oligonucleotides attached thereto is provided. The first type of oligonucleotide has a sequence that is complementary to a second portion of the sequence of the nucleic acid. The second type of oligonucleotide has a sequence that is complementary to a portion of the sequence of the oligonucleotide attached to at least one of the at least two types of nanoparticles of the core probe. The nucleic acid, the nanoparticle, the substrate and the core probe are effectively hybridized with the oligonucleotide on the nanoparticle and the oligonucleotide on the substrate and the nucleic acid, and the oligonucleotide on the core probe is effectively hybridized with the oligonucleotide on the nanoparticle. Under hybridizing conditions and observe for detectable changes.
[0019]
Another embodiment of the method comprises providing a substrate having an oligonucleotide having a sequence complementary to a first portion of the sequence of the nucleic acid to be detected attached. A core probe comprising at least two types of nanoparticles is provided. Each type of nanoparticle has an oligonucleotide attached thereto that is complementary to the oligonucleotide on at least one other type of nanoparticle. The nanoparticles of the aggregate probe are linked together as a result of the hybridization of the oligonucleotide attached to the nanoparticles. A sequence complementary to a second portion of the sequence of the nucleic acid and a sequence complementary to a portion of the sequence of the oligonucleotide attached to at least one of the at least two nanoparticles of the core probe. A linking oligonucleotide is provided. The nucleic acid, the linking oligonucleotide, the substrate and the core probe effectively hybridize the linking oligonucleotide and the oligonucleotide on the substrate with the nucleic acid, and the oligonucleotide on the core probe effectively hybridizes the oligonucleotide on the linking oligonucleotide. Contact under soy conditions and observe for detectable changes.
[0020]
In yet another embodiment, a method includes providing nanoparticles having oligonucleotides attached thereto and providing one or more types of binding oligonucleotides. Each binding oligonucleotide has two parts, the sequence of one part being complementary to the sequence of one of several parts of the nucleic acid. Also, the sequence of the other part is complementary to the sequence of the oligonucleotide on the nanoparticle. The nanoparticle-oligonucleotide conjugate and the binding oligonucleotide are contacted under conditions that allow the oligonucleotide on the nanoparticle to effectively hybridize with the binding oligonucleotide. The nucleic acid and the binding oligonucleotide are contacted under conditions that allow the binding oligonucleotide to effectively hybridize to the nucleic acid. Thereafter, a detectable change is observed. The nanoparticle-oligonucleotide conjugate may be contacted with the binding oligonucleotide prior to contacting the nucleic acid, or all three may be contacted simultaneously.
[0021]
In another embodiment, the method comprises contacting the nucleic acid with at least two types of particles having oligonucleotides attached thereto. The oligonucleotide on the first type of particle has a sequence that is complementary to the first portion of the sequence of the nucleic acid and has an energy donor molecule at the end that is not attached to the particle. The oligonucleotide on the second type of particle has a sequence complementary to the second portion of the sequence of the nucleic acid and has an energy acceptor molecule at the end not attached to the particle. Contacting occurs under conditions that effectively hybridize the oligonucleotide on the particle to the nucleic acid. Also observe the detectable change caused by such hybridization. The energy donor and acceptor molecules may be fluorescent molecules.
[0022]
In a further embodiment, the method comprises providing a type of microsphere having an oligonucleotide attached thereto. The oligonucleotide has a sequence that is complementary to the first portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule. Certain types of nanoparticles that produce a detectable change have oligonucleotides attached thereto that have a sequence complementary to a second portion of the sequence of the nucleic acid. Contacting the nucleic acid with the microspheres and nanoparticles under conditions that effectively hybridize to the oligonucleotides on the latex microspheres and nanoparticles, and then observing a change in fluorescence, another detectable change, or both .
[0023]
In another embodiment, the method includes providing a first type of metal or semiconductor nanoparticle to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the first portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule. Also provided is a second type of metal or semiconductor nanoparticles having oligonucleotides attached thereto. The oligonucleotide has a sequence complementary to the second portion of the sequence of the nucleic acid and is similarly labeled with a fluorescent molecule. The nucleic acid is contacted with the two types of nanoparticles under conditions that effectively hybridize the nucleic acid and the oligonucleotides on the two types of nanoparticles, and then the change in fluorescence is observed.
[0024]
In a further embodiment, the method includes providing a type of particle having oligonucleotides attached thereto. The oligonucleotide has a first portion and a second portion, both portions being complementary to a portion of the sequence of the nucleic acid. Certain types of probe oligonucleotides comprising a first portion and a second portion are also provided. The first portion has a sequence complementary to the first portion of the oligonucleotide attached to the particle, and both portions are complementary to a portion of the sequence of the nucleic acid. The probe oligonucleotide is labeled at one end with a reporter molecule. Thereafter, the particle and the probe oligonucleotide are contacted under conditions that effectively hybridize the oligonucleotide on the particle with the probe oligonucleotide to produce a satellite probe. Thereafter, the satellite probe is contacted with the nucleic acid under conditions that effectively hybridize the probe oligonucleotide and the nucleic acid. The particles are removed and the reporter molecule is detected.
[0025]
In yet another embodiment of the method of the invention, the nucleic acid is detected by contacting the nucleic acid with a substrate having oligonucleotides attached thereto. The oligonucleotide has a sequence that is complementary to a first portion of the sequence of the nucleic acid. The oligonucleotide is located between a pair of electrodes located on a substrate. Contacting occurs under conditions that allow the oligonucleotides on the substrate to effectively hybridize to the nucleic acid. Thereafter, the nucleic acids bound to the substrate are contacted with certain types of nanoparticles. The nanoparticles are formed of a material that can conduct electricity. One or more types of oligonucleotides are attached to the nanoparticles, and at least one of the one or more types of oligonucleotides has a sequence that is complementary to a second portion of the sequence of the nucleic acid. Contacting occurs under conditions that effectively hybridize the oligonucleotide on the nanoparticle to the nucleic acid. If a nucleic acid is present, it is possible to detect a change in conductivity. In a preferred embodiment, multiple pairs of electrodes are arranged on the substrate in an array to enable detection of multiple portions of a nucleic acid, detection of multiple different nucleic acids, or both. . Each electrode pair in the array will have some kind of oligonucleotide attached to the substrate between the two electrodes.
[0026]
The invention further provides a method for detecting a nucleic acid, wherein the method is performed on a substrate. The method includes detecting the presence, amount, or both, of the nucleic acid with an optical scanner.
[0027]
The present invention further provides a kit for detecting a nucleic acid. In one embodiment, the kit includes at least one container containing at least two types of nanoparticles having oligonucleotides attached thereto. The oligonucleotide on the first type of nanoparticles has a sequence that is complementary to the sequence of the first portion of the nucleic acid. The oligonucleotide on the second type of nanoparticles has a sequence that is complementary to the sequence of the second portion of the nucleic acid.
[0028]
Alternatively, the kit may include at least two containers. The first container contains nanoparticles to which oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached. The second container contains nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid.
[0029]
In a further embodiment, the kit comprises at least one container. The container contains metal nanoparticles or semiconductor nanoparticles having oligonucleotides attached thereto. The oligonucleotide has a sequence that is complementary to a portion of the nucleic acid, and the oligonucleotide that is not attached to the nanoparticle has a fluorescent molecule attached to the end.
[0030]
In yet another embodiment, the kit includes a substrate. Nanoparticles are attached to the substrate, and oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached to the nanoparticles. The kit further includes a first container containing nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid. The kit further includes a second container containing a binding oligonucleotide having a selected sequence having at least two portions. The first of the two portions is complementary to at least one portion of the sequence of the oligonucleotide on the nanoparticle in the first container. The kit further includes a third container containing nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the binding oligonucleotide.
[0031]
In another embodiment, the kit comprises a substrate having an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid attached thereto, and an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid attached thereto. And a second container containing nanoparticles having oligonucleotides having a sequence complementary to at least a part of the oligonucleotides attached to the nanoparticles in the first container. .
[0032]
In yet another embodiment, the kit comprises a substrate, a first container containing the nanoparticles, and a second container containing a first type of oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid. A third container containing a second type of oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid; and a third container having a sequence complementary to at least a part of the sequence of the second type oligonucleotide. And a fourth container containing oligonucleotides of the type described above.
[0033]
In a further embodiment, the kit comprises a substrate having oligonucleotides attached having a sequence complementary to the sequence of the first portion of the nucleic acid. The kit also includes a first container containing a liposome having an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid, and a second container having nanoparticles having at least a first type of oligonucleotide attached thereto. And two containers. The first type of oligonucleotide has a hydrophobic group at the end that is not attached to the nanoparticle so that the nanoparticle can be attached to the liposome by hydrophobic interaction. The kit comprises a third type of nanoparticle containing an oligonucleotide having a sequence complementary to at least one portion of the sequence of the second type of oligonucleotide attached to the first type of nanoparticles. It may further include a container. The second type of oligonucleotide attached to the first type of nanoparticle has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticle.
[0034]
In another embodiment, the kit includes a substrate with the nanoparticles attached. Oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached to the nanoparticles. The kit also includes a first container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles having oligonucleotides attached thereto. The nanoparticles of the aggregate probe are bound together as a result of the hybridization of a portion of the oligonucleotide attached to each. An oligonucleotide having a sequence complementary to the second portion of the sequence of the nucleic acid is attached to at least one of the at least two types of nanoparticles of the aggregate probe.
[0035]
In yet another embodiment, the kit includes a substrate having the oligonucleotide attached thereto. The oligonucleotide has a sequence that is complementary to the sequence of the first portion of the nucleic acid. The kit further includes a first container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles with oligonucleotides attached. The nanoparticles of the aggregate probe are bound together as a result of the hybridization of a portion of the oligonucleotide attached to each. An oligonucleotide having a sequence complementary to the second portion of the sequence of the nucleic acid is attached to at least one of the at least two types of nanoparticles of the aggregate probe.
[0036]
In a further embodiment, the kit comprises a substrate having the oligonucleotide attached thereto and a first container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles with oligonucleotides attached. The nanoparticles of the aggregate probe are bound together as a result of the hybridization of a portion of the oligonucleotide attached to each. An oligonucleotide having a sequence complementary to the first portion of the nucleic acid sequence is attached to at least one of the at least one nanoparticle of the aggregate probe. The kit also includes a second container containing the nanoparticles. At least two types of oligonucleotides are attached to the nanoparticles. The first type of oligonucleotide has a sequence that is complementary to a second portion of the sequence of the nucleic acid. The second type of oligonucleotide has a sequence complementary to at least a part of the sequence of the oligonucleotide attached to the substrate.
[0037]
In another embodiment, the kit includes a substrate with the oligonucleotide attached. The oligonucleotide has a sequence that is complementary to the sequence of the first portion of the nucleic acid. The kit also includes a first container containing the liposome to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the sequence of the second portion of the nucleic acid. The kit further includes a second container containing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto. The nanoparticles of the aggregate probe are bound together as a result of the hybridization of a portion of the oligonucleotide attached to each. At least one of the at least two types of nanoparticles of the aggregate probe has an oligonucleotide having a hydrophobic group attached to an end not attached to the nanoparticles.
[0038]
In a further embodiment, the kit may include a first container containing nanoparticles having oligonucleotides attached thereto. The kit also includes one or more additional containers, each container containing a binding oligonucleotide. Each binding oligonucleotide has a first portion having a sequence complementary to at least a portion of the sequence of the oligonucleotide on the nanoparticle, and a second portion having a sequence complementary to the sequence of a portion of the nucleic acid to be detected. The sequence of the second portion of the binding oligonucleotide may be different, as long as each sequence is complementary to a portion of the sequence of the nucleic acid to be detected. In another embodiment, the kit includes a container containing one type of nanoparticle to which the oligonucleotide is attached and one or more types of binding oligonucleotide. Each of the one or more types of binding oligonucleotides has a sequence that includes at least two portions. The first part is complementary to the sequence of the oligonucleotide on the nanoparticle, so that in the container the bound oligonucleotide is hybridized to the oligonucleotide on the nanoparticle. The second portion is complementary to the sequence of a portion of the nucleic acid.
[0039]
In another embodiment, the kit may include one or two containers holding two types of particles. An oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid is attached to the first type of particle. The oligonucleotide is labeled with an energy donor at the end that does not attach to the particle. An oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid is attached to the second type of particle. The oligonucleotide is labeled with an energy acceptor at the end that does not attach to the particle.
The energy donor and acceptor may be fluorescent molecules.
[0040]
In a further embodiment, the kit includes a first container containing nanoparticles having oligonucleotides attached thereto. The kit also includes one or more additional containers, with the binding oligonucleotide in each container. Each binding oligonucleotide has a first portion having a sequence complementary to at least a portion of the sequence of the oligonucleotide on the nanoparticle, and a second portion having a sequence complementary to the sequence of the portion of the nucleic acid to be detected. The sequence of the second portion of the binding oligonucleotide may be different, as long as each sequence is complementary to a portion of the sequence of the nucleic acid to be detected. In yet another embodiment, the kit comprises a container containing one type of nanoparticle to which the oligonucleotide is attached and one or more types of binding oligonucleotide. Each of the one or more types of binding oligonucleotides has a sequence that includes at least two portions. The first portion is complementary to the sequence of the oligonucleotide on the nanoparticle, so that in the container the bound oligonucleotide is hybridized to the oligonucleotide on the nanoparticle. The second portion is complementary to the sequence of a portion of the nucleic acid.
[0041]
In another alternative embodiment, the kit includes at least three containers. The first container contains the nanoparticles. The second container contains a first oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid. The third container contains a second oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid. The kit further includes a fourth container containing a binding oligonucleotide having a selected sequence having at least two portions (the first portion is complementary to at least a portion of the sequence of the second oligonucleotide); A fifth container containing an oligonucleotide having a sequence complementary to the sequence of the second portion is also included.
[0042]
In another embodiment, the kit includes one or two containers holding two types of particles. The first type of particles has oligonucleotides attached thereto that have a sequence complementary to the first portion of the sequence of the nucleic acid and have an energy donor molecule attached to the end that does not attach to the nanoparticles. The second type of particles has oligonucleotides attached thereto that have a sequence that is complementary to the second portion of the sequence of the nucleic acid and have an energy acceptor molecule attached to the end that does not attach to the nanoparticles. The energy donor and acceptor may be fluorescent molecules.
[0043]
In a further embodiment, the kit includes a first container containing a type of microsphere having an oligonucleotide attached thereto. The oligonucleotide has a sequence that is complementary to a first portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule. A second container containing certain types of nanoparticles having oligonucleotides attached thereto. The oligonucleotide has a sequence that is complementary to the second portion of the sequence of the nucleic acid.
[0044]
In another embodiment, the kit includes a first container containing a first type of metal or semiconductor nanoparticles having oligonucleotides attached thereto. The oligonucleotide has a sequence that is complementary to a first portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule. The kit also includes a second container containing a second type of metal or semiconductor nanoparticles having the oligonucleotide attached thereto. The oligonucleotide has a sequence that is complementary to a second portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule.
[0045]
In another embodiment, the kit includes a container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles having oligonucleotides attached thereto. The nanoparticles of the aggregate probe are bound together as a result of the hybridization of a portion of the oligonucleotide attached to each. An oligonucleotide having a sequence complementary to a part of the nucleic acid sequence is attached to at least one of the at least two types of nanoparticles of the aggregate probe.
[0046]
In a further embodiment, the kit includes a container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles having oligonucleotides attached thereto. The nanoparticles of the aggregate probe are bound together as a result of the hybridization of a portion of the oligonucleotide attached to each. At least one of the at least two types of nanoparticles of the aggregate probe has an oligonucleotide attached with a hydrophobic group at the end not attached to the nanoparticles.
[0047]
In a further embodiment, the kit comprises a container containing the satellite probe. Satellite probes include particles to which oligonucleotides have been attached. The oligonucleotide has a first portion and a second portion, both portions having a sequence complementary to a portion of the sequence of the nucleic acid. Satellite probes also include probe oligonucleotides that hybridize to oligonucleotides attached to nanoparticles. The probe oligonucleotide has a first portion and a second portion. The first portion has a sequence that is complementary to the sequence of the first portion of the oligonucleotide attached to the particle. Also, both portions have a sequence complementary to the sequence portion of the nucleic acid. The probe oligonucleotide further has a reporter molecule at one end.
[0048]
In another embodiment, the kit includes a container containing the core probe. The core probe includes at least two types of nanoparticles having oligonucleotides attached thereto. The nanoparticles of the core probe are linked to each other as a result of hybridization of a part of the oligonucleotide attached to the nanoparticles.
[0049]
In yet another embodiment, the kit comprises a substrate having at least one pair of electrodes attached thereto, with the oligonucleotide attached to the substrate between the electrodes. The oligonucleotide has a sequence that is complementary to a first portion of the sequence of the nucleic acid to be detected.
[0050]
The present invention further provides satellite probes, aggregate probes and core probes.
The present invention further provides a substrate having nanoparticles attached thereto. An oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid can be attached to the nanoparticles.
[0051]
The present invention further provides metal or semiconductor nanoparticles having oligonucleotides attached thereto. Oligonucleotides are labeled with fluorescent molecules at the ends that do not attach to the nanoparticles.
[0052]
The present invention further provides a method for nanofabrication. The method includes providing at least one type of linking oligonucleotide having a selected sequence, wherein the sequence of each type of linking oligonucleotide has at least two portions. The method comprises providing one or more types of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotides on each type of nanoparticles have a sequence complementary to a portion of the sequence of the linking oligonucleotide. Further included. The linking oligonucleotide and the nanoparticle are contacted under conditions that allow the oligonucleotide on the nanoparticle to effectively hybridize to the linking oligonucleotide such that the desired nanomaterial or nanostructure is formed.
[0053]
The present invention provides another method of nanofabrication. The method includes providing at least two types of nanoparticles having oligonucleotides attached thereto. The oligonucleotide on the first type of nanoparticles has a sequence that is complementary to the sequence of the oligonucleotide on the second type of nanoparticles. The oligonucleotide on the second type of nanoparticle has a sequence that is complementary to the sequence of the oligonucleotide on the first type of nanoparticle-oligonucleotide conjugate. The first and second types of nanoparticles are contacted under conditions that allow the oligonucleotides on the nanoparticles to effectively hybridize to each other such that a desired nanomaterial or nanostructure is formed.
[0054]
The invention further provides a nanomaterial or nanostructure composed of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles are held together by an oligonucleotide connector.
[0055]
The invention further provides a composition comprising at least two types of nanoparticles having oligonucleotides attached thereto. The oligonucleotide on the first type of nanoparticles has a sequence that is complementary to the sequence of the first portion of the nucleic acid or the linking oligonucleotide. The oligonucleotide on the second type of nanoparticle has a sequence that is complementary to the sequence of the second portion of the nucleic acid or the linking oligonucleotide.
[0056]
The present invention further provides a container assembly comprising a first container containing nanoparticles having oligonucleotides attached thereto and a second container containing nanoparticles having oligonucleotides attached thereto. The oligonucleotide attached to the nanoparticles in the first container has a sequence complementary to the sequence of the oligonucleotide attached to the nanoparticles in the second container. The oligonucleotide attached to the nanoparticles in the second container has a sequence complementary to the sequence of the oligonucleotide attached to the nanoparticles in the first container.
[0057]
The invention further provides a nanoparticle having a plurality of different oligonucleotides attached thereto.
The invention further provides a method for separating a selected nucleic acid having at least two parts from other nucleic acids. The method comprises providing one or more types of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotide on each of the one or more nanoparticles comprises at least one of the at least two portions of the selected nucleic acid. Having a sequence complementary to one sequence of The selected nucleic acid and other nucleic acids are contacted with the nanoparticles under conditions that effectively hybridize the selected nucleic acids and the oligonucleotides on the nanoparticles such that the nanoparticles hybridized with the selected nucleic acids aggregate and precipitate.
[0058]
Furthermore, the present invention provides a method for making a unique nanoparticle-oligonucleotide conjugate. A first such method involves attaching an oligonucleotide to a charged nanoparticle to produce a stable nanoparticle-oligonucleotide conjugate. To do so, the oligonucleotide having a moiety having a functional group capable of binding to the nanoparticle is covalently linked to the oligonucleotide for a time sufficient for at least a portion of the oligonucleotide to be linked to the nanoparticle by the functional group. Contact the particles with water. Next, at least one salt is added to the water to form a salt solution. The ionic strength of the salt solution is determined by the electrostatic repulsion of the oligonucleotides relative to each other, the electrostatic attraction of the negatively charged oligonucleotides to the positively charged nanoparticles, or the positively charged oligonucleotides to the negatively charged nanoparticles. After the salt has been added, the oligonucleotides and nanoparticles must be of sufficient strength to at least partially overcome one of the electrostatic attraction, and the additional oligonucleotides are bound to the nanoparticles to provide a stable nanoparticle. Incubate in salt solution for an additional period sufficient to generate particle-oligonucleotide conjugate. The present invention further provides stable nanoparticle-oligonucleotide conjugates, methods for using the conjugates for detecting and separating nucleic acids, kits containing the conjugates, methods for nanofabrication using the conjugates, and , Nanomaterials and nanostructures comprising the conjugate.
[0059]
The present invention provides another method of attaching an oligonucleotide to a nanoparticle to produce a nanoparticle-oligonucleotide conjugate. The method includes providing an oligonucleotide comprising a type of recognition oligonucleotide and a type of diluent oligonucleotide. The oligonucleotide and the nanoparticles are contacted under conditions effective for at least a portion of each of the certain types of oligonucleotides to bind to the nanoparticles and form a conjugate. The present invention further provides nanoparticle-oligonucleotide conjugates produced by the method, methods of using the conjugate to detect and separate nucleic acids, kits containing the conjugate, and nanofabrics using the conjugate Applications, as well as nanomaterials and nanostructures comprising the conjugate. A "recognition oligonucleotide" is an oligonucleotide that comprises a sequence that is complementary to at least a portion of the sequence of a nucleic acid or oligonucleotide target. A “diluent oligonucleotide” can have any sequence that does not interfere with the ability of the recognition oligonucleotide to bind to the nanoparticles or the ability of the recognition oligonucleotide to bind to its target.
[0060]
The present invention provides yet another method of attaching an oligonucleotide to a nanoparticle to produce a nanoparticle-oligonucleotide conjugate. The method comprises providing an oligonucleotide comprising at least one recognition oligonucleotide. The recognition oligonucleotide includes a recognition portion and a spacer portion. The recognition portion of the recognition oligonucleotide has a sequence that is complementary to at least a portion of the sequence of the nucleic acid or oligonucleotide target. The spacer portion of the recognition oligonucleotide is designed such that the spacer portion can be attached to the nanoparticle. As a result of the attachment of the spacer portion of the recognition oligonucleotide to the nanoparticle, the recognition portion leaves the surface of the nanoparticle and is more accessible for hybridizing with its target. To generate a conjugate, the nanoparticles are contacted with an oligonucleotide comprising the recognition oligonucleotide under conditions effective to allow at least a portion of the recognition oligonucleotide to bind to the nanoparticle. The invention further provides a nanoparticle-oligonucleotide conjugate produced by this method, a method of using the conjugate to detect and separate nucleic acids, a kit comprising the conjugate, a nanofabric using the conjugate Applications, as well as nanomaterials and nanostructures comprising the conjugate.
[0061]
The invention includes a method of attaching an oligonucleotide to a nanoparticle by a linker comprising a cyclic disulfide. Suitable cyclic disulfides have 5 or 6 atoms, including 2 sulfur atoms, in the ring. Suitable cyclic disulfides are commercially available. It is also possible to use reduced forms of cyclic disulfides. Preferably, the linker further comprises a hydrocarbon moiety attached to the cyclic disulfide. Suitable hydrocarbons are commercially available and are attached to cyclic disulfides (eg, as described in the Appendix). Preferably, the hydrocarbon moiety is a steroid residue. A linker is attached to the oligonucleotide and the oligonucleotide linker is attached to the nanoparticles as described herein.
[0062]
The invention further relates to novel nanoparticle probes that utilize specific binding interactions, such as antibody-antigen binding, methods for preparing and using such probes, and kits containing such probes.
[0063]
In one embodiment, the invention provides a method for detecting an analyte, comprising contacting the oligonucleotide-associated nanoparticles with the analyte. At least a portion of the oligonucleotide attached to the nanoparticle binds to a second oligonucleotide associated with the specific binding complement of the analyte as a result of the hybridization. Contacting occurs under conditions that allow for specific binding interactions between the analyte and the specific binding complement associated with the nanoparticle conjugate. As a result of the specific binding interaction between the analyte and the probe, a detectable change can be observed.
[0064]
In another embodiment of the present invention, a method of detecting an analyte comprises providing contact of the oligonucleotide-associated nanoparticles with the analyte. At least a portion of the oligonucleotide attached to the nanoparticle binds to the first portion of the linker oligonucleotide as a result of the hybridization. The second portion of the linker oligonucleotide, upon hybridization, binds to the oligonucleotide bound to the specific binding complement of the analyte. Contacting occurs under conditions that allow the analyte and the nanoparticle conjugate to effectively and specifically bind and interact, and a detectable change can be observed.
[0065]
In another embodiment, a method of detecting an analyte comprises providing an analyte having a first oligonucleotide attached thereto, and contacting the oligonucleotide associated with the analyte with a first type of nanoparticles. Contacting occurs under conditions effective for the oligonucleotide associated with the analyte to hybridize to the oligonucleotide attached to the first type of nanoparticle to form a nanoparticle analyte conjugate. The method further comprises contacting the nanoparticle analyte with a second type of nanoparticle to which the oligonucleotide is associated. At least a portion of the oligonucleotide associated with the second type of nanoparticle binds to the oligonucleotide associated with the specific binding complement of the analyte upon hybridization. The oligonucleotide bound to the analyte has a sequence complementary to at least a portion of the oligonucleotide associated with the first type of nanoparticle. The contacting occurs under conditions that allow the analyte to specifically bind to the specific binding complement bound to the second type of nanoparticle conjugate, and the detectable change causes a specific binding interaction to occur. As a result it can be observed.
[0066]
In yet another embodiment, a method of detecting an analyte comprises providing an analyte having an oligonucleotide attached thereto, an oligonucleotide linker having two parts, and a first type of nanoparticles having the oligonucleotide attached thereto. The oligonucleotide associated with the analyte has a sequence that is complementary to a first portion of the sequence of the linker oligonucleotide. At least a portion of the oligonucleotide that binds to the first type of nanoparticles has a sequence that is complementary to a second portion of the linker oligonucleotide. The oligonucleotide linked to the analyte, the oligonucleotide linked to the nanoparticle, and the linker oligonucleotide are effective for the linker nucleotide, the oligonucleotide linked to the analyte and the oligonucleotide linked to the first type of nanoparticle Under a condition that hybridizes to a nanoparticle analyte conjugate. The method further comprises contacting the analyte conjugate with a second type of nanoparticle conjugate associated oligonucleotide. At least a portion of the oligonucleotide bound to the second type of nanoparticle binds to the oligonucleotide bound to the specific binding complement of the analyte as a result of the hybridization. Contacting occurs under conditions effective to permit a specific binding interaction between the nanoparticle analyte conjugate and a specific binding complement of the second type of nanoparticle. A detectable change may be observed as a result of the specific binding interaction.
[0067]
In another embodiment, a method for detecting an analyte comprises providing a support to which the analyte is attached. The method further includes contacting the analyte bound to the support with the nanoparticles. At least a portion of the oligonucleotide binds to the second oligonucleotide to which the specific binding complement of the analyte is bound as a result of the hybridization. Contacting occurs under conditions effective to permit a specific binding interaction between the analyte and the specific binding complement associated with the nanoparticle conjugate. At this point, a detectable change can be observed.
[0068]
In another embodiment, a method of detecting an analyte comprises providing a support having the oligonucleotide attached thereto and an analyte having the oligonucleotide attached thereto. The oligonucleotide attached to the analyte has a sequence complementary to the linker oligonucleotide attached to the support. The method is further effective for allowing the support-bound oligonucleotide and the analyte-bound oligonucleotide to allow hybridization between the support-bound oligonucleotide and the analyte-bound oligonucleotide. Contacting under suitable conditions. Thereafter, certain types of nanoparticles with attached oligonucleotides are provided. At least a portion of the oligonucleotide associated with the nanoparticle binds to the second oligonucleotide associated with the specific binding complement of the analyte as a result of the hybridization. Finally, the analyte bound to the support and the specific binding complement bound to the nanoparticle conjugate are moved between the analyte bound to the support and the specific binding complement bound to the nanoparticle. Are contacted under conditions effective to tolerate the specific binding interaction, and a detectable change is observed.
[0069]
In yet another embodiment, a method of detecting an analyte comprises providing a support to which the oligonucleotide is attached and a linker oligonucleotide having at least two portions in sequence. The oligonucleotide attached to the support has a sequence complementary to the first portion of the linker oligonucleotide. Thereafter, the support-bound oligonucleotide is contacted with the linker oligonucleotide under conditions effective to permit hybridization between the support-bound oligonucleotide and the first portion of the linker oligonucleotide. . Next, an oligonucleotide-bound specimen is provided. The oligonucleotide associated with the analyte has a sequence that is complementary to the second portion of the linker oligonucleotide. The linker oligonucleotides bound to the support are then converted to the oligonucleotides bound to the analyte and effective to permit hybridization between the oligonucleotides bound to the analyte and the second portion of the linker oligonucleotide. Contact under conditions. Thereafter, a type of nanoparticle conjugate having the oligonucleotide attached thereto is provided. At least a portion of the oligonucleotide bound to the nanoparticle conjugate will bind to the oligonucleotide bound to the specific binding complement of the analyte as a result of hybridization. The support-bound analyte is then contacted under conditions effective to permit a specific binding interaction between the support-bound analyte and the specific binding complement bound to the nanoparticles, Observe detectable changes.
[0070]
In yet another embodiment, a method for detecting an analyte comprises contacting the oligonucleotide-associated support with the oligonucleotide associated with the analyte. The sequence of the oligonucleotide bound to the analyte is complementary to the sequence of the oligonucleotide bound to the support. Contacting occurs under conditions effective to permit hybridization between the oligonucleotide attached to the support and the oligonucleotide attached to the analyte. Thereafter, certain types of nanoparticles with attached oligonucleotides are provided. At least a portion of the oligonucleotide attached to the nanoparticle binds to the first portion of the linker oligonucleotide as a result of the hybridization. Furthermore, the second portion of the linker oligonucleotide binds to the oligonucleotide having the oligonucleotide to which the specific binding complement of the analyte is bound as a result of the hybridization. Next, the support-bound analyte is subjected to conditions effective to permit a specific binding interaction between the support-bound analyte and the specific binding complement bound to the nanoparticles. Contact with nanoparticles. Thereafter, a detectable change is observed.
[0071]
In another embodiment, a method for detecting an analyte comprises providing a support to which the analyte is attached. The support is contacted with an aggregate probe comprising at least two types of nanoparticles associated with the oligonucleotide. Aggregate probe nanoparticles are associated with each other as a result of a portion of the oligonucleotides attached to them hybridizing. At least one of the at least two nanoparticles of the aggregate probe is attached with some oligonucleotide that binds to a second oligonucleotide to which the specific binding complement of the analyte is bound as a result of hybridization. . Contacting occurs under conditions effective to permit a specific binding interaction between the analyte bound to the support and the specific binding complement bound to the aggregate probe. At this point, a detectable change can be observed.
[0072]
In another embodiment, the method of detecting an analyte comprises contacting the oligonucleotide-bound support with the oligonucleotide-bound analyte. The oligonucleotide tied to the analyte has a sequence that is complementary to the sequence of the oligonucleotide tied to the support. Contacting occurs under conditions effective to permit hybridization of the oligonucleotide attached to the support with the oligonucleotide attached to the analyte. Next, an aggregate probe including at least two types of nanoparticles linked to an oligonucleotide is provided. Aggregate probe nanoparticles are associated with each other as a result of a portion of the oligonucleotides attached to them hybridizing. At least one of the at least two nanoparticles of the aggregate probe is attached with an oligonucleotide that binds to a second oligonucleotide to which the specific binding complement of the analyte is bound as a result of hybridization. Contacting occurs under conditions effective to permit a specific binding interaction between the analyte bound to the support and the specific binding complement bound to the aggregate probe. Thereafter, a detectable change is observed.
[0073]
In yet another embodiment, the method of detecting an analyte provides that the oligonucleotide-bound support is contacted with a linker oligonucleotide wherein the sequence of the second linker oligonucleotide has at least two portions. Including. Contacting occurs under conditions effective to permit hybridization between the oligonucleotide attached to the support and the first portion of the linker oligonucleotide. Thereafter, a sample having the oligonucleotide attached thereto is provided. The oligonucleotide associated with the analyte has a sequence that is complementary to the second portion of the linker oligonucleotide. Next, the support-attached linker oligonucleotide is combined under conditions effective to permit hybridization between the second portion of the support-attached linker oligonucleotide and the analyte-attached oligonucleotide. Make contact. Next, an aggregate probe including at least two types of nanoparticles linked to an oligonucleotide is provided. Aggregate probe nanoparticles are associated with each other as a result of a portion of the oligonucleotides attached to them hybridizing. At least one of the at least two nanoparticles of the aggregate probe is attached with an oligonucleotide that binds to a second oligonucleotide to which the specific binding complement of the analyte is bound as a result of hybridization. The support-bound analyte is then subjected to conditions effective to permit specific binding interactions between the support-bound analyte and the specific binding complement bound to the aggregate probe. To contact the aggregate probe. At this point, a detectable change can be observed.
[0074]
In yet another embodiment, the method of detecting an analyte comprises contacting the support-bound oligonucleotide with an analyte-bound oligonucleotide that is complementary to the sequence of the support-bound oligonucleotide. Including. Contacting occurs under conditions effective to permit hybridization of the oligonucleotide attached to the analyte with the oligonucleotide attached to the support. Next, an aggregate probe including at least two types of nanoparticles linked to an oligonucleotide is provided. Aggregate probe nanoparticles are bound together as a result of the hybridization of a portion of the oligonucleotide attached to them. In at least one of the at least two nanoparticles of the aggregate probe, a portion of the oligonucleotide is associated with the first portion of the linker oligonucleotide as a result of hybridization. The second portion of the second linker oligonucleotide binds to the oligonucleotide to which the specific binding complement of the analyte is bound as a result of the hybridization. The support-bound analyte is then subjected to conditions effective to permit a specific binding interaction between the support-bound analyte and the specific binding complement bound to the aggregate probe. To contact the aggregate probe. Thereafter, a detectable change is observed.
[0075]
In yet another embodiment, a method of detecting an analyte comprises contacting the analyte-associated support with an oligonucleotide-associated nanoparticle conjugate. At least a portion of the oligonucleotide associated with the nanoparticle will bind to the second oligonucleotide associated with the specific binding complement of the analyte as a result of the hybridization. Contacting occurs under conditions effective to permit a specific binding interaction between the analyte bound to the support and the specific binding complement bound to the nanoparticle conjugate. Thereafter, the substrate is contacted with a silver dye to produce a detectable and the detectable change is observed.
[0076]
In yet another embodiment of the present invention, a method for detecting an analyte comprises contacting a multivalent analyte with a nanoparticle probe having an oligonucleotide attached thereto. At least a portion of the oligonucleotide attached to the nanoparticle is bound to the first portion of the reporter oligonucleotide as a result of the hybridization. The second portion of the reporter oligonucleotide binds to the oligonucleotide to which the specific binding complement for the analyte is bound as a result of the hybridization. Contacting occurs under conditions effective to permit specific binding interactions between the analyte and the nanoparticle probe and to aggregate the nanoparticles. Thereafter, the aggregate is separated, the aggregate is dehybridized, and subjected to conditions effective to release the reporter oligonucleotide. Thereafter, the reporter oligonucleotide is separated. Detection of the analyte occurs by confirming the presence of the reporter oligonucleotide by conventional means, such as a DNA chip.
[0077]
The present invention provides for the specificity of (i) nucleic acids, (ii) one or more types of nanoparticles associated oligonucleotides, and (iii) two or more biotin molecules each having an oligonucleotide associated therewith. A method for detecting a nucleic acid is provided, comprising providing a complex comprising streptavidin or avidin linked by a binding interaction. The sequence of the nucleic acid has at least two parts. Oligonucleotides linked to the nanoparticles have a sequence complementary to the first portion of the nucleic acid, while oligonucleotides linked to biotin have a sequence complementary to the second portion of the nucleic acid. The nucleic acid is then contacted with the nanoparticle conjugate and complex under conditions effective to permit hybridization of the nanoparticle, complex and nucleic acid. Observe detectable changes resulting from subsequent aggregation.
[0078]
In another embodiment, provided are (i) a nucleic acid, (ii) one or more types of nanoparticles associated with the oligonucleotide, (iii) an oligonucleotide associated with biotin, and (iv) streptavidin or avidin. A method for detecting a nucleic acid is provided. The sequence of the nucleic acid has at least two parts. Oligonucleotides linked to the nanoparticles have a sequence complementary to the first portion of the nucleic acid, while oligonucleotides linked to biotin have a sequence complementary to the second portion of the nucleic acid. The nucleic acid is then combined with the oligonucleotide conjugated to the nanoparticle conjugate and biotin under conditions effective to permit hybridization of the nucleic acid with the oligonucleotide attached to the nanoparticle and the oligonucleotide attached to biotin. Make contact. Observe detectable changes resulting from subsequent aggregation.
[0079]
In another embodiment, a method for detecting a nucleic acid comprises contacting the first type of nanoparticles, attached to the oligonucleotide, with the nucleic acid. The sequence of the nucleic acid has at least two parts. At least a portion of the oligonucleotide attached to the nanoparticle has a sequence that is complementary to the first portion of the nucleic acid. Contacting occurs under conditions effective to permit hybridization between the oligonucleotide attached to the nanoparticle and the first portion of the nucleic acid. Thereafter, an oligonucleotide is provided that has an sbp member (eg, biotin) attached thereto. The sequence of the oligonucleotide that is linked to the sbp member has a sequence that is complementary to the second portion of the sequence of the nucleic acid. The oligonucleotide attached to the sbp member is then combined with the first type of nanoparticles under conditions effective to permit hybridization between the oligonucleotide attached to the sbp member and a second portion of the nucleic acid. Contacting the bound nucleic acid. Thereafter, a second type of nanoparticle conjugate having the oligonucleotide attached thereto is provided. At least a portion of the oligonucleotide attached to the second type of nanoparticle binds to the oligonucleotide attached to the specific binding complement of the analyte (eg, streptavidin or avidin) as a result of hybridization. The contact involves a specific binding interaction between an sbp member (eg, biotin) associated with the first type of nanoparticle and an sbp complement (eg, streptavidin or avidin) associated with the second type of nanoparticle. Occurs under conditions effective to tolerate A detectable change can occur.
[0080]
In another embodiment, the method of detecting a nucleic acid comprises contacting the oligonucleotide-associated support with a nucleic acid having at least two portions in sequence. The oligonucleotide attached to the support has a sequence complementary to the first portion of the nucleic acid. Contacting occurs under conditions effective to permit hybridization between the oligonucleotide attached to the support and the first portion of the nucleic acid. Next, an oligonucleotide having an sbp member (eg, biotin) attached thereto is provided. Oligonucleotides linked to sbp members have sequences complementary to the second portion of the nucleic acid. Next, the nucleic acid bound to the support is combined with the oligonucleotide bound to the sbp member under conditions effective to permit hybridization between the oligonucleotide bound to the sbp member and a second portion of the nucleic acid. Contact with nucleotide. Thereafter, a type of nanoparticle conjugate having the oligonucleotide attached thereto is provided. At least a portion of the oligonucleotide conjugated to the nanoparticle conjugate binds to the oligonucleotide conjugated to the specific binding complement of the sbp member (eg, streptavidin or avidin) upon hybridization. The support-bound sbp member is then subjected to conditions effective to permit specific binding interaction between the support-bound sbp member and the specific binding complement bound to the nanoparticle. Then, contact the nanoparticles and observe the detectable change.
[0081]
In another embodiment, a method for detecting a nucleic acid comprises contacting the nucleic acid with a nanoparticle conjugate attached to an oligonucleotide. The sequence of the nucleic acid has at least two parts. At least a portion of the oligonucleotide attached to the nanoparticle has a sequence that is complementary to the first portion of the nucleic acid. Contacting occurs under conditions effective to permit hybridization of the oligonucleotide attached to the nanoparticle with the first portion of the nucleic acid. Thereafter, an oligonucleotide having an sbp member (eg, streptavidin) is provided. The oligonucleotide to which the sbp member is attached has a sequence that is complementary to the second portion of the nucleic acid. The oligonucleotide having the sbp member is then contacted with the nucleic acid associated with the nanoparticles under conditions effective to permit hybridization between the oligonucleotide having the sbp member and the nucleic acid. Next, a support is provided that has bound thereto a specific binding complement (eg, biotin) for the sbp member. The support is then contacted with the nanoparticle-associated sbp member under conditions effective to allow a specific binding interaction to occur between the sbp member and the sb complement. Observe detectable events.
[0082]
In another embodiment, the method of detecting a nucleic acid comprises providing for contacting the nucleic acid with a support to which the oligonucleotide is attached. The sequence of the nucleic acid has at least two portions, and the oligonucleotide attached to the support has a sequence that is complementary to the first portion of the nucleic acid. Contacting occurs under conditions effective to permit hybridization between the oligonucleotide attached to the support and the first portion of the nucleic acid. Thereafter, an oligonucleotide conjugated to an sbp member (eg, biotin) is provided. The oligonucleotide attached to the sbp member has a sequence that is complementary to the second portion of the nucleic acid. The support-attached nucleic acid is then converted to the oligo under conditions effective to permit hybridization between the second portion of the support-attached nucleic acid and the oligonucleotide attached to the sbp member. Contact with nucleotide. Next, an aggregate probe including at least two types of nanoparticles having oligonucleotides attached thereto is provided. Aggregate probe nanoparticles are associated with each other as a result of a portion of the oligonucleotides attached to them hybridizing. At least one of the at least two nanoparticles of the aggregate probe is attached with an oligonucleotide that binds to a second oligonucleotide to which the complement of the sbp member (eg, streptavidin) is bound as a result of hybridization. . The support-bound sbp member is then subjected to conditions effective to permit specific binding interaction between the support-bound sbp member and the aggregate-bound specific binding complement. To contact the aggregate probe. At this point, a detectable change can be observed.
[0083]
In another embodiment, a method of detecting a nucleic acid comprises contacting the nucleic acid with an aggregate probe comprising at least two types of nanoparticles associated with the oligonucleotide. Nucleic acids have two parts. Aggregate probe nanoparticles are associated with each other as a result of a portion of the oligonucleotides attached to them hybridizing. At least one of the at least two nanoparticles of the aggregate probe is attached with a number of oligonucleotides that bind to the first portion of the nucleic acid as a result of the hybridization. Thereafter, an oligonucleotide having an sbp member (eg, streptavidin) is provided. The oligonucleotide to which the sbp member is attached has a sequence that is complementary to the second portion of the nucleic acid. The oligonucleotide having the sbp member is then contacted with the nucleic acid associated with the aggregate probe under conditions effective to permit hybridization between the oligonucleotide having the sbp member and the nucleic acid attached to the probe. Let it. Next, a support is provided that has bound thereto a specific binding complement (eg, biotin) for the sbp member. The support is then allowed to aggregate under conditions effective to allow a specific binding interaction to occur between the sbp member bound to the aggregate probe and the sb complement bound to the support. Contacting the sbp member associated with the probe. Observe detectable events.
[0084]
The invention further provides nanoparticle-oligonucleotide-sbp member conjugates (nanoparticle sbp conjugates) and compositions comprising the same. An oligonucleotide is bound to the nanoparticle sbp conjugate, and at least a portion of the oligonucleotide is hybridized to the first oligonucleotide to which the specific binding pair (sbp) member is bound by a covalent bond. The oligonucleotide attached to the nanoparticle has a sequence that is complementary to at least a portion of the first oligonucleotide.
[0085]
The invention further provides a nanoparticle conjugate having an oligonucleotide attached thereto and an oligonucleotide having an sbp member attached thereto, wherein the oligonucleotide attached to the nanoparticle is contacted with the oligonucleotide attached to the sbp member. Also provided is a method for producing a nanoprobe sbp conjugate, comprising: At least a portion of the oligonucleotide associated with the nanoparticle has a sequence that is complementary to the sequence of the oligonucleotide associated with the sbp member. Contacting occurs under conditions effective to permit hybridization of the oligonucleotide on the nanoparticle with the first oligonucleotide.
[0086]
The present invention further provides a kit for detecting a specimen. In one embodiment, the kit comprises at least one container. The container holds the nanoparticle sbp conjugate, including the nanoparticles with which the oligonucleotides are associated. At least a portion of the oligonucleotide hybridizes to the first oligonucleotide to which the specific binding pair (sbp) member is bound by a covalent bond. The oligonucleotide attached to the nanoparticle has a sequence that is complementary to at least a portion of the first oligonucleotide. The kit may further comprise a substrate for observing the detectable change.
[0087]
In another embodiment of the present invention, the kit comprises at least two containers. The first container contains the nanoparticles associated oligonucleotide. At least a portion of the oligonucleotide has a sequence that is complementary to a portion of the first oligonucleotide. The second container contains the first oligonucleotide to which the sbp member is covalently attached. The kit may further comprise a substrate for observing the detectable change.
[0088]
In yet another embodiment of the present invention, the kit comprises at least two containers. The first container contains the nanoparticles associated oligonucleotide. At least a portion of the oligonucleotide has a sequence that is complementary to a portion of the first oligonucleotide. The second container contains a first oligonucleotide having a moiety that can be used to covalently attach an sbp member. The kit may further comprise a substrate for observing the detectable change.
[0089]
In yet another embodiment of the present invention, the kit comprises at least three containers. The first container contains the nanoparticles associated oligonucleotide. The second container contains a linker oligonucleotide having at least two parts. A third container contains a first oligonucleotide having a moiety that can be used to covalently bind an sbp member. At least a portion of the oligonucleotide has a sequence that is complementary to the first portion of the linker oligonucleotide. The first oligonucleotide has a sequence that is complementary to at least a second portion of the linker oligonucleotide. The kit may further comprise a substrate for observing the detectable change.
[0090]
The present invention further provides a method for nanofabrication. The method comprises providing at least one linking oligonucleotide having a selected sequence, wherein the sequence of each type of linking oligonucleotide has at least two portions. The method further comprises providing one or more types of nanoparticles attached to the oligonucleotide, wherein the oligonucleotide on each type of nanoparticle has a sequence complementary to a first portion of the sequence of the linked oligonucleotide. including. The method further comprises attaching a first oligonucleotide to each molecule, wherein the first oligonucleotide is attached to two or more biotin molecules having a sequence complementary to a second portion of the sequence of the linking oligonucleotide. Providing a complex composed of streptavidin or avidin boundaries. Allow the linking oligonucleotides, complexes and nanoparticles to hybridize the oligonucleotides on the nanoparticles and the first oligonucleotide conjugated to biotin to the linking oligonucleotide to form the desired nanomaterial or nanostructure. Under conditions effective for
[0091]
The present invention provides another nanofabrication method. The method comprises: (a) at least two types of nanoparticles attached with an oligonucleotide; and (b) at least two types of connecting oligonucleotides having a selected sequence, wherein the sequence of each type of connecting oligonucleotide has at least two portions. And (c) providing a complex composed of streptavidin or avidin conjugated to two or more biotin molecules, each molecule having a first oligonucleotide conjugated thereto. The oligonucleotide on the first type of nanoparticle has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticle and a sequence complementary to the first portion of the sequence of the linking oligonucleotide. The oligonucleotide on the second type of nanoparticles has a sequence complementary to the sequence of the oligonucleotide on the first type of nanoparticle-oligonucleotide conjugate and a sequence complementary to the second portion of the sequence of the linking oligonucleotide. Having. Hybridization of the first and second types of nanoparticles, linking oligonucleotides, and the conjugates to the oligonucleotides on the nanoparticles and to each other and to the linking oligonucleotides to form the desired nanomaterial or nanostructure. The contact is made under conditions effective to permit hybridization of the conjugated oligonucleotide to the nucleotide.
[0092]
The present invention also provides yet another nanofabrication method. The method comprises: (a) at least one type of linking oligonucleotide having a selected sequence, wherein the sequence of each type of linking oligonucleotide has at least two portions; One or more types of nanoparticles attached to an oligonucleotide having a sequence complementary to a first portion of the sequence of nucleotides; (c) a nanoparticle having a sequence complementary to a second portion of the sequence of linked oligonucleotides; Providing one of the oligonucleotides associated with biotin, and (d) streptavidin or avidin. The linking oligonucleotide, biotin, conjugate, nanoparticle is contacted under conditions effective to permit hybridization of the oligonucleotide on the nanoparticle and the first oligonucleotide conjugated to biotin to the linking oligonucleotide. The resulting complex is then combined with streptavidin or avidin under conditions effective to permit a specific binding interaction between biotin and streptavidin or avidin to form the desired nanomaterial or nanostructure. Make contact.
[0093]
The present invention further provides a nanomaterial or nanostructure composed of oligonucleotide-attached nanoparticles, wherein the nanoparticles are grouped together by sbp interaction with the oligonucleotide connector.
[0094]
The invention further provides a method for separating a selected target nucleic acid having at least two portions from other nucleic acids. The method comprises: (a) one or more types of nanoparticles attached with oligonucleotides, wherein the oligonucleotides on each type of nanoparticle have a sequence complementary to the sequence of the first portion of the selected nucleic acid; and (b) A) a streptavidin or avidin linked to two or more biotin molecules, wherein a first oligonucleotide is linked to each molecule, the first oligonucleotide having a sequence complementary to the sequence of the second portion of the selected nucleic acid; Providing a composite that is constructed. The selected nucleic acid and other nucleic acids are hybridized with the oligonucleotide on the nanoparticle and the selected nucleic acid of the first oligonucleotide of the complex, and the selected nucleic acid hybridized to the selected nucleic acid and the complex aggregate and precipitate. Contact with the nanoparticles under conditions effective for
[0095]
The invention further provides a method for separating a selected target acid having at least two moieties from other nucleic acids. The method comprises: (a) one or more types of nanoparticles attached with oligonucleotides, wherein the oligonucleotides on each type of nanoparticle have a sequence complementary to the sequence of the first portion of the selected nucleic acid; Providing a biotin associated with the first oligonucleotide, wherein the first oligonucleotide has a sequence complementary to the sequence of the second portion of the selected nucleic acid; and (c) streptavidin or avidin. Contacting the selected nucleic acid and other nucleic acids with the nanoparticle and biotin construct under conditions effective to permit hybridization of the oligonucleotide and the first oligonucleotide of the biotin construct on the nanoparticle with the selected nucleic acid Let it. The resulting complex is treated with streptavidin or streptavidin under conditions effective to permit a specific binding interaction between biotin and streptavidin or avidin and subsequent aggregation and sedimentation of the resulting selected nucleic acid complex. Contact with avidin.
[0096]
The present invention relates to a method for actively transferring and concentrating a nanoparticle probe conjugated to a biomolecule from / to an electrode surface having a captured target molecule or a nanoparticle conjugated in solution to a captured target. Also provided are methods for actively transferring and enriching a set of probes to a surface of interest.
[0097]
Another object of the present invention is a method for accelerating the migration of nanoparticles to an electrode surface, comprising the nanoparticles bound to a charged first member of a specific binding pair and a second member of the specific binding pair. Providing a contact between the nanoparticles and the electrode surface under conditions effective to permit binding between the first and second members of the specific binding pair; and Subjecting the nanoparticles to an electric field to accelerate migration of the nanoparticles to the surface and promote binding between the first and second members of the binding pair.
[0098]
Yet another object of the invention is a method for detecting a nucleic acid having at least two moieties bound to an electrode surface, the method comprising providing one or more types of nanoparticles having oligonucleotides attached thereto. Wherein the oligonucleotide on each of the one or more types of nanoparticles has a sequence complementary to a sequence of one of the at least two portions of the nucleic acid; Contacting the oligonucleotide with the nucleic acid under conditions that effectively hybridize the nucleic acid; applying an electric field to the nanoparticle to accelerate the movement of the nanoparticle to the electrode surface; and Observing a detectable change caused by the hybridization of
[0099]
Yet another object of the present invention is a method for detecting a nucleic acid having at least two moieties attached to a surface, the method comprising contacting the nucleic acid with at least two types of nanoparticles having oligonucleotides attached thereto. The oligonucleotide on the first type of nanoparticles has a sequence complementary to the sequence of the first portion of the nucleic acid, and the oligonucleotide on the second type of nanoparticles has a sequence complementary to the sequence of the second portion of the nucleic acid. Having a sequence, wherein the contacting occurs under conditions that effectively hybridize the oligonucleotide on the nanoparticle to the nucleic acid, and subjecting the nanoparticle to an electric field to accelerate migration of the nanoparticle to the surface; Observing a detectable change caused by hybridization of the oligonucleotide and the nucleic acid on the nanoparticle. It is. Contacting conditions may include freezing and thawing, as well as heating. A detectable change may be observed on the solid surface, and may include a visually observable change in color.
[0100]
As used herein, the phrase "-type oligonucleotide" refers to a plurality of oligonucleotide molecules having the same sequence. Certain "types" of nanoparticles, conjugates, particles, latex microspheres, etc., having oligonucleotides attached thereto, refer to a plurality of such substances to which the same type of oligonucleotide is attached. Further, the “nanoparticle to which the oligonucleotide is attached” is “nanoparticle-oligonucleotide conjugate”, or, in the case of the detection method of the present invention, “nanoparticle-oligonucleotide probe” "Or simply" probe ".
[0101]
The term "analyte" refers to a compound or composition to be detected, including drugs, metabolites, pesticides, contaminants, and the like. An analyte can be composed of members of a specific binding pair (sbp), is a monovalent (monoepitope) or multivalent (polyepitope) ligand, usually an antigen or hapten, and may be a single compound or A plurality of compounds that share at least one common epitope or determinant. The specimen may be a cell such as a modern cell or a cell such as a cell carrying a blood group antigen such as A, B, D or an HLA antigen, or a microorganism (eg, bacteria, fungi, protozoa) , Virus).
[0102]
Multivalent ligand analytes are typically poly (amino acids), ie, polypeptides, proteins, polysaccharides, nucleic acids, and combinations thereof. Such combinations include components of bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes and the like.
[0103]
For the most part, the polyepitope ligand analytes to which the invention can be applied will have a molecular weight of at least about 5,000, more usually at least about 10,000. In the classification of poly (amino acids), the poly (amino acid) in question generally has a molecular weight of about 5,000 to 5,000,000, and more usually a molecular weight of about 20,000 to 1,000,000. Especially for the hormone in question, the molecular weight generally ranges from about 5,000 to 60,000.
[0104]
A variety of proteins can be considered with respect to families of proteins with similar structural characteristics, proteins with specific biological functions, proteins for particular microorganisms (particularly disease-causing microorganisms), and the like. Such proteins include, for example, immunoglobulins, cytokines, enzymes, hormones, cancer antigens, nutritional markers, tissue-specific antigens, and the like.
[0105]
Proteins, blood clotting factors, protein hormones, antigenic polysaccharides, microorganisms, and other pathogens of interest in the present invention are disclosed, inter alia, in US Pat. No. 4,650,770. The entire disclosure is incorporated herein by reference.
[0106]
Monoepitope ligand analytes will generally have a molecular weight of about 100-2,000, more usually about 125-1,000.
Such an analyte can be a molecule found directly in a sample, such as a body fluid from a host. The sample may be tested directly or may be pre-processed to make the analyte more easily detectable. Further, the analyte of interest can be determined by detection of an agent that demonstrates the presence of the analyte of interest, such as a specific binding pair member that is complementary to the analyte of interest. The presence of the agent is detected only when the analyte in question is present in the sample. Thus, an agent that demonstrates the presence of an analyte is the analyte that is detected in the assay. The bodily fluid may be, for example, urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, and the like.
[0107]
The term "specific binding pair (sbp) member" refers to one of two different molecules that specifically binds to a particular spatial and polar tissue of the other molecule and is thus complementary to that tissue. Refers to a molecule that has a defined area on its surface or in a cavity. This specific binding pair member is also called a ligand and a receptor (antiligand). These are usually immunological pairs such as antigen-antibody, and polynucleotide pairs such as biotin-avidin, hormone-hormone receptor, nucleic acid duplex, IgG-protein A, DNA-DNA and DNA-RNA. Other specific bindings such as are not immunological pairs but are usually included in the present invention and definition of sbp members.
[0108]
The term “ligand” refers to any organic compound for which a receptor naturally exists or for which a receptor can be prepared. The term "ligand" further includes ligand analogs, which are modified ligands having a molecular weight typically greater than 100 and capable of competing with similar ligands for the receptor, usually being organic radicals or analyte analogs. Modifications provide a means of linking a ligand analog to another molecule. The ligand analog will usually, but need not be, different from the ligand beyond the substitution of hydrogen and the bond connecting the ligand to the hub or tableware. A ligand analog can bind to a receptor in a manner similar to a ligand. A ligand analog can be, for example, an antibody directed to a ligand for the idiotype of the antibody.
[0109]
The term “receptor” or “antiligand” refers to any compound or composition that can recognize, for example, a particular spatial and polar organization of a molecule (eg, an epitope or antigenic determinant). Exemplary receptors include natural receptors such as thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids, avidin, protein A, barstar, complement component Clq, and the like. Is included. Avidin includes egg white avidin and biotin binding proteins obtained from other sources such as streptavidin.
[0110]
The term "specific binding" refers to one of the two different molecules specifically recognizing another molecule as compared to essentially less recognition of the other molecule. Generally, molecules have regions on their surface or within cavities that give rise to specific recognition between the molecules. Typical examples of specific binding are antibody-antigen interactions, enzyme-substrate interactions, polynucleotide interactions, and the like.
[0111]
The term "non-specific binding" refers to non-covalent bonding between molecules that is relatively independent of the specific surface structure. Non-specific binding can occur due to several factors, including hydrophobic interactions between the molecules.
[0112]
The term "antibody" refers to an immunoglobulin that specifically binds to a particular spatial and polar tissue of another molecule and is therefore defined as complementary to that tissue. Antibodies can be monoclonal or polyclonal, can be prepared by techniques well known in the art, such as host immunization or serum collection (polyclonal), or by continuous hybrid cell line preparation and secretion protein secretion. It can be prepared by collection (monoclonal) or by cloning and expressing a nucleotide sequence encoding an amino acid sequence required for specific binding of a natural antibody or a mutant sequence thereof. Antibodies include whole immunoglobulins or fragments thereof, including various classes and isotypes such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM. The fragments include Fab, Fv, and F (ab ') sub. 2, Fab 'and the like. In addition, aggregates, polymers, and conjugates of immunoglobulins or fragments thereof can be used as long as they maintain the binding affinity for a particular molecule.
[0113]
(Detailed description of preferred embodiments)
Nanoparticles useful in the practice of the present invention include metals (eg, gold, silver, copper and platinum), semiconductors (eg, CdSe, CdS, and CdSe coated with CdS or ZnS) and magnetic (eg, ferromagnetic) Colloidal materials. Other nanoparticles useful in the practice of the present invention include ZnS, ZnO, TiO.₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs. The size of the nanoparticles is preferably about 5 to about 150 nm (average diameter), more preferably about 5 to about 50 nm, and most preferably about 10 to about 30 nm. The nanoparticles may be rods.
[0114]
Methods for preparing metal, semiconductor and magnetic nanoparticles are well known in the art. For example, Schmid, G .; (Ed.), Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M .; A. (Ed.), Colloidal Gold: Principles, Methods, and Applications (Academic Press, San Diego, 1991); , IEEE Transactions On Magnetics, Vol. 17, p. 1247 (1981); Ahmadi, T .; S. Et al., Science, 272, 1924 (1996); Henglein, A .; J. et al. Phys. Chem. 99, 14129 (1995); Curtis, A .; C. Angew. Chem. Int. Ed. Engl. 27, p. 1530 (1988).
[0115]
ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs nanoparticles are also known in the art. See, for example, Weller, Angew. Chem. Int. Ed. Engl. 32, 41 (1993); Henglein, Top. Curr. Chem. 143: 113 (1988); Henglein, Chem. Rev .. 89, 1861 (1989); Brus, Appl. Phys. A. 53, p. 465 (1991); Bahncmann, Photochemical Conversion and Storage of Solar Energy (Pelizetti and Schiavelo, eds., 1991), p. 251; Wang and Herron. Phys. Chem. 95, p. 525 (1991); Olshavsky et al. Am. Chem. Soc. 112, 9438 (1990); Ushida et al. Phys. Chem. 95, 5382 (1992).
[0116]
Suitable nanoparticles are also commercially available, for example, from Ted Pella, Inc. (Gold), Amersham Corporation (Gold) and Nanoprobes, Inc. (Gold). ing.
[0117]
Preferred for use in detecting nucleic acids are gold nanoparticles. Colloidal gold particles have a high extinction coefficient for bands that emit beautiful colors. These striking colors vary depending on particle size, concentration, interparticle spacing, and the degree of aggregation and shape (geometry) of the aggregates, which makes these materials particularly attractive for colorimetric assays. It is assumed. For example, as soon as the oligonucleotide attached to the gold nanoparticle and the oligonucleotide and the nucleic acid hybridize, discoloration visible to the naked eye occurs (for example, see Examples).
[0118]
Gold nanoparticles are for the same reasons as listed above, as well as for their stability, ease of imaging by electron microscopy, and modification with well-characterized thiol functional groups (see below). Therefore, it is particularly preferable to use it for nanofabrication. Also, semiconductor nanoparticles are preferred for nanofabrication because of their inherent electronic and luminescent properties.
[0119]
The nanoparticles or oligonucleotides or both are functionalized to attach the oligonucleotide to the nanoparticles. Such methods are known in the art. For example, an oligonucleotide functionalized at the 3 'or 5' end with an alkanethiol will readily attach to gold nanoparticles. Whitesides, Proceedings of the Robert A. See Welch Foundation 39th Conference On Chemical Research Nanophase Chemistry, Houston, TX, pp. 109-121 (1995). Further, Mucic et al., Chem. Commun. , 555-557 (1996), which describes a method for attaching 3 'thiol DNA to flat gold surfaces; this method can be used to attach oligonucleotides to nanoparticles. The alkanethiol method can also be used to attach oligonucleotides to other metals, semiconductors and magnetic colloids, and other nanoparticles listed above. Other functional groups for attaching oligonucleotides to solid surfaces include phosphorothioate groups (eg, for attaching oligonucleotide-phosphorothioate to a gold surface, see US Pat. No. 5,472,881), substituted alkyl Siloxane (e.g., for the attachment of oligonucleotides to silica and glass surfaces, see Burwell, Chemical Technology, 4, 370-377 (1974) and Matteuci and Caruthers, J. Am. Chem. Soc., 103). Vol., 3185-3191 (1981), for the binding of aminoalkylsiloxanes and similar mercaptoalkylsiloxanes, see Grabar et al., Anal. Chem., 67, 735. 745 see page) is included. To attach the oligonucleotide to the solid surface, a 5 'thionucleoside or 3' thionucleoside-terminated oligonucleotide may be used. The following references describe other methods that can be used to attach oligonucleotides to nanoparticles: Nuzzo et al. Am. Chem. Soc. 109, p. 2358 (1987) (disulfide on gold); Allara and Nuzzo, Langmuir, Vol. 1, p. 45 (1985) (carboxylic acid on aluminum); Allara and Tompkins, J. Mol. Colloid Interface Sci. 49: 410-421 (1974) (carboxylic acid on copper); Iler, The Chemistry of Sica, Chapter 6, (Wiley, 1979) (carboxylic acid on silica); Timmons and Zisman, J. Phys. Chem. 69, 984-990 (1965) (Carboxylic acid on platinum); Soriaga and Hubbard, J. Mol. Am. Chem. Soc. 104, 3937 (1982) (aromatic ring compounds on platinum); Hubbard, Acc. Chem. Res. 13, 177 (1980) (sulfolane, sulfoxide and other functionalized solvents on platinum); Hickman et al. Am. Chem. Soc. Vol. 111, p. 7271 (1989) (isonitrile on platinum); Maoz and Sagiv, Langmuir, Vol. 3, page 1045 (1987) (silane on silica); Maoz and Sagiv, Langmuir, vol. 1034 (1987) (silane on silica); Wasserman et al., Langmuir, Vol. 5, p. 1074 (1989) (silane on silica); Eltekova and Eltekov, Langmuir, 3, 951 (1987). Y.) (aromatic carboxylic acids, aldehydes, alcohols and methoxy groups on titanium dioxide and silica); Lec et al. Phys. Chem. 92, p. 2597 (1988) (hard phosphate on metal).
[0120]
Oligonucleotides functionalized with cyclic disulfides are within the scope of the present invention. Cyclic disulfides preferably have 5 or 6 atoms, including 2 sulfur atoms, in the ring. Suitable cyclic disulfides are commercially available or synthesized by known methods. Reduced forms of cyclic disulfides can also be used.
[0121]
Preferably, the linker further comprises a hydrocarbon moiety attached to the cyclic disulfide. Suitable hydrocarbons are commercially available and are attached to cyclic disulfides. Preferably, the hydrocarbon moiety is a steroid residue. Oligonucleotide-nanoparticle conjugates prepared using a linker containing a steroid residue attached to a cyclic disulfide were unexpectedly prepared using an alkanethiol or acyclic disulfide as a linker. It was found to be significantly more stable to thiols (eg, dithiothreitol used in polymerase chain reaction (PCR) solutions) compared to the body. Indeed, the oligonucleotide-nanoparticle conjugate of the present invention was found to be 300 times more stable. This unexpected stability is probably due to the fact that each oligonucleotide is anchored to the nanoparticle by two sulfur atoms instead of one. In particular, it is believed that two adjacent sulfur atoms of the cyclic disulfide have a chelating effect that is effective in stabilizing the oligonucleotide-nanoparticle conjugate. Large hydrophobic steroid residues in the linker also appear to contribute to the stability of the conjugate by blocking the nanoparticle from approaching water-soluble molecules to the surface of the nanoparticle.
[0122]
In view of the above, the two sulfur atoms of the cyclic disulfide should preferably be sufficiently close so that both sulfur atoms can stick to the nanoparticles simultaneously. More preferably, the two sulfur atoms are adjacent to each other. Also, the hydrocarbon portion must be large to show a large hydrophobic surface that blocks the surface of the nanoparticles.
[0123]
Oligonucleotide-cyclic nanoparticle conjugates using cyclic disulfide linkers can be used as probes in diagnostic assays for nucleic acid detection and in the nanofabrication methods described herein. Such conjugates of the invention have been unexpectedly found to increase the sensitivity of diagnostic assays using them. In particular, assays using oligonucleotide-nanoparticle conjugates prepared using a linker containing a steroid residue attached to a cyclic disulfide use an approximately 10-fold alkanethiol or acyclic disulfide as the linker. Was found to be 10 times more sensitive than assays using conjugates prepared in this manner.
[0124]
The surprising stability of the oligonucleotide-nanoparticle conjugates of the present invention against the thiols described above allows them to be used directly in PCR solutions. Thus, oligonucleotide-nanoparticles of the invention added as a probe to a DNA target to be amplified by PCR undergo 30 or 40 PCR heating-cooling cycles, and the amplicon can be obtained without opening the tube. Can be detected. Opening sample tubes for additional probes after PCR can cause serious problems due to contamination of equipment used for subsequent tests.
[0125]
Finally, the invention provides a kit comprising a container containing one type of oligonucleotide cyclic disulfide linker of the invention or a container containing one type of oligonucleotide-nanoparticle conjugate of the invention. The kit may further include other reagents or items effective for nucleic acid detection or nanofabrication.
[0126]
Multiple oligonucleotides are attached to each nanoparticle. As a result, each nanoparticle-oligonucleotide conjugate can bind to multiple oligonucleotides or nucleic acids having complementary sequences.
[0127]
In practicing the present invention, oligonucleotides having a defined sequence are used for various purposes. Methods for preparing oligonucleotides having a predetermined sequence are well known. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989) and F.M. See Eckstein (eds.) Oligonucleotides and Analogues, 1st edition (Oxford University Press, New York, 1991). Solid phase synthesis is preferred for both oligonucleotide and oligodeoxyribonucleotide synthesis (well-known DNA synthesis methods are also useful for RNA synthesis). Oligoribonucleotides and oligodeoxyribonucleotides can also be prepared enzymatically.
[0128]
The present invention provides a nucleic acid detection method. These methods can also detect any type of nucleic acid and can be used, for example, in diagnosing disease or sequencing nucleic acids. Examples of nucleic acids that can be detected by the method of the present invention include genes (eg, genes involved in a particular disease), viral RNA and DNA, bacterial DNA, fungal DNA, cDNA, mRNA, RNA and DNA fragments, oligonucleotides, There are synthetic oligonucleotides, modified oligonucleotides, single- and double-stranded nucleic acids, natural and synthetic nucleic acids, and the like. For example, examples of applications of nucleic acid detection methods include viral diseases (eg, human immunodeficiency virus, hepatitis virus, herpes virus, cytomegalovirus and Epstein-Barr virus), bacterial diseases (eg, tuberculosis, Lyme disease, H. pylori, E. coli infection, Legionella infection, Mycoplasma infection, Salmonella infection) Sexually transmitted diseases (eg, gonorrhea), genetic diseases (eg, cystic fibrosis, Duchenne muscular dystrophy, phenylketonuria) Sickle cell anemia), and for the diagnosis and / or monitoring of cancer (eg, genes involved in cancer development); forensic medicine; for DNA sequencing; for paternity testing; for confirming cell lines; for monitoring gene therapy; As well as many other purposes.
[0129]
The nucleic acid detection method based on the observation of discoloration by the naked eye is inexpensive, fast, simple, and robust (reagent is stable), does not require specialized equipment or expensive equipment, and requires almost no instrument measurement. is there. In these respects, the above methods require rapid identification of infectious diseases to assist in the formulation of therapeutics, for example, in research and analysis experiments in DNA sequencing, in the field of detecting the presence of specific pathogens. Particularly suitable for use in clinics and in homes and insurance centers for inexpensive frontline screening.
[0130]
The nucleic acid to be detected can be isolated by known methods, or can be a cell, tissue sample, biological fluid (eg, saliva, urine, blood, serum), a solution containing PCR components, a large excess of oligonucleotide or It can also be detected directly in solutions containing high molecular weight DNA, and other samples known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); D. Hames and S.M. J. See, Higgins, Gene Probes 1 (IRL Press, New York, 1995). Methods for preparing nucleic acids for detection using hybridization probes are well known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); D. Hames and S.M. J. See, Higgins, Gene Probes 1 (IRL Press, New York, 1995).
[0131]
If the nucleic acid is present in small amounts, methods known in the art can be used. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); D. Hames and S.M. J. See, Higgins, Gene Probes 1 (IRL Press, New York, 1995). Polymerase chain reaction (PCR) amplification is preferred.
[0132]
One nucleic acid detection method of the present invention comprises the step of contacting a nucleic acid with one or more types of nanoparticles having oligonucleotides attached thereto. The nucleic acid to be detected has at least two parts. The lengths of these moieties, and their spacing, if any, are selected such that a detectable change occurs when the oligonucleotide on the nanoparticle hybridizes to the nucleic acid. These lengths and spacings can be empirically determined and include the type of particles used and their size, as well as the type of electrolyte present in the solution used for the assay (as is known in the art, for certain types of electrolytes). The electrolyte will affect the conformation of the nucleic acid).
[0133]
Also, when detecting a nucleic acid in the presence of another nucleic acid, the portion of the nucleic acid to which the oligonucleotide on the nanoparticle should bind is selected to include a unique sequence sufficient to make the nucleic acid detection specific. Must. Guidelines for doing so are well-known in the art.
[0134]
Nucleic acids may contain repetitive sequences that are sufficiently close together to use only one type of oligonucleotide-nanoparticle conjugate, but this rarely occurs. Generally, selected portions of the nucleic acid have different sequences and are preferably attached to different nanoparticles, wherein the selected portion will contact nanoparticles bearing two or more different oligonucleotides. Would. One example of a system for detecting nucleic acids is shown in FIG. As can be seen, the first oligonucleotide attached to the first nanoparticle has a sequence that is complementary to the first portion of the target sequence in the single-stranded DNA. The second oligonucleotide attached to the second nanoparticle has a sequence complementary to the second portion of the target sequence in the single-stranded DNA. Additional portions of the DNA can be targeted by the corresponding nanoparticles. See FIG. Targeting some portion of the nucleic acid increases the magnitude of the detectable change.
[0135]
The step of contacting the nucleic acid with the nanoparticle-oligonucleotide conjugate is performed under conditions effective to hybridize the oligonucleotide on the nanoparticle with the target sequence of the nucleic acid. These hybridization conditions are well known in the art and can be easily optimized for the particular system used. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989). It is preferred to use stringent hybridization conditions.
[0136]
Freezing and thawing the solution containing the nucleic acid to be detected and the nanoparticle-oligonucleotide conjugate allows for faster hybridization. The solution may be frozen by any conventional method, such as by placing it in a dry ice-alcohol bath for a time sufficient to freeze (generally, about 1 minute for a 100 μl solution). The solution must be thawed at a temperature below the heat denaturation temperature, but room temperature may be advantageous for most combinations of nanoparticle-oligonucleotide conjugates and nucleic acids. After thawing the solution, hybridization is complete and a detectable change is observed after thawing the solution.
[0137]
The solution containing the nucleic acid to be detected and the nanoparticle-oligonucleotide conjugate is warmed to a temperature below the dissociation temperature (Tm) of the complex formed between the oligonucleotide on the nanoparticle and the target nucleic acid, and the hybridization rate Can also be increased. Alternatively, high-speed hybridization can be achieved by heating to a temperature equal to or higher than the dissociation temperature (Tm) and cooling the solution.
[0138]
The rate of hybridization can also be increased by increasing the salt concentration (eg, from 0.1 M to 0.3 M NaCl).
The detectable change that occurs upon hybridization of the oligonucleotide and the nucleic acid on the nanoparticles can be a color change, the formation of nanoparticle aggregates, or the precipitation of aggregated nanoparticles. Discoloration can be observed visually or by spectroscopic analysis. The formation of nanoparticle aggregates can be observed by electron microscopy or nephelometry. Precipitation of aggregated nanoparticles can be observed visually or by microscopy. Preferred are changes observable to the naked eye. Particularly preferred are discolorations that are observable with the naked eye.
[0139]
Observation of discoloration by the naked eye can be more easily performed with a contrasting color background. For example, when using gold nanoparticles, observing the discoloration can be accomplished by placing the hybridization solution on a solid white surface (such as silica or alumina TLC plates, filter paper, celluloid membranes, and nylon membranes, preferably C-18 silica TLC plates). Spotting is facilitated by drying the spots. Initially, the spots assume the color of the hybridization solution (ranging from pink / red in the absence of hybridization to purplish red / purple in the presence of hybridization). When dried at room temperature or at 80 ° C. (temperature is not critical), if the nanoparticle-oligonucleotide conjugate hybridizes and binds to the target nucleic acid before spotting, a blue spot results. If no hybridization occurs (eg, due to the absence of the target nucleic acid), the spot is pink. The blue and pink spots are stable and do not change after cooling, heating, or over time. Those spots provide a convenient permanent record of the test. No other steps are required to observe the color change (such as separation of those that are not hybridized to the hybridized nanoparticle-oligonucleotide conjugate).
[0140]
An alternative method to easily visualize the assay results is to use a nanoparticle probe sample hybridized to the target nucleic acid with a glass fiber filter (eg, with 13 nm size gold nanoparticles) while aspirating the liquid and passing it through the filter. When used, it is a method of spotting on a borosilicate microfiber filter (pore size 0.7 μm, grade FG75). The excess non-hybridized probe is then filtered, leaving an observable spot containing aggregates formed by hybridization of the nanoparticle probe (retained because it is larger than the pores of the filter) with the target nucleic acid. And rinse with water. This technique can provide high sensitivity because an excess of nanoparticle probes can be used. Unfortunately, nanoparticle probes stick to many other solid surfaces that have been tried (silica slides, reversed-phase plates, nylon, nitrocellulose, cellulose, and other membranes), and these surfaces cannot be used.
[0141]
An important aspect of the detection system shown in FIG. 2 is that the step of obtaining a detectable change depends on the cooperative hybridization of two different oligonucleotides with a given target sequence in the nucleic acid. Mismatches in either of the two oligonucleotides will destabilize the binding between the particles. It is well known that mismatches in base pairs have a much greater destabilizing effect on the binding of shorter oligonucleotide probes than on longer oligonucleotide probes. The advantage of the system shown in FIG. 2 is that despite the fact that this system takes advantage of the long target sequence and the base discrimination associated with the probe (18 base pairs in the example shown in FIG. 2), the system is short. The oligonucleotide probe (9 base pairs in the example shown in FIG. 2) has characteristic sensitivity.
[0142]
The target sequence of the nucleic acid can be contiguous as in FIG. 2 or, as shown in FIG. 3, a third portion in which the two portions of the target sequence are not complementary to the oligonucleotide on the nanoparticle. May be separated by In the latter case, there is also the option of using a filler oligonucleotide that is free in solution and has a sequence complementary to the sequence of this third part (see FIG. 3). When the filler oligonucleotide hybridizes to the third portion of the nucleic acid, it forms a double-stranded segment, thereby changing the average spacing between the nanoparticles and consequently the color. The system shown in FIG. 3 can increase the sensitivity of the detection method.
[0143]
Some embodiments of the nucleic acid detection method utilize a substrate. By using a substrate, a detectable change (signal) can be amplified and the sensitivity of the assay can be increased.
[0144]
Any substrate capable of observing a detectable change may be used. Suitable substrates include transparent solid surfaces (eg, glass, quartz, plastic and other polymers), opaque solid surfaces (eg, TLC silica plates, filter paper, glass fiber filters, white solid surfaces such as celluloid membranes, nylon membranes, etc.). ), And conductive solid surfaces (eg, indium-tin-oxide (ITO)). The substrate may be of any shape or thickness, but is generally flat and thin. Preferred is a transparent substrate such as glass (eg, a glass slide) or plastic (eg, a microtiter plate well).
[0145]
In one embodiment, the oligonucleotide is attached to a substrate. Oligonucleotides are described, for example, in Crisey et al., Nucleic Acids Res. 24, 3031-3039 (1996); Crisey et al., Nucleic Acids Res. 24: 3040-3047 (1996); Mucic et al., Chem. Commun. 555 (1996); Zimmermann and Cox, Nucleic Acids Res. 22, Vol. 492 (1994); Bottomley et al. Vac. Sci. Technol. A, Volume 10, 591 (1992); and Hegner et al., FEBS Lett. 336, p. 452 (1993).
[0146]
The oligonucleotide attached to the substrate has a sequence complementary to the sequence of the first portion of the nucleic acid to be detected. The nucleic acid and the substrate are contacted under conditions effective to hybridize the oligonucleotide on the substrate and the nucleic acid. Thus, the nucleic acid is in a state of being bound to the substrate. Unbound nucleic acid is preferably washed off the substrate before adding the nanoparticle-oligonucleotide conjugate.
[0147]
Next, the nucleic acid bound to the substrate is brought into contact with the first type of nanoparticles to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the sequence of the second portion of the nucleic acid, and the contacting step is performed under conditions effective to hybridize the oligonucleotide on the nanoparticle with the nucleic acid. In this way, the first type of nanoparticles is bound to the substrate. After binding the nanoparticle-oligonucleotide conjugate to the substrate, the substrate is washed to remove unbound nanoparticle-oligonucleotide conjugate and nucleic acid.
[0148]
The oligonucleotides on the first type of nanoparticles may all have the same sequence or may have different sequences that hybridize to different parts of the nucleic acid to be detected. If oligonucleotides with different sequences are used, all different oligonucleotides may be attached to each nanoparticle, but it is preferred that different oligonucleotides be attached to different nanoparticles. FIG. 17 illustrates the use of a nanoparticle-oligonucleotide conjugate designed to hybridize to multiple portions of a nucleic acid. Alternatively, the oligonucleotide on each first type of nanoparticles may have a plurality of different sequences, at least one of which must hybridize to a portion of the nucleic acid to be detected ( See FIG. 25B).
[0149]
Finally, the first type of nanoparticle-oligonucleotide conjugate bonded to the substrate is brought into contact with the second type of nanoparticle to which the oligonucleotide is attached. The oligonucleotide has a sequence complementary to at least a portion of the sequence of the oligonucleotide attached to the first type of nanoparticles, and the contacting step comprises: This is performed under conditions effective to hybridize the nucleotide and the oligonucleotide on the second type of nanoparticle. After binding the nanoparticles, the substrate is preferably washed to remove unbound nanoparticle-oligonucleotide conjugates.
[0150]
The combination of hybridizations results in a detectable change. The detectable change is the same as described above, except that multiplex hybridization amplifies the detectable change. In particular, since a plurality of oligonucleotides (having the same or different sequences) are attached to each first type of nanoparticles, each first type of nanoparticle-oligonucleotide conjugate is composed of a plurality of second types of nanoparticles. Can hybridize to the nanoparticle-oligonucleotide conjugate of Also, the first type of nanoparticle-oligonucleotide conjugate can hybridize to more than one portion of the nucleic acid to be detected. The amplification obtained by multiplex hybridization may make the change detectable for the first time or may increase the magnitude of the detectable change. This amplification increases the sensitivity of the assay and allows detection of small amounts of nucleic acids.
[0151]
If desired, the first and second types of nanoparticle-oligonucleotide conjugates can be added sequentially to deposit additional nanoparticle layers. In this way, the number of immobilized nanoparticles per target nucleic acid molecule can be increased, while the corresponding signal intensity can be enhanced.
[0152]
Also, instead of the first and second types of nanoparticle-oligonucleotide conjugates designed to hybridize directly to each other, oligos that serve to bind the nanoparticles together as a result of hybridization with the linking oligonucleotide. Nanoparticles carrying nucleotides may be used.
[0153]
Methods for preparing nanoparticles and oligonucleotides and attaching oligonucleotides to nanoparticles are described above. Hybridization conditions are well known in the art and can be readily optimized for the particular system used (see above).
[0154]
One embodiment of this nucleic acid (analyte DNA) detection method is shown in FIG. 13A. As shown, the combination of hybridizations results in dark regions where the nanoparticle aggregates are bound to the substrate via the analyte DNA. These dark areas are easily observable with the naked eye using ambient light, preferably by looking at the substrate against a white background. As can be readily seen from FIG. 13A, this method provides a means of amplifying a detectable change.
[0155]
Another embodiment of this nucleic acid detection method is shown in FIG. 25B. As in the example shown in FIG. 13A, the combination of hybridizations creates a dark area where the nanoparticle aggregates are bound to the substrate via the analyte DNA, which can be observed with the naked eye. .
[0156]
In another embodiment, the nanoparticles are attached to a substrate. Nanoparticles are described, for example, in Grabar et al., Analyt. Chem. 67, 73-743 (1995); Bethell et al. Electroanal. Chem. 409, 137 (1996); Bar et al., Langmuir, 12, 1172 (1996); Colvin et al. Am. Chem. Soc. 114, 5221 (1992).
[0157]
After attaching the nanoparticles to the substrate, the oligonucleotide is attached to the nanoparticles. This can be accomplished in the same manner as described above for attaching oligonucleotides to nanoparticles in solution. The oligonucleotide attached to the nanoparticle has a sequence that is complementary to the sequence of the first portion of the nucleic acid.
[0158]
The substrate and the nucleic acid are contacted under conditions effective to hybridize the oligonucleotide on the nanoparticle with the nucleic acid. In this way, the nucleic acid is bound to the substrate. Unbound nucleic acid is preferably washed off the substrate before adding further nanoparticle-oligonucleotide conjugates.
[0159]
Next, a second type of nanoparticle to which the oligonucleotide is attached is prepared. These oligonucleotides have a sequence that is complementary to the sequence of the second portion of the nucleic acid, and the nucleic acid bound to the substrate and the second type of nanoparticle-oligonucleotide conjugate are The oligonucleotide on the nanoparticle-oligonucleotide conjugate is contacted with the nucleic acid under conditions effective to hybridize. In this way, the second type of nanoparticle-oligonucleotide conjugate is bound to the substrate. After binding the nanoparticles, the unbound nanoparticles-oligonucleotide conjugate and the nucleic acid are washed off the substrate. At this point, some change (eg, discoloration) may be detected.
[0160]
The oligonucleotides on the second type of nanoparticles may all have the same sequence or may have different sequences that hybridize to different parts of the nucleic acid to be detected. If oligonucleotides with different sequences are used, all different oligonucleotides may be attached to each nanoparticle, but it is preferred that different oligonucleotides be attached to different nanoparticles. See FIG.
[0161]
A binding oligonucleotide having a selected sequence that includes at least two portions wherein the first portion is complementary to at least a portion of the sequence of the oligonucleotides on the second type of nanoparticles was then bound to the substrate. The second type of nanoparticle-oligonucleotide conjugate is contacted under conditions effective to hybridize the bound oligonucleotide to the oligonucleotide on the nanoparticle. In this way, the binding oligonucleotide is bound to the substrate. After binding the bound oligonucleotide, the unbound bound oligonucleotide is washed off the substrate.
[0162]
Finally, a third type of nanoparticle to which the oligonucleotide is attached is prepared. These oligonucleotides have a sequence that is complementary to the sequence of the second portion of the binding oligonucleotide. The nanoparticle-oligonucleotide conjugate and the binding oligonucleotide bound to the substrate are contacted under conditions effective to hybridize the binding oligonucleotide to the oligonucleotide on the nanoparticle. After binding the nanoparticles, the unbound nanoparticle-oligonucleotide conjugate is washed off the substrate.
[0163]
The combination of hybridizations results in a detectable change. The detectable change is the same as described above, except that multiplex hybridization amplifies the detectable change. In particular, since a large number of oligonucleotides (having the same or different sequences) are attached to each of the second type of nanoparticles, each second type of nanoparticle-oligonucleotide conjugate (via a binding oligonucleotide) 3.) Can hybridize to multiple third type nanoparticle-oligonucleotide conjugates. Also, the second type of nanoparticle-oligonucleotide conjugate can hybridize to more than one portion of the nucleic acid to be detected. The amplification resulting from the multiplex hybridization may make the change detectable for the first time, or may increase the magnitude of the detectable change. This amplification increases the sensitivity of the assay and allows the detection of small amounts of nucleic acids.
[0164]
If desired, the binding oligonucleotide and the second and third types of nanoparticle-oligonucleotide conjugates can be added sequentially to deposit an additional nanoparticle layer. In this way, the number of immobilized nanoparticles per target nucleic acid molecule can be further increased, while the corresponding signal intensity can be increased.
[0165]
It is also possible to eliminate the use of binding oligonucleotides and the second and third types of nanoparticle-oligonucleotide conjugates can be designed to hybridize directly to each other.
[0166]
Methods for preparing nanoparticles and oligonucleotides and attaching oligonucleotides to nanoparticles are described above. Hybridization conditions are well known in the art and can be readily optimized for the particular system used (see above).
[0167]
One embodiment of this nucleic acid (analyte DNA) detection method is shown in FIG. 13B. As shown in the figure, the combination of hybridizations results in dark regions where the nanoparticle aggregates are bound to the substrate via the analyte DNA. These dark areas can be easily observed with the naked eye as described above. As can be seen from FIG. 13B, this embodiment of the method of the present invention provides another means of amplifying a detectable change.
[0168]
Another amplification scheme is the use of liposomes. In this scheme, oligonucleotides are attached to a substrate. Suitable substrates are described above, and the oligonucleotides can be attached to the substrate as described above. For example, if the substrate is glass, this can be achieved by condensing the oligonucleotide via a phosphoryl or carboxylic acid group to an aminoalkyl group on the substrate surface (for related chemistry, see Grabar et al., Anal. Chem. 67, 735-743 (1995)).
[0169]
The oligonucleotide attached to the substrate has a sequence complementary to the sequence of the first part of the nucleic acid to be detected. The nucleic acid and the substrate are contacted under conditions effective to hybridize the oligonucleotide on the substrate and the nucleic acid. Thus, the nucleic acid is in a state of being bound to the substrate. Unbound nucleic acid is preferably washed off the substrate before adding additional components of the system.
[0170]
Next, the nucleic acid bound to the substrate is brought into contact with the liposome to which the oligonucleotide is attached. The oligonucleotide has a sequence complementary to the sequence of the second portion of the nucleic acid, and the contacting step is performed under conditions effective to hybridize the oligonucleotide on the liposome to the nucleic acid. In this way, the liposome becomes bound to the substrate. After binding the liposomes to the substrate, the substrate is washed to remove unbound liposomes and nucleic acids.
[0171]
The oligonucleotides on the liposome may all have the same sequence or may have different sequences that hybridize to different parts of the nucleic acid to be detected. When using oligonucleotides having different sequences, each liposome may have all the different oligonucleotides attached, or different oligonucleotides may be attached to different nanoparticles.
[0172]
To prepare an oligonucleotide-liposome conjugate, the oligonucleotide is conjugated to a hydrophobic group such as cholesteryl (see Letsinger et al., J. Am. Chem. Soc., 115, 7535-7536 (1993)). ), Mixing the hydrophobic-oligonucleotide conjugate with the liposome solution to form liposomes with the hydrophobic-oligonucleotide conjugate immobilized on the membrane (Zhang et al., Tetrahedron Lett., Vol. 37, 6243). -6246 (1996)). Loading of the hydrophobic-oligonucleotide conjugate on the liposome surface can be controlled by adjusting the ratio of the hydrophobic-oligonucleotide conjugate to the liposome in the mixture. Liposomes carrying oligonucleotides attached by hydrophobic interaction of cholesteryl side groups were found to be effective at targeting polynucleotides immobilized on nitrocellulose membranes (Id.). A fluorescein group immobilized in the liposome membrane was used as a reporter group. Although the fluorescein group worked effectively, sensitivity was limited by the fact that the signal from fluorescein in high local concentration regions (eg, on liposome surfaces) was weakened by self-quenching.
[0173]
Liposomes are prepared by methods well known in the art. Zhang et al., Tetrahedron Lett. 37, 6243 (1996). Generally, the size of the liposome is about 5 to 50 times the size (diameter) of the nanoparticles used in the subsequent step. For example, for nanoparticles having a diameter of about 13 nm, it is preferable to use liposomes having a diameter of about 100 nm.
[0174]
The liposome bound to the substrate is brought into contact with the first type of nanoparticles to which at least one first type of oligonucleotide is attached. A hydrophobic group is attached to the end of the first type of oligonucleotide that is not attached to the nanoparticle, and the contacting step involves attaching the oligonucleotide on the nanoparticle to the liposome as a result of the hydrophobic interaction. Perform under effective conditions. At this point, a detectable change can be observed.
[0175]
The method further includes contacting the first type of nanoparticle-oligonucleotide conjugate attached to the liposome with a second type of nanoparticle to which the oligonucleotide is attached. An oligonucleotide having a sequence complementary to at least a part of the sequence of the oligonucleotide on the second type of nanoparticles is attached to the first type of nanoparticles. The oligonucleotide has a sequence that is complementary to at least a portion of the sequence of the second type of oligonucleotide on the first type of nanoparticle. The contacting step is performed under conditions effective to hybridize the oligonucleotides on the first and second types of nanoparticles. This hybridization is generally performed at a mild temperature (for example, 5 to 60 ° C.), and therefore, conditions (for example, 0.3 to 1.0 M NaCl) that can induce hybridization at room temperature are used. After hybridization, the unbound nanoparticle-oligonucleotide conjugate is washed off the substrate.
[0176]
The combination of hybridizations results in a detectable change. The detectable change is the same as described above, except that multiplex hybridization amplifies the detectable change. In particular, since each liposome has a plurality of oligonucleotides (having the same or different sequences) attached to it, each liposome can hybridize to a plurality of the first type of nanoparticle-oligonucleotide conjugate. Similarly, since multiple oligonucleotides are attached to each first type of nanoparticle-oligonucleotide conjugate, each first type of nanoparticle-oligonucleotide conjugate has a plurality of second type of nanoparticles. -Capable of hybridizing with the oligonucleotide conjugate. A liposome can also hybridize to more than one portion of the nucleic acid to be detected. The amplification resulting from the multiplex hybridization may make the change detectable for the first time or may increase the magnitude of the detectable change. This amplification increases the sensitivity of the assay and allows the detection of small amounts of nucleic acids.
[0177]
If desired, the first and second types of nanoparticle-oligonucleotide conjugates can be added sequentially to deposit additional nanoparticle layers. In this way, the number of immobilized nanoparticles per target nucleic acid molecule can be further increased, while the corresponding signal intensity can be increased.
[0178]
Also, instead of the second and third types of nanoparticle-oligonucleotide conjugates designed to hybridize directly to each other, oligonucleotides that serve to bind the nanoparticles as a result of hybridization with the binding oligonucleotide May be used.
[0179]
Methods for preparing nanoparticles and oligonucleotides and attaching oligonucleotides to nanoparticles are described above. Oligonucleotide mixtures with or without a hydrophobic group at the other end, functionalized at one end for binding to the nanoparticles, may be used on the first type of nanoparticles. The relative ratio of these oligonucleotides bound to the average nanoparticles is controlled by the concentration ratio of the two oligonucleotides in the mixture. Hybridization conditions are well known in the art and can be readily optimized for the particular system used (see above).
[0180]
One example of this nucleic acid detection method is shown in FIG. Hybridization of the first type of nanoparticle-oligonucleotide conjugate with the liposome can cause a detectable change. For gold nanoparticles, pink / red can be observed, and if the nanoparticles are sufficiently close together, purple / blue can be observed. Hybridization of the second type of nanoparticle-oligonucleotide conjugate with the first type of nanoparticle-oligonucleotide conjugate results in a detectable change. For gold nanoparticles, purple / blue will be observed. Any of these discolorations can be observed with the naked eye.
[0181]
In still other embodiments utilizing a substrate, an "aggregate probe" may be used. Aggregate probes may be prepared by hybridizing two nanoparticles to which complementary oligonucleotides (a and a ') are attached to form a core (shown in FIG. 28A). Since a plurality of oligonucleotides are attached to each type of nanoparticles, each type of nanoparticle can hybridize to a plurality of other types of nanoparticles. Thus, the core is an aggregate containing a large number of nanoparticles of both types. The core is then capped with a third type of nanoparticles having at least two types of oligonucleotides attached thereto. The first type of oligonucleotide has a sequence b complementary to a partial sequence b 'of the nucleic acid to be detected. The second type of oligonucleotide has the sequence a or a 'such that the third type of nanoparticles hybridizes to the nanoparticles outside the core. Aggregate probes can also be prepared utilizing two types of nanoparticles (see FIG. 28B). At least two types of oligonucleotides are attached to each type of nanoparticles. The first type of oligonucleotide present on each of the two types of nanoparticles has a sequence b that is complementary to some sequence b 'of the nucleic acid to be detected. The second type of oligonucleotide on the second type of nanoparticles is such that the second type of oligonucleotide on the first type of nanoparticles hybridizes to each other to form an aggregate probe. (See FIG. 28B). Since each type of nanoparticle has a plurality of oligonucleotides attached to it, each type of nanoparticle hybridizes with a plurality of other types of nanoparticle and contains a large number of both types of nanoparticle. Will form an aggregate.
[0182]
Aggregate probes can also be used to detect nucleic acids in any of the assay formats described above performed on a substrate, thereby attaching a separate nanoparticle layer to obtain or enhance a detectable change. There is no need to deposit. To further enhance the detectable change, two types of aggregate probes can be used to deposit the aggregate probe layer, with the first type of aggregate probe containing the other type of aggregate probe. An oligonucleotide complementary to the above oligonucleotide is attached. In particular, when the aggregate probes are prepared as shown in FIG. 28B, the aggregate probes can hybridize to one another to form a multilayer. Some possible assay formats utilizing aggregate probes are shown in FIGS. 28C-D. For example, one type of oligonucleotide containing sequence c is attached to a substrate (see FIG. 28C). Sequence c is complementary to part sequence c 'of the nucleic acid to be detected. After the target nucleic acid is added and hybridized with the oligonucleotide attached to the substrate, an aggregate probe is added and hybridized to the target nucleic acid portion having sequence b ', resulting in a detectable change. Alternatively, first, the target nucleic acid may be hybridized with the aggregate probe in the solution, and then hybridized with the oligonucleotide on the substrate, or the target nucleic acid may be simultaneously hybridized with the aggregate probe and the oligonucleotide on the substrate. May be soy. In another embodiment, the target nucleic acid is reacted with an aggregate probe and another type of nanoparticle in solution (see FIG. 28D). Some of the oligonucleotides attached to this additional type of nanoparticles have the sequence c such that they hybridize to the sequence c 'of the target nucleic acid, and Some of the oligonucleotides that are included include sequence d so that they can hybridize to oligonucleotides having sequence d 'that are attached to a substrate.
[0183]
The core itself can be used as a probe for nucleic acid detection. One possible assay format is shown in FIG. 28E. As shown in this figure, one type of oligonucleotide containing sequence b is attached to a substrate. Sequence b is complementary to some sequence b 'of the nucleic acid to be detected. The target nucleic acid is brought into contact with the substrate and hybridized with the oligonucleotide attached to the substrate. Then another type of nanoparticles is added. Some of the oligonucleotides attached to this additional type of nanoparticles have a sequence c complementary to the sequence c 'of the target nucleic acid such that the nanoparticles hybridize to the target nucleic acid bound to the substrate. There is. Also, some oligonucleotides attached to additional types of nanoparticles include sequences a or a 'that are complementary to sequences a and a' on the core probe. A core probe is added and hybridized with the oligonucleotide on the nanoparticle. Since each core probe has sequences a and a 'attached to the core-containing nanoparticles, the core probes hybridize to each other to form a multi-layer attached to the substrate and are detectable. Significant changes can be greatly enhanced. In alternative embodiments, the target nucleic acid may be contacted with an additional type of nanoparticles in solution prior to contacting the substrate, or the target nucleic acid, nanoparticles and substrate may all be contacted simultaneously. In yet another embodiment, an additional type of nanoparticles can be replaced with a linking oligonucleotide that includes both sequence c and sequence a or a '.
[0184]
With a substrate, multiple initial types of nanoparticle-oligonucleotide conjugates or oligonucleotides can be used to detect multiple portions in a single target nucleic acid, to detect many different nucleic acids, or both. Can be attached to the substrate in an array. For example, one substrate may be provided with rows of spots containing different types of oligonucleotides or oligonucleotide-nanoparticle conjugates, each spot designed to bind to a portion of the target nucleic acid. A sample containing one or more nucleic acids is added to each spot, and using the appropriate oligonucleotide-nanoparticle conjugates, oligonucleotide-liposome conjugates, aggregate probes, core probes and binding oligonucleotides, Perform the rest of the assay in one.
[0185]
Finally, the use of a substrate can produce a detectable change or enhance a change detectable by silver staining. Silver staining can be used with any type of nanoparticles that catalyze the reduction of silver. Preferred are nanoparticles made of noble metals (eg, gold and silver). Bassell et al. Cell Biol. 126, 863-876 (1994); Braun-Howland et al., Biotechniques, 13, 928-931 (1992). If the nanoparticles used to detect the nucleic acid do not catalyze the reduction of silver, a complex of silver ions and the nucleic acid can be formed to catalyze the reduction. See Braun et al., Nature, 391, 775 (1998). Silver dyes that can react with phosphate groups on nucleic acids are known.
[0186]
Silver staining can be used to generate or enhance detectable changes in any of the assays performed on the substrate, including those described above. In particular, silver staining has been found to significantly enhance the sensitivity of assays using a single type of nanoparticles, such as those shown in FIG. 25A, so that the nanoparticle layer, aggregate probes and Often there is no need to use a core probe.
[0187]
In a nucleic acid detection assay performed on a substrate, the detectable change can be observed using an optical scanner. Suitable scanners include those used to scan documents to a computer that can operate in a reflective mode (eg, a flatbed scanner), other devices that can perform this function or utilize the same type of optics, Any type of gray scale susceptibility measurement device, and a standard scanner modified to scan a substrate in accordance with the present invention (eg, a flatbed scanner modified to include a container for the substrate) (to date, It has been found impossible to use a scanner operating in transmission mode). The resolution of the scanner must be large enough to make the reaction area on the substrate larger than one pixel of the scanner. The scanner can be used with any substrate, except that the detectable changes caused by the assay must be observed against the substrate in the background (e.g., gray spots such as those generated by silver staining may have a white spot on them). It can be observed with a background, but not with a gray background). The scanner may be a black and white scanner, but is preferably a color scanner. The scanner is most preferably a standard color scanner of the type used to scan documents into a computer. Such scanners are inexpensive and can be easily purchased. For example, Epson Expression 636 (600 × 600 dpi), UMAX Astra 1200 (300 × 300 dpi), or Microtec 1600 (1600 × 1600 dpi) may be used. The scanner connects to a computer loaded with software for processing images obtained by scanning the substrate. The software may be standard software, such as Adobe Photoshop 5.1 and Corel Photopaint 8.0, that are readily available for purchase. The use of grayscale measurement calculation software provides a means of quantifying assay results. The software can provide the number of colors in a color spot and generate a scanned image (eg, a printout) that can be observed to quantify the presence of nucleic acids, the amount of nucleic acids, or both. Can be. In addition, the sensitivity of the assay including the assay described in Example 5 was improved by reducing the color representing a negative result (red in Example 5) from the color representing a positive result (blue in Example 5). can do. The computer may be a standard personal computer that can be easily purchased. Thus, using a standard scanner coupled to a standard computer loaded with standard software provides a convenient, easy and inexpensive means of detecting and quantifying nucleic acids when performing assays on a substrate. Scanned images and calculated results can be stored on a computer to maintain a record of the results for subsequent reference and use. Of course, if desired, more precise equipment and software can be used.
[0188]
Nanoparticle-oligonucleotide conjugates that can be used in any nucleic acid assay are shown in FIGS. 17D-E. The "universal probe" has an oligonucleotide having a single sequence attached thereto. These oligonucleotides are capable of hybridizing to a binding oligonucleotide having a sequence comprising at least two parts. The first portion is complementary to at least some sequence on the nanoparticle. The second portion is complementary to some sequence of the nucleic acid to be detected. A plurality of binding oligonucleotides having the same first portion and a different second portion can be used, in which case the "universal probe" will hybridize with the binding oligonucleotide and then multiplex or different portions of the nucleic acid to be detected. Can bind to a nucleic acid target.
[0189]
In many other embodiments of the invention, the detectable change is a molecule that produces a detectable change when the oligonucleotide, the nanoparticle, or both, hybridizes the oligonucleotide on the nanoparticle to the target nucleic acid. (Eg, fluorescent molecules and dyes). For example, fluorescent molecules can be attached to the ends of oligonucleotides attached to metal and semiconductor nanoparticles that are not attached to the nanoparticles. Metal and semiconductor nanoparticles are known fluorescent quenchers, and the magnitude of the quenching effect depends on the spacing between the nanoparticles and the fluorescent molecules. Oligonucleotides attached to the nanoparticles, when unhybridized, interact with the nanoparticles, resulting in significant quenching. When the fluorescent molecule hybridizes to the target nucleic acid, it becomes spaced apart from the nanoparticle, thereby reducing fluorescence quenching. See FIG. 20A. As the length of the oligonucleotide increases, the change in fluorescence should increase, at least until the fluorescent group moves away from the nanoparticle surface at least so far that the increase in change is no longer observable. Useful lengths of oligonucleotides can be determined empirically. Metal and semiconductor nanoparticles to which fluorescently labeled oligonucleotides are attached may be used in any of the assay formats described above, including those performed in solution or on a substrate.
[0190]
Methods of labeling oligonucleotides with fluorescent molecules and measuring fluorescence are well known in the art. Suitable fluorescent molecules are also well known in the art and include fluorescein, rhodamine and Texas Red. The oligonucleotide will be attached to the nanoparticle as described above.
[0191]
In yet another embodiment, two types of fluorescently labeled oligonucleotides attached to two different particles may be used. Suitable particles include polymer particles (such as polystyrene particles, polyvinyl particles, acrylate and methacrylate particles), glass particles, latex particles, sepharose beads and other similar particles well known in the art. Methods for attaching oligonucleotides to such particles are well known in the art. Chrisey et al., Nucleic Acids Research, Vol. 24, p. 3031-3039 (1996) (glass) and Charreyre et al., Langmuir, Vol. 13, pp. 3103-3110 (1997), Fahy et al., Nucleic Acids Research, Vol. Vol., Pp. 1819-1826 (1993); Elaisari et al. Colloid Interface Sci. Vol. 202, pp. 251-260 (1998); Kolarova et al., Biotechniques, Vol. 20, pp. 196-198 (1996); and Wolf et al., Nucleic Acids Research, Vol. 15, pp. 2911-2926 (1987). ) (Polymer / latex). In particular, a variety of functional groups are available on these particles and can be incorporated into such particles. Functional groups include carboxylic acids, aldehydes, amino groups, cyano groups, ethylene groups, hydroxyl groups, mercapto groups, and the like. Nanoparticles including metal and semiconductor nanoparticles may be used.
[0192]
The two fluorophores are referred to as d and a as donor and acceptor. Various fluorescent molecules useful for such combinations are well known in the art and are available, for example, from Molecular Probes. An attractive combination is fluorescein as a donor and Texas Red as an acceptor. The two types of nanoparticle-oligonucleotide conjugates to which d and a are attached are mixed with the target nucleic acid and the fluorescence is measured with a fluorimeter. The mixture is excited with light of a wavelength that excites d and the mixture is monitored for fluorescence from a. When hybridized, d and a will be close (see FIG. 20B). In the case of non-metallic, non-semiconductor particles, hybridization will be indicated by a shift in fluorescence from that of d to that of a, or the appearance of fluorescence of d in addition to that of a. In the absence of hybridization, the fluorophores are too far apart to cause significant energy transfer and only fluorescence at d will be observed. In the case of metal and semiconductor nanoparticles, the lack of hybridization will be indicated by the lack of fluorescence due to quenching due to d or a (see above). Hybridization is indicated by an increase in fluorescence attributable to a.
[0193]
The above-described particles and nanoparticles having oligonucleotides labeled with acceptor and donor fluorescent molecules attached thereto can be used in the above-described assay formats, including those performed in solution and on substrates. In the case of a solution format, the oligonucleotide sequences are preferably selected to bind to the target nucleic acid as shown in FIGS. 15A-G. In the format shown in FIGS. 13A-B and FIG. 18, binding oligonucleotides can be used to bring the acceptor and donor fluorescent molecules on two nanoparticles into close proximity. Also, in the format shown in FIG. 13A, the oligonucleotide attached to the substrate can be labeled with d. In addition, other labels than fluorescent molecules may be used, for example, chemiluminescent molecules that upon hybridization produce a detectable signal or cause a change in the detectable signal.
[0194]
Another embodiment of the detection method of the present invention is a very sensitive system that utilizes fluorescence and color change detection (shown in FIG. 21). This system uses latex microspheres having oligonucleotides labeled with fluorescent molecules attached thereto, and gold nanoparticles having oligonucleotides attached thereto. Oligonucleotide-nanoparticle conjugates can be prepared as described above. Methods for attaching oligonucleotides to latex microspheres (e.g., Charreyre et al., Langmuir, Vol. 13, pp. 3103-3110 (1997); Elaisari et al., J. Clliod Interface Sci., Vol. 202, pp. 251-260 ( 1998)) is well known, as is the method of labeling oligonucleotides with fluorescent molecules (see above). The oligonucleotide on the latex microsphere and the oligonucleotide on the gold nanoparticle have a sequence that can hybridize to different portions of the sequence of the target nucleic acid, but cannot hybridize to each other. When the two probes are contacted with a target nucleic acid containing a sequence complementary to the sequence of the oligonucleotides on the latex microspheres and gold nanoparticles, a network is formed (see FIG. 21). Due to the quenching properties of the gold nanoparticles, the fluorescence of the oligonucleotides attached to the latex microspheres is quenched between parts of this network. In fact, one gold nanoparticle can quench many fluorophore molecules, because gold nanoparticles have a very large absorption coefficient. Thus, it is possible to monitor the fluorescence of the solution containing the nucleic acid and the two particles and detect the result, with a decrease or elimination of the fluorescence indicating a positive result. However, the assay results are preferably detected by arranging droplets of the solution side by side on the microporous material (see FIG. 21). The microporous material must be transparent or colored (eg, white) so that the pink / red color of the gold nanoparticles can be detected. Also, the microporous material is large enough to allow the gold nanoparticles to pass through the pores when the microporous material is washed and sufficient to retain the latex microspheres on the surface of the microporous material. It must also have a small pore size. Therefore, when using such microporous materials, the size (diameter) of the latex microspheres must be larger than the size (diameter) of the gold nanoparticles. Also, the microporous material must be inert to biological media. Many suitable microporous materials are known in the art, and such materials are commercially available from various filters and membranes, such as modified polyvinylidene fluoride (Millipore Corp.). Durapore^TMAcetatePlus commercially available from Micron Separations Inc. (PVDF such as membrane filters)^TMPure cellulose acetate (such as a membrane filter). Such a microporous material retains a network consisting of the target nucleic acid and the two probes, with a positive result (the presence of the target nucleic acid) being red / pink (attributable to the presence of gold nanoparticles), Demonstrated by the lack of fluorescence (attributable to quenching of the fluorescence by the gold nanoparticles) (see FIG. 21). Negative results (absence of target nucleic acid) indicate that when the microporous material is washed, the gold nanoparticles pass through the pores of the microporous material (thus, no quenching of fluorescence occurs) and white latex microspheres It is evidenced by white and fluorescence to be captured thereon (see FIG. 21). Further, in the case of a positive result, fluorescence and discoloration can be observed as a function of temperature. For example, as the temperature is increased, fluorescence will be observed upon reaching the dehybridization temperature. Thus, looking at color or fluorescence as a function of temperature can provide information on the degree of complementarity between the oligonucleotide probe and the target nucleic acid. As described above, this detection method shows high sensitivity. Small amounts of a 3 fmol (femtomole) single-stranded target nucleic acid having a length of 24 bases and a 20 fmol double-stranded target nucleic acid having a length of 24 bases were detected with the naked eye. This method is also very simple to use. Fluorescence can be generated by simply illuminating the solution or microporous material with a UV lamp, and the fluorescent and colorimetric signals can be monitored visually. Alternatively, to obtain more quantitative results, the fluorimeter may use a front-face mode to measure the fluorescence of the solution with a short path length.
[0195]
The above embodiments have been described with particular reference to latex microspheres and gold nanoparticles. Instead of these particles, any other microspheres or nanoparticles having the other properties mentioned above and to which oligonucleotides can be attached may be used. Many suitable particles and nanoparticles are described above, along with methods for attaching oligonucleotides to them. In addition, microspheres and nanoparticles with other measurable properties may be used. For example, polymer modified particles and nanoparticles that can modify the polymer to have any desired properties, such as fluorescence, color, or electrochemical activity, may be used. Watson et al. Am. Chem. Soc. 121, 462-463 (1999) (polymer-modified gold nanoparticles). Also, magnetic particles, polymer-coated magnetic particles, and semiconductor particles can be used. See Chan et al., Science, 281, 2016 (1998); Bruchez et al., Science, 281, 2013 (1998); Kolarova et al., Biotechniques, 20, 196-198 (1996). .
[0196]
In yet another embodiment, two probes comprising metal or semiconductor nanoparticles to which oligonucleotides labeled with fluorescent molecules are attached (shown in FIG. 22). Oligonucleotide-nanoparticle conjugates can be prepared and labeled with fluorescent molecules as described above. The oligonucleotides on the two types of oligonucleotide-nanoparticle conjugates have sequences that can hybridize to different portions of the sequence of the target nucleic acid but cannot hybridize to each other. When a target nucleic acid containing a sequence complementary to the sequence of the oligonucleotide on the nanoparticle is brought into contact with the two probes, a network structure is formed (see FIG. 22). Due to the quenching properties of metal or semiconductor nanoparticles, the fluorescence of oligonucleotides attached to the nanoparticles is quenched between parts of this network. Thus, it is possible to monitor the fluorescence of the solution containing the nucleic acid and the two probes and detect the result, with a decrease or elimination of the fluorescence indicating a positive result. However, the results of the assay are preferably detected by lining up droplets of the solution on the microporous material (see FIG. 22). The microporous material must have a pore size that, when washed, is large enough to allow the nanoparticles to pass through the pores and small enough to retain the network on its surface ( See FIG. 22). Many suitable microporous materials are known in the art, and such materials include those described above. Such a microporous material retains a network consisting of the target nucleic acid and the two probes, and a positive result (the presence of the target nucleic acid) results in a fluorescence (due to the quenching of the fluorescence by the metal or semiconductor nanoparticles). Proof by lack (see FIG. 22). A negative result (absence of target nucleic acid) is evidenced by fluorescence because the nanoparticles pass through the pores of the microporous material when washing the microporous material (thus, no quenching of fluorescence occurs). (See FIG. 22). Background fluorescence is low because unbound probe is washed away from the detection area. Further, in the case of a positive result, a change in fluorescence can be observed as a function of temperature. For example, as the temperature is increased, fluorescence will be observed upon reaching the dehybridization temperature. Thus, looking at the fluorescence as a function of temperature can provide information about the degree of complementarity between the oligonucleotide probe and the target nucleic acid. Fluorescence can be generated by simply illuminating the solution or microporous material with a UV lamp, and the fluorescence signal can be monitored visually. Alternatively, the fluorescence of the solution may be measured with a short path length using a fluorimeter in frontal mode for more quantitative results.
[0197]
In yet another embodiment, a "satellite probe" is used (see FIG. 24). Satellite probes include central particles having one or more physical properties (eg, dark color, fluorescence quenching, magnetism) that can be used for detection in nucleic acid assays. Suitable particles include nanoparticles and other particles described above. Oligonucleotides (all having the same sequence) are attached to the particles (see FIG. 24). Methods for attaching oligonucleotides to the particles are described above. The oligonucleotide includes at least a first portion and a second portion, both of which are complementary to portions of the sequence of the target nucleic acid (see FIG. 24). Satellite probes further include a probe oligonucleotide. Each probe oligonucleotide has at least a first portion and a second portion (see FIG. 24). The sequence of the first part of the probe oligonucleotide is complementary to the sequence of the first part of the oligonucleotide immobilized on the central particle (see FIG. 24). As a result, when the central particle is brought into contact with the probe oligonucleotide, the oligonucleotide on the central particle hybridizes with the probe oligonucleotide to form a satellite probe (see FIG. 24). Both the first and second portions of the probe oligonucleotide are complementary to portions of the sequence of the target nucleic acid (see FIG. 24). Each probe oligonucleotide is labeled with a reporter molecule, as described in detail below (see FIG. 24). The amount of hybridization overlap between probe oligonucleotide and target (the length of the hybridized portion) is the same as the hybridization overlap between the probe oligonucleotide and the oligonucleotide attached to the particle. Or larger (see FIG. 24). Thus, temperature cycling leading to dehybridization and rehybridization facilitates the transfer of the probe oligonucleotide from the central particle to the target. The particles are then separated from the probe oligonucleotide hybridized to the target, and the reporter molecule is detected.
[0198]
Satellite probes can be used for various detection methods. For example, if the central particle has a magnetic core and is coated with a material capable of quenching the fluorescence of the fluorophore attached to the surrounding probe oligonucleotide, the system will provide in situ fluorescence for nucleic acids. Can be used for detection planning. Functionalized polymer-coated magnetic particles (Fe₃O₄) Is Dynal (Dynabeads)^TM) And Bangs Laboratories (Estapor)^TM) Are available from a number of manufacturers, including silica-coated magnetic Fe₃O₄The nanoparticles are modified using well-developed silica surface chemistry (Chrisey et al., Nucleic Acids Research, Vol. 24, pp. 3031-3039 (1996)) (Liu et al., Chem. Mater., No. 10). Vol., 3936-3940 (1998), which can also be used as a magnetic probe, and a dye molecule, 4-((4- (dimethylamino) phenyl) -azo) benzoic acid (DABCYL), is added to the oligonucleotide. It has been demonstrated to be a potent fluorescence quencher for a variety of fluorophores (Tyagi et al., Nature Biotech., 16, 49-53 (1998)). Imidyl ester (molecular probe) reacts with primary alkylamino group Thus, any magnetic particles or magnetic particles polymer-coated with primary alkylamino groups can be modified with these quencher molecules in addition to both oligonucleotides. Alternatively, the DABCYL quencher may be attached directly to the surface bound oligonucleotide, instead of an alkylamino modified surface, and a satellite probe containing the probe oligonucleotide is contacted with the target. The temperature is circulated to cause the probe oligonucleotide to move from the central particle to the target by applying a magnetic field, removing the particles from solution and leaving the probe oligonucleotide fluorescence in the solution hybridized to the target. The detection is reached by measuring It is.
[0199]
This method uses a probe oligonucleotide labeled with a dye that has different optical properties than the dye on the magnetic nanoparticles, or that perturbs the optical properties of the dye on the magnetic nanoparticles, with the magnetic particles having a dye coating. It can also be applied to colorimetric assays. If both the particles and the probe oligonucleotide are in solution, the solution will exhibit one color from the combination of the two dyes. However, cycling the temperature in the presence of the target nucleic acid will cause the probe oligonucleotide to move from the satellite probe to the target. Once this occurs, the application of a magnetic field removes the dye-coated magnetic particles from solution, leaving a single dye-labeled probe oligonucleotide hybridized to the target. This system can be followed with a colorimeter or with the naked eye, depending on the target level or color strength.
[0200]
In addition, this method can be applied to an electrochemical assay by using a probe oligonucleotide to which a redox active molecule is attached together with an oligonucleotide-magnetic particle conjugate. Any modifiable redox active species can be used, such as a well-studied redox active ferrocene derivative. Ferrocene-derivatized phosphoramidites can be attached directly to oligonucleotides using standard phosphoramidite chemistry. Mucic et al., Chem. Commun. Vol. 555 (1996); Edckstein, edited by Oligonucleotides and Analogues, 1st edition, Oxford University, New York, NY (1991). Ferrocenyl phosphoramidites are prepared from 6-bromohexyl ferrocene in a two-step synthesis. In a typical preparation, 6-bromohexylferrocene is stirred in an aqueous HMPA solution at 120 ° C. for 6 hours to produce 6-hydroxyhexylferrocene. After purification, 6-hydroxyhexyl ferrocene is added to a THF solution of N, N-diisopropylethylamine and β-cyanoethyl-N, N-diisopropylchlorophosphoramide to produce ferrocenyl phosphoramidite. Oligonucleotide-modified polymer-coated gold nanoparticles in which the polymer contains electrochemically active ferrocene molecules may be utilized. Watson et al. Am. Chem. Soc. 121, 462-463 (1999). The polymer may incorporate a copolymer of amino-reactive sites (eg, anhydride) for reaction with an amino-modified oligonucleotide. Moller et al., Bioconjugate Chem. 6, Vol. 174-178 (1995). Cycling the temperature in the presence of the target will cause the redox active probe oligonucleotide to move from the satellite probe to the target. Once this occurs, application of a magnetic field will remove the magnetic particles from solution, leaving the redox-active probe oligonucleotide hybridized to the target nucleic acid. The amount of target can then be quantified by cyclic voltammetry or any electrochemical technique that can examine Redox active molecules.
[0201]
In still another embodiment of the present invention, the nucleic acid is detected by contacting the nucleic acid with a substrate to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the sequence of the first portion of the nucleic acid. The oligonucleotide is disposed between a pair of electrodes disposed on the substrate. The substrate must be made of a non-conductive material (eg, glass, quartz, polymer, plastic). The electrodes may be made of any standard material (eg, a metal such as gold, platinum, tin oxide, etc.). The electrodes can be made using conventional microfabrication techniques. See, for example, Induction To Microlithography (LF Thompson et al., Eds., ACS, Washington, DC, 1983). On the substrate, multiple electrode pairs may be arranged in an array to allow detection of multiple portions of a single nucleic acid, multiple different nucleic acids, or both. Electrode arrays can be purchased (e.g., from Abbtech Scientific, Inc., Richmond, Virginia) or made using conventional microfabrication techniques. You can also. See, for example, Induction To Microlithography (LF Thompson et al., Eds., ACS, Washington, DC, 1983). Photomasks suitable for making arrays can be purchased (eg, from Phototronics, Inc., Milpitas, Calif., Photronics, Milpitas, Calif.). Each array electrode pair has some type of oligonucleotide attached to the substrate between the two electrodes. The contacting step is performed under conditions effective to hybridize the oligonucleotide and nucleic acid on the substrate. The nucleic acid bound to the substrate is then contacted with certain types of nanoparticles. The nanoparticles must be conductive. Such nanoparticles include nanoparticles made of metal, such as gold nanoparticles, and nanoparticles made of semiconductor materials. The nanoparticle will have one or more types of oligonucleotides attached, where at least one type of oligonucleotide has a sequence that is complementary to the sequence of the second portion of the nucleic acid. The contacting step is performed under conditions effective to hybridize the oligonucleotide on the nanoparticle with the nucleic acid. If nucleic acid is present, the circuit between the electrodes should close because nanoparticles attach between the electrodes on the substrate and a change in conductivity is detected. When one type of nanoparticles binds, circuit closure does not occur, and the situation may be to use a narrower interelectrode spacing, use larger nanoparticles, or use another material to close the circuit (but (Only when the nanoparticles are bound between the electrodes of the substrate). For example, when using gold nanoparticles, the circuit is closed by contacting the substrate with a silver dye (as described above) and depositing silver between the electrodes, producing a detectable change in conductivity. Another method of closing the circuit when the addition of one type of nanoparticles is not sufficient is to complement the first type of nanoparticles attached to the substrate to oligonucleotides on the first type of nanoparticles. This is a method of contacting a second type of nanoparticle to which an oligonucleotide having a specific sequence is attached. The contacting step is performed under conditions effective to hybridize the oligonucleotide on the second type of nanoparticles with the oligonucleotide on the first type of nanoparticles. If necessary or desired, additional layers of nanoparticles can be deposited by alternately adding nanoparticles of the first and second types until a sufficient number of nanoparticles have been deposited on the substrate to close the circuit. Another alternative for depositing individual nanoparticles is the use of aggregate probes (see above).
[0202]
The present invention further provides kits for detecting nucleic acids. The kit, in one embodiment, includes at least one container that holds at least two types of nanoparticles to which the oligonucleotide is attached. The oligonucleotide on the first type of nanoparticles has a sequence that is complementary to the sequence of the first portion of the nucleic acid. The oligonucleotide on the second type of nanoparticle has a sequence that is complementary to the sequence of the second portion of the nucleic acid. The container may further include a filler oligonucleotide having a sequence complementary to a third portion of the nucleic acid, wherein the third portion is located between the first and second portions. The filler oligonucleotide may be in a separate container.
[0203]
In a second embodiment, the kit includes at least two containers. The first container holds nanoparticles to which oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached. The second container holds nanoparticles to which oligonucleotides having a sequence complementary to the sequence of the second portion of the nucleic acid are attached. The kit may further include a third container containing a filler oligonucleotide having a sequence complementary to the third portion of the nucleic acid, wherein the third portion is disposed between the first and second portions. I have.
[0204]
In another alternative embodiment, the kit comprises the oligonucleotide and the nanoparticles in separate containers, wherein the oligonucleotide must be attached to the nanoparticles before performing the nucleic acid detection assay. Oligonucleotides and / or nanoparticles can be functionalized such that oligonucleotides can be attached to the nanoparticles. Alternatively, the oligonucleotides and / or nanoparticles may be provided in the kit without functional groups, in which case the oligonucleotides must be functionalized before performing the assay.
[0205]
In another embodiment, the kit includes at least one container. The container holds metal or semiconductor nanoparticles to which the oligonucleotide is attached. The oligonucleotide has a sequence complementary to a part of the nucleic acid, and a fluorescent molecule is attached to an end of the oligonucleotide that is not attached to the nanoparticles.
[0206]
In yet another embodiment, the kit comprises a substrate, to which the nanoparticles are attached. Oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached to the nanoparticles. The kit further includes a first container containing nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid. The oligonucleotides may have the same or different sequences, but each oligonucleotide has a sequence that is complementary to a portion of the nucleic acid. The kit further includes a second container containing a binding oligonucleotide having the selected sequence comprising at least two portions, wherein the first portion of the selected sequence comprises the oligonucleotides on the nanoparticles in the first container. Complementary to at least a portion of the sequence. The kit also includes a third container containing nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the binding oligonucleotide.
[0207]
In another embodiment, the kit includes a substrate to which an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid is attached. The kit further includes a first container containing nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid. The oligonucleotides can have the same or different sequences, but each oligonucleotide has a sequence that is complementary to a portion of the nucleic acid. The kit further includes a second container containing the nanoparticles attached to the oligonucleotide having a sequence complementary to at least a portion of the oligonucleotide attached to the nanoparticles in the first container. .
[0208]
In yet another embodiment, the kit may have the substrate, the oligonucleotide and the nanoparticles in separate containers. The substrate, oligonucleotide and nanoparticles must be properly attached to each other before performing a nucleic acid detection assay. Substrates, oligonucleotides and / or nanoparticles can be functionalized to facilitate this attachment. Alternatively, the substrate, oligonucleotides and / or nanoparticles may be provided without functional groups in the kit, but must be functionalized before performing the assay.
[0209]
In a further embodiment, the kit comprises a substrate to which an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid is attached. The kit also includes a first container containing a liposome to which an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid is attached, and a nanoparticle to which at least a first type of oligonucleotide is attached. A cholesteryl group is attached to the end of the first type oligonucleotide that is not attached to the nanoparticle so that the nanoparticle can be attached to the liposome by hydrophobic interaction. Have been. The kit further includes a third container containing the second type of nanoparticles to which the oligonucleotide is attached, wherein the oligonucleotide comprises the second type of oligonucleotide attached to the first type of nanoparticles. It has a sequence that is complementary to at least a portion of the sequence. The second type of oligonucleotide attached to the first type of nanoparticles has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticles.
[0210]
In another embodiment, the kit may include a substrate to which the nanoparticles are attached. Oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached to the nanoparticles. The kit further includes a first container containing the aggregate probe. The aggregated probe contains at least two types of nanoparticles to which oligonucleotides are attached. The nanoparticles of the aggregate probe bind to each other as a result of the hybridization of a portion of the oligonucleotide attached to each. An oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid is attached to at least one type of nanoparticle of the aggregate probe.
[0211]
In yet another embodiment, the kit can include a substrate to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the sequence of the first portion of the nucleic acid. The kit further includes a first container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles to which oligonucleotides are attached. The nanoparticles of the aggregate probe bind to each other as a result of the hybridization of a portion of the oligonucleotide attached to each. An oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid is attached to at least one type of nanoparticle of the aggregate probe.
[0212]
In an additional embodiment, the kit includes a substrate to which the oligonucleotide is attached, and a first container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles to which oligonucleotides are attached. The nanoparticles of the aggregate probe bind to each other as a result of the hybridization of a portion of the oligonucleotide attached to each. An oligonucleotide having a sequence complementary to the first portion of the nucleic acid sequence is attached to at least one type of nanoparticle of the aggregate probe. The kit further includes a second container containing the nanoparticles. At least two types of oligonucleotides are attached to the nanoparticles. The first type of oligonucleotide has a sequence that is complementary to the sequence of the second portion of the nucleic acid. The second type of oligonucleotide has a sequence complementary to at least a part of the sequence of the oligonucleotide attached to the substrate.
[0213]
In another embodiment, the kit includes a substrate to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the sequence of the first portion of the nucleic acid. The kit further includes a first container containing the liposome to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the sequence of the second portion of the nucleic acid. The kit further includes a second container containing an aggregate probe comprising at least two types of nanoparticles to which the oligonucleotide is attached. The nanoparticles of the aggregate probe bind to each other as a result of the hybridization of a portion of the oligonucleotide attached to each. At least one type of nanoparticle of the aggregate probe has an oligonucleotide having a hydrophobic group attached to the end that is not attached to the nanoparticle.
[0214]
In a further embodiment, the kit can include a first container containing the nanoparticles to which the oligonucleotide is attached. The kit further includes one or more additional containers, each container holding a binding oligonucleotide. Each binding oligonucleotide has a first portion having a sequence complementary to at least a portion of the sequence of the oligonucleotide on the nanoparticle, and a sequence complementary to a portion of the sequence of the nucleic acid to be detected. A second portion. The sequence of the second portion of the binding oligonucleotide may be different as long as each sequence is complementary to a portion of the sequence of the nucleic acid to be detected. In another embodiment, the kit includes a container containing one type of nanoparticle to which the oligonucleotide is attached and one or more types of binding oligonucleotide. Each type of binding oligonucleotide has a sequence that includes at least two parts. The first portion is complementary to the sequence of the oligonucleotide on the nanoparticle, such that the binding oligonucleotide hybridizes to the oligonucleotide on the nanoparticle in the container. The second part is complementary to some sequence of the nucleic acid.
[0215]
In another embodiment, the kit may include one or more containers containing the two types of particles. An oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid is attached to the first type of particle. The other end of the oligonucleotide not attached to the particle is labeled with an energy donor. An oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid is attached to the second type of particle. The end of the oligonucleotide not attached to the particle is labeled with an energy acceptor. The energy donor and acceptor may be fluorescent molecules.
[0216]
In a further embodiment, the kit comprises a first container containing a type of latex microsphere to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the sequence of the first portion of the nucleic acid and is labeled with a fluorescent molecule. The kit further includes a second container containing a type of gold nanoparticle to which the oligonucleotide is attached. These oligonucleotides have a sequence that is complementary to the sequence of the second portion of the nucleic acid.
[0217]
In another embodiment, the kit includes a first container containing a first type of metal or semiconductor nanoparticles to which the oligonucleotide is attached. The oligonucleotide has a sequence that is complementary to the first portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule. The kit further includes a second container containing the second type of metal or semiconductor nanoparticles to which the oligonucleotide is attached. These oligonucleotides have a sequence complementary to the sequence of the second portion of the nucleic acid and are labeled with a fluorescent molecule.
[0218]
In a further embodiment, the kit includes a container containing the satellite probe. Satellite probes include particles to which oligonucleotides are attached. The oligonucleotide has a first portion and a second portion, both of which have a sequence that is complementary to a portion of the sequence of the nucleic acid. The satellite probe further includes a probe oligonucleotide hybridized to the oligonucleotide attached to the nanoparticle. The probe oligonucleotide has a first portion and a second portion. The first portion has a sequence complementary to the sequence of the first portion of the oligonucleotide attached to the nanoparticle, and both portions have a sequence complementary to a portion of the sequence of the nucleic acid. have. A reporter molecule is attached to one end of the probe oligonucleotide.
[0219]
In another embodiment, the kit includes a container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles to which oligonucleotides are attached. The nanoparticles of the aggregate probe bind to each other as a result of the hybridization of a portion of the oligonucleotide attached to each. An oligonucleotide having a sequence complementary to a part of the sequence of the nucleic acid is attached to at least one kind of nanoparticles of the aggregate probe.
[0220]
In additional embodiments, the kit may include a container containing the aggregate probe. Aggregate probes include at least two types of nanoparticles to which oligonucleotides are attached. The nanoparticles of the aggregate probe bind to each other as a result of the hybridization of a portion of the oligonucleotide attached to each. At least one type of nanoparticle of the aggregate probe has an oligonucleotide having a hydrophobic group attached to the end that is not attached to the nanoparticle.
[0221]
In yet another embodiment, the invention includes a substrate having at least one pair of electrodes disposed thereon with oligonucleotides attached between the electrodes of the substrate. In a preferred embodiment, a plurality of pairs of electrodes are attached to the substrate in an array to enable detection of multiple portions of a single nucleic acid, detection of multiple different nucleic acids, or both.
[0222]
The kit may further include other reagents and items useful for detecting nucleic acids. Reagents can include PCR reagents, silver staining reagents, hybridization reagents, buffers and the like. Other items that may be provided as part of the kit include solid surfaces such as TLC silica plates, microporous materials, syringes, pipettes, cuvettes, containers, and (hybridization and A thermocycler (to control the dehybridization temperature). The kit may include reagents for functionalizing the nucleotides or nanoparticles.
[0223]
Precipitation of aggregated nanoparticles provides a means of separating selected nucleic acids from other nucleic acids. This separation can be used as a step in nucleic acid purification. Hybridization conditions are as described above for nucleic acid detection. If the temperature at which the oligonucleotide on the nanoparticle binds to the nucleic acid is lower than the Tm (the temperature at which half of the oligonucleotide binds to its complementary strand), sufficient time is required to precipitate the aggregate. The hybridization temperature (as measured, for example, by Tm) depends on the type of salt (NaCl or MgCl₂) And its concentration. The composition and concentration of the salt is selected to promote the hybridization of the oligonucleotide and the nucleic acid on the nanoparticles with the nucleic acid at a convenient operating temperature without inducing aggregation of the colloid in the absence of the nucleic acid.
[0224]
The present invention further provides a nanofabrication method. The method includes providing at least one linking oligonucleotide having a selected sequence. The linking oligonucleotide used for nanofabrication may contain any desired sequence and may be single-stranded or double-stranded. Linking oligonucleotides can further include chemical modifications at the base, sugar, or backbone portion. The sequence selected for the linking oligonucleotides, and their length and number of chains, will contribute to the stiffness or flexibility of the resulting nanomaterial or nanostructure, or portions of the nanomaterial or nanostructure. In addition to a single type of linking oligonucleotide, it is also conceivable to use two or more different types of linking oligonucleotides. The number of different linking oligonucleotides used and their length will contribute to the shape, pore size and other structural features of the resulting nanomaterials and nanostructures.
[0225]
The sequence of the linking oligonucleotide will have a first portion and a second portion for attachment to the oligonucleotide on the nanoparticle. The first, second or more binding moieties of the linking oligonucleotide can have the same or different sequences.
[0226]
If the binding moieties of the linking oligonucleotides all have the same sequence, only one type of nanoparticle to which the oligonucleotide having the complementary sequence is attached can be used to form the nanomaterial or nanostructure. Good. If two or more binding moieties of a linking oligonucleotide have different sequences, two or more nanoparticle-oligonucleotide conjugates must be used. For example, see FIG. The oligonucleotide on each nanoparticle will have a sequence complementary to one of the two or more binding portions of the sequence of the connecting oligonucleotide. The number, arrangement, and length, if any, of the binding moieties will contribute to the structural properties and properties of the resulting nanomaterials and nanostructures. If the linking oligonucleotide comprises more than one moiety, the sequence of the binding moiety must be selected so that it is not complementary to each other to avoid binding one part of the binding nucleotide to another. is there.
[0227]
Linking the oligonucleotide and the nanoparticle-oligonucleotide conjugate to the oligonucleotide attached to the nanoparticle so that the desired nanomaterial or nanostructure in which the nanoparticle is grouped via the oligonucleotide connector is formed Contact is made under conditions effective to hybridize with the oligonucleotide. These hybridization conditions are well known in the art and can be optimized for a particular nanofabrication scheme (see above). Stringent hybridization conditions are preferred.
[0228]
Further, the present invention provides another nanofabrication method. The method includes providing at least two types of nanoparticle-oligonucleotide conjugates. The oligonucleotide on the first type of nanoparticles has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticles. The oligonucleotide on the second type of nanoparticle has a sequence complementary to the sequence of the oligonucleotide on the first type of nanoparticle. The nanoparticle-oligonucleotide conjugate is effective to hybridize the oligonucleotides on the nanoparticles to one another such that the desired nanomaterial or nanostructure in which the nanoparticles are grouped via the oligonucleotide connector is formed. Contact under conditions. Again, these hybridization conditions are well known in the art and can be optimized for a particular nanofabrication scheme.
[0229]
In both nanofabrication methods of the present invention, the use of nanoparticles having one or more different types of oligonucleotides attached is envisioned. The number of different oligonucleotides attached to the nanoparticles and the length and sequence of one or more oligonucleotides will contribute to the stiffness and structural characteristics of the resulting nanomaterials and nanostructures.
[0230]
Also, the size, shape, and chemical composition of the nanoparticles will contribute to the properties of the resulting nanomaterials and nanostructures. These properties include optical, optoelectronic, electrochemical, and electronic properties, stability in various solutions, changes in pore and channel size, and the ability to separate bioactive molecules while acting as a filter. And so on. In addition to the use of a mixture of nanoparticles having different sizes, shapes and / or chemical compositions, the use of nanoparticles having a uniform size, shape and chemical composition is also envisioned.
[0231]
In any of the fabrication methods, the nanoparticles in the resulting nanomaterial or nanostructure are attached via an oligonucleotide connector. The sequence, length and number of oligonucleotide connectors and the number of different oligonucleotide connectors present will contribute to the stiffness and structural properties of the nanomaterial or nanostructure. Where the oligonucleotide connector is partially double-stranded, its stiffness may be enhanced by using filler oligonucleotides as described above in connection with nucleic acid detection methods. The stiffness of a fully double stranded oligonucleotide connector is enhanced using one or more reinforcing oligonucleotides having complementary sequences to bind to the fully double stranded oligonucleotide connector and form a connector for the triple stranded oligonucleotide. obtain. The use of quadruplex oligonucleotide connectors based on deoxyquanosine or deoxycytidine quadruplexes is also envisioned.
[0232]
Some of the various systems for organizing nanoparticles based on oligonucleotide hybridization are shown in the figures. In a simple system (FIG. 1), one set of nanoparticles has an oligonucleotide with a defined sequence and another set of oligonucleotides has an oligonucleotide with a sequence complementary to it. When the two sets of nanoparticle-oligonucleotides are mixed under hybridization conditions, the two types of nanoparticles are connected via a double-stranded oligonucleotide connector that acts as a spacer that places the nanoparticles at selected intervals. Join.
[0233]
An attractive system for spacing nanoparticles involves the addition of one free-ligating oligonucleotide as shown in FIG. The sequence of the linking oligonucleotide will have at least a first portion and a second portion for binding to the oligonucleotide on the nanoparticle. This system is basically the same as that used for the nucleic acid detection method, except that the length of the connecting oligonucleotide added is the total length of the oligonucleotide attached to the nanoparticles. May be chosen to be equal. The related system shown in FIG. 3 provides a convenient means of adjusting the spacing between nanoparticles as required without having to change the set of nanoparticle-oligonucleotide conjugates used.
[0234]
A more sophisticated scheme for creating defined spacing between nanoparticles is shown in FIG. In this case, a double-stranded DNA or RNA segment containing an overhanging end is used as the linking oligonucleotide. Hybridization of the single stranded overhang segment of the linking oligonucleotide with the oligonucleotide attached to the nanoparticles results in multiple double stranded oligonucleotide bridges between the nanoparticles.
[0235]
Stiffer nanomaterials and nanostructures or portions thereof can be prepared by using triple-stranded oligonucleotide connectors between the nanoparticles. In generating triple strands, pyrimidine: purine: pyrimidine motifs (Moser, HE and Dervan, PB, Science, 238, 645-650 (1987)) or purine: purine: Any of the pyrimidine motifs (Pilch, DS, et al., Biochemistry, Vol. 30, pp. 6081-6087 (1991)) can be used. One example of nanoparticle organization by preparation of a triple-stranded connector using a pyrimidine: purine: pyrimidine motif is shown in FIG. In the system shown in FIG. 10, one set of nanoparticles is attached to a defined strand containing pyrimidine nucleotides, and the other set is attached to a complementary oligonucleotide containing purine nucleotides. The oligonucleotide attachment is designed so that the nanoparticles are separated by double-stranded oligonucleotides formed during hybridization. The free pyrimidine oligonucleotide is then added to the system before, simultaneously with, or immediately after mixing the nanoparticles, with the orientation of the pyrimidine chains attached to the nanoparticles being opposite to the orientation of the pyrimidine chains. Because the third strand in this system is retained via Hoogsteen base pairs, the triple strand is relatively thermally unstable. Covalent cross-links across the width of the duplex are known to stabilize the triple-stranded complex (Sarunke, M., Wu, T., Letsinger, RL, J. Am. Chem. Soc. 114, 8768-8772 (1992); Letsinger, RL and Wu, t., J. Am. Chem. Soc., 117, 7323-7328 (1995); Prakash. , G. and Kool, J. Am. Chem. Soc., 114, 3523-3527 (1992)).
[0236]
In order to construct nanomaterials and nanostructures, it may be desirable to “lock” the assembly in place by covalent crosslinking after formation of the nanomaterial or nanostructure by hybridization of the oligonucleotide components. This can be achieved by incorporating into the oligonucleotide a functional group that triggers an irreversible reaction. One example of a functional group for this is a stilbene dicarboxylic acid group. Irradiation of ultraviolet light (340 nm) to two stilbenedicarboxamide groups aligned in the hybridized oligonucleotide proved to be easily achieved by cross-linking (Lewis, FD et al., J. Am. Chem. Soc., 117, 8785-8792 (1995)).
[0237]
Alternatively, the 5'-O-tosyl group of the oligonucleotide held at the 3'-position of the nanoparticle via the mercaptoalkyl group is replaced with the 3'-O-tosyl group of the oligonucleotide held at the nanoparticle via the mercaptoalkyl group. A method of substitution with a terminal thiophosphoryl group may be used. In the presence of an oligonucleotide that hybridizes to both oligonucleotides, thereby bringing the thiophosphoryl group close to the tosyl group, the tosyl group is replaced with a thiophosphoryl group, and the oligonucleotide with both ends bound to two different nanoparticles. Generate. For this type of substitution reaction, see Herrlein et al. Am. Chem. Soc. 177, pp. 10151-10152 (1995). Due to the fact that thiophosphoryl oligonucleotides do not react with gold nanoparticles under the conditions used to attach mercaptoalkyl-oligonucleotides to gold nanoparticles, they bind to the nanoparticles via the mercapto group and are used for coupling reactions The preparation of gold nanoparticle-oligonucleotide conjugates containing possible terminal thiophosphoryl groups is now possible.
[0238]
Related coupling reactions for locking assembled nanoparticle systems in place are described by Gryaznov and Letsinger, J. et al. Am. Chem. Soc. 115, p. 3808, utilizing the substitution of a bromide at the terminal bromoacetylaminonucleoside with a terminal thiophosphoryl-oligonucleotide. The reaction proceeds in the same manner as the tosylate substitution described above, but at a faster rate. Nanoparticles bearing oligonucleotides terminated with thiophosphoryl groups are prepared as described above. In order to prepare a nanoparticle carrying an oligonucleotide having a bromoacetylamino group as a terminal group, an aminonucleoside (for example, 5'-amino-5'-deoxythymidine or 3'-amino- An oligonucleotide having 3'-deoxythymidine) as a terminal group and the other terminal having a mercaptoalkyl group as a terminal group is prepared. The oligonucleotide molecule is then immobilized on the nanoparticles via a mercapto group, and the nanoparticle-oligonucleotide conjugate is then reacted with a bromoacetyl acylating agent to convert it to an N-bromoacetylamino derivative.
[0239]
A fourth coupling scheme to lock the assembly in place utilizes the oxidation of thiophosphoryl-terminated oligonucleotide-carrying nanoparticles. Mild oxidants such as potassium triiodide, potassium ferricyanide (Gryaznov and Letsinger, Nucleic Acids Research, Vol. 21, p. 1403) or oxygen are preferred.
[0240]
In addition, the properties of the nanomaterials and nanostructures can be modified by incorporating organic and inorganic functional groups into the continuous oligonucleotide strand that are covalently attached to the oligonucleotide strand and held in place. A variety of backbone, base and sugar modifications are well known (see, for example, Uhlmann, E. and Peyman, A., Chemical Reviews, 90, 544-584 (1990)). Oligonucleotide chains can also be replaced with “peptide nucleic acid” chains (PNA) in which nucleotide bases are carried by the polypeptide backbone (Wittung, P. et al., Nature, 367, 561-563 (1994). Year)).
[0241]
As can be seen from the above, the nanofabrication method of the present invention is extremely versatile. Length, sequence and number of linking oligonucleotides, length, sequence and number of oligonucleotides attached to nanoparticles, size, shape and chemical composition of nanoparticles, number of different linking oligonucleotides and nanoparticles used By varying the type and number of strands of the oligonucleotide connector, nanomaterials and nanostructures with a wide variety of structures and properties can be prepared. These structures and properties can be further altered by crosslinking oligonucleotide connectors, functionalizing oligonucleotides, modifying the oligonucleotide backbone, bases or sugars, or using peptide nucleic acids.
[0242]
Nanomaterials and nanostructures that can be prepared by the nanofabrication method of the present invention include nanoscale mechanical devices, separation membranes, biofilters and biochips. The nanomaterials and nanostructures of the present invention can be used as chemical sensors, for computers, for drug delivery, for protein engineering, and as templates for biosynthesis / nanostructure fabrication / designated assemblies of other structures. Is assumed. For other possible uses, see generally, Seeman et al., New J. Am. Chem. 17: 739 (1993). Nanomaterials and nanostructures that can be prepared by the nanofabrication method of the present invention can further include electronic devices. There has been considerable debate as to whether nucleic acids can transfer electrons. As shown in Example 21 below, nanoparticles assembled via DNA conduct electricity (the DNA connector functions as a semiconductor).
[0243]
Finally, the present invention provides several unique methods for preparing nanoparticle-oligonucleotide conjugates. In the first such method, an oligonucleotide is attached to a charged nanoparticle to produce a stable nanoparticle-oligonucleotide conjugate. Charged nanoparticles include metallic nanoparticles such as gold nanoparticles.
[0244]
The method includes providing an oligonucleotide having a moiety containing a functional group capable of binding to a nanoparticle covalently bound. Such moieties and functional groups are as described above for the linkage (ie, chemisorption or covalent attachment) of the oligonucleotide to the nanoparticle. For example, oligonucleotides having an alkanethiol, alkane disulfide, or cyclic disulfide covalently attached at the 5 'or 3' end can be attached to various nanoparticles, including gold nanoparticles.
[0245]
The oligonucleotide and the nanoparticles are contacted in water for a time sufficient to allow at least a portion of the oligonucleotide to bind to the nanoparticles via a functional group. Such time can be determined empirically. For example, it has been found that good results can be obtained in about 12 to 24 hours. Other suitable oligonucleotide binding conditions can also be determined empirically. For example, good results are obtained with a nanoparticle concentration of about 12-20 nM and incubation at room temperature.
[0246]
Then, at least one salt is added to the water to form a salt solution. The salt can be any water-soluble salt. For example, the salt may be sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, a combination of two or more of these salts, or one of these salts in a phosphate buffer. . The salt is preferably added as a concentrated solution, but may be added as a solid. The salt may be added to the water all at once or gradually over time. By "gradual over time" is meant that the salt is added in at least two portions and at regular time intervals. Suitable time intervals can be determined empirically.
[0247]
The ionic strength of the salt solution is determined by the electrostatic repulsion of the oligonucleotides from each other, the electrostatic attraction of the negatively charged oligonucleotides to the positively charged nanoparticles, or the negatively charged oligonucleotides from the negatively charged nanoparticles. It must be sufficient to at least partially overcome the electrostatic repulsion of the nucleotide. It was found that progressively decreasing the electrostatic attraction and repulsion by the progressive addition of salt over time resulted in the highest surface density of oligonucleotides on the nanoparticles. The appropriate ionic strength for each salt or combination of salts can be determined empirically. Preferably, the concentration of sodium chloride is preferably increased gradually over time, and a final concentration of about 0.1 M to about 1.0 M sodium chloride in phosphate buffer has been found to give good results.
[0248]
After the salt is added, the oligonucleotide and the nanoparticles are incubated in the salt solution for an additional time sufficient to allow additional oligonucleotides to bind to the nanoparticles and form a stable nanoparticle-oligonucleotide conjugate. As described in detail below, it was found that increasing the surface density of the oligonucleotide on the nanoparticle stabilized the conjugate. The time of this incubation can be determined empirically. It has been found that good results are obtained with a total incubation time of about 24 to 48 (preferably 40 hours), which is the total incubation time. As mentioned above, the salt concentration is progressive over this total time. Can be increased). This second incubation time in salt solution is referred to herein as the "aging" step. Other conditions suitable for this "aging" step may be determined empirically. For example, good results are obtained at room temperature incubation and pH 7.0.
[0249]
Conjugates formed using the "age" step were found to be significantly more stable than those produced without the "age" step. As mentioned above, this increase in stability is due to the increase in oligonucleotide density on the nanoparticle surface achieved by the "aging" step. The surface density achieved by the "aging" step will depend on the size and type of the nanoparticles, as well as the length, sequence and concentration of the oligonucleotide. The surface density suitable for stabilizing the nanoparticles and the conditions necessary to obtain the desired combination of nanoparticles and oligonucleotides can be determined empirically. Generally, to obtain a stable nanoparticle-oligonucleotide conjugate, at least 10 pmol / cm²Would be appropriate. Surface density of at least 15 pmol / cm²Is preferred. The surface density is about 35-40 pmol / cm, since the ability of the conjugated oligonucleotide to hybridize to nucleic acids and oligonucleotide targets is reduced if the surface density is too high.²The following is preferred.
[0250]
As used herein, “stable” means that the majority of the oligonucleotides remain attached to the nanoparticles for at least 6 months after conjugate formation, and the oligonucleotides are used in nucleic acid detection and nanofabrication methods. Means that it can hybridize to nucleic acid and oligonucleotide targets under standard conditions encountered in the application method.
[0251]
Nanoparticle-oligonucleotide conjugates prepared in this way show, besides their stability, other excellent properties. See, for example, Examples 5, 7, and 19 of the present application. In particular, due to the high surface density of the conjugate, the conjugate will assemble as large aggregates in the presence of the target nucleic acid or oligonucleotide. Although the temperature range at which aggregates form and dissociate has been found to be unexpectedly very narrow, this unique feature has important practical consequences. In particular, this increases the selectivity and sensitivity of the detection method of the invention. Using this conjugate, one base mismatch and a target as small as about 20 fmol can be detected. Although these features were originally discovered in assays performed in solution, the advantages of using these conjugates are that they can be performed on substrates, including assays that use only one conjugate. It has been found that the assay can be performed.
[0252]
It has been found that the hybridization efficiency of the nanoparticle-oligonucleotide conjugate can be dramatically increased by using a recognition oligonucleotide that includes a recognition moiety and a spacer moiety. A "recognition oligonucleotide" is an oligonucleotide that comprises a sequence that is complementary to at least a portion of the sequence of a nucleic acid or oligonucleotide target. In this embodiment, the recognition oligonucleotide comprises a recognition moiety and a spacer moiety, wherein the recognition moiety hybridizes to the nucleic acid or oligonucleotide target. The spacer portion of the recognition oligonucleotide is designed such that the spacer portion can bind to the nanoparticles. For example, a moiety containing a functional group capable of binding to a nanoparticle is covalently bound to the spacer moiety. These are the same moieties and functional groups as described above. As a result of the attachment of the spacer portion of the recognition oligonucleotide to the nanoparticle, the recognition portion moves away from the surface of the nanoparticle and becomes more accessible for hybridization with its target. The length and arrangement of the spacer moiety that provides good spacing of the recognition moiety from the nanoparticles can be determined empirically. A spacer moiety comprising at least about 10 nucleotides, preferably 10-30 nucleotides, has been found to give good results. The spacer moiety may have any sequence that does not interfere with the ability of the recognition oligonucleotide to bind to the nanoparticles or to the nucleic acid or oligonucleotide target. For example, the spacer portion should not have a sequence that is complementary to the sequence of the recognition oligonucleotide, the nucleic acid of the recognition oligonucleotide or the sequence of the oligonucleotide target. Preferably, the bases of the nucleotides in the spacer moiety are all adenines, all thymines, all cytidines, or all guanines, otherwise the problems described above may occur. More preferably, the bases are all adenine or all thymine. Preferably, all bases are thymine.
[0253]
It is further known that the use of a diluent oligonucleotide in addition to the recognition oligonucleotide to provide the desired level of hybridization provides a means of tailoring the conjugate. . The diluent oligonucleotide and the recognition oligonucleotide were found to attach to the nanoparticles in approximately the same ratio as in solution when contacted with the nanoparticles to prepare the conjugate. Thus, the ratio of diluent oligonucleotide to recognition oligonucleotide associated with the nanoparticles can be controlled such that the conjugate participates in the desired number of hybridization events. The diluent oligonucleotide may have any sequence that does not interfere with the ability of the recognition oligonucleotide to bind to the nanoparticles or to the nucleic acid or oligonucleotide target. For example, the diluent oligonucleotide should not have a sequence that is complementary to the sequence of the recognition oligonucleotide or the sequence of the nucleic acid or oligonucleotide target of the recognition oligonucleotide. The diluent oligonucleotide preferably has a shorter length than the recognition oligonucleotide so that the recognition oligonucleotide can bind to its nucleic acid or oligonucleotide target. If the recognition oligonucleotide includes a spacer moiety, the diluent oligonucleotide is most preferably about the same length as the spacer moiety. Thus, the diluent oligonucleotide does not interfere with the ability of the recognition portion of the recognition oligonucleotide to hybridize to the nucleic acid or oligonucleotide target. Even more preferably, the diluent oligonucleotide has the same sequence as the sequence of the spacer portion of the recognition oligonucleotide.
[0254]
Protein receptors and other specific binding pair members are functionalized by oligonucleotides and immobilized on oligonucleotide-modified nanoparticles, providing the molecular recognition properties dictated by protein receptors rather than DNA. A new class of hybrid particles (nanoparticle-receptor conjugates) can be generated that exhibit a high degree of particle stability. Alternatively, a protein having multiple receptor binding sites with receptor-modified oligonucleotides can be functionalized so that in the original nanomaterial assembly scheme discussed above, the protein receptor complex can be substituted for one of the inorganic nanoparticles. It can be used as one of the partial structures. The use of these novel nanoparticle-receptor conjugates in analyte detection strategies has been evaluated in a number of ways, including target identification and protein-protein interaction screening.
[0255]
In one embodiment of the present invention, a novel hybrid that is stable over a wide range of aqueous salt concentrations, has a long shelf life, and allows the use of less soluble as well as more soluble proteins A particle or nanoparticle receptor conjugate is provided. An array of proteins immobilized at discrete sites on the surface can be screened using the probe system (see exemplary structure II in FIG. 47) (FIG. 48). Proteins, peptides, carbohydrates, sugars, oligonucleotides, polynucleotides, and small molecules (in FIG. 49,Aas well asBA variety of specific binding pairs, including proteins, peptides, carbohydrates, sugars, oligonucleotides, polynucleotides, synthetic polymers, synthetic oligomers, or small molecules, or any combination thereof. Similar principles for probe construction and application can be applied to study binding reactions. The basic concept is that the oligonucleotide that is complementary to the oligonucleotide attached to the nanoparticle has a “receptor unit” (FIG.A). The hybridization then produces a nanoparticle probe containing an oligonucleotide sheath around the nanoparticle matrix that acts to increase the water solubility of the system and stabilize the colloids involved in aggregation. Then, using the probe II ', the target immobilized on the solid surface (B).AofBBinding to is monitored by detection of nanoparticles bound to the solid surface (eg, by colorimetry, fluorescence, silver staining, etc.). This scheme isACan be conjugated to the oligonucleotide,BAny molecule that can immobilize on the surface can be used.
[0256]
An embodiment of the present invention is shown in FIG. 47, where I is a gold nanoparticle comprising a number of oligonucleotides tightly linked (eg, by sulfur-gold bonds) to a gold surface, where 1 is a nanoparticle. It is a protein containing a side group oligonucleotide with a sequence complementary to the sequence of the oligonucleotide bound to the particles. Procedures for constructing nanoparticle-oligonucleotide conjugates with high surface density oligonucleotides are described herein. For example, in Example 3 and C.I. A. Mirkin et al., Nature, 382, 607-609 (1996); Elghanian et al., Science, 277, 1078-1081 (1997); J. Storhoff et al. Am. Chem. Soc. 120, 1959-1964 (1998). Methods for conjugating oligonucleotides to proteins are well known and details of one method are described in R. Hendrickson et al., Nucleic Acids Research, 23, 522-529 (1995). When both the oligonucleotide conjugated to the nanoparticle and the oligonucleotide-protein conjugate are subjected to hybridization conditions, one or more oligonucleotide-protein conjugate molecules bind to a given nanoparticle by hybridization. To produce a nanoparticle protein conjugate. See FIGS. 47 and 50 (a). Another embodiment of a nanoparticle receptor conjugate is shown in FIG. 50 (b), which requires the use of a tethered oligonucleotide, and uses a nanoparticle-oligonucleotide construct and an oligonucleotide-protein conjugate. Both are placed under hybridization conditions. In this embodiment, the linker oligonucleotide has one segment (a ') complementary to the oligonucleotide (a) attached to the nanoparticle and another segment complementary to the oligonucleotide (b) attached to the receptor. (B ′).
[0257]
The resulting nanoparticle-protein complex can be separated from unbound proteins by centrifugation or gel filtration and used as a probe for a target analyte such as an antigen. FIG. 51 shows several exemplary detection schemes that can use nanoparticle-protein conjugates as probes. Nanoparticle-protein conjugates can be used to identify target analytes, to screen libraries of molecules such as drugs aligned and immobilized on a support (FIG. 51 (a)), or in histochemical applications to cells. Alternatively, it has a wide variety of applications, including identifying the presence of an analyte in a tissue. The probe can also be used in spot tests where the analyte, eg, protein or drug, has multiple binding sites.
[0258]
The gold probe-receptor-ligand target combination resulting from these tests can be observed visually (eg, spot test, aggregate formation, or silver staining), or by any known procedure. For example, a nanoparticle-protein conjugate system can be used to develop a colorimetric test for nucleic acids that depends on the aggregation properties of the nanoparticle / DNA / protein composite (FIG. 52 (A)). In a typical experiment, a complex comprising four biotin-oligonucleotide conjugates (1-STV) with or without target nucleic acid (3) and streptavidin conjugated to a gold nanoparticle DNA conjugate (2-Au) A body solution was prepared. The sequence of the target nucleic acid has two parts. The oligonucleotide bound to the complex is complementary to the first portion of the nucleic acid, while the bound oligonucleotide of the nanoparticle is complementary to the second portion of the nucleic acid. The solution was heated at 53 ° C. for 30 minutes, after which an aliquot of 3 L of the solution was spotted on a C18 reverse phase thin layer chromatography plate. The solution containing the target nucleic acid showed a blue spot, and the solution containing no target showed a red spot. The results and the sharp melting curve show that one of the gold nanoparticle probes in the spot test (FIG. 52 (B)) could be replaced by a more easily prepared streptavidin / DNA conjugate than the gold probe. Has been demonstrated.
[0259]
The system shown in FIG. 52 (a) can also be used to prepare the novel three-dimensional nanomaterials or assemblies discussed above. In a simple variation of FIG. 52 (a), as shown in FIG. 52 (c), no linking oligonucleotide is used and the oligonucleotide bound to the complex is complementary to the oligonucleotide bound to the nanoparticles. is there.
[0260]
These new nanoparticle-protein probes (II) have unique features, which provide significant advantages over previously described nanoparticle protein conjugates. In particular, the high-density oligonucleotide on the surface of the nanoparticles acts as a sheath for the polyanion, increasing the solubility and stability of the nanoparticle-protein conjugate in water over a very wide range of solubilities, and over a very wide range of salt concentrations. Increase the stability of the nanoparticle-protein conjugate in water. In addition, the reversibility of the oligonucleotide-oligonucleotide linkage provides a unique approach for characterizing the complex formed with the target. In addition, the nanoparticle-protein is more resistant to non-specific binding (see FIG. 53).
[0261]
Preferred nanoparticle-protein complexes are based on gold nanoparticles, but based on a wide variety of other materials (eg, silver, platinum, mixtures of gold and silver, magnetic particles, semiconductors, quantum dots). Other particles described above may be used, and the particle size may be in the range of 2 to 100 nm as described above. US patent application Ser. No. 09 / 344,667 and PCT application WO 98/04740 are both fully incorporated herein by reference and provide suitable nanoparticles and oligonucleotides attached thereto. The method is described.
[0262]
In another embodiment, the nanoparticle-oligonucleotide-protein complex (II) is used as a probe in detecting a specific target molecule such as a protein immobilized on a smooth surface. See, for example, FIGS. 48, 49, 51, and 54. For this purpose, a number of different proteins may be immobilized as arrays on glass slides at different discrete spots. Numerous coupling methods may be used. One method well known in the art is to treat the glass with 3-aminopropyltriethoxysilane followed by 1,4-phenylenediisothiocyanate. This arrangement creates a surface that carries pendant isothiocyanate groups that can be used to attach to materials containing one or more amino groups. A target containing an amino group can be directly immobilized on this modified surface. In other materials, aminoalkyl groups can be attached to the target to provide the required reactive groups. After immobilization of the target on the surface, inactivation of residual isothiocyanate groups, and washing, the surface is exposed to a colloid solution of II. Another method for immobilizing proteins involves "printing" the proteins on glass slides with attached aldehyde groups to produce a protein chip. G. FIG. MacBeath et al., Science, 289, 1760-1763 (2000); Nature, 2000, 405, 837; and Anal. Biochem. 278, page 123, all are fully incorporated by reference. Amino groups on proteins react to form covalent bonds without significantly destroying the tertiary structure of the protein, so that the protein can also react with receptors and other target molecules. Alternatively, the specific target molecule or analyte binds to the oligonucleotide to form an oligonucleotide-analyte conjugate in which the oligonucleotide bound to the analyte has a sequence complementary to the sequence of the oligonucleotide bound to the support. There are many ways to covalently attach proteins and other substances to oligonucleotides. One method involves the use of SMPB (sulfosuccinimidyl 4- [p-maleimidophenyl] butyrate) to attach the thiol-modified oligonucleotide to streptavidin. Nucleic Acids Research, 1994, Vol. 22, p. 5530, is fully incorporated by reference. The sample may be bound to the support by contacting the oligonucleotide bound to the support with the oligonucleotide bound to the sample under hybridization conditions.
[0263]
Thereafter, the probe is brought into contact with the support. After sufficient time for the protein-protein specific binding interaction to occur, the excess nanoparticle conjugate is washed away and the nanoparticles are detected. Metallic nanoparticles may be detected directly by color or as gray spots with silver staining. Thus, specific protein / receptor binding interactions may be exploited as a way to indicate the presence of any one on a glass slide (FIG. 54). In a typical experiment, a portion of a glass slide was exposed by exposing the glass slide to a solution containing streptavidin bound to biotinylated DNA hybridized to a high DNA density gold nanoparticle probe described herein. Alternatively, the hybridized biotinylated DNA containing the complementary DNA can be probed. The probe binds to a portion of the slide containing biotin. The signal associated with the conjugated probe can be further developed by treatment with a silver enhancement solution (see FIG. 54).
[0264]
As indicated above, useful Type II probes can be made using a wide variety of materials as functional recognition elements. The probe synthesis and application schemes shown in FIGS. 49 and 50 are quite similar to those shown in FIG. 48 for studying protein-protein interactions. As a general example of FIG. 49, the conjugated probe is called II ′, and the receptor unit isACalled the target immobilized on the solid surfaceBCalled. The system of FIG. 48 and the variations described herein are specific examples of the general system shown in FIG.
[0265]
In some cases, it may be preferable to use a Type II oligonucleotide-nanoparticle probe constructed from non-metallic nanoparticles. Type II probes based on intrinsically fluorescent nanoparticles can be observed by their fluorescence. Other nanoparticles may be made fluorescent by adding a fluorescent label to the particles either before or after the oligonucleotide sheath is added.
[0266]
In another embodiment of the present invention, a novel protein detection method is provided. If linked DNA can be used as a marker for a specific protein, the protein binding molecule is connected to the gold nanoparticle-oligonucleotide conjugate by a linking oligonucleotide. FIG. 55 describes a protein detection method that utilizes a ligated oligonucleotide as the reporter DNA. In this method, the oligonucleotides are modified with protein binding molecules in such a way that each protein particle has a different oligonucleotide sequence, after which the protein-oligonucleotide conjugate is immobilized on the Au particles as a result of hybridization. . For example, biotinylated DNA can be immobilized on a gold nanoparticle-oligonucleotide conjugate to produce a biotin nanoparticle conjugate. In the presence of streptavidin, the biotin-modified nanoparticles form aggregates, which can be easily separated from other particles. The aggregate is then dissociated by DNA dehybridization and the reporter DNA is isolated from the Au particles and proteins. Since each protein binding molecule has a unique DNA sequence, proteins can be identified by established DNA detection methods such as using a DNA chip. Once the reporter DNA has been isolated, the detection limit can be very low since the reporter DNA can be amplified by PCR.
[0267]
A feature common to all of these nanoparticle-oligonucleotide-receptor conjugate systems is that the components to be tested, such as the signal for the interaction of receptors A and B in FIG. , And the interaction of oligonucleotide a with oligonucleotide a '. The dissociation temperature of the oligonucleotide duplex segment can be varied by controlling the length and sequence of the complementary strand, as well as the salt concentration, so that, depending on the researcher's intention, the It is often possible to design the system to be dissociated at low or high temperatures. As discussed herein, complexes formed by hybridization of oligonucleotide gold nanoparticle probes to oligonucleotides linked to other nanoparticles or to a glass surface typically dissociate over a narrow temperature range. To our knowledge, such a sharp melting transition is unprecedented for a complex derived from an oligonucleotide of this size. This property sets these nanoparticle oligonucleotide probes in FIG. 47 apart from other probe systems, including other probes based on nanoparticles and oligonucleotides. Therefore, if the type IV (FIG. 48) or V type (FIG. 54) system is designed such that the observed signal reflects the dissociation of the oligonucleotide duplex, the dissociation transition will be extremely sharp. This feature provides specific dimensions to characterize the complex of types IV and V. Or,AWhenBTo get information about the strength of the interaction withA−BSystems can also be designed that favor the initial dissociation of the complex.
[0268]
As can be readily appreciated, highly desirable nanoparticle-oligonucleotide conjugates can be prepared using any of the methods described above. By doing so, it is possible to produce a stable conjugate having the hybridization ability that meets the requirements.
[0269]
Any of the above conjugates may be, and preferably are, used in any of the nucleic acid detection methods described above. The present invention also provides a kit including a container containing any of the above conjugates. In addition, the conjugate may be, and preferably is, used in any of the nanofabrication methods and nucleic acid separation methods of the present invention.
[0270]
The invention further relates to the use of an electric field to facilitate hybridization of the nanoparticle-oligonucleotide conjugate of the invention to a complementary oligonucleotide on the electrode surface. Here, in addition to the salts, pH, temperature, chaotropic agents, and other factors discussed above, the electric field strength (especially current levels and current densities) is precisely controllable and continuous for regulating nucleic acid interactions. Can be provided. In addition to facilitating nucleic acid hybridization interactions, electric fields include most molecular biological methods such as specific binding pair interactions, antibody / antigen reactions, cell separation, and related clinical diagnostic methods. Can be extended to facilitate other interactions with nanoparticle-charged molecule conjugates, including but not limited to: Application of an electric field facilitates the design, construction, and use of various types of nanoparticle-based biosensors to monitor (or measure) the progress or generation of a selected biological reaction. For biosensors, see Protein Immobilization, Fundamentals & Applications, R.A. F. Taylor (1991) (chapter 8) and Immobilized Affinity Ligand Technologies, Hermanson et al. (1992) (chapter 5) are discussed in considerable detail. See also U.S. Patent No. 5,965,452 and the references cited therein.
[0271]
Recently, Nanogen et al. Have developed microelectronic nucleic acid array methods and devices that use electric fields as independent parameters to control the transport, hybridization and stringency of nucleic acid interactions. They are "active" array devices in that they use microelectronics as well as microfabrication. See, for example, Edman et al., Nucleic Acid Res. US Pat. No. 6,051,380; 6,068,818; 5,929,208; 5,632,957; 1997, 25 (24), 4907-14; See also 5,565,322; 5,605,662; 5,849,486; 6,048,690; 6,013,166; and 5,965,452. I want to. These are incorporated by reference in their entirety. These documents describe nucleic acid arrays based on microelectronics that use electric fields as independent parameters to control the transport, hybridization and stringency of nucleic acid interactions. Such nucleic acid arrays are "active" array devices in that they use microelectronics as well as microfabrication technology. However, prior to the present invention, Applicants have identified an interaction between a nanoparticle-first specific binding pair member conjugate and a second binding pair member, eg, a nanoparticle-oligonucleotide conjugate. It is believed that the prior art does not explain or suggest that the interaction between complementary nucleotides attached to the surface can be facilitated by an electric field.
[0272]
The term "being" presents "one or more" of its beings. For example, "a feature" refers to one or more features or at least one feature. Accordingly, the terms "a", "one or more" and "at least one" are used interchangeably herein. Note that the terms "comprising," "including," and "having" are used interchangeably.
[0273]
(Example)
Example 1Oligonucleotide-modified gold nanoparticles
A:Preparation of gold nanoparticles
Frens, Nature Phys. Sci. 241: 20 (1973) and Grabar, Anal. Chem. 67, p. 735 (1995).₄Was reduced with citrate to prepare colloidal gold (13 nm in diameter). Briefly, all glassware was treated with aqua regia (3 parts HCl, 1 part HNO₃) And washed with Nanopure H₂Rinse with O and then oven dried before use. HAuCl₄And sodium citrate were purchased from Aldrich Chemical Company. Aqueous HAuCl₄(1 mM, 500 ml) was stirred and refluxed. Then 38.8 mM sodium citrate (50 ml) was added quickly. The color of the solution changed from light yellow to dark red, but reflux was continued for 15 minutes. After cooling to room temperature, the red solution was filtered through a 1 micron filter from Micron Separations Inc. Au colloids were characterized by UV-visible spectroscopy using a Hewlett Packard 8452A diode array spectrophotometer and transmission electron microscopy (TEM) using a Hitachi 8100 transmission electron microscope. 13 nm diameter gold particles produce a visible discoloration when aggregated with the target and probe oligonucleotides in the 10-35 nucleotide range.
[0274]
B.Oligonucleotide synthesis
Oligonucleotides were synthesized on a 1 μmol scale using a Milligene Expedite DNA synthesizer in single column mode using phosphoramidite chemistry. Eckstein, F.C. (Ed.), Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991). All solutions were purchased from Milligene (DNA synthesis grade). Average coupling efficiencies ranged from 98 to 99.8%, and the final dimethoxytrityl (DMT) protecting group was not cleaved from the oligonucleotide to aid purification.
[0275]
To obtain the 3'-thiol-oligonucleotide, the thiol modifier C3 S-SCPG support was purchased from Glen Research and used in an automated synthesizer. During normal cleavage from the solid support (16 hours at 55 ° C.)₄0.05M dithiothreitol (DTT) was added to the OH solution to reduce 3 'disulfide to thiol. Excess DTT was removed by extraction with ethyl acetate before purification by reverse phase high performance liquid chromatography (HPLC).
[0276]
In order to obtain a 5'-thiol oligonucleotide, a 5'-thiol modifier C₆-Phosphoramidite reagent was purchased from Glen Research, 44901 Falcon Place, Sterling, Va 20166, Falcon Place 44901, Stirling, VA. Oligonucleotides were synthesized and the final DMT protection was removed. Then, 100 μmol of 5 ′ thiol modifier C₆-1 ml of anhydrous acetonitrile was added to the phosphoramidite. Mix 200 μl of amidite solution with 200 μl of activator (freshly made from the synthesizer), introduce by syringe onto column still containing synthetic oligonucleotide on solid support and reciprocate in column for 10 minutes I let it. The support was then washed with anhydrous acetonitrile for 30 seconds (2 × 1 ml). Add 700 μl of 0.016 M I to the column₂/ H₂An O / pyridine mixture (oxidant solution) was introduced and then reciprocated in the column using two syringes for 30 seconds. Then the support is CH₃After a 1 minute wash with a 1: 1 mixture of CN / pyridine (2 × 1 ml), a final wash with anhydrous acetonitrile (2 × 1 ml), then the column was dried with a stream of nitrogen. The trityl protecting group was not removed to aid purification.
[0277]
0.03M Et₃NH⁺OAc⁻1% / min gradient 95% CH with buffer (TEAA), pH 7₃Reversed phase HPLC was performed on a Dionex DX500 system equipped with a Hewlett Packard ODS hypersil column (4.6 × 200 mm, 5 mm particle size) using CN / 5% TEAA. The flow rate was 1 ml / min and UV detection was 260 nm. For purification of the DMT-protected unmodified oligonucleotide, preparative HPLC (elution time 27 minutes) was used. After the buffer was recovered and evaporated, DMT was cleaved from the oligonucleotide by treatment with 80% acetic acid at room temperature for 30 minutes. The solution was then evaporated to near dryness, water was added and the cleaved DMT was extracted from the aqueous oligonucleotide solution using ethyl acetate. The amount of oligonucleotide was quantified by absorbance at 260 nm and the final purity was assessed by reverse phase HPLC (elution time 14.5 minutes).
[0278]
The same protocol was used for purification of the 3'-thiol oligonucleotide except that DTT was added after DMT was extracted to reduce the amount of disulfide formed. After 5 hours at 4 ° C., DTT was extracted using ethyl acetate and the oligonucleotide was repurified by HPLC (elution time 15 minutes).
[0279]
To purify the 5'-thiol modified oligonucleotide, preparative HPLC was performed under the same conditions as for the unmodified oligonucleotide. After purification, 150 μl of 50 mM AgNO was added to the anhydrous oligonucleotide sample.₃The solution was added to remove the trityl protecting group. The sample turned milky as soon as the cut occurred. After 20 minutes, 200 μl of a 10 mg / ml solution of DTT was added to complex the Ag (reaction time 5 minutes) and the sample was centrifuged to precipitate a yellow complex. The oligonucleotide solution (<50 OD) is then applied to a desalted NAP-5 column (with Swedish DNA Grade Sephadex D-25 Medium for desalting and buffer exchange of more than 10 bases) (Swedish). Transferred to Pharmacia Biotech, Uppsala, Uppsala, Sweden. The amount of 5'-thiol modified oligonucleotide was quantified by measuring the absorbance at 260 nm by UV-visible spectroscopy. Ion exchange HPLC was performed on a Dionex Nucleopac PA-100 (4 × 250) column using a 2% / min gradient of 10 mM NaOH, 1M NaCl solution with 10 mM NaOH solution (pH 12) to assess final purity. Typically, two peaks were obtained with elution times of about 19 and 25 minutes (elution time depends on oligonucleotide chain length). These peaks corresponded to thiol and disulfide oligonucleotides, respectively.
[0280]
C.Attachment of oligonucleotides to gold nanoparticles
An aqueous solution of 17 nM (150 μl) Au colloid prepared as described in Part A above was mixed with 3.75 μM (46 μl) 3′-thiol-TTTGCTGA prepared as described in Part B and capped in a 1 ml volume. It was left at room temperature for 24 hours in an Eppendorf vial. The second colloid solution was reacted with 3.75 μM (46 μl) 3′-thiol-TACCGTTTG. Note that these oligonucleotides are non-complementary. Just before use, equal amounts of the two nanoparticle solutions were combined. No reaction occurred because the oligonucleotides were non-complementary.
[0281]
The oligonucleotide-modified nanoparticles were stable at high temperature (80 ° C.) and high salt concentration (1 M NaCl) for many days, and no particle growth was observed. Stability under high salt concentrations is important because such conditions are required for the hybridization reactions that form the basis of the detection methods and nanofabrication of the present invention.
[0282]
Example 2:Formation of nanoparticle aggregates
A.Preparation of linked oligonucleotides
Two (non-thiolated) oligonucleotides were synthesized as described in Part B of Example 1. These oligonucleotides had the following sequences:
3'ATATGCGCGA TCTCAGCAAA (SEQ ID NO: 1); and
3 'GATCGCCAT ATCAACGGTA (SEQ ID NO: 2).
When these two oligonucleotides are mixed in a 1 M NaCl, 10 mM phosphate buffer (pH 7.0) solution, they hybridize to form a duplex with 12 base pair overlap and two 8 base pair cohesive ends. Was done. Each of the sticky ends had a sequence complementary to one sequence of the oligonucleotide attached to the Au colloid prepared in Part C of Example 1.
[0283]
B.Formation of nanoparticle aggregates
The ligated oligonucleotide (final concentration 0.17 μM after dilution with NaCl) prepared in Part A of this example was mixed with the nanoparticle-oligonucleotide conjugate (diluted with NaCl) prepared in Part C of Example 1 at room temperature. Later final concentration of 5.1 nM). The solution was then diluted with aqueous NaCl (to a final concentration of 1 M) and buffered with 10 mM phosphoric acid, pH 7, which is suitable for oligonucleotide hybridization. Immediately thereafter, a color change from red to purple was observed, followed by a precipitation reaction. See FIG. Within a few hours, the solution became clear and a pinkish gray precipitate settled at the bottom of the reaction vessel. See FIG.
[0284]
To confirm that the process contained both oligonucleotide and colloid, the precipitate was collected and resuspended (with shaking) in 1M aqueous NaCl buffered at pH7. In this way, oligonucleotides that did not hybridize to the nanoparticles are removed. Next, a temperature / time dissociation experiment was performed by monitoring the absorbance (260 nm) characteristic of the hybridized oligodeoxyribonucleotide and the absorbance (700 nm) characteristic of the aggregated colloid indicating the distance between the gold particles. See FIG.
[0285]
Record the change in absorbance at 260 and 700 nm on a Perkin-Elmer Lambda2 UV-Visible spectrophotometer using a Peltier PTP-1 Temperature Controlled Cell Holder while circulating the temperature between 0 and 80 ° C. at a rate of 1 ° C./min. did. The DNA solution, buffered at pH 7 with a 10 mM phosphate buffer and at 1 M NaCl concentration, was about 1 absorbance unit (OD).
[0286]
The result is shown in FIG. 8A. The temperature is from 0 ° C. to (the dissociation temperature (T_m) (T_mCirculating between 80 ° C. (38 ° C. higher than = 42 ° C.), there was a good correlation between the optical signatures of both colloids and oligonucleotides. The UV-visible spectrum of the unbound Au colloid was much less temperature dependent (FIG. 8B).
[0287]
Heating the polymer oligonucleotide-colloid precipitate above its melting point resulted in substantial optical changes visible to the naked eye. The clear solution turned dark red as the polymer biomaterial dehybridized to form unbound colloids that were soluble in aqueous solution. This process was reversible, as evidenced by the temperature trace in FIG. 8A.
[0288]
In control experiments, it was revealed that the 14-T: 14-A duplex was not effective in inducing aggregation of reversible Au colloid particles. In another control experiment, a ligated oligonucleotide duplex with 4 base pair mismatches at the cohesive end was prepared using the oligonucleotide (prepared as described in Part C of Example 1 and reacted as described above). It was found that the modified nanoparticles did not induce reversible particle aggregation. In a third control experiment, the non-thiolated oligonucleotide having a sequence complementary to the cohesive end of the linking oligonucleotide and reacting with the nanoparticles, reversibly aggregated when the nanoparticles were combined with the linking oligonucleotide. Did not generate.
[0289]
Further facts about the polymerization / assembly process became apparent from transmission electron microscopy (TEM) studies of the precipitate. TEM was performed with a Hitachi 8100 transmission electron microscope. Spot 100 μl colloid solution on a porous carbon grid
[0290]
To prepare a typical sample. The grid was then vacuum dried to form an image. A TEM image of Au colloid bound via the hybridized oligonucleotide showed a large assembled Au colloid network (FIG. 9A). Unbound Au colloids disperse or undergo a particle growth reaction without aggregation under comparable conditions. Hayat, Colloidal Gold: Principles, Methods, and Applications (Academic Press, San Diego, 1991). Note that there is no fact of colloid particle growth in the experiments performed to date; the hybridized colloids appear to have a remarkably constant size with an average diameter of 13 nm.
[0291]
TEM provides a layer stack that makes it difficult to evaluate the order of the three-dimensional aggregates. However, smaller images of monolayer two-dimensional aggregates provided more facts about the self-assembly process (FIG. 9B). Dense agglomerates where uniform particles are about 60 ° apart are seen. This interval is somewhat shorter than the 95 ° interval expected for colloids bound via a rigid oligonucleotide hybrid having the sequence used. However, due to nicks in the duplex obtained after hybridizing the oligonucleotides on the nanoparticles with the linking oligonucleotides, these aggregates were not rigid hybrids and were very flexible . Note that this is a variable that can be controlled by reducing the number of system overlap strands from four to three (thus reducing the number of nicks), or by using triple strands instead of double strands. I want to be.
[0292]
Example 3Preparation of oligonucleotide-modified gold nanoparticles
A gold colloid (13 nm in diameter) was prepared as described in Example 1. Thiol-oligonucleotide [HS (CH₂)₆OP (O) (O⁻) -Oligonucleotides] were also prepared as described in Example 1.
[0293]
It has been found that the method of attaching thiol-oligonucleotides to gold nanoparticles as described in Example 1 may not give satisfactory results. In particular, when long oligonucleotides were used, the oligonucleotide-colloid conjugate was not stable in the presence of a large excess of high molecular weight salmon sperm DNA used as a model for background DNA normally present in diagnostic systems. Prolonged exposure of the colloid to thiol-oligonucleotides produced oligonucleotide-colloid conjugates that were stable to salmon sperm DNA, but the resulting conjugates did not hybridize satisfactorily. Further experiments yielded the following procedure for attaching thiol-oligonucleotides of any length to colloidal gold so that the conjugate was stable and hybridized satisfactorily to high molecular weight DNA.
[0294]
A 1 ml solution of colloidal gold (17 nM) in water was mixed with excess (3.68 μM) thiol-oligonucleotide (28 bases long) in water and the mixture was left at room temperature for 12-24 hours. Then, 100 μl of 0.1 M sodium hydrogen phosphate buffer, pH 7.0 and 100 μl of 1.0 M NaCl were premixed and added. After 10 minutes, 10 μl of 1% aqueous NaN₃Was added and the mixture was left for a further 40 hours. This "aging" step was designed to increase the surface coverage by the thiol-oligonucleotide and displace the oligonucleotide base from the gold surface. In a subsequent assay, the solution was frozen in a dry ice bath after 40 hours of incubation and then thawed at room temperature, resulting in a somewhat more clear and more demarcated red spot. In either case, the solution was then centrifuged at 14,000 rpm for about 15 minutes in an Eppendorf Centrifuge 5414, with most of the oligonucleotides (indicated by absorbance at 260 nm), as indicated by the absorbance at 520 nm. A very pale pink supernatant containing 7-10% colloidal gold and a dense, dark gelatinous residue at the bottom of the tube were obtained. The supernatant was removed and the residue was resuspended in about 200 μl of buffer (10 mM phosphoric acid, 1.0 M NaCl) and recentrifuged. After removing the supernatant solution, the residue was washed with 1.0 ml of buffer (10 mM phosphoric acid, 0.1 M NaCl) and 10 μl of 1% NaN.₃Placed in aqueous solution. The solution was pipetted up and down several times to aid dissolution. The resulting red master solution was left at room temperature for several months, placed on a silica thin phase chromatography (TLC) plate (see Example 4), and placed on 2M NaCl, 10 mM MgCl2.₂, Or a solution containing a high concentration of salmon sperm DNA was stable (that is, it remained red and did not aggregate).
[0295]
Example 4:Enhanced Nanoparticle-Oligonucleotide Hybridization Hybridization
Oligonucleotide-colloidal gold conjugates I and II shown in FIG. 11 were prepared as described in Example 3. Hybridization of these two conjugates was extremely slow. In particular, aqueous 0.1 M NaCl or 10 mM MgCl₂Mixing the conjugate I and II samples in +0.1 M NaCl and allowing the mixture to stand at room temperature for 1 day resulted in little or no discoloration.
[0296]
Two methods have been found to improve hybridization. In the first method, a faster mixture of conjugates I and II (containing 15 nM each in 0.1 M NaCl solution) is frozen in a dry ice-isopropyl alcohol bath for 5 minutes and then thawed at room temperature for faster speed. Was obtained. The thawed solution showed a bluish color. When 1 μl of the solution was spotted on a standard C-18 TLC silica plate (Alltech Associates), a dark blue color was immediately seen. Hybridization and the resulting color change caused by the freeze-thaw procedure were reversible. Upon heating the hybridized solution to 80 ° C., the solution turned red and produced a pink spot on the TLC plate. Upon freezing and thawing, the system returned to the (blue) hybridized state (both solution and spot on C-18 TLC plate). In a similar experiment where the solution was not frozen, the spot obtained on the C-18 TLC plate was pink.
[0297]
A second way to get fast results is to warm the conjugate and the target. For example, in another experiment, the oligonucleotide-gold colloid conjugate and the oligonucleotide target sequence were rapidly warmed to 65 ° C. in a 0.1 M NaCl solution and cooled to room temperature over 20 minutes. Spotting on a C-18 silica plate and drying resulted in a blue spot indicating hybridization. In contrast, incubation of the conjugate and target for 1 hour at room temperature in 0.1 M NaCl did not produce a blue color indicative of hybridization. Hybridization in 0.3 M NaCl is even faster.
[0298]
Example 5:Assay using nanoparticle-oligonucleotide conjugate
The oligonucleotide-gold colloid conjugates 1 and 2 shown in FIGS. 12A-F were made as described in Example 3, and the oligonucleotide target 3 shown in FIG. It was produced as follows. Mismatch and deletion targets 4, 5, 6 and 7 were purchased from the Northwestern University Biotechnology Facility, Chicago, IL, Chicago, Illinois. These oligonucleotides were synthesized on a 40 nmol scale and purified on a reverse phase C18 cartridge (OPC). Their purity was quantified by performing ion exchange HPLC.
[0299]
Rapid heating to stringent temperatures followed by rapid cooling achieved selective hybridization. For example, 100 μl of 0.1 M NaCl + 5 mM MgCl containing 15 nM of each oligonucleotide-colloid conjugate 1 and 2 and 3 nmol of target oligonucleotide 3, 4, 5, 6 or 7₂Was heated to 74 ° C., cooled to the temperature indicated in the table below, and the mixture was incubated at this temperature for 10 minutes to effect hybridization. A 3 μl sample of each reaction mixture was then spotted on a C-18 TLC silica plate. Upon drying (5 minutes), a dark blue color appeared if hybridization had occurred.
[0300]
The results are shown in Table 1 below. A pink spot indicates a negative test (i.e., the nanoparticles did not bind by hybridization), and a blue spot indicates a positive test (i.e., the nanoparticles were closer to hybridization for both oligonucleotide-colloid conjugates). ).
[0301]
[Table 1]

As can be seen from Table 1, hybridization at 60 ° C. resulted in blue spots only for perfectly matched target 3. Hybridization at 50 ° C. produced blue spots on both targets 3 and 6. Hybridization at 45 ° C. resulted in blue spots on targets 3, 5 and 6.
[0302]
In a related series, targets containing one mismatched T nucleotide produced a positive test (blue) at 58 ° C, and conjugates 1 and 2 produced a negative test (red) at 64 ° C. Under the same conditions, perfectly matched target (3) produced a positive test under both temperatures, this test discriminating between perfectly matched target and target containing one mismatched base. It is possible to do.
[0303]
Similar results were obtained using different hybridization methods. In particular, selective hybridization was achieved by freezing, thawing and then rapidly warming to stringent temperatures. For example, 100 μl of 0.1 M NaCl containing 15 nM of each oligonucleotide-colloid conjugate 1 and 2 and 10 pmol of target oligonucleotide 3, 4, 5, 6 or 7 are placed in a dry ice-isopropyl alcohol bath for 5 minutes. Min, thawed at room temperature, then rapidly cooled to the temperature indicated in Table 2 below and the mixture was incubated at this temperature for 10 minutes to perform hybridization. A 3 μl sample of each reaction mixture was then spotted on a C-18 TLC silica plate. Table 2 shows the results.
[0304]
[Table 2]

An important feature of these systems is that the discoloration associated with the temperature change is very rapid and occurs over a temperature range of about 1 ° C. This demonstrates a high degree of cooperativity in the melting and association processes involving the colloid conjugate, indicating that oligonucleotide targets containing a perfect match sequence and a single base pair mismatch can be readily identified.
[0305]
The high degree of discrimination is considered to be due to two features. First, obtaining a positive signal requires alignment of two relatively short probe oligonucleotide segments (15 nucleotides) on the target. In equivalent binary detection systems, mismatches in either segment are less stable than mismatches in longer probes (eg, 30 base length oligonucleotides). Second, the signal at 260 nm obtained from hybridization of the target oligonucleotide and the nanoparticle conjugate in solution is based on nanoparticles, rather than DNA. This signal is dependent on the dissociation of nanoparticle assemblies organized as a polymer network by multiple oligonucleotide duplexes. This narrows the temperature range observed for aggregate dissociation compared to standard DNA thermal denaturation. Briefly, some duplexes in cross-linked aggregates can dissociate without dispersing the nanoparticles in solution. Therefore, the temperature range at which the aggregates melt is extremely narrow (4 ° C.) compared to the temperature range (12 ° C.) associated with melting of an equivalent system without nanoparticles. An even more significant and advantageous aspect of this detection method is the temperature range of the colorimetric response (<1 ° C.) observed on C-18 silica plates. In principle, this three-component nanoparticle-based method would be more selective than any two-component detection system based on a single-stranded probe hybridizing to the target nucleic acid.
[0306]
A master solution containing 1 nmol of target 3 was prepared in 100 μl of hybridization buffer (0.3 M NaCl, 10 mM phosphoric acid, pH 7). 1 μl of this solution corresponds to 10 pmol of target oligonucleotide. Serial dilutions were performed by taking aliquots from the master solution and diluting with hybridization buffer to the desired concentration. Table 3 shows the sensitivities obtained with 3 μl of a mixture of probes 1 and 2 with different amounts of target 3. After hybridization using freeze-thaw conditions, 3 μl aliquots of these solutions were spotted on C-18 TLC plates to quantify color. In Table 3 below, pink indicates a negative test and blue indicates a positive test.
[0307]
[Table 3]

This experiment indicates that 10 fmol is the lower limit of detection for this particular system.
[0308]
Example 6:Assay using nanoparticle-oligonucleotide conjugate
As shown in FIG. 13B, the DNA-modified nanoparticles were adsorbed on the modified transparent substrate. The method comprises using DNA hybridization interactions to attach DNA-modified nanoparticles to the nanoparticles attached to a glass substrate.
[0309]
Glass microscope slides were purchased from Fisher Scientific. The slide was cut into approximately 5 x 15 mm pieces using a scribe pen with a diamond tip. Slides are run at 50 ° C for 4: 1H.₂SO₄: H₂It was cleaned by immersion in an O solution for 20 minutes. The slides were then rinsed with plenty of water and then ethanol and dried under a stream of dry nitrogen. To functionalize the slide surface with a thiol-terminated silane, the slide was immersed in a degassed ethanol 1% (by weight) mercaptopropyl-trimethoxysilane solution for 12 hours. Slides were removed from the ethanol solution and rinsed with ethanol and then water. The nanoparticles were immersed in a solution containing 13 nm diameter gold nanoparticles (the formulation described in Example 1) and adsorbed on the thiol end surface of the slide. After 12 hours in the colloid solution, the slide was removed and rinsed with water. The resulting slide has a pink color attributable to the adsorbed nanoparticles and shows a UV-visible absorbance profile (surface plasmon absorbance peak at 520 nm) similar to the aqueous gold nanoparticle colloid solution. See FIG. 14A.
[0310]
Glass slides were prepared using a 0.2 OD (1.7 μM) solution containing freshly purified 3 ′ thiol oligonucleotide (3 ′ thiol ATGCTCAACTCT [SEQ ID NO: 33] (synthesized as described in Examples 1 and 3)). After immersion for 12 hours, the slide was removed and rinsed with water.
[0311]
To demonstrate the ability of the analyte DNA strand to bind the nanoparticles to the modified surface, ligated oligonucleotides were prepared. The ligated oligonucleotide (prepared as described in Example 2) was a 24 bp long (5'TACGAGTTTGAGAATCCTGGAATGCG) containing a sequence with a 12 bp end complementary to DNA already adsorbed on the substrate surface (SEQ ID NO: 33). [SEQ ID NO: 34]. Next, the substrate was immersed in a hybridization buffer (0.5 M NaCl, 10 mM phosphate buffer, pH 7) solution containing a linking oligonucleotide (0.4 OD, 1.7 μM) for 12 hours. After removing the substrate and rinsing it with a similar buffer, the substrate was complemented with an oligonucleotide (TAGGACTTACGC5 ′ thiol [SEQ ID NO: 35]) complementary to the non-hybridized portion of the connecting oligonucleotide attached to the substrate (Example) 3) was immersed in a solution containing 13 nm diameter gold nanoparticles modified as described in (3). After immersion for 12 hours, the substrate was taken out and rinsed with a hybridization buffer. The color of the substrate turned purple and the UV-visible absorbance at 520 nm almost doubled (FIG. 14A).
[0312]
Melting curves were generated to confirm that the oligonucleotide-modified gold nanoparticles were attached to the oligonucleotide / nanoparticle-modified surface by DNA hybridization interaction with the ligated oligonucleotide. For thawing experiments, the substrates were placed in cuvettes containing 1 ml of hybridization buffer and the same equipment used in Part B of Example 2 was used. The absorbance signal (520 nm) attributable to the nanoparticles was monitored while increasing the temperature of the substrate at a rate of 0.5 ° C./min. When the temperature exceeded 60 ° C., the signal of the nanoparticles dropped dramatically. See FIG. 14B. The first derivative of the signal showed a melting temperature of 62 ° C., which is consistent with the temperatures seen for the three DNA sequences hybridized in a solution without nanoparticles. See FIG. 14B.
[0313]
Example 7:Assay using nanoparticle-oligonucleotide conjugate
The detection system shown in FIGS. 15A-G was designed such that the two probes 1 and 2 were end-to-end aligned on the complementary target 4. This differs from the system described in Example 5 where the two probes are closely aligned on the target strand (see FIGS. 12A-F).
[0314]
The oligonucleotide-gold nanoparticle conjugates shown in FIGS. 15A-G were prepared as described in Example 3, except that the nanoparticles were prepared in a hybridization buffer (0.3 M NaCl, 10 mM phosphate). , PH 7). The final nanoparticle-oligonucleotide conjugate concentration was predicted to be 13 nM, as measured by the decrease in surface plasmon band intensity at 522 nm that produced red nanoparticles. Oligonucleotide targets shown in FIGS. 15A-G were purchased from the Northwestern University Biotechnology Facility, Evanston, Ill.
[0315]
Upon mixing 150 μl of hybridization buffer containing 13 nM oligonucleotide-nanoparticle conjugates 1 and 2 with 60 pmol (60 μl) of target 4, the color of the solution immediately changed from red to purple. This discoloration results from the formation of a large oligonucleotide-bound polymer network of gold nanoparticles, which results in a red shift in the surface plasmon resonance of the nanoparticles. When the solution was allowed to stand for 2 hours, precipitation of aggregates of large macroscopic nature was observed. A “melting analysis” of the solution containing the suspended aggregates was performed. To perform the "melting analysis", the solution was diluted to 1 ml with hybridization buffer and the aggregates at 260 nm with a retention time of 1 min / 1 C while increasing the temperature from 25 C to 75 C. Optical signatures were recorded at one minute intervals. "Melting temperature" of 53.5 ° C (T_m), A characteristic abrupt transition (full width at half maximum, FW of first derivative), consistent with the characterization of aggregates as oligonucleotide-nanoparticle polymers_1/2= 3.5 ° C) was observed. This is due to the wider transition observed for oligonucleotides without nanoparticles (T_m= 54 ° C, FW_1/2= ~ 13.5 ° C)_mComparable to enough. A “melting analysis” of the oligonucleotide solution without nanoparticles was performed under similar conditions as the analysis with nanoparticles, except that the temperature was increased from 10 ° C. to 80 ° C. The content of each oligonucleotide component in the solution was 1.04 μM.
[0316]
To test the selectivity of the system, the T of the aggregate formed from the complete complement 4 of probes 1 and 2_mIs determined by the T of aggregates formed from targets containing one single base mismatch, deletion or insertion._m'(See FIGS. 15A-G). All gold nanoparticle-oligonucleotide aggregates, including incomplete targets, have different aggregate T_mAs demonstrated by the values, they exhibited significant measurable destabilization as compared to aggregates formed from perfect complements (see FIGS. 15A-G). Solutions containing incomplete targets can be easily distinguished from solutions containing perfect complement by their color when placed in a water bath maintained at 52.5 ° C. This temperature is the T of the mismatched polynucleotide._mHigher, and therefore only the solution containing the complete target, turned purple at this temperature. "Melting analysis" was also performed on probe solutions containing semi-complementary targets. It was observed that the absorbance at 260 nm increased very slightly.
[0317]
Then, 2 μl (20 pmol) of each oligonucleotide target (see FIGS. 15A-G) was added to a solution containing 50 μl of each probe (13 nM) in hybridization buffer. After standing at room temperature for 15 minutes, the solution was transferred to a temperature-controlled water bath and incubated at the temperature shown in Table 4 below for 5 minutes. A 3 μl sample of each reaction mixture was then spotted on a C-18 silica plate. Two control experiments were performed to demonstrate that both probes needed to be aligned on the target in order to induce aggregation and thus discoloration. The first control experiment consisted of Probe 1 and Probe 2 with no target present. The second control experiment consisted of probe 1 and probe 2 containing target 3 complementary to only one probe sequence (see FIG. 15B). The results are shown in Table 4 below. Pink spots indicate a negative test and blue spots indicate a positive test.
[0318]
It should be noted that the visually detectable colorimetric change occurred over less than 1 ° C., whereby complete target 4 could have mismatched (5 and 6), truncated (7) and two oligos. It can be easily distinguished from targets having a single base insertion (8) at the target location where the nucleotide probe meets (see Table 4). Colorimetric change T_cIs T_mAnd temperature are close but not the same. For both controls, there is no sign of aggregation or instability of particles in solution, as evidenced by the pinkish red observed under all temperatures, and a negative spot in the plate test at all temperatures (Pink) (Table 4).
[0319]
The observation that one single base insertion target 8 can be distinguished from perfect complementarity target 4 is truly remarkable given the perfect complementarity of the insert strand with the two probe sequences. The destabilization of aggregates formed from 8 and nanoparticle probes is due to the use of two short probes and a base stack between the two thymidine bases that the ends of the probes meet when hybridized to a perfectly complementary target. It seems to be due to loss. Equivalent condition (T_mA similar result was observed when a target containing a three base pair insertion (CCC) was hybridized to the probe under (= 51 ° C.). In the system described above in Example 5, targets with base insertions could not be distinguished from perfectly complementary targets. Therefore, the system described in this example is highly preferred in terms of selectivity. This system also showed the same sensitivity as the system described in Example 5 at about 10 fmol without using the amplification technique.
[0320]
These results indicate that any single base mismatch along the target strand, as well as any insertions into the target strand, can be detected. The temperature range over which discoloration can be detected is very steep, and the change occurs over a very narrow temperature range. This abrupt change indicates that there is a great degree of cooperativity in the melting process involving a large colloidal network attached via the target oligonucleotide chain. This results in significant selectivity as shown by the data.
[0321]
[Table 4]

Example 8:Assay using nanoparticle-oligonucleotide conjugate
One set of experiments was performed involving hybridization with "filled" double-stranded oligonucleotides. The nanoparticle-oligonucleotide conjugates 1 and 2 shown in FIG. 16A were combined with various length targets (24, 48 and 72 bases long) and complementary filler oligos as shown in FIGS. 16A-C. Incubated with nucleotides. The conditions were otherwise as described in Example 7. Oligonucleotides and nanoparticle-oligonucleotide conjugates were prepared as described in Example 7.
[0322]
As expected, the different reaction solutions had significantly different optical properties after hybridization due to the spacing-dependent optical properties of the gold nanoparticles. See Table 5 below. However, when these solutions were spotted on C-18 TLC plates and dried at room temperature or at 80 ° C., they developed a blue color regardless of the length of the target oligonucleotide and the spacing between the gold nanoparticles. See Table 5. This is thought to occur because the solid support promotes aggregation of the hybridized oligonucleotide-nanoparticle conjugate. This demonstrates that spotting the solution on a TLC plate significantly increases the spacing between gold nanoparticles (at least 72 bases) but still allows colorimetric detection.
[0323]
[Table 5]

The discoloration observed in this and other examples occurs when the spacing between gold nanoparticles (interparticle spacing) is about the same as or smaller than the diameter of the nanoparticles. Thus, the size of the nanoparticles, the size of the oligonucleotides attached to the nanoparticles, and the spacing of the nanoparticles when the nanoparticles hybridize to the target nucleic acid, depends on the oligonucleotide-nanoparticle conjugate hybridizing to the nucleic acid target. Affects whether discoloration is observable when forming aggregates. For example, a gold nanoparticle having a diameter of 13 nm produces a discoloration when aggregated with an oligonucleotide attached to a nanoparticle designed to hybridize to a target sequence 10 to 35 nucleotides in length. Will. The suitable inter-nanoparticle spacing to produce a color change when the nanoparticles are hybridized to the target nucleic acid will depend on the degree of aggregation, as evidenced by the results. These results further indicate that the solid surface further promotes aggregation of already agglomerated samples, bringing the gold nanoparticles closer together.
[0324]
The discoloration observed for the gold nanoparticles may be due to the shift or broadening of the gold surface plasmon resonance. It is unlikely that this discoloration will occur for gold nanoparticles less than about 4 nm in diameter. This is because the length of the oligonucleotide required for specific detection of the nucleic acid exceeds the diameter of the nanoparticle.
[0325]
Example 9:Assay using nanoparticle-oligonucleotide conjugate
5 μl of each of probes 1 and 2 (FIG. 12A) was combined with buffer (10 mM phosphoric acid, pH 7) to a final concentration of 0.1 M NaCl, and 1 μl of human urine was added to this solution. The solution was frozen, thawed, and then spotted on a C-18 TLC plate, but no blue color developed. To a similar solution containing 12.5 μl of each probe and 2.5 μl of human urine, 0.25 μl (10 pmol) of target 3 (FIG. 12A) was added. The solution was frozen, thawed and then spotted on a C-18 TLC plate to give a blue spot.
[0326]
Similar experiments were performed in the presence of human saliva. A solution containing 12.5 μl of each probe 1 and 2 and 2 and 2.5 μl of target 3 was heated to 70 ° C. After cooling to room temperature, 2.5 μl of saliva solution (human saliva diluted 1:10 with water) was added. After freezing and thawing the resulting solution and then spotting on a C-18 TLC plate, a blue spot indicating hybridization of the probe and target was obtained. No blue spots were observed in control experiments where no target was added.
[0327]
Example 10:Assay using nanoparticle-oligonucleotide conjugate
The assay was performed as shown in FIG. 13A. First, a glass microscope slide purchased from Fisher Scientific was cut into approximately 5 x 15 mm pieces with a scribe pen with a diamond tip. Slides at 50 ° C with 4: 1 H₂SO₄: H₂O₂It was immersed in the solution for 20 minutes to be cleaned. The slides were then rinsed with plenty of water and then ethanol and dried under a stream of dry nitrogen. Thiol-modified DNA was adsorbed on slides using a modified literature procedure (Chrisey et al., Nucleic Acids Res., Vol. 24, pp. 3031-3039 (1996)). First, slides were purchased at room temperature from a 1% solution of trimethoxysilylpropyldiethyltriamine in 1 mM acetic acid in Nanopure water (DETA, United Chemical Technology, Bristol, PA). ) For 20 minutes. Slides were rinsed with water and then ethanol. After drying with a stream of dry nitrogen, it was baked at 120 ° C. for 5 minutes using a temperature controlled heating block. The slides were allowed to cool and then immersed in 1 mM succinimidyl-4- (maleimidophenyl) -butyrate (SMPB, purchased from Sigma Chemicals) in 80:20 methanol: dimethoxysulfoxide for 2 hours at room temperature. . After taking out from the SMPB solution and rinsing with ethanol, a cap was formed on an amine site not bound to the SMPB crosslinking agent as follows. First, the slides were immersed in an 8: 1 THF: pyridine solution containing 10% 1-methylimidazole for 5 minutes. The slide was then immersed in a 9: 1 THF: acetic anhydride solution for 5 minutes. These capping solutions were purchased from Glen Research, Sterling, VA. Slides were rinsed with THF, then ethanol, and finally water.
[0328]
The modified glass slide was immersed in a 0.2 OD (1.7 μM) solution containing the newly purified oligonucleotide (3 ′ thiol ATGCTCAACTCT [SEQ ID NO: 33]) to attach the DNA to the surface. After soaking for a period of time, the slide was removed and rinsed with water.
[0329]
To demonstrate the ability of the test DNA strand to bind the nanoparticles to the modified substrate surface, a ligated oligonucleotide was prepared. The linking oligonucleotide was 24 bp long (5'TACGAGTTTGAGAATCCTGAATGCCG) with a sequence containing a 12 bp end complementary to the DNA already adsorbed on the substrate surface [SEQ ID NO: 34]. Next, the substrate was immersed in a hybridization buffer (0.5 M NaCl, 10 mM phosphate buffer, pH 7) solution containing a linking oligonucleotide (0.4 OD, 1.7 μM) for 12 hours. After removing the substrate and rinsing with a similar buffer, the substrate is modified with an oligonucleotide (TAGGACTTACGC5 'thiol [SEQ ID NO: 35]) complementary to the unhybridized portion of the linking oligonucleotide attached to the substrate. Immersed in a solution containing 13 nm diameter gold nanoparticles. After immersion for 12 hours, the substrate was taken out and rinsed with a hybridization buffer. The color of the glass substrate changed from a clear colorless to a transparent pink. See FIG. 19A.
[0330]
The slide was immersed in a ligated oligonucleotide solution as described above, and then immersed in a solution containing 13 nm diameter gold nanoparticles to which the oligonucleotide (3 ′ thiol ATGCTCAACTCT [SEQ ID NO: 33]) was attached. An additional nanoparticle layer was added. After soaking for 12 hours, the slides were removed from the nanoparticle solution, rinsed and soaked in the hybridization solution as described above. The color of the slide was so deep red that it was clear. See FIG. 19A. The ligated oligonucleotide and nanoparticle soaking procedure was repeated using 13 nm gold nanoparticles modified with oligonucleotide (TAGGACTTACGC5'thiol [SEQ ID NO: 35]) as the final nanoparticle layer to add the final nanoparticle layer. Again, the color became darker and the UV-visible absorbance at 520 nm increased. See FIG. 19A.
[0331]
Melting curves were created to confirm that the oligonucleotide-modified gold nanoparticles were attached to the oligonucleotide-modified surface by DNA hybridization interaction with the ligated oligonucleotide. For thawing experiments, slides were placed in cuvettes containing 1.5 ml of hybridization buffer and an apparatus similar to that used in Example 2, Part B was used. While increasing the temperature of the substrate from 20 ° C. to 80 ° C., the absorbance signal (520 nm) attributable to the nanoparticles was monitored at a retention time of 1 ° C./1 minute. When the temperature exceeded 52 ° C., the signal of the nanoparticles dropped dramatically. See FIG. 19B. The first derivative of the signal showed a melting temperature of 55 ° C., which is consistent with the temperature seen for the oligonucleotide-nanoparticle conjugate and the ligated oligonucleotide hybridized in solution. See FIG. 19B.
[0332]
Example 11:Polyribonucleotide assay using nanoparticle-oligonucleotide conjugate as probe
The previous examples utilized oligo-deoxyribonucleotides as targets in the assay. This example demonstrates that nanoparticle-oligonucleotide conjugates can be used as probes in polyribonucleotide assays. Poly (rA) (0.004₂₆₀1) of the solution of dT via a 5 'terminal mercaptoalkyl linker.₂₀The experiment was performed in addition to 100 μL of gold nanoparticles (〜１０10 nM in the particles) conjugated to (20 mer oligonucleotide containing thymidylate residue). The coupling procedure was as described in Example 3. As described in Example 4, after freezing in a dry ice / isopropyl alcohol bath, thawing at room temperature, and spotting on a C18 TLC plate, a blue spot characteristic of aggregation of nanoparticles by hybridization is observed. Was done. Control experiments performed in the absence of target yielded pink spots instead of blue spots.
[0333]
Example 12:Assay of anthrax protective antigen segments using nanoparticle-oligonucleotide conjugates
In many cases, amplification of a double-stranded DNA target by PCR requires providing enough material for the assay. This example uses a nanoparticle-oligonucleotide conjugate to assay a DNA strand in the presence of its complement (i.e., assay for single strand after thermal dehybridization of double stranded target). And recognizes the amplicon obtained from the PCR reaction and demonstrates that it can specifically bind.
[0334]
A PCR solution containing a 141 base pair double stranded amplicon of the protective antigen segment of B. anthracis was provided by the Navy (Navy) (sequence shown in FIG. 12). The amplicon assay was performed using a Qiaquick Nucleotide Removal Kit (Qiagen, Santa Clarita, Calif.) (Qiagen, Inc., Santa Clarita, Calif.) And standard protocols for this kit from 100 μl of PCR solution. , Except that the DNA was eluted using a 10 mM phosphate buffer at pH 8.5 instead of using the buffer provided in the kit. The eluent was then evaporated to dryness on a Speed Vac (Savant). To this residue was added 5 μl of a master mixture prepared by mixing equal volumes of each of the two solutions consisting of two different oligonucleotide-nanoparticle probes (see FIG. 23). Each oligonucleotide-nanoparticle probe was prepared as described in Example 3. The combined probe solution to form the master mixture was 10 μl of 2 M NaCl and 5 μl of oligonucleotide blocker solution (50 pmol of each blocker oligonucleotide in 0.3 M NaCl, 10 mM phosphoric acid, pH 7.0 solution (FIG. 23 and below). ) Was prepared by adding 5 μl of a nanoparticle-oligonucleotide solution of full strength (about 10 nM). The amplicon-probe mixture was heated to 100 ° C. for 3 minutes, then frozen in a dry ice / ethanol bath and thawed to room temperature. Small aliquots (2 μl) were spotted on C18 TLC plates and dried. A dark blue spot indicating hybridization was obtained.
[0335]
Control tests were performed in the same manner in the absence of the amplicon target, in the absence of probe 1, in the absence of probe 2, or in the absence of sodium chloride, but all were negative, ie pink spots were obtained. . Similarly, tests performed with probes 1 and 2 containing a PCR amplicon from the anthrax lethal factor segment instead of the protective antigen segment are negative (pink spots). These controls confirmed that both probes were essential, that appropriate salt conditions were required for hybridization, and that the test was specific for a particular target sequence.
[0336]
An oligonucleotide blocker was added to prevent the second strand of the initial double-stranded target (ie, the strand complementary to the target strand) from binding to the target nucleic acid region outside the segment that binds to the probe (with respect to the sequence). Is shown in FIG. 23). Because such binding interferes with the binding between the nanoparticle oligonucleotide probe and the target strand. In this example, the blocker oligonucleotide was complementary to the single-stranded target in a region not covered by the probe. An alternative strategy is to use a blocker oligonucleotide that is complementary to the PCR complementary strand (the strand that is complementary to the target strand) outside of the region that competes with the probe oligonucleotide.
[0337]
Example 13:Direct PCR amplicon assay without isolating amplicons from PCR solutions
The procedure described in Example 12 involved the step of separating the PCR amplicon from the PCR solution before adding the nanoparticle-oligonucleotide probe. For many purposes, it would be desirable to be able to perform assays directly in PCR solutions without prior isolation of the polynucleotide product. Protocols for such assays have been developed and are described below. This protocol has been successfully performed using the GeneAmp PCR Reagent Kit containing Amplitaq DNA polymerase and using several PCR products derived under standard conditions.
[0338]
Two kinds of gold nanoparticles-oligonucleotide probes (each 0.008 A₅₂₀5 μl of the mixture (unit), then 1 μl of the blocker oligonucleotide (10 pmol each), 5 μl of 5 M NaCl and 2 μl of 150 mM MgCl 2₂Was added. The mixture is heated at 100 ° C. for 2 minutes to separate the strands of the double-stranded target, the tube is immersed directly in a cold bath (eg, dry ice / ethanol) for 2 minutes, then the tube is removed and the solution is removed. Thawed at room temperature (freeze-thaw cycles facilitate hybridization of probe to target oligonucleotide). Finally, several μl of the solution was spotted on a plate (eg, C18 RP TLC plate, silica plate, nylon membrane, etc.). As an example, blue indicates the presence of the target nucleic acid in the PCR solution, and pink is negative for this target.
[0339]
Example 14:Direct recognition of double-stranded oligonucleotides without dehybridization using aggregates of nanoparticle-oligonucleotide conjugates
In previous examples, the double-stranded target was heated and dehybridized to generate a single strand that interacted with the single-stranded oligonucleotide probe attached to the nanoparticle. This example demonstrates that a double-stranded oligonucleotide sequence can be recognized by a nanoparticle when a triple-stranded complex can be formed without prior target dehybridization.
[0340]
Two different systems, poly A: poly U and dA₄₀: DT₄₀And 0.8 A in 100 μl of buffer (0.1 M NaCl, 10 mM phosphoric acid, pH 7.0).₂₆₀One microliter of a solution containing units of target duplex is added to Au-sdT in 0.3 M NaCl, 10 mM phosphate buffer (pH 7.0).₂₀The test was performed by adding 100 μl of a colloid solution of the nanoparticle-oligonucleotide conjugate (共 役 10 nM in the particles; see Example 11). The tube was then immersed in a dry ice / isopropyl alcohol bath for quick freezing, the tube was removed from the bath, thawed and left at room temperature (22 ° C.), then 3 μl of the solution was placed on a C18 TLC plate. Spotting resulted in a blue spot characterizing the hybridization and aggregation of the nanoparticles.
[0341]
The principle of this test is that a nanoparticle probe (in this example carrying a pyrimidine oligonucleotide) binds in a sequence-specific manner at a purine oligonucleotide / pyrimidine oligonucleotide site along a double-stranded target. The binding forms nanoparticle aggregates because many binding sites on each duplex are available. These results indicate that this assay, based on a triple-stranded complex containing a nanoparticle probe, works with both oligoribonucleotide and oligodeoxyribonucleotide duplex targets.
[0342]
Example 15:Assays using fluorescence and colorimetric detection
All hybridization experiments were performed in 0.3 M NaCl, 10 mM phosphoric acid, pH 7.0, buffer. AcetatePlus^TMFiltration membrane (0.45 μm) was purchased from Micron Separations Inc., Westboro, Mass., Westborough, Mass. Alkylamine functionalized latex microspheres (3.1 μm) were purchased from Bangs Laboratories, Fishers, IN (Bangs Laboratories, Fishers, IN). Standard phosphoramidite chemistry (Eckstein ed.) Using Amino-Modifier C7 CPG solid support (Glen Research) and 5'-fluorescein phosphoramidite (6-FAM, Glen Research) on an Expedite 8909 synthesizer. , Oligonucleotides and Analogues, 1st edition, Oxford University, New York, NY, 1991) to synthesize a fluorophore-labeled oligonucleotide having a 3 'terminal functionalized with an alkylamino group, Purified by HPLC. These oligonucleotides are coupled to amine-functionalized latex microspheres using diisothiocyanate coupling to create dithiourea linkages as described in Charreyre et al., Langmuir, Vol. 13, pages 3103-3110 (1997). Attached. Briefly, a 1000-fold excess of a solution of 1,4-phenylenedithiocyanate in DMF was added to an aqueous borate buffer of amino-modified oligonucleotides (0.1 M, pH 9.3). After several hours, the excess 1,4-phenylenediisothiocyanate was extracted with butanol and the aqueous solution was lyophilized. The activated oligonucleotide was redissolved in borate buffer and reacted with amino-functionalized latex microspheres in carbonate buffer (0.1 M, pH 9.3, 1 M NaCl). After 12 hours, the particles were centrifuged and washed three times with a buffered saline solution (0.3 M NaCl, 10 mM phosphoric acid, pH 7.0). 5'-oligonucleotide modified gold nanoparticles were prepared as described in Example 3.
[0343]
The target oligonucleotide (1-5 μl, 3 nM) was added to 3 μl of a fluorophore-labeled oligonucleotide-modified latex microsphere probe solution (3.1 μm; 100 fM). After 5 minutes, 3 μl of 5 ′ oligonucleotide-modified gold nanoparticle probe solution (13 nm; 8 nM) was added to the solution containing target and latex microsphere probe. After standing for an additional 10 minutes, the solution containing the probe and target was vacuum filtered through an AcetatePlus membrane. The membrane retained relatively large latex particles and allowed all unhybridized gold nanoparticle probes to pass through. Red spots were observed on the membrane in the presence of sufficient concentration of target and latex microspheres and gold nanoparticles hybridized to the target (positive result). Control experiments in which an aliquot of the solution containing the target oligonucleotide was replaced with an equal volume of water were always performed. In this case, a white spot was left on the membrane (negative result). In the case of the 24-base pair model system, 3 fmol of the target oligonucleotide could be visually detected with a colorimeter.
[0344]
Double-stranded target oligonucleotide (1-5 μl, 20 nM), 3 μl fluorophore-labeled oligonucleotide-latex microspheres (3.1 μm; 100 fM) and 3 μl 5′-oligonucleotide-gold nanoparticles (13 nm; 8 nM) And heated to 100 ° C. for 3 minutes. The reaction vessel containing the solution was then immediately flushed with liquid N₂Immerse in the bath for 3 minutes and freeze. This solution was then thawed at room temperature and filtered as described above. For a 24-base pair model system, 20 fmol of double-stranded oligonucleotide could be detected visually with a colorimeter.
[0345]
When monitored by fluorescence, the above detection method proved to be difficult due to background fluorescence from the membrane. This problem was overcome by centrifuging and "washing" the latex microspheres to remove excess gold nanoparticle probes before spotting aliquots on reversed-phase TLC plates. Hybridization experiments were performed as described above. After performing hybridization between the probe and the target, 10 μl of buffer was added to the solution, followed by centrifugation at 10,000 × g for 2 minutes. The supernatant was removed and 5 μl of buffer was added to help resuspend the precipitate. A 3 μl aliquot was then spotted on a reverse phase TLC plate. For both the single-stranded oligonucleotide and the double-stranded target oligonucleotide, 25 fmol could be visually detected with a colorimeter. The fluorescent spot was visible to the naked eye using a hand-held UV lamp until the amount of target in the 3 μl aliquot used to form the spot was as low as 50 fmol. Optimizing this system will also allow detection of lower amounts of target nucleic acid.
[0346]
Example 16:Assay using silver staining
In order to detect the presence of a specific DNA sequence in a solution, a DNA hybridization test on an oligonucleotide-modified substrate is generally used. The increasing promise of combinatorial DNA sequences for scrutinizing genetic information illustrates the importance of these heterologous sequence assays for future science. In most assays, hybridization of a fluorophore-labeled target with a surface-bound probe is monitored by fluorescence microscopy or densitometry. Although fluorescence detection is extremely sensitive, its use is limited by laboratory equipment costs and background emission from most conventional substrates. In addition, the surface hybridization test to detect single nucleotide polymorphisms cannot be used because of the lower selectivity of a completely complementary probe to a labeled oligonucleotide target as compared to one having a single base mismatch. The detection scheme with improved simplicity, sensitivity and selectivity of the fluorescence method can embody all the possibilities of combinatorial sequences. The present invention provides such an improved detection scheme.
[0347]
For example, in a three-component sandwich assay, oligonucleotide-modified gold nanoparticles and an unmodified DNA target could be hybridized to an oligonucleotide probe attached to a glass substrate (see FIGS. 25A-B). The nanoparticles can be individual (see FIG. 25A) or a “tree” of multiple nanoparticles (see FIG. 25B). The "tree" increases the signal sensitivity compared to the individual nanoparticles, and the "tree" of hybridized gold nanoparticles is often visible to the naked eye as a dark region on the glass substrate. Without a "tree" or to amplify the signal generated by the "tree", the hybridized gold nanoparticles can be treated with a silver staining solution. The "tree" facilitates the staining process and makes detection of target nucleic acids faster than individual nanoparticles.
[0348]
The following is a description of one particular system (illustrated in FIG. 25A). The capture oligonucleotide (3'-HS (CH₂)₃-A₁₀ATGCTCAACTCT; SEQ ID NO: 43) was immobilized on a glass substrate as described in Example 10. The target oligonucleotide (5'-TACGAGTTTGAGAATCCTGAATGCCG-3 ', SEQ ID NO: 44, the concentration shown in Table 6 below for each experiment) and the capture oligonucleotide were compared to 0.3 M as described in Example 10. Hybridization was performed in NaCl, 10 mM phosphate buffer. The substrate was rinsed twice with the same buffer, and the target complementary DNA (5'-HS (CH₂)₆A₁₀(CGCATTCAGGAT, SEQ ID NO: 45) (formulation described in Example 3) was immersed in a solution containing a gold nanoparticle probe functionalized for 12 hours. Then, the substrate was₃Rinse many times with Cl⁻Was removed. The substrate was then developed with a silver staining solution (Silver Enhancer Solutions A and B 1: 1 mixture, Sigma Chemical Co., # S-5020 and # S-5145) for 3 minutes. Scan the substrate with a flatbed scanner (typically used to scan documents into a computer) coupled to a computer loaded with software capable of calculating grayscale measurements (eg, Adobe Photoshop), and perform the grayscale measurements. went. The results are shown in Table 6 below.
[0349]
[Table 6]

Example 17:Aggregates containing quantum dots
This example illustrates the immobilization of synthetic single-stranded DNA on semiconductor nanoparticle quantum dots (QDs). Since natural CdSe / ZeS core / shell QDs (ｎｍ4 nm) are soluble only in organic media, they do not readily react directly with alkyl thiol-terminated single-stranded DNA. This problem was avoided by first capping the QD with 3-mercaptopropionic acid. The carboxylic acid group was then deprotected with 4- (dimethylamino) pyridine to render the particles water soluble and to facilitate the reaction of the QD with the 3'-propylthiol or 5'-hexylthiol modified oligonucleotide sequence. After DNA modification, particles were separated from unreacted DNA by dialysis. The "linker" DNA strand was then hybridized with the surface binding sequence to produce a long nanoparticle assembly. QD assemblies characterized by TEM, UV / visible spectroscopy and fluorescence microscopy can be reversibly assembled by controlling solution temperature. Temperature-dependent UV-visible spectra of the novel QD aggregates and the composite aggregates formed between QDs and gold nanoparticles (粒子 13 nm) were obtained.
[0350]
A.General law
Nanopure water (18.1 MΩ) prepared using a NANOpure ultrapure water purification system was used consistently. Fluorescence spectra were obtained using a Perkin Elmer LS 50B fluorescence spectrometer. Melting analysis was performed using an HP 8453 diode array spectrophotometer equipped with an HP 9090a Peltier Temperature Controller. Centrifugation was performed using an Eppendorf 5415 centrifuge or a Beckman Avanti 30 centrifuge. TEM images were obtained using a Hitachi HF-2000 field emission TEM operating at 200 kV.
[0351]
B.Preparation of oligonucleotide-QD conjugate
The synthesis of semiconductor quantum dots (QDs) has improved significantly in recent years, making it relatively easy now to prepare monodisperse samples of predetermined sizes for certain materials, most notably CdSe. I can do it. Murray et al. Am. Chem. Soc. 115, 8706, 1993; Hines et al. Phys. Chem. 100, 468, 1996. As a result, QDs can be combined with light emitting diodes (Schlamp et al., J. Appl. Phys., 82, 5837, 1997; Dabbousi et al., Appl. Phys. Lett., 66, 1316, 1995) and non-light emitting diodes. Possibilities for use in a variety of technologies such as radiobiological labels (Bruchez et al., Science, 281, 2013, 1998; Chan et al., Science, 281, 2016, 1998). The intrinsic electronic and fluorescent properties of some of these particles have been extensively studied (Aliviatos, J., Phys. Chem., 100, 13226, 1996, and references therein; Klein et al.). , Nature, 699, 1997; K. . No, et al., J.Chem.Phys, Vol. 106, 9869 pages, 1997; Nirmal et al., Nature, 383, pp. 802 pages, see 1996). However, many applications will require that these particles be spatially arranged on a surface or organized in a three-dimensional material (Vossmeyer et al., J. Appl. Phys. 84, 3664, 1998). In addition, the ability to organize one or more nanoparticles into a superlattice structure (Murray et al., Science, 270, 1335, 1995) would provide a complete and potentially interesting and useful property. New types of hybrid materials will be possible.
[0352]
DNA is an ideal synthon for programming nanoscale building block assemblies into periodic two- and three-dimensional extended structures. Many properties of DNA, including ease of synthesis, very high binding specificity and virtually unlimited programmability by nucleotide sequence, can be exploited for use with QD assemblies.
[0353]
Modification of QDs with DNA has proven more difficult than with gold nanoparticles. The general method for preparing highly fluorescent CdSe QDs results in materials coated with a mixture of trioctylphosphine oxide (TOPO) and trioctylphosphine (TOP). As a result, these QDs are only soluble in non-polar solvents, which makes it difficult to functionalize QDs by direct reaction with highly charged DNA strands. This difficulty was overcome by the method described below, which succeeded in modifying semiconductor nanoparticles with single-stranded DNA for the first time. Others have considered the interaction of QDs with double-stranded DNA in rigorous studies, but these studies exploit the sequence-specific binding properties of DNA to induce assemblies of extended QD structures. Note that there was no. Coffer et al., Appl. Phys. Lett. 69, 3851, 1996; Mahtab et al. Am. Chem. Soc. 148, 7028, 1996.
[0354]
Since the surface of the CdSe / ZnS core / shell QD binds to an organic thiol, it is desirable to modify these semiconductor particles with a DNA chain having an alkylthiol as a terminal group by a substitution reaction. However, such methods were hampered because these QDs were not water-soluble. Recently, two different methods have been reported for making QDs water soluble and immobilizing protein structures on QD surfaces. One involves encapsulating the core / shell structure with a silica layer (Bruchez et al., Science, Vol. 281, 2013, 1998) and the other is mercaptoacetic acid to stabilize the particles and make them water-soluble. (Chan et al., Science, Vol. 281, 2016, 1998). The procedure described in this example, which produces a significantly stable colloid under DNA hybridization conditions, utilizes 3-mercaptopropionic acid to passivate the QD surface.
[0355]
-20 mg (prepared as described in Hines et al., J. Phys. Chem., 100, 468, 1996) in 1.0 ml of N, N-dimethylformamide (DMF; Aldrich). To a suspension of TOP / TOPO stabilized CdSe / ZnS QD was added an excess of 3-mercaptopropionic acid (0.10 ml, 1.15 mmol; Aldrich) using a syringe to give 3-mercaptopropionic acid. A clear dark orange solution containing the functionalized QD was produced. The reaction took place rapidly. For subsequent reactions, the excess 3-mercaptopropionic acid was not removed and the particles were stored at room temperature in DMF.
[0356]
However, for characterization of QD, unreacted 3-mercaptopropionic acid was removed from a portion of the sample and purified as follows. A 0.50 ml sample was centrifuged (4 hours at 30,000 rpm) and the supernatant was removed. The remaining solution was washed with ０．３0.3 ml of DMF and recentrifuged. After repeating this step two more times, the FTIR spectrum was recorded. FTIR (polyethylene card, 3M): 1710cm^-1(S), 1472cm^-1(M), 1278cm^-1(W), 1189cm^-1(M), 1045cm^-1(W), 993cm^-1(M), 946cm^-1(W), 776cm^-1(M), 671cm^-1(M). Unlike TOP / TOPO stabilized native QDs, 3-mercaptopropionic acid modified QDs have a characteristic 1710 cm^-1V_coThe band showed.
[0357]
Although 3-mercaptopropionic acid-modified QDs are substantially insoluble in water, their solubility is determined by adding a surface-bound mercaptopropionic acid moiety to 4- (dimethylamino) pyridine (DMAP; old) as described in the following paragraph. Rich Co., Ltd.) and significantly increased. The QD was then readily dispersed in water, resulting in an orange solution that was stable at room temperature for up to one week.
[0358]
To attach the oligonucleotide to the QD, 150 μl of a solution of 3-mercaptopropionic acid-functionalized particles in DMF (optical density at 530 nm = 21.4) was added to DMAP (8.0 mg, 0 mg in 0.4 ml DMF). 0.065 mmol). An orange precipitate formed. This was separated by centrifugation (〜30 sec at 3,000 rpm) and then 3′-propylthiol- or 5′hexylthiol-terminated oligonucleotides (1.0-2.0 OD / ml; Prepared as described in 1; the sequence is described below). The precipitate (dissolved in water) was characterized by IR spectroscopy (polyethylene card, 3M). IR (cm^-1): 1647 (m), 1559 (s), 1462 (m), 1214 (w), 719 (w), 478 (s). After standing for 12 hours, the oligonucleotide containing solution was added to 0.15 M NaCl and the particles were aged for another 12 hours. The concentration of NaCl was then increased to 0.3 M and the mixture was left for a further 24 to 40 hours before using a 100 kDa membrane (Spectra / Por Cellulose Ester Membrane) with PBS (0.3 M NaCl, 10 mM phosphate buffer, (pH 7, 0.01% sodium azide). Dialysis was performed for 48 hours, during which time the dialysis bath was refreshed three times.
[0359]
Oligonucleotide-QD conjugates prepared in this manner exhibited indefinite water stability. In addition, colloids exhibit a sharp [full width at half maximum (FWHM) at 546 nm (FWHM) = 33 nm (Murray et al., J. Am. Chem. Soc., 115, 8706, 1993). ] It had a symmetric emission and remained strongly fluorescent.
[0360]
With this protocol, two different oligonucleotide-QD conjugates were prepared and stored in PBS. One is a propylthiol function at the 3 'end, a 12 mer capture sequence, and an intervening 10 base (all A) spacer: 5'-TCTCAACTCGTAA.₁₀− (CH₂)₃-SH [SEQ ID NO: 46]. The other is a 5'-hexylthiol-terminated sequence, a further 10 bases (all A) spacer, and a 12-mer capture sequence non-complementary to the 3'-propylthiol sequence: 5'-SH- (CH₂)₆-A₁₀CGCATTCAGGAT-3 '[SEQ ID NO: 47] was used.
[0361]
C.Preparation of QD assembly
Approximately equal amounts of these two oligonucleotides (200 μl each, OD₅₃₀= 0.224 and 0.206) and then combined with 6 μl (60 pmol) of a solution of the complementary ligated 24mer sequence (5′-TACGAGTTTGAGAATCCT-GAATGCCG-3 ′, SEQ ID NO: 48) and mixed at room temperature for 20 minutes. A QD aggregate formed within ~ 30 minutes (Figure 26). The mixture was frozen (−78 ° C.) and slowly warmed to room temperature, resulting in faster ligation.
[0362]
The cluster formed was not large enough to precipitate from solution. However, the clusters could be separated by centrifugation (10,000 rpm for 10 minutes) at a relatively low speed compared to the unconnected particles (2,3 hours at 30,000 rpm).
[0363]
The decrease in fluorescence immediately after hybridization was quantified by integration of the fluorescence signals from 475 nm to 625 nm (excitation wavelength 320 nm) of four pairs of samples. Each pair was prepared in the following manner. In an Eppendorf centrifuge tube, a solution of 3′-propylthiol-terminated DNA-modified particles (30 μl, optical density at 530 nm = 0.224) was modified with 5′-hexylthiol-terminated DNA. Combined with QD (30 μl, optical density at 530 nm = 0.206) and then diluted with 140 μl of PBS. The mixture was then divided into two equal parts, one containing complementary "linker" DNA (3 μl, 30 pmol) and the other a non-complementary "linker" DNA (5'-CTACTTAGATCCGAGTGCCCACAT-3 ', SEQ ID NO: 49) (3 μl, 30 pmol) was added. All eight samples were then frozen in a dry ice / acetone bath (−78 ° C.), after which the samples were removed from the bath and slowly warmed to room temperature. To predict the change in fluorescence efficiency upon hybridization, the fluorescence intensity of the "target" (complementary "linker") sample is determined by the difference in absorbance at 320 nm from the corresponding control sample containing the non-complementary "linker". Adjusted to clarify.
[0364]
The results indicate that the QD / QD assembly averages 26.4 ± 6.1% reduction in integrated fluorescence intensity and a red shift of ２2 nm in emission maximum attributed to a cooperative event between QDs. Indicated. Interestingly, Bawendi et al. Noticed a similar but slightly larger red shift when comparing the fluorescence of densely separated QDs and isolated dots that are widely separated in frozen matrices (Murray et al., Science). 270, 1335, 1995). Although these changes in the fluorescence spectrum may indicate that excimers have been formed between the QDs, the exact nature of such complexes is largely still out of analogy. . As expected, no aggregation was observed without the "linker" or not complementary, or in the absence of either of the two particles.
[0365]
The "melting" behavior of the DNA was monitored by observing the UV-visible spectrum of the aggregate as a function of temperature. For this “thaw” analysis, the precipitate containing the QD / QD aggregates was centrifuged at 10,000 rpm for 10 minutes, washed with 7 μl of PBS, re-centrifuged and suspended in 0.7 ml of PBS. The UV / visible spectral signature of the assemblage was recorded at 2 ° C intervals with a 1 minute hold time before each measurement as the temperature was raised from 25 ° C to 75 ° C. The mixture was stirred at a speed of 500 rpm to ensure homogeneity throughout the experiment. A temperature vs. extinction profile was then created from the extinction at 600 nm. The "melting" temperature was determined using the first derivative of these profiles.
[0366]
As a result, FIG. 27B (T_m= 57 ° C.) clearly demonstrated that the DNA was immobilized on the QD surface and that hybridization was involved in the assembly process. The shift when compared to DNA alone is very steep (FWHM of each first derivative: 4 ° C vs. 9 ° C), which is consistent with the formation of aggregate structures with multiple DNA bonds per particle. An increase in quenching due to denaturation was observed, most likely due to a screening effect where particles in the aggregate were prevented from absorbing light by the surrounding QDs.
[0367]
D.Preparation of QD / gold assembly
Having DNA-functionalized QDs, the construction of hybrid assemblies made from multiple types of nanoparticle building blocks has become feasible. To make these hybrid assemblies, 13 nm gold nanoparticles modified with １７17 nM 3′-hexylthiol (30 μl, ５5 fmol; prepared as described in Example 3) were prepared in an Eppendorf centrifuge tube. The solution was mixed with 5'-hexylthiol-terminated DNA-modified QDs (15 μl, optical density at 530 nm 0.206). “Linker” DNA (5 μl, 50 pmol) was added and the mixture was cooled to −78 ° C. and then slowly warmed to room temperature, producing a reddish purple precipitate. No aggregation behavior was observed in the absence of both types of particles and the complementary target. After centrifugation (3,000 rpm for 1 minute) and removal of the supernatant, the precipitate was washed with 100 μl of PBS and centrifuged again.
[0368]
The washed precipitate was suspended in 0.7 ml of PBS for "melting" analysis. Changes in surface plasmon resonance of the gold nanoparticles were tracked using UV-visible spectroscopy to create a temperature vs. extinction profile at 525 nm. Using surface plasmon resonance of gold nanoparticles provides a much more sensitive probe for monitoring hybridization than using a QD-only UV-visible spectral signature. Thus, "melting" experiments can be performed on much smaller samples (requires -10% of the QD solution), but the intensity of the plasmon band blurs the UV / visible signal from the QD. As with the pure QD system described above, a sharp (FWHM of first derivative = 4.5 ° C) melting transition occurred at 58 ° C (see Figure 27D).
[0369]
High resolution TEM images of these assemblies showed a gold nanoparticle network interconnected by multiple QDs (FIG. 27C). QDs that have much lower contrast than gold nanoparticles in TEM images can be identified by their grating fringes. QDs can barely be resolved with high-resolution TEM, but clearly show the periodic structure of these complex assemblies and the role that DNA plays in their formation.
[0370]
E. FIG.wrap up
The results described in this example most reliably demonstrate that immobilization of DNA on the QD surface has been achieved and that these particles can now be used in combination with DNA under hybridization conditions. . Using DNA-functionalized QDs, initial DNA-induced QD formation and mixed gold / QD nanoparticle structures were demonstrated. The successful modification of semiconductor QDs with DNA has important implications for materials research, and these unique building blocks are now being incorporated into new and functional multicomponent nanostructures and nanoscale materials, and are now The door has been opened to extensive study of the fluorescent, electronic, and chemical properties of these blocks.
[0371]
Example 18:Synthetic methods of oligonucleotide-nanoparticle conjugates and conjugates produced by those methods
A.General law
HAuCl₄・ 3H₂O and trisodium citrate were purchased from Aldrich Chemical Company, Milwaukee, WI, Milwaukee, WI. 99.999% pure gold and titanium wires were purchased from Goldsmith Inc., Evanston, Ill. Silicon wafers (100) with a 1 micron thick oxide layer were purchased from Silicon Quest International, Santa Clara, CA, Santa Clara, CA. 5'-thiol modifier C6-phosphoramidite reagent, 3'-propyl thiol modifier CPG, fluorescein phosphoramidite, and other reagents required for oligonucleotide synthesis were purchased from Glen Research, Inc., Stirling, VA. did. All oligonucleotides were developed using an automated DNA synthesizer (Expedited) using standard phosphoramidite chemistry (Eckstein, F., Oligonucleotides and Analogues; First Edition; Oxford University Press, New York, 1991). Prepared. Oligonucleotides containing only the 5'hexylthiol modification were prepared as described in Example 1. 5- (and 6-)-Carboxyfluorescein, succinimidyl ester, was purchased from Molecular Probes, Eugene, OR, Eugene, Oregon. NAP-5 columns (Sephadex G-25 Medium, DNA grade) were purchased from Pharmacia Biotech. For all experiments, Nanopure H purified using a Barnstead NANOpure ultrapure water system₂O (> 18.0 MΩ) was used. For centrifugation of the Au nanoparticle solution, an Eppendorf 5415C or Beckman Avanti 30 centrifuge was used. High performance liquid chromatography was performed using HP series 1100 HPLC.
[0372]
B.Physical measurement
Electronic absorption spectra of oligonucleotide and nanoparticle solutions were recorded using a Hewlett-Packard (HP) 8452a diode array spectrophotometer. Fluorescence spectroscopy was performed using a Perkin-Elmer LS50 fluorimeter. Transmission electron microscopy (TEM) was performed on a Hitachi 8100 transmission electron microscope operating at 200 kV. The atomic concentration of gold in the nanoparticle solution was determined using an AtomScan 25 atomic spectrometer equipped with an inductively coupled plasma (ICP) source (gold emission was monitored at 242.795 nm).
[0373]
C.Synthesis and purification of fluorescently labeled alkanethiol-modified oligonucleotides
A thiol-modified oligonucleotide chain containing 12 bases was prepared using a 5 'hexyl thiol moiety and a 3' fluorescein moiety. The sequence of 12mer (S12F) is HS (CH₂)₆5-CGC-ATT-CAG-GAT-3 '-(CH₂)₆-F [SEQ ID NO: 50] and 32mer (SA₂₀12F) had the same 12mer sequence and an additional 20dA spacer sequence at the 5 'end [SEQ ID NO: 51]. Thiol-modified oligonucleotides are described in Storhoff et al. Am. Chem. Soc. 120: 1959-1964 (1998). The automated synthesis used the amino modifier C7 CPG solid support and the 5 'end was manually modified with hexylthiol phosphoramidite as described above. A 3 'amino, 5' trityl protected thiol modified oligonucleotide was prepared using 0.03 M triethylammonium acetate (TEAA), pH 7 and 95% CH with a 1% / min gradient while monitoring the UV signal of the DNA at 254 nm.₃Purified by reverse phase HPLC using a HP ODS Hypersil column (5 mm, 250 × 4 mm) containing CN / 5% 0.03 M TEAA at a flow rate of 1 ml / min. The retention times of the 5'-S-trityl, 3 'amino modified 12 base and 32 base oligonucleotides were 36 minutes and 32 minutes, respectively.
[0374]
The lyophilized product was added to 1 ml of 0.1 M Na₂CO₃And 10 mg / ml fluorescein succinimidyl ester (5,6 FAM-SE, Molecular Probes) in anhydrous DMF according to the manufacturer's instructions (Molecular Probes literature) with stirring in the dark while stirring in the dark. 100 μl was added over 1.5 hours. The solution was stirred at room temperature for a further 15 hours and then precipitated from 100% ethanol at -20 ° C. The precipitate is collected by centrifugation and₂The bound product was separated from unreacted amino-terminated oligonucleotides by ion exchange HPLC. A Dionex Nucleopac PA-100 column (250 × 4 mm) was operated at a flow rate of 0.8 ml / min using a 10 mM NaOH aqueous eluent and a 1% / min gradient of 1 M NaCl / 10 mM NaOH. The retention times of the 5'-S-trityl, 3 'fluorescein modified 12mer and 32mer were 50 minutes and 49 minutes, respectively. Oligonucleotide products were desalted by reverse phase HPLC. Removal of the trityl protecting group of a fluorescein-terminated trityl oligonucleotide is described previously (Storhoff et al., J. Am. Chem. Soc., 120, 1959-1964 (1998)). Performed using silver nitrate and dithiothreitol (DTT). Oligonucleotide yield and purity were evaluated using the techniques described previously for alkylthiol oligonucleotides (Storhoff et al., J. Am. Chem. Soc., 120: 1959-1964 (1998)). . Oligonucleotides were used immediately after trityl removal of the thiol groups.
[0375]
3 ′ is propylthiol and the 5 ′ fluorescein moiety (HS (CH₂)₃-3'- (W)₂₀-TAG-GAC-TTA-CGC-5 '-(CH₂)₆-F, W = A or T), a thiol-modified oligonucleotide containing 32 bases [SEQ ID NO: 52] was synthesized with an automatic synthesizer using a 3 ′ thiol modifier CPG. The 5 'end of each oligonucleotide was manually ligated to fluorescein phosphoramidite (6-FAM (6-FAM, Glen Research). The modified oligonucleotide was purified by ion exchange HPLC (1% of 1 M NaCl). / Min gradient, 10 mM NaOH; retention time (Rt) -48 min (W = T), Rt-29 min (W = A)) After purification, the oligonucleotide solution was desalted by reverse phase HPLC. Portions were deprotected with dithiothreitol by the method previously described (Storhoff et al., J. Am. Chem. Soc. 120: 1959-1964 (1998)).
[0376]
D.Synthesis and purification of fluorescein-labeled oligonucleotide
Fluorophore-labeled complement (12'F) was obtained from S12F and SA₂₀12 bases 3′-GCG-TAA-GTC-CTA-5 ′-(CH₂)₆-F [SEQ ID NO: 53]. Oligonucleotides were synthesized using standard methods and fluorescein phosphoramidites (6-FAM, Glen Research) and CPG-linked oligonucleotides were synthesized using a syringe-based procedure similar to that described above for 5'alkylthiol modification. The 5 'end was coupled. Purification was performed using reverse phase HPLC as described above. The retention time of the fluorescein-labeled oligonucleotide was 18 minutes. Using an amino modifier C7 CPG solid support for an automatic synthesizer, a fluorophore-labeled complement, 3′12F (5′-ATC-CTG-AAT-GCG-F; [SEQ ID NO: 54]) was prepared. The 5- (6) -carboxyfluorescein succinimidyl ester was then coupled to the 3 'amine using the procedure described above.
[0377]
E. FIG.Preparation and characterization of gold nanoparticles
The gold nanoparticles were HAuCl2, as described in Example 1.₄Was prepared by reduction with citrate. Transmission electron microscopy (TEM) performed using a Hitachi 8100 TEM was used to quantify the size distribution of the obtained nanoparticles. At least 250 particles were sized from the TEM negative using graphic software (IamgeTool). The average diameter of a typical particle formulation was 15.7 ± 1.2 nm. The spherical nanoparticles and density are the bulk gold density (19.30 g / cm²) Was calculated and the average molecular weight per particle was calculated (2.4 × 10⁷g / mol). The concentration of gold atoms in the gold nanoparticle solution was determined by ICP-AES (Inductively Coupled Plasmon Atomic Emission Spectroscopy). For calibration, a gold atom adsorption standard solution (Aldrich) was used. Comparison of the concentration of gold atoms in the particle solution with the average particle mass obtained by TEM analysis gave the molar concentration of gold particles in a given formulation, typically 〜１０10 nM. Measuring the UV-visible absorbance of the nanoparticle solution at the surface plasmon frequency (520 nm) and calculating the molar extinction coefficient (ε at 520 nm) of the particles typically yields a 15.7 ± 1.2 nm diameter. 4.2 × 10 for particles⁸M^-1cm^-1Met.
[0378]
F.Preparation of gold thin film
A silicon wafer is cut into 10 mm × 6 mm pieces, and a piranha corrosion solution (4: 1 concentrated H₂SO₄： 30% H₂O₂) For 30 minutes and then rinsed with plenty of water and then ethanol. (Warning: piranha etch solution reacts violently with organic substances and must be handled with great care.) Edwards Auto 306 evaporator equipped with Edwards FTM6 quartz crystal microbalance (3 x 10-7 mbar reference pressure) ) Was used to deposit metal at a rate of 0.2 nm / sec. The oxidized side of the silicon was coated with a 5 nm Ti adhesion layer followed by 200 nm gold.
[0379]
G. FIG.Preparation of 5'alkylthiol oligonucleotide modified gold nanoparticles
The newly deprotected oligonucleotide was added to the aqueous nanoparticle solution (particle concentration 〜１０10 nM) to a final oligonucleotide concentration of 3 μM, and the gold nanoparticles were modified with fluorescein-alkylthiol oligonucleotide. After 24 hours, the solution was buffered at pH 7 (0.01 M phosphoric acid) and NaCl solution was added (to a final concentration of 0.1 M). The solution was "aged" under these conditions for an additional 40 hours. Then, it was centrifuged at 14,000 rpm for 30 minutes to remove excess reagent. After removing the supernatant, the red oily precipitate was washed twice with 0.3 M NaCl, 10 mM phosphate buffer, pH 7 (PBS), re-dispersed by continuous centrifugation, and finally in fresh buffer. Was redispersed. During the washing procedure, always a small amount (-10% as determined by UV-visible spectroscopy) of the nanoparticles is discarded with the supernatant. Therefore, final nanoparticle concentration was measured by TEM, ICP-AES, and UV-visible spectroscopy (see above). The extinction coefficient and the particle size distribution did not change significantly as a result of the oligonucleotide modification.
[0380]
H. 5 'Preparation of alkylthiol oligonucleotide modified gold thin film
The silicon-supported gold film was immersed in a deposition solution of the deprotected alkylthiol-modified oligonucleotide for the same number of times and under the same buffer conditions as for the gold nanoparticles. After oligonucleotide deposition, the membrane was rinsed well with 0.3 M PBS and stored in buffer. Only one side of the gold was evaporated, leaving the non-passivated silicon / silicon oxide side. However, the alkylthiol-modified DNA did not significantly adsorb to the unbound silicon oxide surface immersed in PBS.
[0381]
I.Determination of alkylthiol oligonucleotides loaded on nanoparticles
To displace the oligonucleotide, mercaptoethanol (ME) was added (to a final concentration of 12 mM) to the nanoparticles or thin films modified with the fluorophore-labeled oligonucleotide in 0.3 M PBS. After 18 hours at room temperature with intermittent shaking, the gold nanoparticles were centrifuged or the gold film was removed to separate the solution containing the substituted oligonucleotide from the gold. An aliquot of the supernatant was diluted 2-fold by adding 0.3 M PBS, pH7. Care was taken to maintain the pH and ionic strength of the sample and calibration standard solution the same for all measurements due to the sensitivity of the optical properties of fluorescein to these conditions (Zhao et al., Spectrochimica Acta, 45A: 1113-1116). Page (1989)). The maximum fluorescence (measured at 520 nm) was converted to the molarity of the fluorescein-alkylthiol modified oligonucleotide by interpolation from a standard linear calibration curve. A standard curve was generated using known concentrations of fluorophore-labeled oligonucleotide using the same buffer and salt concentrations. Finally, the measured oligonucleotide molarity was divided by the original gold nanoparticle concentration to obtain the average number of oligonucleotides per particle. The normal surface coverage value was then calculated by dividing by the expected particle surface area (assuming spherical particles) in the nanoparticle solution. The roundness assumption is based on the calculated average roundness of 0.93. The roundness factor is calculated as (4 x pi x area) (perimeter x 2) quoted from Baxes, Gory, Digital Image Processing, page 157 (1994).
[0382]
J.Quantification of hybridized target surface density
To measure the hybridization activity of the attached oligonucleotide, the oligonucleotide labeled with a fluorophore (complementary to the surface-bound oligonucleotide (12'F)) was hybridized to the hybridization conditions (3 μM complementary oligonucleotide, 0.1 μM). 3M PBS, pH 7, 24 hours) with oligonucleotide-modified surfaces (gold nanoparticles or thin films). Unhybridized oligonucleotide was removed from the gold by rinsing twice with buffered saline as described above. Thereafter, the oligonucleotide labeled with a fluorophore was dehybridized by the addition of NaOH (final concentration ５０50 mM, pH 11-12, 4 hours). After separation of the 12'F-containing solution from the nanoparticle solution by centrifugation, the solution was neutralized by the addition of 1 M HCl, and the concentration of hybridized oligonucleotide and corresponding hybridized target surface density were determined by fluorescence spectroscopy. Was measured by
[0383]
K.Quantification and hybridization of surface coverage
Citrate-stabilized gold nanoparticles were converted to 12-mer fluorescein-modified alkylthiol DNA (HS- (CH₂)₆5'-CGC-ATT-CAG-GAT- (CH₂)₄-F [SEQ ID NO: 50]). The fluorophore-labeled oligonucleotide was then removed from the gold surface after a complete wash-off of the non-adsorbed oligonucleotide, and a surface coverage study was performed, using fluorescence spectroscopy (as described above). The oligonucleotide concentration was quantified.
[0384]
Removing all oligonucleotides from the gold surface and then the gold nanoparticles from the solution is important for obtaining accurate fluorescence coverage data for several reasons. First, the fluorescent signal of the labeled surface-bound DNA is efficiently quenched as a result of fluorescence resonance energy transfer (FRET) to the gold nanoparticles. Indeed, after the fluorescein-modified oligonucleotide (12-32 nucleotide strand, sequence described above) is immobilized on 15.7 ± 1.2 nm gold nanoparticles and the residual oligonucleotide in solution is washed away, There are few measurable signals for fluorescein modified oligonucleotides. Second, the gold nanoparticles absorb a significant amount of light between 200-530 nm, so the presence of the gold nanoparticles in the solution when measuring the fluorescence acts as a filter and can be used It reduces not only the excitation energy but also the intensity of the emitted radiation. At maximum emission of fluorescein, the gold surface plasmon band at 520 nm decreases.
[0385]
The mercaptoethanol (ME) was used to rapidly displace the surface bound oligonucleotide by an exchange reaction. To examine displacement kinetics, oligonucleotide-modified nanoparticles were exposed to ME (12 mM) for an increasing time before centrifugation and fluorescence measurements. The fluorescence intensity associated with the solution without nanoparticles can be used to determine how much oligonucleotide is released from the nanoparticles. The amount of oligonucleotide released in exchange for ME increased up to about 10 hours of exposure (FIG. 29), indicating complete displacement of the oligonucleotide. The displacement reaction is fast, presumably because the oligonucleotide membrane cannot block access of the ME to the gold surface (Biebuyck et al., Langmuir, Vol. 9, p. 1766 (1993)).
[0386]
The average oligonucleotide surface coverage of the alkylthiol-modified 12-mer oligonucleotide (S12F) on the gold nanoparticles was 34 ± 1 pmol / cm.²(Average of 10 individual measurements of the sample). For particles with a diameter of 15.7 ± 1.2 nm, this corresponds to approximately 159 thiol-bound 12-mer chains per gold particle. Although the particle size varied slightly from batch to batch, the area normalized surface coverage was similar for the different nanoparticle formulations.
[0387]
To confirm that this method is useful for obtaining an accurate oligonucleotide surface coverage, use a method to replace fluorophore-labeled oligonucleotides from a thin gold film and obtain information similar to surface coverage data The aim was to compare with experiments using different techniques. In these experiments, gold thin films were subjected to similar oligonucleotide modification and ME replacement procedures as citrate stabilized gold nanoparticles (see above). The oligonucleotide displacement versus time curve of the gold film is very similar to that measured for the gold nanoparticles. This suggests that the replacement rates of the films are similar, despite the typical surface coverage values measured for these films being somewhat lower than the oligonucleotide coverage on the gold nanoparticles. . Oligonucleotide surface coverage on gold thin film measured by our technique (18 ± 3 pmol / cm)²) Is within the previously reported coverage area on the oligonucleotide thin film (10 pmol / cm for a 25 base oligonucleotide on a gold electrode measured using electrochemical or surface plasmon resonance spectroscopy (SPRS)).²(Steel et al., Anal. Chem., 70, 4670-4677 (1998)). It is considered that the difference in the surface coverage is due to the difference in the sequence and length of the oligonucleotide and the difference in the method of preparing the thin film.
[0388]
The extent of hybridization of the complementary fluorophore-labeled oligonucleotide (12'F)) to the nanoparticle with the surface-bound 12mer oligonucleotide was determined as described above. Briefly, S12F-modified nanoparticles were exposed to 12F 'at a concentration of 3 μM under hybridization conditions (0.3 M PBS, pH 7) for 24 hours and then thoroughly rinsed with buffer. Again, it was necessary to remove the hybridized strand from the gold before the fluorescence measurement. This was accomplished by denaturing the double stranded DNA in a high pH solution (NaOH, pH 11) followed by centrifugation. The hybridized 12'F had a total of 1.3 ± 0.2 pmol / cm²(About 6 duplexes per 15.7 nm particle; the average number of duplexes per particle is the normalized hybridized surface coverage (pmol / cm²) Was multiplied by the average particle surface area determined from the particle size distribution measured by TEM. ) Reached. To determine the extent of non-specific adsorption, S12F-modified gold nanoparticles were exposed to a fluorophore-labeled non-complementary 12-base oligonucleotide (12F ') in 0.3 M PBS. After careful rinsing (continuous centrifugation / redispersion step) followed by high pH treatment, the coverage of non-specifically adsorbed oligonucleotides on the nanoparticles is about 0.1 pmol / cm²It turned out to be. To compare the hybridization results with the reported values for the gold electrode, a similar method was used to measure hybridization to the S12F modified gold film. Hybridization 6 + 2 pmol / cm²The extent of the hybridization (2-6 pmol / cm) was reported for the mixed base 25mer on the gold electrode.²) (Steel et al., Anal. Chem. 70: 4670-4677 (1998)).
[0389]
Table 7 summarizes the surface coverage and hybridization values of the S12F / 12F 'system for both the nanoparticle system and the thin films. The most striking result is low hybridization efficiency (less than 4% of the surface-bound strands on the nanoparticles, 33% of the strands on the membrane hybridized). Previous studies have shown similarly low hybridization to well-packed oligonucleotide monolayers. This may reflect the low accessibility of the incoming hybridizing strand due to the combination of steric compactness of the bases (especially near the gold surface) and electrostatic repulsion interactions.
[0390]
L. Effect of oligonucleotide spacer on surface coverage and hybridization
Although the high coverage of the S12F oligonucleotide is advantageous in terms of nanoparticle stabilization, its low hybridization efficiency allows us to reduce steric congestion around the hybridizing sequence. Was prompted to devise. An oligonucleotide (32mer) was synthesized in which a 20dA spacer sequence was inserted between the alkylthiol group and the first 12 base recognition sequence. This strategy was chosen based on the following two assumptions. That is, 1) bases near the nanoparticle surface are sterically inaccessible due to weak interaction between the nitrogen-containing base and the gold surface and steric crowding between chains; For the roughly spherical particles of 0.7 nm, a 12-mer sequence attached to the end of a 20-mer spacer unit that is generally perpendicular to its surface (Levicky et al., J. Am. Chem. Soc. 120: 9787-9792 (1998)). Will lead to a film having a larger free volume compared to a film formed from the same 12mer tied directly to the surface.
[0391]
Single chain SA₂₀Surface density of 12F chain (15 ± 4 pmol / cm²) Is Sl2F (± 1 pmol / cm of 34)²), But the particles modified with the 32mer using the same surface modification showed a significant stability wp compared to the particles modified with the 12mer. As expected, SA₂₀Hybridization efficiency of 12F / 12F 'system "(6.6 ± 0.2 pmol / cm², 44%) increased about 10 times the hybridization efficiency of the original S12F / 12F 'system (Table 7).
[0392]
M.Effect of electrolyte concentration on oligonucleotide attachment
In studies using the S12F sequence, the salt ripening step was found to be important for obtaining stable oligonucleotide-modified nanoparticles (see Example 3). When the gold nanoparticles modified with S12F in pure water were irreversibly melted and centrifuged, a black precipitate was formed, but those aged in salt did not aggregate when centrifuged in a high ionic strength solution. Was. The increased stability is believed to be due to the high oligonucleotide surface coverage that provides steric and electrostatic protection. SA₂₀Using 12F-modified particles, the effect of electrolyte conditions on oligonucleotide surface addition was investigated. As shown in FIG. 8, thiol-modified oligonucleotides having 5 ′ hexyl thiol and 3 ′ fluorescein moieties were prepared and attached to gold nanoparticles as described above to reduce the surface coverage of these oligonucleotides on the nanoparticles. Quantification was as above. The results are shown in Table 7 below. The final surface coverage of the gold nanoparticles exposed to the oligonucleotides in water for 48 hours was either "aged" with salt or increased salt concentration progressively over the last 24 hours of the experiment (final concentration was 1. 0M NaCl), which was much lower (7.9 ± 0.2 pmol / cm)²) (See above).
[0393]
It is noteworthy that the synthesized gold nanoparticles aggregate irreversibly even in extremely low ionic strength media. Indeed, gold nanoparticles are inherently incompatible with salts, especially polyanions such as oligonucleotides. This aging treatment is essential for preparing stable oligonucleotide particles. Therefore, the particles must first be modified with an alkylthiol oligonucleotide in water before the ionic strength is gradually increased. The oligonucleotides appear to lie initially flat tied together via a weak interaction of the nitrogen-containing base with gold. A similar mode of interaction has been proposed for oligonucleotides attached to thin films (Herne et al., J. Am. Chem. Soc., 119, 8916-8920 (1997)). However, the interaction of the oligonucleotide with the positively charged nanoparticle surface is expected to be even stronger (Weitz et al., Surf. Sci., 158, 147-164 (1985)). In the aging step, the high ionic strength medium effectively shields not only the charge repulsion between adjacent oligonucleotides, but also the attraction between the polyanionic oligonucleotides and the positively charged gold surface. This allows more oligonucleotide to bind to the nanoparticle surface, thereby increasing the oligonucleotide surface coverage.
[0394]
N.Effect of oligonucleotide spacer sequence on surface coverage
To investigate how the sequence of the spacer affects the coverage of the oligonucleotide on the Au nanoparticles, a fluorescein-modified 32mer with a 20dA spacer and a 20dT spacer inserted between the 3 'propylthiol and the fluorescein-labeled 12mer sequence The strand was prepared. S3'T₂₀12F and S3'A₂₀The most striking results from the surface coverage and hybridization studies on the 12F-modified nanoparticles are that the 20dA spacer (24 ± 1 pmol / cm²) Compared to the 20dT spacer (35 ± 1 pmol / cm²), A larger surface coverage was achieved. The percentage of surface bound chains hybridized is ST₂₀For 12mer nanoparticles (79%), SA₂₀Twelve nanoparticles (lower than "-94%", but the number of hybridized strands were comparable. These results indicate that the dT-rich oligonucleotide strands were less nano-sized than the dA-rich oligonucleotides. This suggests that the 20dA spacer segment blocks gold sites by laying flat on the particle surface, while the 20dT spacer segment is non-specifically interacting with the particle surface. It can extend vertically, which promotes higher surface coverage.
[0395]
O.Effect of co-adsorbed diluent oligonucleotides
In addition to efficient hybridization, another important property of oligonucleotide-modified nanoparticles is the ability to regulate the total number of hybridization events. This is most easily accomplished by adjusting the surface density of the recognition strand. Other workers have used co-adsorbed diluent alkylthiols, such as mercaptohexanol, with modified oligonucleotides on gold electrodes to control hybridization (Steel et al., Anal. Chem. 70: 4670-). 4677 (1998); Herne et al., J. Am. Chem. Soc. 119: 8916-8920 (1997)). However, the inherent low stability of unprotected gold nanoparticles has placed significant constraints on the choice of diluent molecules. Thiol-modified 20dA sequence (SA₂₀) [SEQ ID NO: 55] was found to be suitable in that it maintained particle stability in the high ionic strength buffer required for hybridization and protected the surface from non-specific adsorption. .
[0396]
Nanoparticles are combined with various recognition chains (SA₂₀12F) vs. diluent (SA₂₀2.) Modification with a solution having a chain molecular ratio. The obtained particles were treated with SA₂₀Analyzed by the fluorescence method described above for measurement of 12F surface density, and then tested for hybridization efficiency with 12'F.
[0397]
SA₂₀The 12F surface density is determined by the SA in the deposition solution.₂₀12F vs. SA₂₀(Fig. 30). This is due to the SA attached to the nanoparticles.₂₀12F and SA₂₀This is an interesting result, since it suggests that the ratio reflects the ratio of the solutions. This result is in contrast to the results normally seen with mixtures of short-chain alkyl or T-functionalized thiols where solubility and chain length play a critical role in adsorption kinetics (Bain et al., J. Am. Chem. Soc. 111: 7155-7164 (1989); Bain et al., J. Am. Chem. Soc. 111: 7164-7175 (1989).
[0398]
The amount of complementary 12F oligonucleotide hybridized to each different sample was also determined by SA₂₀It increased linearly with increasing 12F surface coverage (FIG. 31). The fact that this relationship is well defined indicates that the degree of hybridization of the nanoparticle-oligonucleotide conjugate can be predicted and controlled. This is because the higher the hybridization of 12F 'SA₂₀The 12F coverage range becomes more difficult, suggesting that steric crowding and electrostatic repulsion between the oligonucleotides is likely to occur.
[0399]
P.wrap up
This study shows that oligonucleotide stabilization is high enough to stabilize the nanoparticles, but low enough that a high percentage of the strands are accessible for hybridization with oligonucleotides in solution, and Demonstrates the importance of achieving balance. It is possible to attach oligonucleotides to nanoparticles to obtain high oligonucleotide surface coverage, to attach oligonucleotide spacer segments to reduce electrostatic interactions, and the average number of hybridization events for each nanoparticle. This is achieved by adjusting the salt conditions while attaching co-adsorbed chains to control reproducibly. It has also been shown that the nature of the strand (spacer) sequence affects the number of oligonucleotide strands loaded on the gold nanoparticles. This study has important implications for understanding the interaction between oligonucleotides and nanoparticles and for optimizing the sensitivity of the nanoparticle-oligonucleotide detection method.
[0400]
[Table 7]

Embedded image

[Table 8]

[Table 9]

[0401]
Example 19:Gene chip assay
This example describes a very selective and very sensitive assay for combinatorial DNA arrays using oligonucleotide-functionalized gold nanoparticles. The very narrow nanoparticle-target complex thermal dissociation temperature range allows a given oligonucleotide sequence to be distinguished from targets with one nucleotide mismatch with very high selectivity. Further, when combined with a signal amplification method based on nanoparticle catalytic reduction of silver (I), the sensitivity of this nanoparticle array detection system is two orders of magnitude better than that of a conventional analogous fluorophore system. I have.
[0402]
Sequence-selective DNA detection is becoming increasingly important as scientists uncover the genetic basis of the disease and use this new information to improve medical diagnosis and treatment. Commonly used heterologous DNA sequence detection systems, such as Southern blots and combinatorial DNA chips, are based on the specific hybridization of surface-bound single-stranded oligonucleotides complementary to target DNA. Both the specificity and sensitivity of these assays depend on the dissociation properties of the capture strand hybridized to all and mismatched sequences. Surprisingly, as described below, it was surprisingly discovered that a single type of nanoparticle hybridized to a substrate exhibited a substantially sharper melting profile than a similar fluorophore-based system or unlabeled DNA. Was done. In addition, the melting temperature of the nanoparticle duplex is 11 ° C. higher than for similar fluorophore systems with identical sequences. Combining these two observations with the development of a quantitative signal amplification method based on nanoparticle catalyzed reduction of silver (I), two orders of magnitude greater than single base mismatch mismatch selectivity and similar conventional fluorophore-based assays In addition, a new chip-based DNA detection system having high sensitivity can be developed.
[0403]
Gold nanoparticles with oligonucleotides prepared as described in Example 3 attached to indicate the presence of a specific DNA sequence hybridized to a transparent substrate in a three component sandwich assay format (see FIG. 32) (Diameter 13 nm) was used. In a typical experiment, a substrate was prepared by functionalizing a float glass microscope slide (Fisher Scientific) with an amine-modified probe oligonucleotide as described in Example 10. Using this method, slides functionalized with one type of oligonucleotide or multiple types of oligonucleotide arrays spotted using a commercially available microarrayer on the entire surface of the slide were made. The nanoparticles with the indicator oligonucleotide attached and a synthetic 30-mer oligonucleotide target (based on anthrax protective antigen sequence) were then co-hybridized on these substrates (see FIG. 32). Thus, the presence of the surface nanoparticles indicated the detection of a specific 30 base sequence. At higher target concentrations (≧ 1 nM), the denser hybridized nanoparticles on the surface made the surface light pink (see FIG. 33). At low target concentrations, the attached nanoparticles were not visible to the naked eye (although images were seen on field emission scanning electron microscopy). To facilitate visualization of the nanoparticles hybridized to the substrate surface, a signal amplification method was used in which silver ions were catalytically reduced by hydroquinone to form silver metal on the slide surface. This method has been used to expand protein- and antibody-bound gold nanoparticles in histochemical microscopy studies (Hacker, Colloidal Gold: Principles, Methods, and Applications, MA Hayat, Ed. (Academic). Press, San Diego, 1989), Volume 1, Chapter 10; Zehbe et al., Am. J. Pathol., 150, 1553 (1997)), the use of which in a quantitative DNA hybridization assay is described. Novel (Tomlinson et al., Anal. Biochem., 171: 217 (1988)). This method not only allowed the extremely low surface coverage of the nanoparticle probe to be visualized with a simple flatbed scanner or with the naked eye (FIG. 33), but also allowed the quantification of target hybridization based on the optical density of the stained area. (FIG. 34). Importantly, no surface staining was observed in the absence of the target, or in the presence of the non-complementary target, indicating that both the non-specific binding of the nanoparticles to the surface and the non-specific silver staining Also proves that nothing happens. This result is an excellent feature of these nanoparticle-oligonucleotide conjugates that allows for very sensitive and very selective detection of nucleic acids.
[0404]
Furthermore, it has been determined that the unique hybridization properties of the oligonucleotide-functionalized nanoparticles of the present invention can be used to further improve the selectivity of combinatorial oligonucleotide arrays (or "gene chips") (Fodor, Science). 27, p. 393 (1997)). The relative ratio of targets hybridized to different elements of the oligonucleotide array will determine the accuracy of the array during target sequencing. This ratio depends on the hybridization properties of the duplex formed between the different capture strands and the DNA target. Surprisingly, these hybridization properties are dramatically improved by using nanoparticle targets instead of fluorophore labels. As shown in FIG. 35, dehybridization of a nanoparticle-labeled target from a surface-bound capture strand is much more temperature-sensitive than that of a fluorophore-labeled target having the same sequence. The fluorophore-labeled target dehybridized from the surface capture strand over a very wide temperature range (first derivative FWHM = 16 ° C.), whereas the same nanoparticle labeled target melted much more rapidly (first derivative FWHM = 3 ° C). These steep dissociation profiles were expected to improve the stringency of chip-based sequence analysis, usually affected by hybridization stringency washes. Indeed, the ratio of target hybridized to a complementary surface probe to target hybridized to a mismatched probe after a stringency wash at a specific temperature (represented by the horizontal line in FIG. 35) is the fluorophore Nanoparticle labels are much higher than labels. This should mean high selectivity in chip detection format. In addition, the nanoparticle label may reduce the surface duplex melting temperature (T_m) Will improve array sensitivity.
[0405]
To evaluate the effectiveness of nanoparticles as a colorimetric indicator for oligonucleotide arrays, test chips were probed with a synthetic target and labeled with a fluorophore indicator and a nanoparticle indicator. Test arrays and oligonucleotide targets were prepared using published protocols (Guo et al., Nucl. Acids Res., Vol. 22, p. 5456 (1994); 175 μm diameter separated by 375 μm using a Genetic Microsystems 417 Microarrayer. The spot array was patterned). The array contained four elements corresponding to each of the four possible nucleotides (N) at position 8 of the target (see FIG. 32). In the hybridization buffer, the synthetic target and the fluorescent or nanoparticle-labeled probe were stepwise hybridized to the array, with a 35 ° C. stringency buffer wash after each step. First, 2 × PBS (0.3 M NaCl, 10 mM Na₂H₂PO₄/ Na₂HPO₄20 μl of a 1 nM synthetic target solution in buffer, pH 7) and the array are hybridized for 4 hours at room temperature in a hybridization chamber (Grace Bio-Labs Cover Well PC20) and then cleaned at 35 ° C. Washed with × PBS buffer. Then, 20 μl of a 100 pM oligonucleotide-functionalized gold nanoparticle solution in 2 × PBS and the array were hybridized for 4 hours at room temperature in a fresh hybridization chamber. The array was washed with clean 2 × PBS at 35 ° C., then 2 × PBS (0.3 M NaNO₃, 10 mM NaH₂PO₄/ Na₂HPO₄Washed twice with buffer, pH 7). Next, the nanoparticle array was immersed in a silver amplification solution (Sigma Chemical Co., Silver Enhancer Solution) for 5 minutes, and washed with water. Due to the silver amplification, the array elements were considerably darkened and the 200 μm diameter elements could be easily seen with a flatbed scanner or even the naked eye.
[0406]
Arrays challenged with the model target and the nanoparticle labeled probe and stained with silver solution clearly showed highly selective hybridization to complementary array elements (FIG. 36A). Duplicate spots of the same capture sequence showed reproducible and consistent hybridization signals. No background adsorption by the nanoparticles or silver dye was observed. The image grayscale values recorded by the flatbed scanner were the same as those observed on clean microscope slides. The dark spot corresponding to adenine at position 8 (N = A) indicates that the oligonucleotide target hybridized at a ratio of more than 3: 1 to the perfectly complementary capture strand over the mismatched strand. ing. Further, the total grayscale value of each spot set is calculated using the Watson-Crick base pair, A: T> G: T> C: T> T: T (Alllawi et al., Biochemistry, 36, 10581 (1988)). ). Usually, it is particularly difficult to distinguish G: T mismatches from A: T complements (Saiki et al., Mutation Detection, Coton et al. (Eds. Oxford University Press, Oxford, 1998), Chapter 7, S. Ikuta et al. , Nucl. Acids Res., Vol. 15, p. 797 (1987), discrimination of these two array elements demonstrates the incredible resolution of nanoparticle labels in detecting single nucleotide mismatches. I have. The selectivity of the nanoparticle-based array was higher than that of the fluorophore indicating array (FIG. 36B), with the fluorophore label providing only a 2: 1 selectivity for adenine at position 8.
[0407]
Assays utilizing nanoparticle labeled probes were significantly more sensitive than those utilizing fluorophore labeled probes. The hybridization signal is as low as 50 fM (or 1 × 10 5 for a hybridization chamber containing 20 μl of solution).⁶(Total copy number) at a target concentration of N = A elements. This represents a dramatic increase in sensitivity over conventional Cy3 / C75 fluorophore-labeled arrays, which typically require target concentrations of 1 pM or more. There is no doubt that the high melting temperature observed for the nanoparticle-target complex immobilized on the surface contributes to the sensitivity of the array. It is believed that the increased stability of the probe / target / surface-oligonucleotide complex in the nanoparticle system compared to the fluorophore system results in less target and probe being lost during the washing step.
[0408]
The colorimetric nanoparticle labels of combinatorial oligonucleotide arrays may be useful in applications such as single nucleotide polymorphism analysis where resolution of one mismatch, sensitivity, cost, and ease of use are important factors. Furthermore, the sensitivity of this system, which needs to be globally optimized, indicates a potential method for detecting oligonucleotide targets without the need for a target amplification scheme such as the polymerase chain reaction.
[0409]
Example 20:Nanoparticle structure
2 illustrates a reversible assembly of a supramolecular multilayer gold nanoparticle structure on a glass support mediated by a hybridized DNA linker. Oligonucleotide-functionalized nanoparticles are sequentially attached to an oligonucleotide-functionalized glass support in the presence of a complementary DNA linker. The unique recognition properties of DNA result in the selective assembly of the nanoparticle structure in the presence of a complementary linker. In addition, these structures can aggregate or dissociate in response to external stimuli that mediate hybridization of the linked double-stranded DNA, including solution temperature, pH, and ionic strength. This system, in addition to providing a very selective and controlled way to construct nanoparticle-based structures on solid supports, combines the optical and melting properties of DNA-linked nanoparticle networks. Enables study of influential factors.
[0410]
Others have described how bifunctional organic molecules (Gittins et al., Adv. Mater., Vol. 11, p. 737 (1999); Brust et al., Langmuir, Vol. 14: 5425 (1998); Bright et al., Langmuir, 14: 5696 (1998); Grabar et al., J. Am. Chem. Soc., 118: 1148 (1996); Freeman et al., Science, 267: 1629. (1995); Schmid et al., Angew. Chem. Int. Ed. Engel., 39: 181 (2000); Marinakos et al., Chem. Mater., 10: 1214 (1998)) or Polymer electrolytes (Storhoff et al., J. Am. Chem. Soc., 120, 1959 (1998); Storhoff et al., J. Cluster Sci., 8: 179 (1997); Elghanian et al., Science, 277: 1078 (1997). Mirkin et al., Nature 382, 607 (1996)) was used to demonstrate whether monolayer and multilayer nanoparticle materials could be controllably constructed from planar structures. An attractive feature of using DNA as interconnects for nanoparticles is that by selecting the DNA sequence, the interparticle spacing, particle periodicity and particle composition can be synthetically programmed. Further, the reversible binding properties of the oligonucleotide can be used to reliably form a thermodynamic structure instead of a dynamic structure. This method not only provides a new and powerful way to control the growth of nanoparticle-based structures from solid supports, but also the nanoparticle aggregate size and the melting and optical properties of the aggregate DNA interconnect structure You can also evaluate the relationship with The relationship between these two physical parameters and their material construction is essential for the use of nanoparticle network materials, especially in the field of biodetection.
[0411]
Oligonucleotide-functionalized gold nanoparticles of 13 nm diameter used in the construction of multilayer assemblies were prepared as described in Examples 1 and 3. These nanoparticles include 5'-hexylthiol-capped oligonucleotide 1 (5'-HS (CH₂)₆O (PO₂ ⁻) O-CGCATTCAGGAT-3 '[SEQ ID NO: 50] and 3'-propanethiol-cap oligonucleotide 2 (3'-HS (CH₂)₃O (PO₂ ⁻) O-ATGCTCAACTCT-5 '[SEQ ID NO: 59] was attached (see FIG. 37). Glass slides were functionalized with 12 mer oligonucleotide 2 as described in Example 10. In order to construct a nanoparticle layer, the substrate was first immersed in a 10 nM solution of 24mer linker 3 (5'-TACGAGTTTGAGAATCCTGAATGCCG-3 '[SEQ ID NO: 60]) and hybridized at room temperature for 4 hours (see FIG. 37). ). The support was washed with clean buffer and then hybridized with a 2 nM solution of Particle a for 4 hours at room temperature to attach the first nanoparticle layer. Similarly, the second nanoparticle layer could be attached to the first nanoparticle layer only by exposing the substrate surface to the solution of the linker 3 and the nanoparticles b. These hybridization steps could be repeated to attach multiple alternating layers of nanoparticles a and b, each layer connected to the previous layer via linker 3. No hybridization of the nanoparticles to the surface was observed in the absence of a linker or in the presence of a non-complementary oligonucleotide. In addition, conditions promoting DNA linker hybridization: neutral pH, mild salt concentration (> 0.05 M NaCl) and duplex melting temperature (T_mMultilayer aggregates were observed only at temperatures below).
[0412]
Each hybridized nanoparticle layer made the substrate darker red, and the supporting glass slide after the ten layers hybridized appeared gold by reflection. Transmission UV-visible spectroscopy of the substrate was used to monitor the continuous hybridization of the nanoparticles to the surface (FIG. 38A). The low absorbance of the initial nanoparticle layer suggests that this layer facilitated the formation of additional layers, with the intensity of the plasmon band increasing almost linearly as more layers were added. (No further increase in absorbance was observed with increasing exposure time or increasing concentration of the linker 3 or nanoparticle solution for each successive nanoparticle layer formation). The linear increase in absorbance after the initial nanoparticle layer formation indicates that the surface was saturated with hybridized nanoparticles as the layer was added continuously (FIG. 38B). This indicates that one (FIG. 39A) and two (FIG. 39B) nanoparticle layers on the surface show low nanoparticle coverage for one layer, but nearly complete coverage for two layers. Supported by field emission scanning electron microscope (FE-SEM) images. Plasmon band λ of multilayer assembly_maxShifts less than 10 nm even after five layers. The detection of this shift is based on other experimental treatments of gold nanoparticle aggregates (Grabar et al., J. Am. Chem. Soc., 118, 1148 (1996)) and theoretical treatments (Quinten et al., Surf) Sci., 172, 557 (1996); Yang et al., J. Chem. Phys., 103: 869 (1995). However, the magnitude of the shift is λ_maxSmall compared to that previously observed for suspensions of oligonucleotide-linked gold nanoparticle networks showing> 570 nm (see previous example). This means that perhaps hundreds or thousands more nanoparticles need to be linked to produce the dramatic red-to-blue color change observed for gold nanoparticle-based oligonucleotide probes. Suggests. (Storhoff et al., J. Am. Chem. Soc., Vol. 120, p. 1959 (1998); Storhoff et al., J. Cluster Sci., Vol. 8, p. 179 (1997); Elghanian et al., Science, Vol. 277, 1078 (1997); Mirkin et al., Nature, 382, 607 (1996)). The surface plasmon shift of aggregated gold nanoparticles was found to be largely dependent on the spacing between particles (Quinten et al., Surf. Sci., 172: 557 (1986); Storhoff et al., J. Am. Chem. Soc., Supra). The large spacing provided by the oligonucleotide linker (8.2 nm in this system) significantly reduces the cumulative effect of nanoparticle aggregation on the gold surface plasmon band.
[0413]
The dissociation properties of the assembled nanoparticle multilayers were highly dependent on the number of layers. The multi-layer coated substrate is suspended in a buffer and the temperature is adjusted to the T_mWhen raised to a higher temperature (53 ° C.), the nanoparticles dissociated into solution, leaving a colorless glass surface. Even if the pH of the buffer suspension was raised or lowered (> 11 or <3) or the salt concentration was reduced (less than 0.01 M NaCl), the nanoparticles were dissociated by dehybridizing the ligated DNA. . The multilayer assembly is completely reversible, and the nanoparticles can hybridize to and dehybridize from the glass substrate (eg, three cycles require no detectable irreversible nanoparticle binding). Not shown).
[0414]
Importantly, all surface-bound nanoparticle aggregates have a T_mAlthough the dissociation occurred at temperatures greater than, the sharpness of these transitions was dependent on the size of the supported aggregates (FIGS. 39D-F). Surprisingly, the dissociation of the first nanoparticle layer from the support resulted in a more abrupt transition (FIG. 39D, FWHM of the first derivative =) than that of the same oligonucleotide without nanoparticles in solution (FIG. 39C). 5 ° C). As the nanoparticle layer further hybridizes to the substrate, the melting transition of the oligonucleotide-linked nanoparticles continues to increase rapidly until it is comparable to the melting transition of the large nanoparticle network aggregate found in solution. (FIG. 39E-F, FWHM of first derivative = 3 ° C.). (Gittins et al., Adv. Mater., Vol. 11: 737 (1999); Brust et al., Langmuir, Vol. 14: 5425 (1998)). These experiments were performed to obtain an optimal rapid melting curve. Have demonstrated the need for multiple DNA interconnects with three or more nanoparticles. These experiments further show that the optical changes in this system are completely decoupled from the melting properties (ie, small agglomerations can result in abrupt transitions, but no discoloration).
[0415]
Example 21:Electrical properties of gold nanoparticle aggregates
Electronic transfer through DNA has been one of the most widely discussed issues in the field of chemistry over the past few years. (Kelley et al., Science, 283: 375-381 (1999); Turro et al., JBIC, 3: 201-209 (1998); Lewis et al., JBIC, 3: 215-221. Ratner, M., Nature, 397, 480-481 (1999); Okahata et al., J. Am. Chem. Soc., 120, 6165-6166 (1998)). . Some claim that DNA can transfer electrons efficiently, while others consider DNA to be an insulator.
[0416]
In the field of research with no apparent similarities, great efforts have been devoted to studying the electrical properties of nanoparticle-based materials (Terrill et al., J. Am. Chem. Soc., 117, 12537). Brust et al., Adv. Mater., Vol. 7, pp. 79-797 (1995); Bethell et al., J. Electronal. Chem., 409: 137-143 (1996). Music, et al., Chem. Mater., 9: 1499-1501 (1997); Brust et al., Langmuir, 14, 5425-5429 (1998); Collier et al., Science, 277: 1978-1981 (1997)). Indeed, many groups have explored ways to assemble nanoparticles into two- and three-dimensional networks, and have investigated the electronic properties of such structures. However, virtually nothing is known about the electrical properties of nanoparticle-based materials linked to DNA.
[0417]
For the first time in this study, the electrical properties of gold nanoparticle assemblies formed by different lengths of DNA interconnects were investigated. As shown below, these hybrid inorganic aggregates behave as semiconductors, despite oligonucleotide particle interconnect lengths ranging from 24-72 nucleotides. The results described herein show that the DNA interconnects do not need to form an insulating barrier between the metal nanoparticles, and thus do not destroy the electrical properties of the particles. It can be used as a scaffold material specific to. These results indicate a novel way in which such hybrid assemblies can be utilized as electronic materials.
[0418]
At the heart of this subject are the following problems: nanoparticles assembled via DNA can still conduct electricity, or the heavily added DNA cross-conjugate on each particle acts as an insulating shell. (Mucic, RC, Synthetically Programmable Nanoparticle Assembly Uisng DNA, Thesis Ph.D., Northwestern University (1999)). The conductivity of these materials was investigated as a function of temperature, oligonucleotide length and relative humidity. DNA-linked nanoparticle structures can be analyzed by field emission scanning electron microscopy (FE-SEM), synchrotron small angle x-ray scattering (SAXS) experiments, thermal denaturation profiles, and UV-visible. Characterized by spectroscopy.
[0419]
In a typical experiment (see FIG. 40), citrate-stabilized 13 nm gold nanoparticles were treated as described in Examples 1 and 3 with 3 'and 5' alkanethiol-capped 12mer oligonucleotide 1 ( 3 'SH (CH₂)₂O (PO₂ ⁻) O-ATGCTCCAACTCT 5 '[SEQ ID NO: 59]) and 2 (5' SH (CH₂)₆O (PO₂ ⁻) O-CGCATTCAGGAT 3 ′ [SEQ ID NO: 50]). 24, 48 or 72 base long DNA strand 3 (5'TACGAGTTGAGAATCCTGAATGCG3 '[SEQ ID NO: 60]), 4 (5'TACGAGTTGAGACCGTTAAGACGAGGCAATC-ATGCAATCCTGAATGCG3' [SEQ ID NO: 61]), and 5 (5'TACGAGTTGAGACCGTTAAGACGAGGCAATCATGCATATATTGGACGCTTTACGGACAACATCCTGAATGCG3 '[SEQ ID NO: 62] ) Was used as a linker. Fillers 6 (3'GGCAATTCTGCTCGTGTTACGTGT5 '[SEQ ID NO: 63]) and 7 (3'GGCAATTCTGCTCTCGTTAGTACGTATATAACCTGCGAAATGCCTGTTG5' [SEQ ID NO: 64]) were used with a 48 base linker and a 72 base linker. DNA modified nanoparticles and DNA linkers and fillers were stored in 0.3 M NaCl, 10 mM phosphate (pH 7) buffer (referred to as 0.3 M PBS) before use. To construct a nanoparticle assembly, gold nanoparticles modified with 1 (652 μl, 9.7 nM) and gold nanoparticles modified with 2 (652 μl, 9.7 μM) were linked to linker DNA 3, 4, or 5 (30 μl, 10 nM). After complete precipitation, the aggregates were quenched with 0.3M CH₃COONH₄The solution was washed to remove excess DNA and NaCl.
[0420]
The aggregate is freeze-dried (10^-3-10^-2Torr) to dryness to obtain pellets, volatile salt CH₃COONH₄Was removed. Unfunctionalized citrate stabilized particles prepared by the Frens method (Frens, Nature Phys. Sci., 241: 20-22 (1973)) were dried as membranes and used for comparison. The resulting dried agglomerates had a color similar to cloudy brass and were very brittle. FE-SEM images showed that the oligonucleotide-modified nanoparticles remained intact when dried, but that the citrate-stabilized nanoparticles were fused together. Importantly, the aggregates ligated through the dried DNA could be redispersed in 0.3 M PBS buffer (1 ml) and exhibited excellent melting properties. When such a dispersion was heated to 60 ° C., hybridization of the DNA cross-conjugate occurred, resulting in a red solution of dispersed nanoparticles. Combining this with the FE-SEM data, it has finally been proven that DNA-modified gold nanoparticles do not aggregate irreversibly when dried.
[0421]
The conductivity of the three samples (dry aggregates linked via 3, 4, and 5, respectively) was measured using a computer controlled four probe technique. The electrical contacts consisted of fine gold wires (25 and 60 μm in diameter) attached to the pellet with gold paste. Apply the sample to a moderate vacuum (10^-3-10^-2The conductivity was measured while cooling under low pressure dry helium gas with increasing temperature. The sample chamber was isolated from light to eliminate possible optoelectronic events. The excitation current was kept below 100 nA and the pressure across the sample was limited to a maximum of 20V. Surprisingly, the conductivity of the aggregate formed from all three linkers is less than 10 at room temperature.^-5-10^-4In the range of S / cm, these aggregates exhibited similar temperature-dependent behavior. The conductivity of the DNA-linked aggregate showed an Arrhenius behavior up to about 190 K, characteristic of a semiconductor material. This is similar to the activated electron hopping behavior observed in discontinuous metal island films (Barwinski, Thin Solid Films, Vol. 128: 1-9 (1985)). The network structure of the gold nanoparticles linked via the alkanedithiol showed a similar temperature dependence (Brust et al., Adv. Mater., 7: 794-797 (1995); Bethell et al., J. Am. Electronal. Chem., 409: 137-143 (1996)). The activation energy for charge transfer is given by equation (1)
[0422]
σ = σ_oexp [-E_α/ KT]] (1)
Can be obtained from a plot of 1nσ versus 1 / T. The average activation energies calculated from the three measurements were 7.4 ± 0.2 meV, 7.5 ± 0.3 meV, 7.6 ± 0.4 meV for the 24, 48 and 72 mer linkers, respectively. For these calculations, conductivity from 50 ° K to 150 ° K was used.
[0423]
Since the electrical properties of these types of materials should depend on the interparticle spacing, the interparticle spacing of the dispersed dry agglomerates was measured using a synchrotron SAXS experiment. The SAXS experiment was carried out in an Advanced Proton Source, Dupont-Northwestern-Dow Collaborative Access Team (DND-CAT) Sector 5 in Argon National Laboratory. Dilution samples of DNA-linked aggregates and DNA-modified colloids were irradiated with a 0.34 micron beam of 1.54 ° radiation and scattered radiation was collected with a CCD detector. The 2D data was cyclically averaged and converted to a scattering vector quantity, s = 2 sin (θ) / λ (where 2θ is the scattering angle and λ is the wavelength of the incident radiation), I (s). All data were corrected for background scatter and sample absorption. When all three aggregate structures were dried, the first peak position of interparticle spacing sensitivity was 0.063 nm for 24 mer, 48 mer and 72 mer linked aggregates, respectively.^-1, 0.048 nm^-1, And 0.037 nm^-10.087 nm from the s value of^-1Significantly changed. This is because upon drying, the spacing between the particles was significantly reduced to the point where the particles almost touched, and such spacing is substantially independent of linker length, but the spacing between particles in solution is This shows that it was largely dependent on the linker length. This shows why similar activation energies were observed for the three different systems in the dried pellet conductivity experiment. Furthermore, it also reveals why relatively high conductivity was observed, independent of the view of the electronic properties of DNA. Unlike the DNA-linked material, the dried citrate-stabilized gold nanoparticle membrane exhibited metal-like behavior. This is consistent with SEM data which showed that such particles fused together.
[0424]
Above 190 ° K, the measured conductivity of the DNA ligated sample showed a very steep drop behavior. For all samples, the conductivity began to drop sharply at about 190 K, continued to drop to approximately 250 K, and then increased again. To investigate this behavior in detail, the conductivity was measured while repeatedly cooling and heating the sample. Interestingly, the drop in conductivity occurred only when the temperature was increasing. The effect of relative humidity on the conductivity of gold aggregates was investigated because DNA is hydrophilic and water can potentially affect the electrical properties of the hybrid structure. Increasing the humidity from 1% to 10% increased the resistance by a factor of 10. Before conducting the conductivity measurement, the sample was evacuated (10^-6Note that the peculiar dip was very weak when maintained under Torr for 48 hours. From these observations, it can be seen that a very sharp drop in conductivity above 190 ° K and a subsequent increase is due to the melting of water and the DNA melting which temporarily increased the interparticle spacing (until evaporation occurred). It was inferred to be related to hygroscopicity. Consistent with this hypothesis, SAXS measurements on dry aggregates moistened with 0.3 M PBS buffer showed a 200% increase in interparticle spacing (〜2 nm).
[0425]
These studies are important for the following reasons: First, these studies demonstrate that using the molecular recognition properties of DNA, nanoparticle-based materials can be passivated and assembled without destroying their discrete structure or electrical properties. These DNA-functionalized particles are used to study electrical migration in three-dimensional macroscopic assemblies or even lithographically patterned structures (Piner et al., Science, 283: 661-663 (1999)). In such cases, it is absolutely necessary to accurately describe their electrical transfer characteristics. Second, these studies indicate that the conductivity of the dried assembly is substantially independent of DNA linker length over fairly long linker spacing (8-24 nm). This is thought to be the result of removing water and using volatile salts in these experiments. Indeed, the free volume created by solvent and salt removal compresses the DNA to the surface and brings the particles in the aggregate closer together. Third, aggregates with DNA-protected nanoparticles behave as semiconductors, whereas membranes formed from citrate-stabilized particles exhibit irreversible particle fusion and metal-like behavior. Finally, these results indicate that sequence-specific binding events between oligonucleotide-functionalized nanoparticles and target DNA can lead to circuit closure and a sharp increase in conductivity (ie, from insulator to semiconductor). The use of these materials in DNA diagnostic applications is illustrated (see next example).
[0426]
Example 22:Detection of nucleic acid using gold electrode
The nucleic acid detection method using a gold electrode is schematically illustrated in FIG. Guo et al., Nucleic Acids Res. , Vol. 22, pp. 5456-5465 (1994), and the glass surface between the two gold electrodes is treated with a 12-mer oligonucleotide 1 (3 'NH₂(CH₂)₇O (PO₂ ⁻) O-ATG-CTC-AAC-TCT [SEQ ID NO: 59]). Oligonucleotide 2 (5'SH (CH₂)₆O (PO₂ ⁻) O-CGC-ATT-CAG-GAT [SEQ ID NO: 50]) was prepared and attached to 13 nm gold nanoparticles as described in Examples 1 and 18 to produce nanoparticles a. Target DNA3 and nanoparticle a were added to the device. The color of the glass surface turned pink, indicating that the target DNA-gold nanoparticle aggregate was formed on the glass substrate. The device is then immersed in 0.3 M NaCl, 10 mM phosphate buffer and heated at 40 ° C. for 1 hour to remove non-specifically bound DNA, and then silver as described in Example 19. Treated with staining solution for 5 minutes. The resistance of the electrode was 67 kΩ.
[0427]
For comparison, the control device was modified by attaching oligonucleotide 4 instead of oligonucleotide 1 between the electrodes. Oligonucleotide 4 has the same sequence as oligonucleotide 2 on the nanoparticle (5'NH₂(CH₂)₆O (PO₂ ⁻) O-CGC-ATT-CAG-GAT [SEQ ID NO: 50]) and will bind to target DNA3 to prevent binding of the nanoparticles. Otherwise, the tests were performed as described above. The resistance was higher than the detection limit of the used multimeter, 40 MΩ.
[0428]
This experiment shows that only the complementary target DNA strand forms a nanoparticle assembly between the two electrodes of the device, and that the circuit can be completed by hybridization of the nanoparticles and subsequent silver staining. Thus, complementary and non-complementary DNA can be distinguished by measuring conductivity. This format can also be extended to substrate arrays (chips) using thousands of pairs of electrodes that can simultaneously test thousands of different nucleic acids.
[0429]
Example 23: Preparation of oligonucleotide-modified gold nanoparticles using cyclic disulfide linker
In this example, a steroid that provides a gold-oligonucleotide conjugate that is simple to prepare, is widely useful, and exhibits greater stability to DTT than a gold-oligonucleotide conjugate prepared using a mercaptohexyl linker A new cyclic disulfide linker for attaching oligonucleotides to a gold surface based on disulfide 1a (FIG. 42) is described. It is known that ester derivatives of 1,2-dithiane-4,5-diol form a monolayer on the gold surface (Nuzzo et al., J. Am. Chem. Soc. 105, 4481-4483). , Cyclic disulfide was selected as the reactive site of the anchor unit. Cyclic disulfides will bind to the surface via both sulfur atoms (Ulman, A., MRS Bulletin, June 46-51), giving a chelating structure that may exhibit improved stability. The linking element is a keto alcohol that is readily available and easily induced to degenerate, and is expected to be useful as a substituent with a large hydrophobic surface to help screen water-soluble molecules approaching the gold surface. Therefore, epiandrosterone was selected (Retsinger et al., J. Am. Chem. Soc. 115, 7535-7536-Bioconjugate Chem. 9, 826-830).
[0430]
Oligonucleotide-gold probes used in previous studies were prepared by reacting oligonucleotides with terminal mercaptohexyl groups with gold nanoparticles in aqueous buffer. The probe was found to be surprisingly robust and perform well even after heating to 100 ° C. and storage at 5 ° C. for 3 years. However, the present inventors have found that this conjugate loses its activity as a hybridization probe when immersed in a solution containing a thiol that acts by removing the derivatized oligonucleotide from the gold surface. . This feature poses a problem when the nanoparticle probe is used in a solution containing thiols (eg, a PCR solution containing dithiothreitol (DTT) as a stabilizer for the polymerase enzyme).
[0431]
(A)General law
NMR spectra were analyzed using CDCl₃, TMS as internal (H) standard, external (³¹P) H as standard₃PO₄Using 500 MHz (¹H) and 400 MHz (³¹P; 161.9 MHz acquisition) Recorded on a Varian spectrometer. Chemical shifts are expressed in δ units. MS data was obtained on a Quattro II triple quadrupole mass spectrometer. Automated oligonucleotide synthesis was performed on a Milligene Expedite DNA DNA synthesizer. Analysis of S was performed by Oneida Research Services.
[0432]
(B)Preparation of steroid-disulfide ketal (1a)
The synthetic scheme is shown in FIG. A solution prepared by dissolving epiandrosterone (0.5 g), 1,2-dithiane-4,5 diol (0.28 g) and p-toluenesulfonic acid (15 mg) in toluene (30 mL) was subjected to dehydration for 7 hours. Reflux (Dean Stark apparatus). Thereafter, the toluene was removed under reduced pressure and the residue was dissolved in ethyl acetate. The solution was washed with water, dried over sodium sulfate and concentrated to a syrup strip residue. This was allowed to stand in pentane / ether overnight to give compound la as a white solid (400 mg). The Rf value (TLC, silica plate, ether as eluent) was 0.5. By comparison, the Rf values of epiandrosterone and 1,2-dithiane-4,5-diol under the same conditions are 0.4 and 0.3, respectively. Recrystallization from pentane / ether gave a white powder (mp 110-112 ° C).¹H NMR, δ 3.6, (1H, C³OH) 3.54-3.39 (2H, m 2CHH of dithiane ring), 3.2-3.0 (4H, m2CH₂S), 2.1-0.7 (29H, m H of steroid); mass spectrum (ES⁺) C₂₃H₃₆O₃S₂(M + H) calcd 425.2179, found 425.2151 (C₂₃H₃₇O₃S₂) Analysis Calculated 15.12, found 15.26.
[0433]
(C)Preparation of steroid-disulfide ketal phosphoramidite derivative (1b)
Compound 1a (100 mg) was dissolved in THF (3 mL) and cooled in a dry ice alcohol bath. N, N-diisopropylethylamine (80 μL) and β-cyanoethylchlorodiisopropyl phosphoramidite (80 μL) were added sequentially. Thereafter, the mixture was warmed to room temperature, stirred for 2 hours, mixed with ethyl acetate (100 mL), and 5% NaHCO₃Washed with an aqueous solution, dried over sodium sulfate and concentrated to dryness. The residue was dissolved in a minimum amount of dichloromethane, precipitated by adding hexane at -70 ° C and dried under vacuum to yield 100 mg. ;³¹P NMR 146.02.
[0434]
(D)Preparation of 5 'modified oligonucleotide-nanoparticle conjugate
Except for using compound 1b (FIG. 42) in the final phosphorylation step, 5 'modified oligonucleotides Ic1 and 1c2 were constructed on a CPG support using conventional phosphoramidite chemistry. The product is concentrated NH₄Release from the support by treatment with OH for 16 hours at 55 ° C. Oligonucleotides were run on a Dionex DX500 system equipped with a Hewlett Packard ODS Hypersil column (4.6 × 200 nm, 5 μm particle size) with a TEAA buffer (pH 7.0) and a 1% / min gradient of 95% CH.₃Purified by reverse phase HPLC using CN / 5% 0.03TEAA at a flow rate of 1 mL / min. An excellent feature of the hydrophobic steroid group is that the capped derivative is easily separated from the uncapped oligomer. Sulfur-derivatized oligonucleotides IIa, IIc1, and IIc2 (FIG. 43) were prepared using commercially available “5′-thiol-modifying reagents” (C6Glen Research) as previously described (Storhoff, JJ. J. Am. Chem. Soc. 120, 1959-1964) dissociation of the trityl protecting group with silver nitrate. For the preparation of disulfide IIc1, "thiol-modified C6SS" (Glen Research) was used for the final phosphorylation and the terminal dimethoxytrityl group was dissociated in 80% aqueous acetic acid.
[0435]
Each modified oligonucleotide was treated with the method used for immobilization of the oligonucleotide via the immobilized mercaptohexyl group (Storhoff et al. (1998) J. Am. Chem. Soc. 120, 1959-1964) to １３13 nm of gold. Immobilized on nanoparticles. This involves immersing stabilized nanoparticles (直径 13 nm in diameter) with citrate in a buffered salt solution containing oligonucleotides with terminal sulfur substituents (HS- or acyclic or cyclic disulfides) for 56 hours, NaCl up to 0.1 M was added and allowed to stand for 24 hours. The nanoparticles were pelleted by centrifugation and the supernatant solution was removed. The nanoparticles were washed, resuspended in buffer, centrifuged again, and suspended in 0.1 M NaCl, 10 mM phosphate. This method resulted in a nanoparticle-oligonucleotide conjugate without excess sulfurized oligonucleotide used in the loading process.
[0436]
(E)Hybridization
To evaluate the integrity of the nanoparticle-oligonucleotide probe containing the disulfide-steroid anchor, probes Ic1, Ic2, IIc1, IIc2, and IIIc1 were made by immobilizing the modified oligonucleotide on gold nanoparticles. The oligomers in a given series have the same nucleotide sequence, but differ in the structure of the 5 'head Y. Ic1 and Ic2 have a steroid-disulfide head group; IIc1, IIc2 have a mercaptohexyl head group, and IIIc1 has an acyclic disulfide head group. (DA)₂₀The strand functions as a spacer between gold and the oligonucleotide recognition moiety to facilitate hybridization. Many of the sulfur-derivatized oligomers bind to each nanoparticle. Hybridization of a nanoparticle probe pair with a target oligonucleotide leads to the formation of a three-dimensional network and a color change from red to blue-grey (Mucic, RC, et al., J. Am. Chem. Soc. 120, 12674-12675).
[0437]
Probe hybridization was investigated using a 79mer oligonucleotide target containing a sequence complementary to the probe. (FIG. 43). The reaction was performed with 0.5 M NaCl, 10 mM phosphate (pH 7.0), a colloidal solution of probe pairs Ic1, Ic2, and IIc1, IIc2, and IIIc1, IIIc2 (50 μL and 1.0 A for each nanoparticle probe).₅₂₀The reaction was performed at room temperature by adding 1 μL of the target solution (10 pmol IV) to the unit. Aliquots (3 μL) were taken at 10 seconds, 5 minutes, and 10 minutes and spotted on C-18 reverse phase TLC plates. The various probe pairs all worked the same. That is, the spot for the 10-second reaction turns red (indicating that the nanoparticles are free); the spot for the 10-minute reaction turns navy blue (characteristic of nanoparticle aggregates); the 5-minute reaction turns reddish brown A blue spot was given (indicating a mixture of unbound and bound nanoparticles). Consistent with previous observations on aggregation of nanoparticles achieved by oligonucleotide hybridization (Storhoff, JJ et al., J. Am. Chem. Soc. 120, 1959-1964; Elghanian, Science, 277, J. Am. Chem. Soc. 120, 12674-12675; Mitchell, GP, J. Am. Chem. Soc. 121, 8122-8123), reaction is reversible. It was a target. The aggregate was warmed to 90 ° C. (above the dissociation temperature of the oligomer linking the nanoparticles together) and spotted while hot, giving a red spot. Control experiments in which the oligonucleotide target was omitted or where the oligonucleotide target was not complementary to the probe were red under all conditions.
[0438]
The present inventors conclude that the nanoparticle conjugate generated by the steroid-disulfide anchor functions effectively as a hybridization probe. In addition, conjugates with different anchor units react with a significant percentage of the target oligonucleotide as judged by spot tests. This feature is consistent with the expectation that the probe will have a significant density of oligonucleotides on the surface of the nanoparticle and have a nucleotide recognition moiety that is relatively far away from the 5 'head group.
[0439]
(F)Reaction of dithiothreitol with nanoparticle probe
The addition of thiols to a colloidal solution of gold nanoparticles loaded with gold nanoparticles or mercaptohexyl-oligonucleotides leads to aggregation of the nanoparticles. The color changes from red to dark blue and upon standing a dark precipitate deposits. Thiols displace mercaptoalkyl-oligonucleotides bound to a gold surface, as demonstrated in experiments with nanoparticles with fluorescently labeled oligonucleotides (Mucic, RC (1999) Synthetic Programmable Nanoparticle Assembly Using). DNA PhD paper, Northwestern University). In contrast to the aggregation caused by the hybridization of the oligonucleotide-nanoparticle conjugate, such a reaction is irreversible, and neither heating nor addition of NaOH can degrade the aggregate.
[0440]
The inventors have used this color to monitor the reaction of DTT with probes made using steroid cyclic disulfide head groups, mercaptohexyl head groups, and acyclic disulfide head groups. The experiment was performed by adding 1 μL of a 1 M DTT solution to 100 μL of a nanoparticle-oligonucleotide probe solution (2A of nanoparticle) of 0.5 M NaCl and 10 mM phosphate (pH 7.0).₅₂₀Units), then 3 μL aliquots were spotted on TLC plates at various times and observed by color. As shown in Table 1, mercaptohexyl (IIc1 and IIc2) and colloidal probes derived from oligonucleotides with acyclic oligonucleotide head groups (IIIc1) reacted rapidly. A red-blue spot was obtained in 20 seconds and a strong blue spot was obtained within 5 minutes. By 100 minutes, most of the gold had precipitated. In contrast, no color change was observed within 40 minutes in the reaction of probes made with steroid cyclic disulfide head groups (Ic1, Ic2). It took 100 minutes to reach the same color as obtained with probes made with IIc1 and IIc2. In IIIc1, it took 20 seconds. Based on this, the present inventors speculate that the reaction rate of the steroid disulfide probe with DTT is about 1/300 of the reaction rate of the other probes, the probe made from the acyclic disulfide anchor 3c. Reacts at about the same rate as a probe made from a mercaptohexyl anchor. This latter result is not surprising given the evidence that the reaction of acyclic disulfides with gold is involved in the cleavage of SS bonds (Zhong, CJ Langmuir, 15, 518). -525). Thus, an oligonucleotide with an acyclic head group will probably be linked to gold by one sulfur atom, as in the case of a mercaptohexyl-oligonucleotide derivative.
[0441]
To ascertain whether the probe made from Icl and Ic2 actually still functions as a hybridization probe after standing in the presence of DTT, we used the probe under the conditions used for the reactions in Table 1. Two samples of the mixture were treated with DTT. After 30 minutes, 1 μL of a 79mer target oligonucleotide solution (10 pmol) was added. Samples were snap frozen, thawed and measured by spot test. Sample spots containing the target were blue and control spots lacking the target were red. This demonstrated that the nanoparticle conjugate was not only stable, but also effective as a probe after exposure to DTT under conditions that caused aggregation of the probe from the mercaptohexyl or linear disulfide anchor group. .
[0442]
[Table 10]

(G)Conclusion
Gold nanoparticle-oligonucleotide conjugates made using this cyclic disulfide linker function as effective probes to detect specific oligonucleotide sequences, replacing conventional mercaptohexyl groups or acyclic disulfide units. It shows much greater stability to dithiothreitol than the corresponding conjugate made using. The high stability against thiol inactivation may be due, at least in part, to the anchoring of each oligonucleotide to gold by two sulfur atoms.
[0443]
Example 24:Preparation of oligonucleotide-modified gold nanoparticles using a simple cyclic disulfide linker
In this example, the present inventors prepared a non-steroidal cyclic disulfide linker and an oligonucleotide-nanoparticle probe from the linker, and provided a thiol-containing solution for the probe prepared with the steroidal cyclic disulfide and alkylthiol linker. Probe stability in the presence of was evaluated. The alkylthiol anchor group I (FIG. 44) [C. A. Mirkin et al., Nature, 382, 607 (1996); Storhoff et al. Am. Chem. Soc. 120, 1959 (1998)] or steroidal cyclic disulfide anchor group II (FIG. 44) [R. L. Letsinger et al., Bioconjugate Chemistry, 11, 289 (2000)] describe a method of preparing a probe for detecting a DNA or RNA sequence by binding an oligonucleotide to a gold nanoparticle. Conjugates prepared using steroidal cyclic disulfide linkers as probes have been developed using alkylthiol anchors in that they are much more stable in the presence of thiol compounds such as mercaptoethanol and dithiothreitol (DTT). It was found to be more free than the prepared conjugate. This feature is important because the PCR solution used to amplify the DNA sample to be detected contains a small amount of DTT to protect the enzyme. For simple and rapid detection of PCR products, it is desirable to use a probe with high stability to DTT so that the test can be performed directly in the PCR solution without separating the first amplified DNA.
[0444]
Two features highlight the steroid cyclic anchor (Compound 1, FIG. 42). (1) cyclic disulfides (which can in principle provide two binding sites that cooperate to hold a given oligonucleotide on the gold surface) and (2) gold by hydrophobic interactions. A steroid unit capable of stabilizing the upper adjacent chain (for example, see RL Letsinger et al., J. Am. Chem. Soc. 115, 7535 (1993)). To evaluate the significance of their contribution, we prepared and examined gold conjugates immobilized by cyclic disulfide compounds IIIc lacking steroid groups (FIG. 44).
[0445]
Compound 2a, prepared by heating trans-1,2-dithiane-4,5-dithiol with acetol in toluene, was converted to cyanoethyl N, N-di-i-propyl phosphoramidite reagent 2b. The reagent 2b was used for the final coupling step in the synthesis of the modified oligonucleotides 2c1 and 2c2. One gold conjugate probe was prepared by treating a colloidal gold solution with 2c1 and an equimolar amount of 2d. 2d functions as a diluent on the gold surface. Companion probes were similarly made from 2c2 and 2d. These nanoparticle conjugates were stable in the range of 0.1, 0.3, 0.5, 0.7 M of sodium chloride solution for both standing and freezing / thawing.
[0446]
(A)Preparation of compounds 2a, 2b, 2c1, 2c2
Compound 2a was prepared as described in Example 23. Phosphorylation of 2a and synthesis of oligonucleotides 2c1 and 2c2 were performed as described above for steroidal cyclic disulfide derivatives in Example 23 and elsewhere [R. L. Letsinger et al., Bioconjugate Chemistry, 11, 289 (2000), the disclosure of which is incorporated by reference in its entirety]. The reaction time for the step involved in condensing IIIb to the oligomer on the CPG support was 10 minutes.
[0447]
(B)Preparation of gold-oligonucleotide conjugate
Equimolar amounts of oligonucleotides 2c1 and 2d or 2c2 and 2d were added to 13 nm colloidal gold (〜１０10 nM) to provide a solution containing 1.7 μmol / mL of each oligonucleotide. The solution was stored in the dark for 24 hours, after which salts were added to make the solution 0.3 M NaCl, 10 mM phosphate (pH 7.0), 0.01% sodium azide. After 24 hours, the NaCl concentration was increased to 0.8 M and the solution was left for another 24 hours. The colloid is then filtered to remove aggregates and the solution is centrifuged to collect the nanoparticles. The pellet was washed with nanopure water, centrifuged again, and redispersed in 0.1 M NaCl, 10 mM phosphate buffer (pH 7.0), 0.01% sodium azide.
[0448]
(C)Reaction of dithiothreitol with nanoparticle probe
Displacement studies were performed at room temperature (22 ° C.) by adding 2 μL of 0.1 M DTT to an equal volume mixture of colloid conjugates from 20 μL of 2c1 and 2c2. Aliquots (3 μL) were taken periodically and spotted on a white nylon membrane. At first, the spot was red. Displacement of the oligonucleotide sulfur derivative from gold with DTT resulted in a mixture giving a blue-grey spot in the spot test. The time of replacement with DTT was taken as the time when the mixture gave a strong blue-grey in the spot test. In the case of a mixture of conjugates derived from 2c1 and 2c2, this time was 10 hours.
[0449]
For comparison, oligonucleotide conjugates were synthesized using oligonucleotide sequences cl and c2 (Figure 44), also using a mercaptohexyl anchor (Compound 2, Figure 42) and a steroid cyclic disulfide anchor (Compound 1, Figure 42). ). The reaction times of the conjugate prepared from the monothiol derivative (2 and FIG. 42) and the steroid cyclic disulfide, as measured by the time giving a blue-gray spot, were 5 minutes and 53 hours, respectively. These values correspond to a relative stability of 1, to 60, to 300 for nanoparticle-oligonucleotide conjugates derived from mercaptoalkyl derivatives, non-steroidal cyclic disulfides and steroidal cyclic disulfides. The results show that the cyclic disulfide anchor unit itself is sufficient to provide high stability to mercaptoalkyl groups in these systems. Large hydrophobic groups in the nanoparticle conjugate derived from Compound 1 also appear to play a role in enhancing the stability of oligonucleotides to thiol substitution.
[0450]
Example 25:Preparation of oligonucleotide-modified gold nanoparticles
In this example, we evaluated the stability of trithiol, a new acyclic disulfide linker, in the presence of a thiol-containing solution, as compared to alkyl thiols and steroidal cyclic disulfide linkers. For comparison, oligonucleotides with a mercaptohexyl anchor (compound 5, FIG. 45) and a steroidal cyclic disulfide anchor (compound 6, FIG. 45) were prepared.
[0451]
(A)Synthesis and characterization of 5'-trimercaptoalkyl oligonucleotides
As shown in FIG. 47, a 5′-trimercaptoalkylthiol oligonucleotide was synthesized. Trembler phosphoramidite 7 (Glen Research, Sterling, VA) (FIG. 45) and thiol modifier C6SS phosphoramidite were sequentially attached to the 5 'end of the protected oligomer bound to the CPG support. The product was dissociated from CPG and purified as described above. The retention time for a trithiol oligonucleotide with three DMT groups is about 64 minutes. Subsequently, the 5'-DMT group was removed by dissolving the oligonucleotide in 80% acetic acid for 30 minutes, followed by evaporation. The oligonucleotide was redissolved in 500 μL of nanopure water, and the solution was extracted with ethyl acetate (3 × 300 μL). After evaporation of the solvent, the oligonucleotide was obtained as a white solid. The retention time of this 5'-tri-disulfide oligonucleotide without DMT group was about 35 minutes on the reverse phase column and 24 minutes on the ion exchange column. All of these peaks accounted for 97% or more of the spectrum area, indicating that the oligonucleotide was highly pure. The formula weight of oligonucleotide 8 (Figure 45) was obtained by electrospray MS (calculated 12242.85, found 122444.1). The three disulfide groups on the DNA strand were reduced to trithiol groups as described above for the 5 'monothiol DNA, and then the oligonucleotide was purified on a NAP-5 column.
[0452]
(B)Preparation of 5'-thiol or disulfide DNA modified gold nanoparticles
Gold nanoparticles purchased from Vector Laboratories (Burlingame, CA) were used. 5 OD of thiol-modified DNA was added to 10 mL of 30 nm gold colloid. The solution was slowly made up to 0.3 M NaCl / 10 mM sodium phosphate buffer (pH 7) (PBS). Thereafter, the nanoparticles were sedimented by centrifugation. After removing the colorless supernatant, the red oily precipitate was redispersed in 10 mL of fresh PBS buffer. By repeating this process, the colloid was washed twice with 10 mL of fresh PBS buffer.
[0453]
(C)Stability test of thiol-DNA modified gold nanoparticles
Solid DTT was added to a 600 μL solution of 30 nm gold nanoparticle colloids modified with various types of thiol or disulfide DNA until the DTT concentration was 0.017M. As DTT displaces the oligonucleotide, the color of the colloid changes from red to blue. UV / VIS spectra were taken as a function of time. The absorbance at ５528 nm associated with the dispersed 30 nm gold particles began to decrease and the broad band at 700 nm began to grow. The 700 nm band is associated with colloid aggregation. As shown in FIG. 48, 30 nm gold particles modified with one thiol oligonucleotide (1) rapidly form aggregates at 0.017 M DTT, and after 1.5 hours the colloid turns completely blue . The solution containing the nanoparticles modified with the disulfide oligonucleotide (4) turns blue after 20 hours under the same conditions. For nanoparticles modified with trithiol-oligonucleotides (cleaved 6), it took 40 hours for the solution to turn blue.
[0454]
Example 26:Detection of analyte using nanoparticle-nucleic acid-sfp member conjugate
In this example, the detection of a specimen (biotin) is demonstrated using a nanoparticle-streptavidin probe (FIGS. 54 and 56).
[0455]
(A)Preparation of nanoparticle-nucleic acid-protein conjugate
The nanoparticle-nucleic acid conjugate is prepared as described in Example 3. The oligonucleotide modifier used to prepare these conjugates has the following sequence: 5 'SH (CH₂ )₆ -A₁₀-CGCATTCAGGAT3 '(2). As shown in FIG. 56, the biotin-labeled oligonucleotide (1) [3′-biotin-TEG-A₁₀-ATGCTCAACTCT5 '] is prepared according to literature procedures using a biotin TEG CPG support (Glen Research, Sterling, Virginia; catalog number 20-29555-01). To prepare a streptavidin / DNA conjugate, streptavidin was reacted with 1 equivalent of a biotin-modified oligonucleotide in a 20 mM Tris (pH 7.2), 0.2 mM EDTA buffer for 2 hours at room temperature in a stirrer. Was. Streptavidin complexed with different numbers of oligonucleotides was separated by ion exchange HPLC using 20 mM Tris (pH 7.2) and a 0.5% / min gradient of 20 mM Tris, 1 M NaCl at a flow rate of 1 mL / min. UV signal was monitored at 260 nM and 280 nM. Streptavidin / biotin modified oligonucleotide mixtures corresponded to 1: 1, 2: 1, 3: 1, and 4: 1 oligonucleotide-streptavidin complexes at 45, 56, 67, and 71 minutes, respectively. 4 peaks. The 1: 1 complex of streptavidin / oligonucleotide (10 μM, 5 μL) was isolated and linker DNA [5′TACGAGTTTGAGAATCCTGATTGCG3 ′] in 0.3 M PBS (0.3 M NaCl, 10 mM phosphate buffer, pH 7). 3) Premix with (10 μM, 5 μL), then add the mixture to the nanoparticle-oligonucleotide conjugate (10 nM, 130 μL) in 0.3 M PBS to add the nanoparticle-oligonucleotide-streptavidin conjugate. Prepared. The solution containing the nanoparticle-oligonucleotide conjugate, streptavidin / oligonucleotide, and linker DNA was frozen in dry ice for 10 minutes and thawed to facilitate DNA hybridization. After 12 hours, the solution was centrifuged at 1000 rpm for 20 minutes to remove the supernatant. The red oily precipitate of the nanoparticle-oligonucleotide-streptavidin conjugate was redispersed in 0.3 M PBS buffer.
[0456]
Thereafter, the presence of the target analyte, ie, biotin immobilized on the glass surface, was detected using the nanoparticle-oligonucleotide-streptavidin conjugate.
[0457]
(B)Biotin detection
Biotin was immobilized on glass slides according to the following procedure: Oligonucleotide-modified glass slides were prepared by a previously reported method (Science, 2000, Vol. 289, pp. 1757-1760), followed by biotin-modified oligos. The nucleotide was hybridized to the surface DNA. (Biotin-modified glass slides can be prepared by a variety of methods. One example is to prepare an amine-modified glass slide and then react the amine on the surface with various commercially available biotinylation reagents that react with the amine. As shown in FIG. 54, the immobilized biotin glass slide was subsequently treated with a medium containing nanoparticles-oligonucleotide-streptavidin (2 nM in 0.3 M PBS). After washing and treatment with a silver-enhanced solution, the appearance of a gray or shiny silver spot demonstrated the presence of the captured probe [Silver-enhanced solution A (catalog number S5020, Sigma Company, Missouri, USA) St. Louis, Iowa), and Silver Enhancement Solution B (Cat. No. S5145, Sigma Company), Science, 2000, vol. 289, p. 1775]. If desired, a nanoparticle-oligonucleotide-spf probe may be prepared using a fluorescently labeled specific binding pair member linked to the oligonucleotide. Thereafter, the presence of the analyte may be detected using the fluorophore-labeled probe.
[0458]
Example 27:Comparison of specific binding of colloidal gold streptavidin conjugate
In this example, a nanoparticle streptavidin probe prepared according to Example 26 and a commercially available Au colloid (20 nm) / streptavidin conjugate (full name: gold-labeled streptavidin-labeled) (Sigma Company, Missouri, USA) Non-specific binding was assessed from St. Louis, catalog number S6514). 5 μl each of a commercial solution containing the conjugate and a solution containing the probe of the invention (2 nM in 0.3 M PBS) were spotted on glass slides. After 20 hours, the slides were washed with water and treated with a silver-enriched solution [see Example 26]. The commercial conjugate shows a gray spot 5 minutes after color development, indicating the presence of Au colloid on the glass surface, whereas the nanoparticle streptavidin probe of Example 3 shows a recognizable silver stain. Not shown (FIG. 53). The difference in signal could be increased by placing the second DNA-modified nanoparticles on a silver stained slide and re-staining it. This demonstrates that the nanoparticle-oligonucleotide-sbp conjugate of the invention is more resistant to non-specific binding than the commercially available Au colloid / streptavidin conjugate.
[0459]
Example 28:Preparation of nanoparticle assembly
As discussed herein, specific binding pair members, eg, receptors or ligands, are functionalized by oligonucleotides and immobilized on oligonucleotide-modified nanoparticles to direct specific binding pair members rather than DNA. A new class of hybrid particles can be generated that exhibit the high stability of oligonucleotide-modified particles in addition to possessing the molecular recognition properties described above. Alternatively, a protein with multiple receptor binding sites with receptor-modified oligonucleotides can be functionalized so that in our original material assembly scheme, the protein receptor complex can be substructured instead of one of the inorganic nanoparticles. Can be used. In this example, we prepared a new nanoparticle assembly (FIG. 52) using 13 nm gold nanoparticles, streptavidin, and biotinylated DNA to obtain the physical and chemical properties of the resulting new bioinorganic material. Study some of the properties.
[0460]
(A)Experiment
Methods for preparing oligonucleotide-modified 13 nm Au particles (2-Au) and linker DNA (3) have been reported elsewhere. The oligonucleotide modifier used to prepare these conjugates has the following sequence: 5 'SH (CH₂ )₆ -A₁₀-CGCATTCAGGAT3 '. Linker DNA (3) has the following sequence: [5'TACGAGTTTGAGAATCCTGATTGCG3 ']. J. J. Storhoff, R .; Elghanian, R .; C. Music, C .; A. Mirkin, R .; L. Letsinger, J. et al. Am. Chem. Soc. See 1998, Vol. 120, p. 1959. Au particles (2-Au) and linker DNA (3) were stored in 0.3 M NaCl, 10 mM phosphate buffer (pH 7, 0.3 M PBS) before use. Biotin-modified DNA (2) was synthesized using a biotin TEG CPG support (Glen Research) and purified by literature methods. J. J. Storhoff, R .; Elghanian, R .; C. Music, C .; A. Mirkin, R .; L. Letsinger, J. et al. Am. Chem. Soc. 1998, 120, 1959; Brown, D.C. J. S. See Brown, in Oligonucleotides and Analogues (edited by F. Eckstein), Oxford University Press, New York, 1991. Streptavidin was purchased from Sigma and dissolved in 20 mM Tris buffer (30 M, pH 7.2). To make streptavidin / DNA conjugate (1-STV), streptavidin was added to 8 equivalents of biotin-oligonucleotide conjugate 1 in 20 mM Tris (pH 7.2), 0.2 mM EDTA buffer and agitator. At room temperature for 2 hours. The biotin-oligonucleotide conjugate has the sequence [3-biotin-TEG-A₁₀-ATGCTCAACTCT5 ']. Mixtures with different ratios of streptavidin to DNA (1: 0.4, 1: 1, 1: 4) were also prepared for HPLC analysis. The streptavidin / DNA conjugate was separated from the excess DNA by ion exchange HPLC at a flow rate of 1 mL / min with 20 mM Tris (pH 7.2) and 0.5% / min gradient 20 mM Tris, 1 M NaCl, while separating from the excess DNA at 260 nm and 280 nm. Was used to monitor the UV signal of the DNA. The 1: 0.4 streptavidin / DNA mixture has two peaks at 45 and 56 minutes, and the 1: 1 mixture has 1: 1, 2: 1 at 45, 56, 67, and 71 minutes, respectively. Four peaks corresponding to the 3: 1, and 4: 1 oligonucleotide-streptavidin complexes were shown. The 1: 4 mixture showed four peaks at similar positions except that the intensity of the third peak (67 min) and the fourth peak (71 min) increased. The HPLC spectrum of the 1: 8 streptavidin / DNA mixture shows two major peaks, one for the unreacted DNA at 59 minutes and one for the 4: 1 oligonucleotide-streptavidin complex at 71/71. Stuff showed. In addition, there was also a 67 minute shoulder corresponding to the 3: 1 complex. The purified streptavidin-biotinylated DNA conjugate was concentrated and dispersed in 0.3 M PBS by ultrafiltration (Centricon 30). The aggregate for TEM, heat denaturation experiments, and SAXS measurement was a solution containing streptavidin-oligonucleotide conjugate (1-STV), gold nanoparticle DNA conjugate (2-Au), and linker DNA (3). Was prepared by freezing in dry ice for 10 minutes and thawed before measurement to facilitate hybridization.
[0461]
(B)result
The nanoparticle / protein assembly reported here (FIG. 52) comprises three partial structures: streptavidin (1-STV) conjugated to four biotinylated oligonucleotides and oligonucleotide-modified gold nanoparticles (2- Au) and a linker oligonucleotide (3) having half of the sequence complementary to 1 and the other half complementary to 2 (see FIG. 52). In a typical experiment, linker DNA3 (10 μM, 21 μL) was introduced into a mixture of 2-Au (9.7 nM, 260 μL) and 1-STV (1.8 μM, 27 μL), or linker DNA3 (10 μM, 21 μL). ) Was premixed with 1-STV (1.8 μM, 27 μL), after which the mixture was added to 2-Au (9.7 nM, 260 μL) in 0.3 M PBS. Aggregates with comparable properties can be formed from both methods, but premixing of 3 and 1-STV promotes aggregate formation. 13 nm gold nanoparticles are substantially larger than streptavidin (4 nm × 4 nm × 5 nm) [P. C. Weber, D.S. H. Ohlendorf, J .; J. Wendoloski, F .; R. Salemme, Science, 1989, 243, 85; M. Green, Methods Enzymol. 1990, 184, 51], utilizing a 1:20 molar ratio of Au nanoparticles to streptavidin, from small aggregates containing several nanoparticles, or to a hybridized layer of streptavidin. The formation of a rather elongated polymer structure was favored over a structure consisting of a single functionalized gold nanoparticle.
[0462]
Interestingly, at room temperature, when 1-STV, 2-Au, and 3 were mixed, the UV-vis spectrum of the solution containing 1-STV, 2-Au, and 3 was shown to be stable. Even after 3 days, no observable particle aggregation occurs. However, raising the temperature of the solution (53 ° C.) a few degrees below the melting point (Tm) of the DNA interconnects led to the growth of millimeter-sized aggregates (FIG. 57A) and the Au plasmon resonance associated with particle assembly. A characteristic red shift and drop was provided. C. A. Mirkin, R .; L. Letsinger, R.A. C. Music, J .; J. See Storhoff, Nature 1996, 382, 607. Transmission electron microscopy (TEM) images (FIG. 57B) show that the particles in the aggregate retain their physical shape before and after annealing, indicating no fusion of the particles, and A stable effect of the surface oligonucleotide layer is demonstrated. The requirement for thermal activation to initiate particle assembly is inconsistent with previously studied systems (FIG. 52b), which show that oligonucleotide-modified gold particles can be stored at room temperature under very similar conditions. And contains two gold nanoparticle conjugates that are incorporated into the aggregate within minutes of adding the linker DNA. C. A. Mirkin, R .; L. Letsinger, R.A. C. Music, J .; J. See Storhoff, Nature 1996, 382, 607. This feature is due to: (1) Au on the streptavidin (DNA: streptavidin = 4: 1) compared to the DNA coating on the Au nanoparticles (DNA: particles =） 110: 1) -Low probability of collision in the STV assembly system [L. M. Demer, C .; A. Mirkin, R .; C. Music, R .; A. Reynolds, R.A. L. Letsinger, R.A. Elghanian, G .; Viswanadham, Anal. Chem. 2000, Vol. 72, p. 5535], or (2) due to the initial formation of a kinetic structure in which most or all of the DNA on a single streptavidin binds to the DNA on a single particle. there is a possibility. Aggregate formation can be promoted by heating as well as freezing the solution. R. Elghanian, J .; J. Storhoff, R .; C. Music, R .; L. Letsinger, C.E. A. See Mirkin, Science 1997, 277, 1078.
[0463]
Melting experiments based on UV-vis spectroscopy were performed on streptavidin / nanoparticle aggregates, confirming that they were formed by DNA hybridization (FIG. 58A). Upon heating, the absorbance at 520 nm initially decreases, resulting in a temporary drop in the melting curve, followed by a sharp rise in absorbance as DNA melting occurs and the particles disperse. This temporary descent behavior was observed in pure gold nanoparticle systems and was attributed to aggregate growth prior to melting, ie, "aging" of certain aggregates. J. J. Storhoff, A .; A. Lazarides, C.I. A. Mirkin, R .; L. Letsinger, R.A. C. Music, G .; C. Schatz, J. et al. Am. Chem. Soc. 2000, vol. 122, p. 4640. After melting, the Au plasmon resonance is centered at 520 nm, which is characteristic of dispersed 13 nm nanoparticles. These experiments consequently demonstrate that sequence-specific hybridization, rather than non-specific interactions, performs particle / protein assembly and that the process is completely reversible.
[0464]
Au nanoparticles / protein assemblies can also be formed by streptavidin / biotin interactions, as opposed to hybridization-inducible assemblies. For example, in a typical experiment, 1 (0.24 mM, 1.3 μL), 3 (10 μM, 32 μL), and 2-modified nanoparticles (Au nanoparticle concentration: 9.7 nM, 260 μL) were combined with 0.3 M PBS. Mixing in buffer produced biotin-modified Au particles. The mixture was heated to 60 ° C. for 10 minutes and then cooled to room temperature to facilitate hybridization. Twenty-four hours later, the solution containing the 1, 3, and 2-modified Au nanoparticles was added to streptavidin (10 μM, 4.2 μL) in 0.3 M PBS and heated to 50 ° C. to precipitate the nanoparticles. Aggregates were formed. The color of the solution changes to purple, indicating the formation of aggregates, and increasing the solution temperature above Tm (65 ° C.) causes the aggregates to become 2-modified Au particles, 1-modified streptavidin, and 3 Could be dismantled. Small angle X-ray scattering (SAXS) [S. J. Park, A .; A. Lazarides, C.I. A. Mirkin, P .; W. Brazis, C .; R. Kannewurf, R .; L. Letsinger, Angew. Chem. Int. Ed. 2000, 39, 3845; Guinier, F.S. G. FIG. B., Small Angle Scattering of X-rays, Wiley, New York, 1995; A. Korgel, D .; Fitzmaurice, Phys. Rev .. B 1999, Vol. 59, page 14191], using Au and streptavidin substructures (1-STV, 2-Au) and two different length DNA linkers (24-mer (3) and 48-mer). ) And aggregates formed from the aggregates based on the Au-Au assembly and similar linking oligonucleotides (2-Au, 4-Au, 24-mer linker (3), 48-mer linker). (Fig. 59). The 48-mer linker is a 5'TACGAGTTTGAGA containing cohesive ends of the 12-mer complementary to 1 and 2.CCGTTAAGACGAGGCAATCATGCAATCCTGAATGCG and 3 'GGCAATTCTGCTCCCGTTAGTACGT(A double-stranded region is underlined). 1,2, and 4A used in the above experiments to design SAXS experiments and provided higher accessibility for DNA hybridization₁₀Spacer [L. M. Demer, C .; A. Mirkin, R .; C. Music, R .; A. Reynolds, R.A. L. Letsinger, R.A. Elghanian, G .; Viswanadham, Anal. Chem. 2000, Vol. 72, p. 5535] to create a more robust system. Aggregates linked by DNA showed relatively clear diffraction peaks, and Au-STV aggregates exhibited diffraction peaks with smaller s values than Au-Au aggregates formed from the same linker. This suggests that the distance between Au particles in the Au-STV system is larger. Moreover, when 48-mer DNA was used instead of 24-mer DNA as the linker in both the Au-Au and Au-STV assemblies, the diffraction peaks shifted to smaller s values (Figure 59).
[0465]
G (r), which is a pair distance distribution function (PDDF), is expressed by equation (1) (where q = (4π / λ) sin (θ / 2), and ρ is a particle number density. Was calculated from the SAXS pattern using [A. Guinier, F .; G. FIG. B., Small Angle Scattering of X-rays, Wiley, New York, 1955; A. Korgel, D .; Fitzmaurice, Phys. Rev .. B 1999, Vol. 59, pp. 14191].
[0466]
g (r) = 1 + (1 / 2B² Δr) Iq (s (q) -1) sin (qr) dq Equation (1)
Here, g (r) indicates the probability of finding the second particle as a function of the distance r from the selected particle. The closest inter-Au particle distances (distance from center to center) obtained from PDDF are 24mer linked Au-Au, 48mer linked Au-Au, 24mer linked Au-STV, and 48mer 19.3 nm, 25.4 nm, 28.7 nm, and 40.0 nm for the coupled Au-STV assembly, respectively. Comparing the interparticle distances of the Au-STV and Au-Au systems, the Au-STV assembly has the expected Au nanoparticle / streptavidin periodicity and the two components (Au nanoparticle and streptavidin) It is clearly shown that the strong DNA double-stranded linker is well separated.
[0467]
(C) Conclusion
This study is important for the following reasons: 1) It has been demonstrated that the DNA-dependent nanoparticle assembly method can be extended to the structure of proteins in addition to the inorganic nanoparticles previously studied. In fact, this is the first report demonstrating a reversible DNA-dependent assembly of Au nanoparticles and a proteinaceous structure functionalized by DNA. 2) It is well known that many proteins, including streptavidin, are adsorbed on the surface of colloidal gold to form strongly bound complexes. Our experiments demonstrate the stable effect of a dense DNA layer on the surface of the nanoparticles and the resistance to streptavidin adsorption without hybridization. Therefore, they are directed to a method of specifically immobilizing the protein structure on the surface of nanoparticles by very specific interactions in a manner that does not substantially disrupt the activity of the protein. Particles with proteins adsorbed on the surface play an important role in immunochemistry [M. A. Hayat, Colloidal Gold: Principles, Methods, and Applications, Academic Press, San Diego, 1991], histochemical studies and immunoassays through the development of stable protein / particle conjugates in which proteins interact directly with the nanoparticle surface. An improved nanoparticle probe for can be derived.
[0468]
Example 29:Acceleration of nanoparticles across an electric field
The present invention describes a method for moving nanoparticles stabilized with citric acid and nanoparticles coated with charged biomolecules in an electric field. Regarding the use of nanoparticles for detecting nanoparticles and nucleic acids, PCT Application Nos. WO 98/04740 (Northwestern University) and WO 00/33079 (Nanosphere LLC), both of which are incorporated herein in their entirety. Is described in detail. Detection techniques that utilize nanoparticle probes functionalized with biomolecules are based on the ability of the probe molecule to find immobilized targets and capture oligonucleotide chains (in the case of chips). This process can accelerate the movement of the probe to the electrode surface with the “captured target” and the movement of the set of probes having the captured target in solution to the target surface. The method of the present invention has important biodiagnostic applications using nanoparticles-based probes. For example, the methods of the invention can be used to accelerate the hybridization of a nanoparticle probe or set of probes to a surface functionalized with complementary DNA or a complementary antigen / antibody.
[0469]
The concept of applying an electric field in a fluorescent hybridization detection system to promote active movement of a biomolecule, such as DNA, in an electric field toward a target site is described, for example, in US Pat. No. 5,849,486. Have been. However, none of these patents mention the application of an electric field to accelerate the movement of the nanoparticles bound to the biomolecules to the target site.
[0470]
To investigate the effect of an electric field on the transport of DNA-modified nanoparticles, 13 nm gold nanoparticles were modified with 3 'propanethiol-capped 22-mer DNA and the modified particles were dispersed in water. DC across two indium-tin oxide (ITO) electrodes using a Keithley 487 picoammeter / voltage source (Keithley Instruments, Inc., Cleveland Aurora Road 28775, Ohio, Cat. No. 487 picometer) An electric field was applied and the current was measured. See Scheme 1 illustrating the movement of the electrodes and nanoparticles. ITO-coated glass (Delta Technologies, Stillwater, MN, USA, number CD-50IN-CUV) was cut into 0.8 x 1 cm pieces. Two indium-tin oxide coated glass substrates were used as cathode (cathode) and anode (anode). The electrodes were immersed in the solution of DNA-modified nanoparticles and a DC electric field of 3 V was applied for 15 minutes (4 μA). After application of the electric field, the electrodes were washed with water. A pink color was observed on the positively biased electrode (anode), with no visible change on the cathode. Capture of the nanoparticles on the anode was confirmed by signal enhancement using silver with a solution of Ag (I) and hydroquinone. Each electrode was immersed in the silver staining solution for 3 minutes. The anode turned black and the cathode had no substantial change. This observation indicates that DNA-modified particles can be transported by an electric field due to the high negative charge on the particles. This also indicates that an electric field can be used to facilitate hybridization of the DNA-modified particles to complementary oligonucleotides on the electrode surface. It further suggests that it may extend to other biorecognition processes involving charged particles.
[0471]
All patents, patent applications, and references cited herein are incorporated by reference in their entirety.
[Brief description of the drawings]
FIG. 1 is a schematic showing formation of nanoparticle aggregates by binding nanoparticles having complementary oligonucleotides attached thereto. The nanoparticles are attached in aggregates as a result of the hybridization of the complementary oligonucleotide. X represents (-S (CH₂)₃OP (O) (O⁻) -Represents any covalent anchor (such as where S is attached to a gold nanoparticle). Although FIG. 1 and several subsequent figures show, for clarity, only a single oligonucleotide attached to each nanoparticle, in practice each nanoparticle has Some oligonucleotides are attached. It is also important to note that the relative sizes of the gold nanoparticles and oligonucleotides are not drawn to scale in FIG. 1 and subsequent figures.
FIG. 2 is a schematic diagram showing a system for detecting nucleic acids using nanoparticles to which oligonucleotides are attached. The oligonucleotides on the two nanoparticles have sequences complementary to two different parts of the single-stranded DNA shown. As a result, when these oligonucleotides hybridize to DNA, a detectable change occurs (forming aggregates and causing discoloration).
FIG. 3 is a schematic diagram of a variation of the system shown in FIG. The oligonucleotide on the two nanoparticles has a sequence complementary to two different portions of the single-stranded DNA separated by a third portion that is not complementary to the oligonucleotide on the indicated nanoparticle. are doing. Also shown are any filler oligonucleotides that can be used to hybridize to non-complementary portions of single-stranded DNA. When the DNA, nanoparticles and filler oligonucleotide are combined, the nanoparticles aggregate to form a nicked double-stranded oligonucleotide connector.
FIG. 4 is a schematic diagram showing the reversible aggregation of nanoparticles with attached oligonucleotides as a result of hybridization and dehybridization with a linking oligonucleotide. The illustrated ligating oligonucleotide is a double-stranded DNA having an overhang end (sticky end) complementary to the oligonucleotide attached to the nanoparticle.
FIG. 5 is a schematic diagram showing the formation of a nanoparticle aggregate by combining a nanoparticle having an oligonucleotide attached thereto and a linking oligonucleotide having a sequence complementary to the oligonucleotide attached to the nanoparticle.
FIG. 6A shows a cuvette containing two types of colloidal gold each having a different oligonucleotide attached thereto, and a ligated double-stranded oligonucleotide having a sticky end complementary to the oligonucleotide attached to the nanoparticle ( (See FIG. 4). 80 ° C. above the Tm of the cuvette A-ligated DNA; dehybridized (heat denatured). The color is dark red.
FIG. 6B shows a cuvette containing two types of colloidal gold to which different oligonucleotides are attached, and a linked double-stranded oligonucleotide having an attached end complementary to the oligonucleotide attached to the nanoparticles ( (See FIG. 4). After cooling to room temperature below the Tm of the cuvette B-linker DNA. Upon hybridization, the nanoparticles aggregated but the aggregates did not precipitate. The color is purple.
FIG. 6C shows a cuvette containing two types of colloidal gold to which different oligonucleotides are attached, and a linked double-stranded oligonucleotide having an attached end complementary to the oligonucleotide attached to the nanoparticles ( (See FIG. 4). Cuvette C-After several hours at room temperature, the aggregated nanoparticles settled at the bottom of the cuvette. The solution is clear and the precipitate is pinkish-grey. Heating B or C produces A.
FIG. 7: As shown in FIG. 4, when the temperature of the gold nanoparticles to which the oligonucleotides are attached is lowered, the gold nanoparticles are hybridized with the linked oligonucleotides, and thus the change in the absorbance when the nanoparticles are aggregated is shown. nm).
FIG. 8A is a graph of absorbance versus temperature / time change for the system shown in FIG. At low temperatures, the gold nanoparticles to which the oligonucleotides are attached aggregate by hybridization with the linked oligonucleotides (see FIG. 4). Under high temperature (80 ° C.), the nanoparticles dehybridize. Changes in temperature over time indicate that this is a reversible process.
FIG. 8B is a graph of the change in absorbance versus temperature / time performed in the same manner as in FIG. 8A using an aqueous solution of unmodified gold nanoparticles. The reversible change seen in FIG. 8A is not observed.
FIG. 9A is a transmission electron microscope (TEM) image. It is a TEM image of the aggregated gold nanoparticle couple | bonded by the hybridization of the oligonucleotide on a gold nanoparticle, and a connection oligonucleotide.
FIG. 9B is a transmission electron microscope (TEM) image. FIG. 4 is a TEM image of a two-dimensional aggregate showing the ordering of bound nanoparticles.
FIG. 10 is a schematic diagram showing the formation of a thermostable triple-stranded oligonucleotide connector between nanoparticles having a pyrimidine: purine: pyrimidine motif. Such a triple-stranded connector has greater rigidity than a double-stranded connector. In FIG. 10, one nanoparticle has an oligonucleotide composed entirely of purines attached to it, and the other nanoparticle has an oligonucleotide composed entirely of pyrimidine attached thereto. The third oligonucleotide for forming a triple-stranded connector (not attached to the nanoparticle) consists of pyrimidine.
FIG. 11 is a schematic diagram illustrating the formation of nanoparticle aggregates by combining nanoparticles to which complementary oligonucleotides are attached, wherein the nanoparticles form aggregates as a result of hybridization of the complementary oligonucleotides. Are combined. In FIG. 11, circles represent nanoparticles, formula is the oligonucleotide sequence, and s is a thio-alkyl linker. Many oligonucleotides on two types of nanoparticles can hybridize to each other to form an aggregate structure.
FIG. 12A is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 3 are shown. The circles represent the nanoparticles, the formula is the oligonucleotide sequence, and the dashed and dashed lines represent the joints connecting the nucleotides.
FIG. 12B is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 are shown. The circles represent the nanoparticles, the formula is the oligonucleotide sequence, and the dashed and dashed lines represent the joints connecting the nucleotides.
FIG. 12C is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 4 are shown. The circles represent the nanoparticles, the formula is the oligonucleotide sequence, and the dashed and dashed lines represent the joints connecting the nucleotides.
FIG. 12D is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 5 are shown. The circles represent the nanoparticles, the formula is the oligonucleotide sequence, and the dashed and dashed lines represent the joints connecting the nucleotides.
FIG. 12E is a schematic diagram showing a nucleic acid detection system using a nanoparticle to which an oligonucleotide is attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 6 are shown. The circles represent the nanoparticles, the formula is the oligonucleotide sequence, and the dashed and dashed lines represent the joints connecting the nucleotides.
FIG. 12F is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 7 are shown. The circles represent the nanoparticles, the formula is the oligonucleotide sequence, and the dashed and dashed lines represent the joints connecting the nucleotides.
FIG. 13A is a schematic diagram showing a DNA (analyte DNA) detection system using nanoparticles and a transparent substrate.
FIG. 13B is a schematic diagram showing a DNA (analyte DNA) detection system using nanoparticles and a transparent substrate.
FIG. 14A shows gold nanoparticles with oligonucleotides attached (one population in solution, one population attached to a transparent substrate as shown in FIG. 13B) linked oligonucleotides 5 is a graph of absorbance vs. wavelength (nm) showing the change in absorbance when aggregated by hybridization with Hg.
FIG. 14B is a graph of the change in absorbance for the hybridized system shown in FIG. 14A with increasing temperature (melting).
FIG. 15A is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 15B is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 3 are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 15C is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 4 are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 15D is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 5 are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 15E is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 6 are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 15F is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 7 are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 15G is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and single-stranded oligonucleotide target 8 are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 16A is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and multiple single-stranded oligonucleotide targets of different lengths are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 16B is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and multiple single-stranded oligonucleotide targets of different lengths are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 16C is a schematic diagram showing a nucleic acid detection system using nanoparticles to which oligonucleotides are attached. Oligonucleotide-nanoparticle conjugates 1 and 2 and multiple single-stranded oligonucleotide targets of different lengths are shown. Circles represent nanoparticles, formula is oligonucleotide sequence, S represents thio-alkyl linker.
FIG. 17A is a schematic diagram showing a nanoparticle-oligonucleotide conjugate and a nucleic acid detection system using a nanoparticle to which an oligonucleotide is attached. Circles represent nanoparticles, straight lines represent oligonucleotide chains (bases not shown), two closely spaced parallel lines represent double-stranded molecular segments, and lower case letters represent specific nucleotide sequences (a is a 'and Complementary, b is complementary to b ', etc.).
FIG. 17B is a schematic diagram showing a nanoparticle-oligonucleotide conjugate and a nucleic acid detection system using a nanoparticle to which an oligonucleotide is attached. Circles represent nanoparticles, straight lines represent oligonucleotide chains (bases not shown), two closely spaced parallel lines represent double-stranded molecular segments, and lower case letters represent specific nucleotide sequences (a is a 'and Complementary, b is complementary to b ', etc.).
FIG. 17C is a schematic diagram showing a nanoparticle-oligonucleotide conjugate and a nucleic acid detection system using a nanoparticle to which an oligonucleotide is attached. Circles represent nanoparticles, straight lines represent oligonucleotide chains (bases not shown), two closely spaced parallel lines represent double-stranded molecular segments, and lower case letters represent specific nucleotide sequences (a is a 'and Complementary, b is complementary to b ', etc.).
FIG. 17D is a schematic diagram showing a nanoparticle-oligonucleotide conjugate and a nucleic acid detection system using a nanoparticle to which an oligonucleotide is attached. Circles represent nanoparticles, straight lines represent oligonucleotide chains (bases not shown), two closely spaced parallel lines represent double-stranded molecular segments, and lower case letters represent specific nucleotide sequences (a is a 'and Complementary, b is complementary to b ', etc.).
FIG. 17E is a schematic diagram showing a nanoparticle-oligonucleotide conjugate and a nucleic acid detection system using a nanoparticle to which the oligonucleotide is attached. Circles represent nanoparticles, straight lines represent oligonucleotide chains (bases not shown), two closely spaced parallel lines represent double-stranded molecular segments, and lower case letters represent specific nucleotide sequences (a is a 'and Complementary, b is complementary to b ', etc.).
FIG. 18 is a schematic diagram showing a nucleic acid detection system using liposomes (large double circles), nanoparticles (small white circles), and a transparent substrate. Black squares represent cholesteryl groups, twisted lines represent oligonucleotides, and ladders represent double-stranded (hybridized) oligonucleotides.
FIG. 19A is a graph of absorbance versus wavelength (nm) showing the change in absorbance when the gold nanoparticle-oligonucleotide conjugate is assembled as a multilayer on a transparent substrate as shown in FIG. 13A.
FIG. 19B is a graph of the change in absorbance for the hybridized system shown in FIG. 19A with increasing temperature (melting).
FIG. 20A is a schematic diagram of a scheme using fluorescently labeled oligonucleotides attached to metal or semiconductor quenching nanoparticles (FIG. 20A).
FIG. 20B is a schematic of a scheme using fluorescently labeled oligonucleotides attached to non-metallic, non-semiconductor particles (FIG. 20B).
FIG. 21 is a schematic diagram showing a target nucleic acid detection system using gold nanoparticles to which oligonucleotides are attached and latex microspheres to which fluorescently labeled oligonucleotides are attached. Small black circles represent nanoparticles, large open circles represent latex microspheres, and large ellipses represent microporous membranes.
FIG. 22 is a schematic diagram showing a target nucleic acid detection system using two types of fluorescently labeled oligonucleotide-nanoparticle conjugates. Black circles represent nanoparticles, large ellipses represent microporous membranes.
FIG. 23. Sequence of materials used in an assay for anthrax protective antigen (see Example 12).
FIG. 24: Magnetic nanoparticles (black spheres) having oligonucleotides (linear) attached thereto, and “satellite probes” including probe oligonucleotides (linear) hybridized to oligonucleotides attached to nanoparticles were used. 1 is a schematic diagram showing a target nucleic acid detection system, in which a probe oligonucleotide is labeled with a reporter group (open square). A, B, C, A ', B', and C 'represent specific nucleotide sequences, with A, B, and C being complementary to A', B ', and C', respectively.
FIG. 25A is a schematic diagram showing a DNA detection system using nanoparticles and a transparent substrate. In these figures, a, b and c represent different oligonucleotide sequences and a ', b' and c 'represent oligonucleotide sequences complementary to a, b and c, respectively.
FIG. 25B is a schematic diagram showing a DNA detection system using nanoparticles and a transparent substrate. In these figures, a, b and c represent different oligonucleotide sequences and a ', b' and c 'represent oligonucleotide sequences complementary to a, b and c, respectively.
FIG. 26 is a schematic diagram showing a system for forming an aggregate of CdSe / ZnS core / shell quantum dots (QD).
FIG. 27A shows a fluorescence spectrum comparing dispersed QD and aggregated QD at 400 nm excitation. Samples were prepared identically, except that one was supplemented with complementary "linker" DNA and the other with equal and equal concentrations of non-complementary DNA.
FIG. 27B shows the UV-visible spectrum of the QD / QD assemblage at different temperatures before, during and after “melting”. The inset in the figure shows the temperature vs. extinction profile for the thermal denaturation of the aggregate. Denaturation experiments were performed with 13 nm gold nanoparticles and / or ４4 nm CdSe / ZnS core / shell QD in 0.3 M NaCl, 10 mM phosphate buffer (pH 7), 0.01% sodium azide. .
FIG. 27C shows a high resolution TEM image of a portion of the hybrid gold / QD assembly. A QD lattice fringe resembling a fingerprint is visible near each gold nanoparticle.
FIG. 27D shows UV-visible spectra of hybrid gold / QD aggregates at different temperatures before, during and after “melting”. The inset in the figure shows the temperature vs. extinction profile for the thermal denaturation of the aggregate. Denaturation experiments were performed with 13 nm gold nanoparticles and / or ４4 nm CdSe / ZnS core / shell QD in 0.3 M NaCl, 10 mM phosphate buffer (pH 7), 0.01% sodium azide. .
FIG. 28A is a schematic diagram showing production of a core probe and an aggregate probe and a DNA detection system using these probes. In these figures, a, b, c and d represent different oligonucleotide sequences and a ', b', c 'and d' represent oligonucleotide sequences complementary to a, b, c and d, respectively. Represent.
FIG. 28B is a schematic diagram showing the preparation of a core probe and an aggregate probe, and a DNA detection system using these probes. In these figures, a, b, c and d represent different oligonucleotide sequences and a ', b', c 'and d' represent oligonucleotide sequences complementary to a, b, c and d, respectively. Represent.
FIG. 28C is a schematic diagram showing the preparation of a core probe and an aggregate probe and a DNA detection system using these probes. In these figures, a, b, c and d represent different oligonucleotide sequences and a ', b', c 'and d' represent oligonucleotide sequences complementary to a, b, c and d, respectively. Represent.
FIG. 28D is a schematic diagram showing production of a core probe and an aggregate probe and a DNA detection system using these probes. In these figures, a, b, c and d represent different oligonucleotide sequences and a ', b', c 'and d' represent oligonucleotide sequences complementary to a, b, c and d, respectively. Represent.
FIG. 28E is a schematic diagram showing production of a core probe and an aggregate probe and a DNA detection system using these probes. In these figures, a, b, c and d represent different oligonucleotide sequences and a ', b', c 'and d' represent oligonucleotide sequences complementary to a, b, c and d, respectively. Represent.
FIG. 29 is a graph of the displacement fraction of oligonucleotides from nanoparticles with attached oligonucleotides (closed circles) or gold thin films (open squares) by mercaptoethanol.
FIG. 30 is a graph of the surface coverage of the recognition oligonucleotide on the nanoparticle obtained for various recognition oligonucleotide: diluent oligonucleotide ratios used in the preparation of the nanoparticle-oligonucleotide conjugate.
FIG. 31 is a graph of the surface coverage of hybridized complementary oligonucleotides for various surface coverages of the recognition oligonucleotide on the nanoparticles.
FIG. 32 is a schematic diagram showing a system for detecting target DNA in a four-element array on a substrate using nanoparticle-oligonucleotide conjugates and amplification by silver staining.
FIG. 33 is an image of a 7 mm × 13 mm oligonucleotide-functionalized float glass slide obtained using a flatbed scanner. (A) Slide before hybridization of DNA target with gold nanoparticle-oligonucleotide indicating conjugate. (B) Slide A after hybridization of 10 nM target DNA and 5 nM nanoparticle-oligonucleotide indicating conjugate. The pink color is due to the attachment of gold nanoparticles having a red diameter of 13 nm. (C) Slide B after exposure to silver amplification solution for 5 minutes. (D) Same as (A). (E) Slide D after 100 pM target and 5 nM nanoparticle-oligonucleotide indicating conjugate hybridized. The absorbance of the nanoparticle layer was too low to be observed with the naked eye or a flatbed scanner. (F) Slide E after exposure to silver amplification solution for 5 minutes. Slide F is much brighter than slide C, indicating a lower target concentration. (G) Control slides exposed to 5 nM nanoparticle-oligonucleotide indicating conjugate and exposed to silver amplification solution for 5 minutes. No darkening of the slide was observed.
FIG. 34. Gray scale (optical) of oligonucleotide-functionalized glass surfaces exposed to various concentrations of target DNA prior to exposure to 5 nM gold nanoparticle-oligonucleotide indicator conjugate and to silver amplification solution for 5 minutes. Density) graph.
FIG. 35A is a graph of% hybridized target versus temperature showing dissociation of the fluorophore-labeled target from the oligonucleotide-functionalized glass surface.
FIG. 35B is a graph of% hybridized target versus temperature showing dissociation of the nanoparticle-labeled target (FIG. 35B) from the oligonucleotide-functionalized glass surface. 35A and B, the measurement was performed by measuring the fluorescence (FIG. 35A) and the absorbance (FIG. 35B) of the dissociated label in a solution on the glass surface. The line labeled "b" shows the dissociation curve of perfectly matched oligonucleotides on glass, and the line labeled "r" is the mismatched oligonucleotide on glass (single base mismatch). Is shown. The vertical line in the graph represents the given temperature (melting temperature T for each curve) for each measurement._m3 shows the expected fractional identification selectivity for the target fraction and the fluorophore and the nanoparticle-based gene chip dissociated in (Middle). Fluorescence (FIG. 35A): complement (69%) / mismatch (38%) = 1.8: 1. Absorbance (FIG. 35B): complement (85%) / mismatch (14%) = 6: 1. The width of the fluorophore labeling curve (FIG. 35A) is characteristic of the dissociation of the fluorophore labeled target from the gene chip (Forman et al., Molecular Modeling of Nucleic Acids, Leontis et al., Eds. (ACS Symposium Series 682, American Chemical Society, Washington, DC, 1998), pp. 206-228).
FIG. 36A is an image of a model oligonucleotide sequence challenged with a synthetic target and a fluorescently labeled nanoparticle-oligonucleotide conjugate probe.
FIG. 36B is an image of a model oligonucleotide sequence challenged with a nanoparticle-labeled nanoparticle-oligonucleotide conjugate probe. In FIGS. 36A and B, C, A, T, and G indicate that a single base change occurs in the oligonucleotide attached to the substrate, resulting in complete pairing with the target (base A) or a single base mismatch (base A). Represents a spot (element) on the sequence that resulted in a base C, T or G) instead of a perfect match with. The gray scale ratio of element C: A: T: G is 9: 37: 9: 11 for FIG. 36A and 3: 62: 7: 34 for FIG. 36B.
FIG. 37 is a schematic diagram showing a system for forming an aggregate (A) or a layer (B) of nanoparticles (a and b) linked via a linking nucleic acid (3).
FIG. 38A. UV-visible spectrum of alternating layers of a and b of gold nanoparticles hybridized to oligonucleotide-functionalized glass microscope slides via complementary linker 3 (see FIG. 37). These spectra are 1 (a, λ_max= 524 nm), 2 (b, λ_max= 529 nm), 3 (c, λ_max= 532 nm), 4 (d, λ_max= 534 nm) or 5 (e, λ)_max= 534 nm). These spectra were measured directly via the slide.
FIG. 38B shows the λ of the nanoparticle assembly (see FIG. 38A) with increasing number of layers._maxGraph of absorbance at.
FIG. 39A shows one of the oligonucleotide-functionalized gold nanoparticles co-hybridized with an oligonucleotide-functionalized conductive indium-tin-oxide (ITO) slide (made in the same manner as an oligonucleotide-functionalized glass slide) with a DNA linker. FE-SEM of layer. The visible absorbance spectrum of this slide was identical to FIG. 38A, indicating that the functionalization on ITO and the nanoparticle coverage were similar to those on glass. The average density of nanoparticles counted from 10 such images is about 800 nanoparticles / μm²Met.
FIG. 39B is an FE-SEM image of two nanoparticle layers on an ITO slide. The average density of nanoparticles counted from 10 such images is about 2800 nanoparticles / μm²Met.
FIG. 39C shows the dissociation of a 0.5 μM solution of an oligonucleotide double-stranded molecule (1 + 2 + 3; see FIG. 35A) from a single strand in 0.3 M NaCl, 10 mM phosphate buffer (pH 7).₂₆₀) Absorbance.
FIG. 39D shows the dissociation of one layer of oligonucleotide functionalized gold nanoparticles from a glass slide immersed in 0.3 M NaCl, 10 mM phosphate buffer at 260 nm (A₂₆₀) Absorbance.
FIG. 39E shows the dissociation of four layers of oligonucleotide-functionalized gold nanoparticles from a glass slide immersed in 0.3 M NaCl, 10 mM phosphate buffer at 260 nm (A₂₆₀) Absorbance.
FIG. 39F shows the dissociation of 10 layers of oligonucleotide-functionalized gold nanoparticles from a glass slide immersed in 0.3 M NaCl, 10 mM phosphate buffer at 260 nm (A₂₆₀) Absorbance. Referring to Figures DF, 520 nm (A) through the slide decreases with increasing temperature.₅₂₀) Was measured to obtain a melting profile. The inset for each of FIGS. 39D-F shows the first derivative of the measured dissociation curve. The FWHM of these curves is 13.2 ° C. (inset of FIG. 39C), 5.6 ° C. (inset of FIG. 39D), 3.2 ° C. (inset of FIG. 39E), (inset of FIG. 39F). 2.9 ° C.
FIG. 40 is a schematic diagram showing a system used for measuring the electrical properties of a gold nanoparticle assembly bound via DNA. For clarity, only a single hybridization event is shown.
FIG. 41 is a schematic diagram showing a nucleic acid detection method using a gold electrode and gold nanoparticles.
FIG. 42 is a schematic illustrating the structure of cyclic disulfide 1, including preferred compound 1 having a steroid moiety used in linking oligonucleotides to nanoparticles. Steroid disulfide molecules are obtained by condensing 4,5-dihydroxy-1,2-dithiane with epiandrosterone. Gold nanoparticle-oligonucleotide conjugates were prepared using steroid disulfide-modified oligonucleotides, and compared to nanoparticle-oligonucleotide conjugates using oligonucleotide-mercaptohexyl linkers for their preparation. Shows greater stability against
FIG. 43 is a schematic showing the synthesis and chemical formula for a steroid cyclic sulfide anchor group.
FIG. 44 is a schematic diagram showing the cyclic disulfide of Formula 2 used to prepare the oligonucleotide cyclic disulfide linker described in Example 24 and similar related cyclic disulfides used as anchor groups.
FIG. 45 is a schematic diagram showing the structure described in Example 25; FIG. 45 (a) shows 5'-monothiol-modified oligonucleotide 5, 35 base 5'-steroid disulfide oligomer 6, Trembler phosphoramide 7, and 5-tri-mercaptoalkyl oligonucleotide 8.
FIG. 46 is a schematic diagram showing a chemical mechanism for producing a novel trithiol oligonucleotide.
FIG. 47. Schematic representation of high density oligonucleotide nanoparticle conjugate I, oligonucleotide protein conjugate 1, nanoparticle-oligonucleotide-protein conjugate II, and probe-target complex III. Oligonucleotide conjugate I has a sequence complementary to the oligonucleotide bound to conjugate I. Probe II is a representative probe for proteins. Complex III results from a specific binding interaction between the protein target and the protein bound to the probe.
FIG. 48 is a schematic diagram of an example of the application of Probe II in detecting specific proteins in an array of different proteins immobilized on a glass surface. Gold nanoparticles can be recognized visually or by silver staining. Fluorescent nanoparticles can be recognized by their fluorescence. As shown in this figure, the glass plate shows three different proteins immobilized on the surface, one of which binds to the protein with probe II. In step 1, the plate is exposed to a solution containing probe II. In step 2, unbound probe II is washed away. In step 3, the presence of bound nanoparticles (IV) at the site occupied by the first protein in the series is observed.
FIG. 49 is a schematic diagram of a nanoparticle-oligonucleotide-receptor probe (II ′) and an application example in which a specific target is detected with an array of substances immobilized on a smooth surface. The recognition receptor unit (specific binding complementary to the target)AAnd the target isBCalled.
FIG. 50: (a) Oligonucleotide linked to receptor (R) is complementary to oligonucleotide bound to nanoparticle and nanoparticle conjugate is hybridized to oligonucleotide bound to receptor by hybridization Schematic of the nanoparticle-oligonucleotide-receptor probe (II) showing binding, and (b) the oligonucleotide linked to the receptor (R) binds to the first portion of the linked oligonucleotide as a result of hybridization And a schematic diagram of the nanoparticle-oligonucleotide-receptor probe (II) showing that the oligonucleotide bound to the nanoparticle binds to the second portion of the linked oligonucleotide as a result of hybridization. For simplicity, only one oligonucleotide-protein conjugate attached to the oligonucleotide-nanoparticle conjugate is shown, but the actual number may be higher.
FIG. 51A is a schematic illustration of an application of a probe (FIG. 50b) in detecting a specific target, eg, a drug or protein, with an array of different drugs or proteins immobilized on a glass surface.
FIG. 51B is a schematic illustration of an application of the probe of FIG. 50b in detecting a specific target in cells having a mixture of different molecules. In FIGS. 51A and 51B, the probe can be used in a spot test even when an analyte, for example, a protein or a drug has a multiple binding site.
FIG. 52. Hybridization of a complex of streptavidin bound to a biotin molecule with bound oligonucleotide 1, nanoparticle oligonucleotide conjugate 2, and target nucleic acid 3.
FIG. 52B shows gold nanoparticle assembly by hybridizing an oligonucleotide of one nanoparticle with a nanoparticle of a second type.
FIG. 52C shows a gold nanoparticle obtained by hybridization of a complex of avidin specifically bound to four types of biotin molecules such that each biotin molecule binds to an oligonucleotide having a sequence complementary to the oligonucleotide bound to the nanoparticles. 5 is a schematic diagram of a three-dimensional gold nanoparticle-streptavidin assembly or aggregate formed as a result of a particle assembly. In all three examples of FIGS. 52A and 52B, when the aggregate was formed, the color of the solution changed from red to purple or blue-gray.
FIG. 53 is a photograph showing a non-specific binding test (Example 27) of a gold nanoparticle / oligonucleotide / streptavidin conjugate of the present invention (left) and a commercially available colloidal gold / streptavidin conjugate (right). The commercial conjugate showed a gray spot, indicating that the conjugate was adhered to the glass surface, whereas the conjugate of the invention did not show significant silver staining.
FIG. 54 is a schematic representation of the binding of gold nanoparticle-oligonucleotide-streptavidin conjugate to surface-bound biotin. Biotin is linked to an oligonucleotide having a sequence complementary to the oligonucleotide bound to the support surface, and the oligonucleotide-biotin conjugate is hybridized to the oligonucleotide bound to the surface.
FIG. 55 is a schematic diagram of a protein detection method using a linker as a DNA receptor for the protein. As shown, the nanoparticle-oligonucleotide-biotin conjugate with bound receptor DNA forms a three-dimensional nanoparticle assembly upon addition of streptavidin. The aggregate is then isolated and dehybridized to form a nanoparticle-oligonucleotide conjugate, a complex of streptavidin that binds to a biotin molecule having an oligonucleotide to bind, and its receptor. Release. The protein is detected by conventional identification of the receptor DNA, including use of a DNA chip.
FIG. 56: (a) Streptavidin binding to a single biotin linked to an oligonucleotide (oligonucleotide linked to biotin further binds to first portion of linker oligonucleotide as a result of hybridization), and b) Gold nanoparticle-oligonucleotide-streptavidin conjugate from a gold nanoparticle with an attached oligonucleotide (the oligonucleotide attached to the nanoparticle has a sequence complementary to the second portion of the linker oligonucleotide) Schematic showing body preparation (Example 26).
FIG. 57: (a) Forming aggregates when a solution of streptavidin-oligonucleotide conjugate (1-STV), 13 nm gold particles (2-Au) and linker DNA (3) is heated to 53 ° C. And (b) Transmission electron microscopy (TEM) images (Example 28) showing that the particles in the aggregates retain their physical shape before and after annealing, indicating that there is no fusion of the particles. And further demonstrate a stable effect of the surface oligonucleotide layer.
FIG. 58A. Melting curves as a function of temperature confirm that streptavidin-nanoparticle aggregates form by DNA hybridization interactions rather than non-specific interactions, and that the process is reversible. See Example 28.
FIG. 58B. Melting curves as a function of wavelength confirm that streptavidin-nanoparticle aggregates form by DNA hybridization interactions rather than non-specific interactions, and that the process is reversible. See Example 28.
FIG. 59 is a small angle X-ray scattering diffraction pattern showing various interparticle distances of various aggregates as described in Example 28.
FIG. 60 is an exemplary view of a set of the ITO electrode device described in the embodiment 29;

Claims (598)

A method for detecting a nucleic acid having at least two parts, comprising:
Providing a type of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotide on each nanoparticle has a sequence complementary to the sequence of at least two portions of the nucleic acid;
Contacting the nucleic acid with the nanoparticle under conditions that effectively hybridize the oligonucleotide on the nanoparticle to two or more portions of the nucleic acid;
Observing a detectable change caused by hybridization of the oligonucleotide on the nanoparticle to the nucleic acid. A method for detecting a nucleic acid having at least two parts, comprising:
Contacting the nucleic acid with at least two types of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotides on the first type of nanoparticles have a sequence complementary to a first portion of the sequence of the nucleic acid; The oligonucleotide on the second type of nanoparticles having a sequence complementary to a second portion of the sequence of the nucleic acid, wherein said contacting occurs under conditions that effectively hybridize the oligonucleotide on the nanoparticle with the nucleic acid. When,
Observing a detectable change caused by hybridization of the oligonucleotide on the nanoparticle to the nucleic acid. 3. The method of claim 2, wherein said contact conditions include freezing and thawing. 3. The method of claim 2, wherein said contacting conditions include heating. 3. The method of claim 2, wherein the detectable change is observed on a solid surface. 3. The method of claim 2, wherein the detectable change is a color change observable to the naked eye. 7. The method of claim 6, wherein the color change is observed on a solid surface. 3. The method of claim 2, wherein the nanoparticles are formed from gold. Claims wherein the non-nanoparticle end of the oligonucleotide attached to the nanoparticle is labeled with a molecule that produces a detectable change based on hybridization of the oligonucleotide on the nanoparticle with nucleic acid. 3. The method according to 2. The method according to claim 9, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles, and the oligonucleotide attached to the nanoparticles is labeled with a fluorescent molecule. The nucleic acid has a third portion located between the first and second portions, and the sequence of the oligonucleotide on the nanoparticle has no sequence complementary to the third portion of the nucleic acid;
3. The nucleic acid of claim 2, wherein the nucleic acid is further contacted with a filler oligonucleotide having a sequence complementary to the third portion of the nucleic acid, wherein the contacting occurs under conditions that effectively hybridize the filler oligonucleotide with the nucleic acid. Method. 3. The method according to claim 2, wherein the nucleic acid is viral RNA or viral DNA. 3. The method according to claim 2, wherein the nucleic acid is a disease-related gene. 3. The method according to claim 2, wherein the nucleic acid is bacterial DNA. 3. The method according to claim 2, wherein the nucleic acid is fungal DNA. 3. The method of claim 2, wherein the nucleic acid is a synthetic DNA, a synthetic RNA, a structurally modified natural or synthetic RNA, or a structurally modified natural or synthetic DNA. 3. The method of claim 2, wherein the nucleic acid is of a biological source. 3. The method of claim 2, wherein the nucleic acid is a product of a polymerase chain reaction amplification method. 3. The method of claim 2, wherein the nucleic acid is contacted simultaneously with the first and second types of nanoparticles. 3. The method of claim 2, wherein the nucleic acid is contacted and hybridized with an oligonucleotide on the first type of nanoparticle before contacting with the second type of nanoparticle. 21. The method of claim 20, wherein the first type of nanoparticles is attached to a substrate. 3. The method of claim 2, wherein the nucleic acid is double-stranded and the hybridization with the oligonucleotide on the nanoparticle results in a triple-stranded complex. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a substrate having a first type of nanoparticles attached thereto, wherein the oligonucleotides are attached to the nanoparticles, the oligonucleotides being complementary to a first portion of the sequence of the nucleic acid to be detected. Having an array;
Contacting the nucleic acid with a nanoparticle attached to a substrate under conditions that effectively hybridize the oligonucleotide on the nanoparticle with the nucleic acid;
Providing a second type of nanoparticles having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to one or more other portions of the sequence of the nucleic acid;
Contacting the nucleic acid molecule bound to the substrate with a second type of nanoparticles under conditions that effectively hybridize the oligonucleotide on the second type of nanoparticle with the nucleic acid;
Observing a detectable change. The substrate has a plurality of types of nanoparticles attached thereto in an array to enable detection of multiple portions of a single nucleic acid, detection of multiple different nucleic acids, or both. 24. The method according to 23. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a substrate having a first type of nanoparticles attached thereto, wherein the oligonucleotide has an oligonucleotide attached thereto, the oligonucleotide having a sequence complementary to a first portion of the sequence of the nucleic acid to be detected. Having steps;
Contacting the nucleic acid with a nanoparticle attached to a substrate under conditions that effectively hybridize the oligonucleotide on the nanoparticle with the nucleic acid;
Providing a second type of nanoparticles having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to one or more other portions of the sequence of the nucleic acid;
Contacting the nucleic acid bound to the substrate with the second type of nanoparticles under conditions that effectively hybridize the oligonucleotides on the second type of nanoparticles with the nucleic acid;
Providing a binding oligonucleotide having a selected sequence having at least two portions, wherein the first portion is complementary to at least a portion of the sequence of the oligonucleotide on a second type of nanoparticles;
Contacting the binding oligonucleotide with a second type of nanoparticle attached to the substrate under conditions that allow the binding oligonucleotide to effectively hybridize to the oligonucleotide on the nanoparticle;
Providing a third type of nanoparticles having an oligonucleotide attached thereto, the oligonucleotide having a sequence complementary to the sequence of the second portion of the binding oligonucleotide;
Contacting the third type of nanoparticles with a binding oligonucleotide associated with the substrate under conditions that allow the binding oligonucleotide to effectively hybridize to the oligonucleotide on the nanoparticle;
Observing a detectable change. The substrate has a plurality of types of nanoparticles attached thereto in an array to enable detection of multiple portions of a single nucleic acid, detection of multiple different nucleic acids, or both. 25. The method according to 25. A method for detecting a nucleic acid having at least two parts, comprising:
Contacting a nucleic acid to be detected with a substrate having an oligonucleotide attached thereto, said oligonucleotide having a sequence complementary to a first portion of the sequence of said nucleic acid, wherein said contacting comprises: Occurring under conditions that effectively hybridize nucleotides to the nucleic acid;
Contacting the nucleic acid bound to a substrate with a first type of nanoparticles having one or more types of oligonucleotides attached thereto, wherein at least one of the one or more types of oligonucleotides is the nucleic acid Having a sequence that is complementary to a second portion of the sequence of claim 1, wherein said contacting occurs under conditions that effectively hybridize an oligonucleotide on a nanoparticle to said nucleic acid;
Contacting the first type of nanoparticles bound to the substrate with the second type of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotides on the second type of nanoparticles are of the first type of nanoparticles Having a sequence complementary to at least a portion of the sequence of one of said one or more types of oligonucleotides, wherein said contacting effectively causes the oligonucleotides on the first and second types of nanoparticles Steps that occur under hybridizing conditions;
Observing a detectable change. The first type of nanoparticles has only one type of oligonucleotide attached thereto, the oligonucleotide comprising a second portion of the nucleic acid sequence and at least a portion of the oligonucleotide sequence on the second type of nanoparticles. 28. The method of claim 27, having a sequence complementary to Contacting a second type of nanoparticles associated with the substrate with the first type of nanoparticles, wherein the contacting conditions effectively hybridize the oligonucleotides on the first and second types of nanoparticles. 29. The method of claim 28, further comprising the steps occurring below. At least two kinds of oligonucleotides are attached to the first kind of nanoparticles, the first kind of oligonucleotide has a sequence complementary to the second part of the sequence of the nucleic acid, and the second kind of oligonucleotide 28. The method of claim 27, wherein the nucleotide has a sequence that is complementary to the sequence of at least a portion of the oligonucleotide on the second type of nanoparticle. Contacting the second type of nanoparticles bound to the substrate with the first type of nanoparticles, wherein the conditions are such that the oligonucleotides on the contacted first and second types of nanoparticles are effectively hybridized. 31. The method of claim 30, further comprising the step of contacting with. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 28. The method according to 27. 33. The method according to any one of claims 23 to 32, wherein the substrate is a transparent substrate or an opaque white substrate. 34. The method of claim 33, wherein the detectable change is the formation of a dark area on the substrate. 33. The method according to any one of claims 23 to 32, wherein the nanoparticles are formed from gold. 33. The method of any one of claims 23 to 32, wherein the substrate is contacted with a silver dye to produce a detectable change. 33. The method according to any one of claims 23 to 32, wherein the detectable change is observed with an optical scanner. A method for detecting a nucleic acid having at least two parts, comprising:
Contacting a nucleic acid to be detected with a substrate having an oligonucleotide attached thereto, said oligonucleotide having a sequence complementary to a first portion of the sequence of said nucleic acid, wherein said contacting comprises: Occurring under conditions that effectively hybridize nucleotides to the nucleic acid;
Contacting said nucleic acid attached to a substrate with a type of nanoparticles having oligonucleotides attached thereto, said oligonucleotide having a sequence complementary to a second portion of the sequence of said nucleic acid, Occurs under conditions that effectively hybridize the oligonucleotide on the nanoparticle to the nucleic acid;
Contacting the substrate with a silver dye to produce a detectable change;
Observing a detectable change. 39. The method of claim 38, wherein the nanoparticles are formed from a noble metal. 40. The method of claim 39, wherein the nanoparticles are formed from gold or silver. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 38. The method of claim 38. 42. The method according to any one of claims 38 to 41, wherein the detectable change is observed with an optical scanner. A method for detecting a nucleic acid having at least two parts, comprising:
Contacting a nucleic acid to be detected with a substrate having an oligonucleotide attached thereto, said oligonucleotide having a sequence complementary to a first portion of the sequence of said nucleic acid, wherein said contacting comprises: Occurring under conditions that effectively hybridize the nucleic acid to the nucleic acid;
Contacting the nucleic acid attached to the substrate with a liposome having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to a portion of the sequence of the nucleic acid, and wherein the contacting comprises: Occurring under conditions that effectively hybridize nucleotides to the nucleic acid;
Contacting a liposome bound to the substrate with a first type of nanoparticles having at least a first type of oligonucleotide attached thereto, the end of the first type of oligonucleotide not being attached to the nanoparticles. Has a hydrophobic group, said contact taking place under conditions effective to allow the oligonucleotide on the nanoparticle to attach to the liposome as a result of the hydrophobic interaction;
Observing a detectable change. A method for detecting a nucleic acid having at least two parts, comprising:
Contacting a nucleic acid to be detected with a substrate having an oligonucleotide attached thereto, said oligonucleotide having a sequence complementary to a first portion of the sequence of said nucleic acid, wherein said contacting comprises: Occurring under conditions that effectively hybridize nucleotides to the nucleic acid;
Contacting the nucleic acid attached to the substrate with a liposome having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to a portion of the sequence of the nucleic acid; Occurring under conditions that effectively hybridize nucleotides to the nucleic acid;
Contacting the liposome bound to the substrate with a first type of nanoparticles having at least one first type of oligonucleotide attached thereto, wherein the first type of oligonucleotide is attached to the nanoparticles. The non-exposed end is provided with a hydrophobic group, said contact taking place under conditions effective to allow the oligonucleotide on the nanoparticle to attach to the liposome as a result of the hydrophobic interaction;
Contacting the first type of nanoparticles associated with the liposome with a second type of nanoparticles having oligonucleotides attached thereto,
A second type of oligonucleotide having a sequence complementary to at least a portion of the sequence of the oligonucleotide on the second type of nanoparticles is attached to the first type of nanoparticles;
The oligonucleotide on the second type of nanoparticles has a sequence complementary to at least a portion of the sequence of the second type of oligonucleotide on the first type of nanoparticles;
Said contacting occurs under conditions that allow the oligonucleotides on the first and second types of nanoparticles to effectively hybridize;
Observing a detectable change. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 44. The method according to 43 or 44. 45. The method of claim 43 or claim 44, wherein the nanoparticles are formed from gold. 45. The method of claim 43 or 44, wherein the substrate is contacted with a silver dye to produce a detectable change. 45. The method of any one of claims 43 or 44, wherein the detectable change is observed with an optical scanner. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a substrate having a first type of nanoparticles attached thereto, wherein the oligonucleotides are attached to the nanoparticles, the oligonucleotides being complementary to a first portion of the sequence of the nucleic acid to be detected. Having an array;
Contacting the nucleic acid with a nanoparticle attached to a substrate under conditions that effectively hybridize the oligonucleotide on the nanoparticle with the nucleic acid;
Providing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are associated with each other as a result of a portion of the oligonucleotides attached thereto hybridizing; At least one of the at least two nanoparticles of the aggregate probe has attached thereto an oligonucleotide having a sequence complementary to the second part of the sequence of the nucleic acid;
Contacting the nucleic acid molecule attached to the substrate with an aggregate probe under conditions that effectively hybridize the oligonucleotide on the aggregate probe with the nucleic acid;
Observing a detectable change. The substrate has a plurality of types of nanoparticles attached thereto in an array to enable detection of multiple portions of a single nucleic acid, detection of multiple different nucleic acids, or both. 49. The method according to 49. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a substrate having an oligonucleotide attached thereto, the oligonucleotide having a sequence complementary to a first portion of the sequence of the nucleic acid to be detected;
Providing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are bound together as a result of a portion of the oligonucleotides attached thereto being hybridized, An oligonucleotide having a sequence complementary to a second portion of the sequence of the nucleic acid attached to at least one of the at least two nanoparticles of the probe;
Contacting the nucleic acid, the substrate, and the aggregate probe under conditions that effectively hybridize the nucleic acid with the oligonucleotide on the aggregate probe and the oligonucleotide on the substrate,
Observing a detectable change. Contacting the nucleic acid with the substrate such that the nucleic acid hybridizes with the oligonucleotide on the substrate, and then coagulating the nucleic acid bound to the substrate such that the nucleic acid hybridizes with the oligonucleotide on the aggregate probe. 52. The method of claim 51, wherein the method is contacted with a collecting probe. Contacting the nucleic acid with the aggregate probe so that the nucleic acid hybridizes with the oligonucleotide on the aggregate probe, and then contacting the nucleic acid with the substrate such that the nucleic acid hybridizes with the oligonucleotide on the substrate, The method of claim 51. 52. The method of claim 51, wherein the nucleic acid is contacted simultaneously with the aggregate probe and the substrate. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 51. The method according to 51. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a substrate having oligonucleotides attached thereto,
Providing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are associated with each other as a result of a portion of the oligonucleotides attached thereto hybridizing; At least one of the at least two nanoparticles of the aggregate probe has attached thereto an oligonucleotide having a sequence complementary to the first portion of the sequence of the nucleic acid to be detected;
Providing at least one type of nanoparticles having at least two types of oligonucleotides attached thereto, wherein the first type of oligonucleotide has a sequence complementary to a second portion of the sequence of the nucleic acid; The oligonucleotide having a sequence complementary to at least a portion of the sequence of the oligonucleotide attached to the substrate;
Contacting the nucleic acid, aggregate probe, nanoparticle, and substrate, wherein the contact effectively hybridizes the oligonucleotide on the aggregate probe and on the nanoparticle with the nucleic acid, and on the nanoparticle. Occurring under conditions that effectively hybridize the oligonucleotide with the oligonucleotide on the substrate;
Observing a detectable change. Contacting the nucleic acid with the aggregate probe and the nanoparticles such that the nucleic acid hybridizes with the oligonucleotide on the aggregate probe and the oligonucleotide on the nanoparticle, after which the oligonucleotide on the nanoparticle is the oligonucleotide on the substrate 57. The method of claim 56, wherein the nucleic acid associated with the aggregate probe and the nanoparticles so as to hybridize with the substrate is contacted with a substrate. The nucleic acid is contacted with an aggregate probe so that the nucleic acid hybridizes with the oligonucleotide on the aggregate probe, and then the nucleic acid is bound to the aggregate probe so that the nucleic acid hybridizes with the oligonucleotide on the nanoparticle. Contacting the nucleic acid with the nanoparticle, and then contacting the nucleic acid associated with the aggregate probe and the nanoparticle with the substrate such that the oligonucleotide on the nanoparticle hybridizes with the oligonucleotide on the substrate. 57. The method according to claim 56. Contacting the nucleic acid with the aggregate probe so that the nucleic acid hybridizes with the oligonucleotide on the aggregate probe, and contacting the nanoparticle with the substrate such that the oligonucleotide on the nanoparticle hybridizes with the oligonucleotide on the substrate 57. The method of claim 56, wherein the nucleic acid is subsequently contacted with an aggregate probe such that the nucleic acid hybridizes with the oligonucleotide on the nanoparticle. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 56. The method according to 56. The method according to any one of claims 49 to 60, wherein the substrate is a transparent substrate or an opaque white substrate. 62. The method of claim 61, wherein the detectable change is the formation of a dark region on the substrate. 61. The method of any one of claims 49-60, wherein the nanoparticles of the aggregate probe are formed from gold. 61. The method of any one of claims 49-60, wherein the substrate is contacted with a silver dye to produce a detectable change. 61. The method of any one of claims 49 to 60, wherein the detectable change is observed with an optical scanner. A method for detecting a nucleic acid having at least two parts, comprising:
Contacting a nucleic acid to be detected with a substrate having an oligonucleotide attached thereto, said oligonucleotide having a sequence complementary to a first portion of the sequence of said nucleic acid, wherein said contacting comprises: Occurring under conditions that effectively hybridize nucleotides to the nucleic acid;
Contacting the nucleic acid attached to the substrate with a liposome having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to a portion of the sequence of the nucleic acid; Occurring under conditions that effectively hybridize the nucleic acid to the nucleic acid;
Providing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are linked together as a result of a portion of the oligonucleotides attached thereto being hybridized; At least one of the at least two types of nanoparticles of the assembly probe is provided with an oligonucleotide having a hydrophobic group at an end not attached to the nanoparticles,
Contacting the liposome bound to the substrate with the aggregate probe under conditions effective to allow the oligonucleotide on the aggregate probe to attach to the liposome as a result of the hydrophobic interaction;
Observing a detectable change. 67. The method of claim 66, wherein the nanoparticles of the aggregate probe are formed from gold. 67. The method of claim 66, wherein the substrate is contacted with a silver dye to produce a detectable change. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 66. The method according to 66. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a substrate having an oligonucleotide, the oligonucleotide having a sequence complementary to a first portion of the sequence of the nucleic acid to be detected;
Providing a core probe having at least two types of nanoparticles, wherein each type of nanoparticles has an oligonucleotide complementary to at least one of the other types of nanoparticles attached thereto; Wherein the aggregate probe nanoparticles are associated with each other as a result of the oligonucleotides attached thereto being hybridized,
Providing a type of nanoparticles having two types of oligonucleotides attached thereto, wherein the first type of oligonucleotide has a sequence complementary to a second portion of the sequence of the nucleic acid; The nucleotide having a sequence complementary to a portion of the sequence of the oligonucleotide attached to at least one of the at least two nanoparticles of the core probe;
The nucleic acid, the nanoparticles, the substrate, and the core probe, the nucleic acid is effectively hybridized with the oligonucleotide on the nanoparticle and the oligonucleotide on the substrate, and the oligonucleotide on the nanoparticle with the oligonucleotide on the core probe. Contacting under conditions that effectively hybridize;
Observing a detectable change. Contacting the nucleic acid with the substrate such that the nucleic acid hybridizes with the oligonucleotide on the substrate, and contacting the nucleic acid with the nucleic acid bound to the substrate so that the nucleic acid hybridizes with the oligonucleotide on the nanoparticle; 71. The method of claim 70, wherein the nanoparticles associated with the nucleic acid are contacted with a core probe such that the oligonucleotides on the core probe hybridize with the oligonucleotides on the nanoparticle. The nucleic acid is contacted with the nanoparticles so that the nucleic acid hybridizes with the oligonucleotide on the nanoparticle, and then the nucleic acid bound to the nanoparticle is hybridized with the oligonucleotide on the substrate. 71. The method of claim 70, wherein the nanoparticles associated with the nucleic acid are contacted with a core probe such that the oligonucleotides on the core probe hybridize with the oligonucleotides on the nanoparticle. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a substrate having an oligonucleotide attached thereto, the oligonucleotide having a sequence complementary to a first portion of the sequence of the nucleic acid to be detected;
Providing a core probe having at least two types of nanoparticles, wherein each type of nanoparticle has an oligonucleotide attached thereto that is complementary to an oligonucleotide on at least one other type of nanoparticle. The nanoparticles of the aggregate probe are linked together as a result of the hybridization of the oligonucleotides attached thereto,
One type comprising a sequence complementary to a second portion of the sequence of the nucleic acid and a sequence complementary to a portion of the sequence of an oligonucleotide attached to at least one of the at least two nanoparticles of the core probe. Providing a linking oligonucleotide;
The nucleic acid, linking oligonucleotide, substrate, and core probe effectively hybridize the nucleic acid with the linking oligonucleotide and the oligonucleotide on the substrate, and make the oligonucleotide on the linking oligonucleotide effective with the oligonucleotide on the core probe. Contacting under conditions that hybridize to
Observing a detectable change. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 73. The method according to any one of 70 to 73. 74. The method according to any one of claims 70 to 73, wherein the substrate is a transparent substrate or an opaque white substrate. 77. The method of claim 76, wherein the detectable change is the formation of a dark region on the substrate. 74. The method of any one of claims 70-73, wherein the core probe nanoparticles are formed from gold. 74. The method of any one of claims 70-73, wherein the substrate is contacted with a silver dye to produce a detectable change. 74. The method of any one of claims 70 to 73, wherein the detectable change is observed with an optical scanner. A method for detecting a nucleic acid having at least two parts, comprising:
Providing nanoparticles with oligonucleotides attached thereto;
Providing one or more types of binding oligonucleotides, each binding oligonucleotide having two portions, wherein the sequence of one portion is complementary to the sequence of one of the portions of the nucleic acid. The sequence of the other part is complementary to the sequence of the oligonucleotide on the nanoparticle;
Contacting the nanoparticle and the binding oligonucleotide under conditions that effectively hybridize the oligonucleotide on the nanoparticle with the binding oligonucleotide;
Contacting the nucleic acid and the binding oligonucleotide under conditions that allow the binding oligonucleotide to effectively hybridize with the nucleic acid;
Observing a detectable change. 81. The method of claim 80, wherein the nanoparticles are contacted with a binding oligonucleotide prior to contacting with the nucleic acid. A method for detecting a nucleic acid having at least two parts, comprising:
Providing nanoparticles with oligonucleotides attached thereto;
Providing one or more binding oligonucleotides, each binding oligonucleotide having two portions, wherein the sequence of one portion is complementary to the sequence of at least two portions of the nucleic acid; The sequence of the portion of is complementary to the sequence of the oligonucleotide on the nanoparticle;
Contacting the nanoparticle and the binding oligonucleotide under conditions that effectively hybridize the oligonucleotide on the nanoparticle with the binding oligonucleotide;
Contacting the nucleic acid and the binding oligonucleotide under conditions that allow the binding oligonucleotide to effectively hybridize with the nucleic acid;
Observing a detectable change. A method for detecting a nucleic acid having at least two parts, comprising:
Contacting the nucleic acid with at least two types of particles having oligonucleotides attached thereto,
The oligonucleotide on the first type of particle has a sequence complementary to the first portion of the sequence of the nucleic acid, is labeled with an energy donor,
The oligonucleotide on the second type of particle has a sequence complementary to the second portion of the sequence of the nucleic acid, is labeled with an energy acceptor,
Contacting under conditions that effectively hybridize the oligonucleotide on the particle with the nucleic acid;
Observing a detectable change caused by hybridization of the oligonucleotide on the particle to the nucleic acid. 84. The method of claim 83, wherein the energy donor and acceptor are fluorescent molecules. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a type of microsphere having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to a first portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule;
Providing a type of nanoparticle having an oligonucleotide attached thereto, the oligonucleotide having a sequence complementary to a second portion of the sequence of the nucleic acid, wherein the nanoparticle is capable of producing a detectable change. One step,
Contacting the nucleic acid with the microspheres and nanoparticles under conditions that effectively hybridize the oligonucleotides on the microspheres and nanoparticles with the nucleic acid;
Observing a change in fluorescence, another detectable change generated by the nanoparticles, or both. 86. The method of claim 85, wherein the detectable change produced by the nanoparticles is a color change. 86. The method of claim 85, wherein the microspheres are latex microspheres and the nanoparticles are gold nanoparticles and observe a change in fluorescence, color, or both. A portion of the mixture of latex microspheres, nanoparticles, and nucleic acid is placed in an observation region located in the microporous material, and the microporous material is processed to remove unbound gold nanoparticles from the observation region, 91. The method of claim 87, further comprising observing a change in fluorescence, color, or both. A method for detecting a nucleic acid having at least two parts, comprising:
Providing a first type of metal or semiconductor nanoparticles having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to a first portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule. Steps,
Providing a second type of metal or semiconductor nanoparticles having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to a second portion of the sequence of the nucleic acid and is labeled with a fluorescent molecule. Steps,
Contacting the nucleic acid with the two types of nanoparticles under conditions that effectively hybridize the oligonucleotide on the two types of nanoparticles with the nucleic acid;
Observing a change in fluorescence. A portion of the mixture of nanoparticles and nucleic acids is placed in an observation area located on the microporous material, the microporous material is processed to remove unbound nanoparticles from the observation area, and then the change in fluorescence is observed 90. The method of claim 89, further comprising the step of: A method for detecting a nucleic acid having at least two parts, comprising:
Providing a type of particle having an oligonucleotide attached thereto, the oligonucleotide having a first portion and a second portion, both portions being complementary to a portion of the sequence of the nucleic acid;
Providing a type of probe oligonucleotide having a first portion and a second portion, wherein the first portion has a sequence complementary to the first portion of the oligonucleotide attached to the particle; The portion is complementary to a portion of the sequence of the nucleic acid, and the probe oligonucleotide is further labeled at one end with a reporter molecule;
Contacting the particle and the probe oligonucleotide under conditions that effectively hybridize the oligonucleotide on the particle with the probe oligonucleotide to produce a satellite probe;
Thereafter, contacting the satellite probe with the nucleic acid under conditions that effectively hybridize the nucleic acid with the probe oligonucleotide,
Removing the particles;
Detecting a reporter molecule. 92. The method of claim 91, wherein the particles are magnetic particles and the reporter molecule is a fluorescent molecule. 92. The method of claim 91, wherein the particles are magnetic particles and the reporter molecule is a dye molecule. 92. The method of claim 91, wherein the particles are magnetic particles and the reporter molecule is a redox active molecule. A kit comprising at least one container, wherein the container contains a composition comprising at least two types of nanoparticles having the oligonucleotide attached thereto, wherein the oligonucleotide on the first type of nanoparticles is a nucleic acid. A kit having a sequence complementary to the sequence of the first portion, wherein the oligonucleotide on the second type of nanoparticle has a sequence complementary to the sequence of the second portion of the nucleic acid. 97. The kit of claim 95, wherein the composition in the container further comprises a filler oligonucleotide having a sequence complementary to a third portion of the nucleic acid located between the first and second portions. 97. The kit of claim 95, wherein the nanoparticles are formed from gold. 97. The kit of claim 95, further comprising a solid surface. A kit comprising at least two containers,
The first container contains nanoparticles to which oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached,
A kit comprising, in a second container, nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid. 100. The kit of claim 99, further comprising a third container holding a filler oligonucleotide having a sequence complementary to the third portion of the nucleic acid located between the first and second portions. 100. The kit of claim 99, wherein the nanoparticles are formed from gold. 100. The kit of claim 99, further comprising a solid surface. A kit comprising at least two containers,
The first container contains nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the first portion of the binding oligonucleotide,
The second container has a sequence comprising at least two portions, wherein the first portion is complementary to the sequence of the oligonucleotide on the nanoparticle and the second portion is complementary to the sequence of a portion of the nucleic acid. A kit containing a plurality of types of binding oligonucleotides. An additional container is provided, each additional container containing an additional binding oligonucleotide, wherein each additional binding oligonucleotide has a first portion complementary to the sequence of the oligonucleotide on the nanoparticle. 104. The kit of claim 103, wherein the second portion has a sequence comprising at least two portions that is complementary to the sequence of another portion of the nucleic acid. 114. The kit of claim 103, wherein the nanoparticles are formed from gold. 114. The kit of claim 103, further comprising a solid surface. A kit,
One type of nanoparticle with the oligonucleotide attached, and the first portion is complementary to the sequence of the oligonucleotide on the nanoparticle so that the bound oligonucleotide is hybridized to the oligonucleotide on the nanoparticle. A container containing one or more types of binding oligonucleotides, each type having a sequence comprising at least two portions, wherein the second portion is complementary to the sequence of one or more portions of the nucleic acid. kit. A kit that may comprise at least one container, wherein the container contains metal or semiconductor nanoparticles having oligonucleotides attached thereto, the oligonucleotide having a sequence complementary to a portion of a nucleic acid, A kit, wherein a fluorescent molecule is attached to an end of the oligonucleotide that is not attached to the nanoparticles. A kit,
A substrate to which nanoparticles having oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached;
A first container containing nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid. A second container containing a binding oligonucleotide having a selected sequence having at least two portions, wherein the first portion is complementary to at least a portion of the sequence of oligonucleotides on the nanoparticles in the first container;
110. The kit of claim 109, further comprising: a third container containing nanoparticles having attached thereto an oligonucleotide having a sequence complementary to the sequence of the second portion of the binding oligonucleotide. A kit comprising at least three containers,
A first container containing nanoparticles,
A second container containing a first oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid;
A third container containing a second oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid. 112. The kit of claim 111, further comprising a fourth container containing a third oligonucleotide having a sequence complementary to the sequence of the third portion of the nucleic acid located between the first and second portions. 112. The kit of claim 111, further comprising a substrate. A fourth container containing a binding oligonucleotide having a selected sequence having at least two portions, wherein the first portion is complementary to at least a portion of the sequence of the second oligonucleotide;
114. The kit of claim 113, further comprising: a fifth container containing an oligonucleotide having a sequence complementary to the sequence of the second portion of the binding oligonucleotide. 112. The kit of claim 111, wherein the oligonucleotide, the nanoparticles, or both carry a functional group for attaching the oligonucleotide to the nanoparticles. 114. The kit of claim 113, wherein the substrate, the nanoparticles, or both carry a functional group for attaching the nanoparticles to the substrate. 114. The kit of claim 113, wherein the nanoparticles are attached to a substrate. 112. The kit of claim 111, wherein the nanoparticles are formed from gold. A kit,
A substrate having an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid attached thereto;
A first container containing nanoparticles having a portion of an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid,
A second container containing nanoparticles having oligonucleotides having a sequence complementary to at least a part of the sequence of oligonucleotides attached to the nanoparticles in the first container. A kit,
Board and
A first container containing nanoparticles,
A second container containing a first oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid;
A third container containing a second oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid;
A fourth container containing a third oligonucleotide having a sequence complementary to at least a portion of the sequence of the second oligonucleotide. 121. The kit of claim 120, wherein the oligonucleotide, nanoparticle, substrate, or all carry a functional group for attaching the oligonucleotide to the nanoparticle or attaching the oligonucleotide to the substrate. 121. The kit of claim 120, wherein the nanoparticles are formed from gold. A kit,
A substrate having an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid attached thereto;
A first container containing a liposome having an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid,
A second container containing nanoparticles to which at least a first type of oligonucleotide is attached, wherein the first type of oligonucleotide has a hydrophobic group at an end not attached to the nanoparticles. A kit comprising: two containers. The second type of oligonucleotide is attached to the nanoparticles of the second container, and the second type of oligonucleotide has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticles. ,
The kit also includes
The method according to claim 1, further comprising a third container containing the second type of nanoparticles having an oligonucleotide having a sequence complementary to at least a part of the sequence of the second type of oligonucleotide on the first type of nanoparticles. 123. The kit according to 123. A kit,
A substrate to which nanoparticles having oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid are attached;
A first container containing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are bound to each other as a result of a portion of the oligonucleotides attached thereto being hybridized. A first container to which an oligonucleotide having a sequence complementary to a second portion of the nucleic acid sequence is attached to at least one of the at least two types of nanoparticles of the aggregate probe. Kit. A kit,
A substrate having an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid attached thereto;
A first container containing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are bound to each other as a result of a portion of the oligonucleotides attached thereto being hybridized. A first container to which an oligonucleotide having a sequence complementary to a second portion of the nucleic acid sequence is attached to at least one of the at least two types of nanoparticles of the aggregate probe. Kit. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 126. The kit according to 126. A kit,
A substrate having an oligonucleotide attached thereto,
A first container containing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are bound to each other as a result of hybridization of a portion of the oligonucleotide attached thereto. A first container having an oligonucleotide having a sequence complementary to a first portion of a nucleic acid sequence attached to at least one of the at least two types of nanoparticles of the aggregate probe;
A second container containing nanoparticles having at least two types of oligonucleotides attached thereto, wherein the first type of oligonucleotide has a sequence complementary to a second portion of the nucleic acid sequence, and wherein the second type of oligonucleotide has A second container, wherein the nucleotide has a sequence complementary to at least a portion of the sequence of the oligonucleotide attached to the substrate. A kit,
A substrate having an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid attached thereto;
A first container containing a liposome having an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid,
A second container containing an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are bound together as a result of a portion of the oligonucleotides attached thereto hybridizing. A second container, wherein an oligonucleotide is attached to at least one of the at least two types of nanoparticles of the aggregate probe, and an end of the oligonucleotide that is not attached to the nanoparticles has a hydrophobic group. And a kit comprising: 130. The kit of any one of claims 125-129, wherein the substrate is a transparent substrate or an opaque white substrate. 130. The kit of any one of claims 125-129, wherein the nanoparticles of the aggregate probe are formed from gold. A kit comprising at least three containers,
A first container containing nanoparticles,
A second container containing a first oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid;
A third container containing a second oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid. 133. The kit of claim 132, further comprising a fourth container containing a third oligonucleotide having a sequence complementary to the sequence of the third portion of the nucleic acid located between the first and second portions. A substrate was further provided. 133. The kit of claim 132. A fourth container containing a binding oligonucleotide having a selected sequence having at least two portions, wherein the first portion is complementary to at least a portion of the sequence of the second oligonucleotide;
135. The kit of claim 134, further comprising: a fifth container containing an oligonucleotide having a sequence complementary to the sequence of the second portion of the binding oligonucleotide. 133. The kit of claim 132, wherein the oligonucleotide, the nanoparticles, or both carry a functional group for attaching the oligonucleotide to the nanoparticles. 135. The kit of claim 134, wherein the substrate, the nanoparticles, or both carry a functional group for attaching the nanoparticles to the substrate. 135. The kit of claim 134, wherein the nanoparticles are attached to a substrate. 133. The kit of claim 132, wherein the nanoparticles are formed from gold. A kit,
A substrate having an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid attached thereto;
A first container containing nanoparticles to which oligonucleotides having a sequence complementary to the sequence of the second portion of the nucleic acid are partially attached;
A second container containing nanoparticles having oligonucleotides having a sequence complementary to at least a part of the sequence of oligonucleotides attached to the nanoparticles in the first container. A kit,
Board and
A first container containing nanoparticles,
A second container containing a first oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid;
A third container containing a second oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid;
A fourth container containing a third oligonucleotide having a sequence complementary to at least a portion of the sequence of the second oligonucleotide. 142. The kit of claim 141, wherein the oligonucleotide, nanoparticle, substrate, or all carry a functional group for attaching the oligonucleotide to the nanoparticle or attaching the oligonucleotide to the substrate. 142. The kit of claim 141, wherein the nanoparticles are formed from gold. A kit,
A substrate having an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid attached thereto;
A first container containing a liposome having an oligonucleotide having a sequence complementary to the sequence of the second portion of the nucleic acid,
A second container containing nanoparticles having at least a first type of oligonucleotide attached thereto, wherein the first type of oligonucleotide has a hydrophobic group at an end not attached to the nanoparticles, And a second container. A second type of oligonucleotide is attached to the nanoparticles in the second container, wherein the second type of oligonucleotide has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticles,
The kit also includes
The method according to claim 1, further comprising a third container containing the second type of nanoparticles having an oligonucleotide having a sequence complementary to at least a part of the sequence of the second type of oligonucleotide on the first type of nanoparticles. 144. The kit according to 144. A kit comprising at least two containers,
The first container contains particles to which oligonucleotides having a sequence complementary to the sequence of the first portion of the nucleic acid and having an end not attached to the particles and labeled with an energy donor are contained. Yes,
The second container contains particles having oligonucleotides having a sequence complementary to the sequence of the second portion of the nucleic acid and having an end not attached to the particles labeled with an energy acceptor. Yes, kit. 149. The kit of claim 146, wherein the energy donor and acceptor are fluorescent molecules. A kit comprising at least one container, wherein the container has a sequence complementary to the sequence of the first portion of the nucleic acid, and the end not attached to the particle is labeled with an energy donor. A particle of the first type having the oligonucleotide attached thereto and a sequence attached thereto, which has a sequence complementary to the sequence of the second portion of the nucleic acid, and the end not attached to the particle has an energy acceptor. And a second type of particle to which the oligonucleotide labeled with (1) is attached. 149. The kit of claim 148, wherein the energy donor and acceptor are fluorescent molecules. A kit,
A first container containing microspheres having a sequence complementary to the first portion of the sequence of the nucleic acid and having oligonucleotides labeled with fluorescent molecules attached thereto;
A second container containing a type of nanoparticle having an oligonucleotide having a sequence complementary to the second portion of the sequence of the nucleic acid attached thereto. The kit of claim 150, wherein the microspheres are latex microspheres and the nanoparticles are gold nanoparticles. 151. The kit of claim 150, further comprising a microporous material. A kit,
A first container containing a first type of metal nanoparticle or semiconductor nanoparticle having a sequence complementary to the first portion of the nucleic acid sequence and having an oligonucleotide labeled with a fluorescent molecule attached thereto;
A second container containing a second type of metal nanoparticle or semiconductor nanoparticle having a sequence complementary to the second portion of the sequence of the nucleic acid and having an oligonucleotide labeled with a fluorescent molecule attached thereto. Kit. 153. The kit of claim 153, further comprising a microporous material. A kit including a container containing a satellite probe, wherein the satellite probe includes:
Particles having oligonucleotides having a first portion and a second portion, wherein both portions have a sequence complementary to a portion of the sequence of the nucleic acid;
A probe oligonucleotide having a first portion and a second portion, hybridizing to an oligonucleotide attached to a nanoparticle, and further having a reporter molecule attached at one end, wherein the first portion is the oligonucleotide attached to a particle. A probe oligonucleotide having a sequence complementary to the sequence of the first portion of nucleotides, both portions having sequences complementary to portions of the sequence of the nucleic acid. A kit comprising a container containing an aggregate probe, wherein the aggregate probe comprises at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe comprise one of the oligonucleotides attached thereto. A kit wherein the oligonucleotides having a sequence complementary to a part of the sequence of the nucleic acid are attached to at least one of the at least two types of nanoparticles of the aggregate probe, as a result of the hybridization of the portions. A kit comprising a container containing an aggregate probe, wherein the aggregate probe comprises at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe comprise one of the oligonucleotides attached thereto. Are hybridized to each other as a result of hybridization, and at least one of the at least two types of nanoparticles of the aggregate probe has an oligonucleotide having a hydrophobic group attached to the end that is not attached to the nanoparticle. ,kit. An aggregate probe, wherein the aggregate probe comprises at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are mutually hybridized as a result of a portion of the oligonucleotides attached thereto being hybridized. An aggregate probe, wherein the oligonucleotide has a sequence complementary to a portion of the sequence of the nucleic acid attached to at least one of the at least two nanoparticles of the aggregate probe. Each of the two types of nanoparticles has two types of oligonucleotides attached thereto, and the first type of oligonucleotides attached to each type of nanoparticles has a sequence complementary to a part of the nucleic acid sequence, 159. The method of claim 158, wherein the second type of oligonucleotide attached to the second type of nanoparticles has a sequence complementary to at least a portion of the sequence of the second type of oligonucleotide attached to the second type of nanoparticles. Gathering probe. The three types of nanoparticles having oligonucleotides attached thereto, the oligonucleotides attached to the first type of nanoparticles have a sequence complementary to at least a part of the sequence of the oligonucleotides attached to the second type of nanoparticles. The oligonucleotide attached to the second type of nanoparticles has a sequence complementary to at least a part of the sequence of the oligonucleotide attached to the first type of nanoparticles, and the third type of nanoparticles has two types of oligonucleotides. The first type of oligonucleotide has a sequence complementary to the portion of the nucleic acid sequence, and the second type of oligonucleotide is attached to the first or second type of nanoparticles. 160. The aggregate probe of claim 158, wherein the probe has a sequence complementary to at least a portion of the sequence of nucleotides. An aggregate probe, wherein the aggregate probe comprises at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles of the aggregate probe are associated with each other as a result of a portion of the oligonucleotides attached thereto being hybridized. An aggregate having an oligonucleotide attached to at least one of the at least two types of nanoparticles of the aggregate probe, and a hydrophobic group attached to an end of the oligonucleotide that is not attached to the nanoparticles. probe. A kit including a container containing a core probe, wherein the core probe includes at least two types of nanoparticles having oligonucleotides attached thereto, and the nanoparticles of the core probe have a part of the oligonucleotide attached thereto. A kit that is tied to each other as a result of hybridization. 163. The kit of claim 162, further comprising a substrate having an oligonucleotide having a sequence complementary to the first portion of the sequence of the nucleic acid to be detected attached. The method further comprises a container containing one type of nanoparticles having two types of oligonucleotides attached thereto, wherein the first type of oligonucleotide has a sequence complementary to the second portion of the nucleic acid, and the second type of oligonucleotide is 163. The kit of claim 162 or 163, wherein the kit has a sequence complementary to a portion of the sequence of an oligonucleotide attached to at least one of said at least two nanoparticles of a core probe. A type of linking oligo having a sequence complementary to a second portion of the sequence of the nucleic acid and a sequence complementary to a portion of the sequence of the oligonucleotide attached to at least one of the at least two nanoparticles of the core probe 163. The kit of claim 162 or 163, further comprising a container containing the nucleotide. A core probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the core probe nanoparticles are associated with each other as a result of a portion of the oligonucleotide attached thereto being hybridized. Substrate with nanoparticles attached. 167. The substrate of claim 167, wherein the nanoparticles have attached thereto an oligonucleotide having a sequence complementary to the sequence of the first portion of the nucleic acid. A metal or semiconductor nanoparticle to which an oligonucleotide is attached, wherein an end of the oligonucleotide that is not attached to the nanoparticle is labeled with a fluorescent molecule. A satellite probe,
Particles having a first portion and a second portion, and both portions having oligonucleotides attached thereto having a sequence complementary to a portion of the sequence of the nucleic acid;
A probe oligonucleotide hybridized to an oligonucleotide attached to a nanoparticle, the probe oligonucleotide having a first portion and a second portion, wherein the first portion is complementary to a sequence of the first portion of the oligonucleotide attached to the particle. A probe oligonucleotide having a sequence, both portions having a sequence complementary to a portion of the sequence of the nucleic acid, and further having a reporter molecule attached at one end. A method of nanofabrication,
Providing at least one linking oligonucleotide having a selected sequence, wherein each type of linking oligonucleotide sequence has at least two portions;
Providing one or more types of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotides on each type of nanoparticles have a sequence complementary to the sequence of the portion of the linking oligonucleotide;
The oligonucleotides on the nanoparticles are effectively hybridized to the linking oligonucleotides such that the linking oligonucleotides and nanoparticles form the desired nanomaterial or nanostructure where the nanoparticles are held together by the oligonucleotide connector. Contacting under conditions. At least two types of nanoparticles having oligonucleotides attached thereto are provided, wherein the oligonucleotides on the first type of nanoparticles have a sequence complementary to a first portion of the sequence of the linking oligonucleotide, and 172. The method of claim 171, wherein the oligonucleotide on the nanoparticle has a sequence that is complementary to a second portion of the sequence of the linking oligonucleotide. 173. The method of claim 171 or 172, wherein the nanoparticles are metal nanoparticles, semiconductor nanoparticles, or a combination thereof. 178. The method of claim 173, wherein the metal nanoparticles are formed from gold and the semiconductor nanoparticles are formed from CdSe / ZnS (core / shell). A method of nanofabrication,
Providing at least two types of nanoparticles having oligonucleotides attached thereto,
The oligonucleotide on the first type of nanoparticles has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticles,
Oligonucleotides on the second type of nanoparticles having a sequence complementary to the sequence of the oligonucleotides on the first type of nanoparticles;
Contacting the first and second types of nanoparticles under conditions that effectively hybridize the oligonucleotides on the nanoparticles to one another such that a desired nanomaterial or nanostructure is formed. 178. The method of claim 175, wherein the nanoparticles are metal nanoparticles, semiconductor nanoparticles, or a combination thereof. 177. The method of claim 176, wherein the metal nanoparticles are formed from gold and the semiconductor nanoparticles are formed from CdSe / ZnS (core / shell). A nanomaterial or nanostructure composed of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles are held together by an oligonucleotide connector. 177. The nanomaterial or nanostructure of claim 178, wherein at least a portion of the oligonucleotide connector is triple-stranded. 177. The nanomaterial or nanostructure of claim 178, wherein the nanoparticles are metal nanoparticles, semiconductor nanoparticles, or a combination thereof. 181. The nanomaterial or nanostructure of claim 180, wherein the metal nanoparticles are formed from gold and the semiconductor nanoparticles are formed from CdSe / ZnS (core / shell). A composition comprising at least two types of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotides on the first type of nanoparticles have a sequence that is complementary to the sequence of the first portion of the nucleic acid or the linking oligonucleotide. And wherein the oligonucleotide on the second type of nanoparticle has a sequence that is complementary to the sequence of the second portion of the nucleic acid or the linking oligonucleotide. 183. The composition of claim 182, wherein the nanoparticles are metal nanoparticles, semiconductor nanoparticles, or a combination thereof. 183. The composition of claim 183, wherein the metal nanoparticles are formed from gold and the semiconductor nanoparticles are formed from CdSe / ZnS (core / shell). An assembly of containers,
A first container containing nanoparticles having oligonucleotides attached thereto,
A second container containing nanoparticles having oligonucleotides attached thereto,
The oligonucleotide attached to the nanoparticles in the first container has a sequence complementary to the sequence of the oligonucleotide attached to the nanoparticles in the second container,
The assembly, wherein the oligonucleotide attached to the nanoparticles in the second container has a sequence that is complementary to the sequence of the oligonucleotide attached to the nanoparticles in the second container. 187. The assembly of claim 185, wherein the nanoparticles are metal nanoparticles, semiconductor nanoparticles, or a combination thereof. 189. The assembly of claim 186, wherein the metal nanoparticles are formed from gold and the semiconductor nanoparticles are formed from CdSe / ZnS (core / shell). Nanoparticles with different oligonucleotides attached. A method for separating a selected nucleic acid having at least two portions from other nucleic acids,
Providing two or more types of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotides on each type of nanoparticle have a sequence complementary to a sequence of one of a plurality of portions of the selected nucleic acid. Steps and
Contacting the nucleic acid and the nanoparticles under conditions that effectively hybridize the oligonucleotide on the nanoparticle with the selected nucleic acid, such that the nanoparticles hybridized to the selected nucleic acid aggregate and precipitate; A method of attaching an oligonucleotide to a charged nanoparticle to produce a stable nanoparticle-oligonucleotide conjugate, comprising:
Providing an oligonucleotide having a moiety containing a functional group capable of binding to the nanoparticle covalently bound,
Contacting the oligonucleotide and the nanoparticle in water for a period of time sufficient for at least a portion of the oligonucleotide to bind to the nanoparticle;
Adding at least one salt to the water to form a salt solution, wherein the ionic strength of the salt solution is determined by the electrostatic attraction or repulsion of the oligonucleotides to the nanoparticles, and the electrostatic attraction of the oligonucleotides to each other. Steps that are strong enough to at least partially overcome the repulsion;
Contacting the oligonucleotide and the nanoparticle in a salt solution for an additional period of time sufficient for the additional oligonucleotide to bind to the nanoparticle and form a stable nanoparticle-oligonucleotide conjugate. 190. The method according to claim 190, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 192. The method of claim 191, wherein the nanoparticles are gold nanoparticles. 192. The method of claim 192, wherein the moiety comprising a functional group capable of binding to a nanoparticle is an alkanethiol. 190. The method of claim 190, wherein all the salts are added to the water in a single addition. 190. The method of claim 190, wherein the salt is added gradually over time. The salt comprises sodium chloride, magnesium chloride, potassium chloride, ammonia, chloride, sodium, acetate, ammonium acetate, a combination of two or more of these salts, one of these salts in a phosphate buffer, and 190. The method of claim 190, wherein the method is selected from the group consisting of a combination of two or more of these salts in a phosphate buffer. 196. The method of claim 196, wherein said salt is sodium chloride in a phosphate buffer. Nanoparticles having oligonucleotides present nanoparticle surfaces with a surface density of at least 10 picomoles / cm ² - oligonucleotide conjugates are produced The method of claim 190. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 15 picomoles / cm ^2, The method of claim 198. Present on the nanoparticle surfaces with a surface density of oligonucleotides of about 15 picomoles / cm ² ~ about 40 picomoles / cm ^2, The method of claim 199. A method of attaching an oligonucleotide to a nanoparticle to produce a nanoparticle-oligonucleotide conjugate, comprising:
Providing an oligonucleotide comprising at least one recognition oligonucleotide, wherein each recognition oligonucleotide has a spacer moiety and a recognition moiety, wherein the spacer moiety is designed to be capable of binding to the nanoparticle. Steps and
Contacting the oligonucleotide and the nanoparticle under conditions effective to bind at least a portion of the recognition oligonucleotide to the nanoparticle to form a nanoparticle-oligonucleotide conjugate. 209. The method of claim 201, wherein a moiety having a functional group capable of binding to the nanoparticle is covalently linked to each spacer moiety of the recognition oligonucleotide. 220. The method of claim 201, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 203. The method of claim 203, wherein the nanoparticles are gold nanoparticles. 202. The method of claim 204, wherein the spacer moiety comprises at least about 10 nucleotides. 210. The method of claim 205, wherein the spacer moiety comprises about 10 to about 30 nucleotides. 220. The method of claim 206, wherein the nucleotide bases of the spacer are all adenines, all thymines, all cytosines, all uracils, or all guanines. A method of attaching an oligonucleotide to a nanoparticle to produce a nanoparticle-oligonucleotide conjugate, comprising:
Providing an oligonucleotide comprising a type of recognition oligonucleotide and a type of diluent oligonucleotide;
Contacting the oligonucleotide with the nanoparticles under conditions effective for at least a portion of each of the certain type of oligonucleotides to bind to the nanoparticles and form a nanoparticle-oligonucleotide conjugate. And a method consisting of: 210. The method of claim 208, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 210. The method of claim 209, wherein the nanoparticles are gold nanoparticles. 210. The method of claim 208, wherein each recognition oligonucleotide has a spacer moiety and a recognition moiety, wherein the spacer moiety is designed to be capable of binding to a nanoparticle. The method according to claim 211, wherein a moiety having a functional group capable of binding to the nanoparticle is covalently bound to each spacer moiety of the recognition oligonucleotide. 222. The method of claim 211, wherein the spacer portion of the recognition oligonucleotide comprises at least about 10 nucleotides. 220. The method of claim 213, wherein the spacer portion of the recognition oligonucleotide comprises from about 10 nucleotides to about 30 nucleotides. 220. The method of claim 211, wherein the nucleotide bases of the spacer are all adenines, all thymines, all cytosines, all uracils, or all guanines. 222. The method of claim 211, wherein the diluent oligonucleotide comprises about the same number of nucleotides as contained in the spacer portion of the recognition oligonucleotide. 218. The method of claim 216, wherein the sequence of the diluent oligonucleotide is the same as the sequence of the spacer portion of the recognition oligonucleotide. 210. The method of claim 208, wherein the oligonucleotide comprises at least two recognition oligonucleotides. A method of attaching an oligonucleotide to a charged nanoparticle to produce a nanoparticle-oligonucleotide conjugate, comprising:
Providing an oligonucleotide, wherein the moiety having a functional group capable of binding to the nanoparticle is covalently linked and comprises a type of recognition oligonucleotide and a type of diluent oligonucleotide;
Contacting the oligonucleotide with the nanoparticle in water for a period of time sufficient for at least a portion of each of the type of oligonucleotide to bind to the nanoparticle;
Adding at least one salt to the water to form a salt solution, wherein the ionic strength of the salt solution is determined by the electrostatic attraction or repulsion of the oligonucleotides to the nanoparticles, and the electrostatic attraction of the oligonucleotides to each other. Steps that are strong enough to at least partially overcome the repulsion;
Contacting the oligonucleotide and the nanoparticle in a salt solution for an additional period of time sufficient for each additional oligonucleotide of the type of oligonucleotide to bind to the nanoparticle to form the nanoparticle-oligonucleotide conjugate. And a method consisting of: 220. The method of claim 219, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 220. The method of claim 220, wherein the nanoparticles are gold nanoparticles. 223. The method of claim 221, wherein the moiety comprising a functional group capable of binding to a nanoparticle is an alkanethiol. 220. The method of claim 219, wherein all salts are added to the water in one addition. 220. The method of claim 219, wherein the salt is added gradually over time. The salt comprises sodium chloride, magnesium chloride, potassium chloride, ammonia, chloride, sodium, acetate, ammonium acetate, a combination of two or more of these salts, one of these salts in a phosphate buffer, and 220. The method of claim 219, wherein the method is selected from the group consisting of a combination of two or more of these salts in a phosphate buffer. 225. The method of claim 225, wherein said salt is sodium chloride in a phosphate buffer. Nanoparticles having oligonucleotides present nanoparticle surfaces with a surface density of at least 10 picomoles / cm ² - oligonucleotide conjugates are produced The method of claim 219. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 15 picomoles / cm ^2, The method of claim 227. Present on the nanoparticle surfaces with a surface density of oligonucleotides of about 15 picomoles / cm ² ~ about 40 picomoles / cm ^2, The method of claim 228. 220. The method of claim 219, wherein each recognition oligonucleotide has a spacer moiety and a recognition moiety, and each spacer moiety is covalently linked to a moiety having a functional group capable of binding to a nanoparticle. 230. The method of claim 230, wherein the spacer moiety comprises at least about 10 nucleotides. 230. The method of claim 231, wherein the spacer moiety comprises about 10 to about 30 nucleotides. 230. The method of claim 230, wherein the nucleotide bases of the spacer are all adenines, all thymines, all cytosines, all uracils, or all guanines. 230. The method of claim 230, wherein the diluent oligonucleotide comprises about the same number of nucleotides as contained in the spacer portion of the recognition oligonucleotide. 235. The method of claim 234, wherein the sequence of the diluent oligonucleotide is the same as the sequence of the spacer portion of the recognition oligonucleotide. 220. The method of claim 219, wherein the oligonucleotide comprises at least two recognition oligonucleotides. A nanoparticle-oligonucleotide conjugate, which is a nanoparticle having an oligonucleotide attached thereto, wherein the oligonucleotide is present on the surface of the nanoparticle with a sufficient surface density such that the conjugate is stable; The moiety is a conjugate having a sequence complementary to at least a portion of the sequence of a nucleic acid or another oligonucleotide. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 10 picomoles / cm ^2, conjugate of claim 237 Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 15 picomoles / cm ^2, nanoparticles of claim 238. Present on the nanoparticle surfaces with a surface density of oligonucleotides of about 15 picomoles / cm ² ~ about 40 picomoles / cm ^2, nanoparticles of claim 239. 237. The nanoparticle of claim 237, wherein the nanoparticle is a metal nanoparticle or a semiconductor nanoparticle. 242. The nanoparticle of claim 241, wherein the nanoparticle is a gold nanoparticle. A nanoparticle having an oligonucleotide, wherein the oligonucleotide comprises at least one recognition oligonucleotide, each of the recognition oligonucleotides having a spacer portion and a recognition portion, wherein the spacer portion is associated with the nanoparticle. A nanoparticle, wherein the recognition moiety has a sequence that is complementary to at least a portion of the sequence of a nucleic acid or another oligonucleotide. 243. The nanoparticle of claim 243, wherein the spacer moiety has a moiety that is covalently linked to the nanoparticle, wherein the moiety includes a functional group that links the spacer moiety to the nanoparticle. 243. The nanoparticle of claim 243, wherein the spacer moiety comprises at least about 10 nucleotides. 245. The nanoparticle of claim 245, wherein the spacer moiety comprises about 10 to about 30 nucleotides. 243. The nanoparticle of claim 243, wherein the bases of the nucleotides in the spacer moiety are all adenine, all thymine, all cytosine, all uracil, or all guanine. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 10 picomoles / cm ^2, nanoparticles of claim 243. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 15 picomoles / cm ^2, nanoparticles of claim 248. Present on the nanoparticle surfaces with a surface density of oligonucleotides of about 15 picomoles / cm ² ~ about 40 picomoles / cm ^2, nanoparticles of claim 249. 243. The nanoparticle of claim 243, wherein the nanoparticle is a metal nanoparticle or a semiconductor nanoparticle. 252. The method of claim 251, wherein the nanoparticles are gold nanoparticles. Nanoparticles having an oligonucleotide attached thereto, wherein the oligonucleotide is
At least one recognition oligonucleotide, each having a sequence complementary to at least a portion of the sequence of the nucleic acid or another oligonucleotide;
Certain diluent oligonucleotides;
And nanoparticles. Each of the recognition oligonucleotides has a spacer moiety and a recognition moiety, wherein the spacer moiety is designed to be associated with the nanoparticle, wherein the recognition moiety has a sequence complementary to at least a portion of the sequence of the nucleic acid or another oligonucleotide. 253. The nanoparticle of claim 253, comprising: 256. The nanoparticle of claim 254, wherein the spacer moiety has a moiety that is covalently attached to the nanoparticle, wherein the moiety includes a functional group that attaches the spacer moiety to the nanoparticle. 256. The nanoparticle of claim 254, wherein the spacer moiety comprises at least about 10 nucleotides. 257. The nanoparticle of claim 256, wherein the spacer moiety comprises about 10 to about 30 nucleotides. 254. The nanoparticle of claim 254, wherein the bases of the nucleotides in the spacer moiety are all adenines, all thymines, all cytosines, all uracils, or all guanines. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 10 picomoles / cm ^2, nanoparticles of claim 253. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 15 picomoles / cm ^2, nanoparticles of claim 259. Present on the nanoparticle surfaces with a surface density of oligonucleotides of about 15 picomoles / cm ² ~ about 40 picomoles / cm ^2, nanoparticles of claim 260. 256. The nanoparticle of claim 254, wherein the diluent oligonucleotide comprises about the same number of nucleotides as in the spacer portion of the recognition oligonucleotide. 273. The nanoparticle of claim 262, wherein the sequence of the diluent oligonucleotide is the same as the sequence of the spacer portion of the recognition oligonucleotide. 253. The nanoparticle of claim 253, wherein the nanoparticle is a metal nanoparticle or a semiconductor nanoparticle. 275. The nanoparticle of claim 264, wherein the nanoparticle is a gold nanoparticle. A method for detecting a nucleic acid, comprising:
Contacting at least one nanoparticle-oligonucleotide conjugate according to any one of claims 237-242 with a nucleic acid under conditions that allow the oligonucleotide on the nanoparticle to effectively hybridize to the nucleic acid;
Observing a detectable change caused by hybridization of the nucleic acid to the oligonucleotide on the nanoparticle. A method for detecting a nucleic acid, comprising:
Contacting at least one nanoparticle according to any one of claims 243-265 with a nucleic acid under conditions that effectively hybridize the at least one recognition oligonucleotide on the nanoparticle with the nucleic acid;
Observing a detectable change caused by hybridization of the recognition oligonucleotide and the nucleic acid. A method for detecting a nucleic acid having at least two parts, comprising:
246. Providing a type of nanoparticle-oligonucleotide conjugate according to any one of claims 237-242, wherein the oligonucleotide on each nanoparticle is complementary to the sequence of at least two portions of the nucleic acid. A step having a dynamic arrangement;
Contacting the nucleic acid with the conjugate under conditions that effectively hybridize the oligonucleotide on the nanoparticle and two or more portions of the nucleic acid;
Observing a detectable change caused by hybridization of the nucleic acid to the oligonucleotide on the nanoparticle. A method for detecting a nucleic acid having at least two parts, comprising:
250. The step of contacting at least two types of nanoparticle-oligonucleotide conjugates according to any one of claims 237-240 with a nucleic acid, wherein the oligonucleotides on the first type of conjugated nanoparticles are nucleic acids. Wherein the oligonucleotide on the nanoparticles of the second type of conjugate has a sequence complementary to the second portion of the sequence of the nucleic acid, wherein the contacting comprises: Occurring under conditions that effectively hybridize the oligonucleotide on the nanoparticle to the nucleic acid;
Observing a detectable change caused by hybridization of the nucleic acid to the oligonucleotide on the nanoparticle. 269. The method of claim 269, wherein said contacting conditions include freezing and thawing. 269. The method of claim 269, wherein said contacting conditions include heating. 269. The method of claim 269, wherein the detectable change is observed on a solid surface. 269. The method of claim 269, wherein the detectable change is a color change observable to the naked eye. 280. The method of claim 273, wherein the color change is observed on a solid surface. 269. The method of claim 269, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 269. The method of claim 269, wherein the nanoparticles are gold nanoparticles. Claims wherein the non-nanoparticle end of the oligonucleotide attached to the nanoparticle is labeled with a molecule that produces a detectable change based on hybridization of the oligonucleotide on the nanoparticle with nucleic acid. 269. 287. The method of claim 277, wherein the nanoparticles are metal or semiconductor nanoparticles and the oligonucleotide attached to the nanoparticles is labeled with a fluorescent molecule. The nucleic acid has a third portion located between the first and second portions, and the sequence of the oligonucleotide on the nanoparticle has no sequence complementary to the third portion of the nucleic acid;
269. The nucleic acid of claim 269, wherein the nucleic acid is further contacted with a filler oligonucleotide having a sequence complementary to the third portion of the nucleic acid, wherein the contacting occurs under conditions that effectively hybridize the filler oligonucleotide with the nucleic acid. Method. 269. The method of claim 269, wherein said nucleic acid is viral RNA or DNA. 269. The method of claim 269, wherein the nucleic acid is a disease associated gene. 269. The method of claim 269, wherein the nucleic acid is bacterial DNA. 269. The method of claim 269, wherein the nucleic acid is fungal DNA. 269. The method of claim 269, wherein the nucleic acid is a synthetic DNA, a synthetic RNA, a structurally modified natural or synthetic RNA, or a structurally modified natural or synthetic DNA. 269. The method of claim 269, wherein the nucleic acid is of a biological source. 269. The method of claim 269, wherein the nucleic acid is a product of a polymerase chain reaction amplification. 269. The method of claim 269, wherein the nucleic acid is contacted simultaneously with the first and second types of conjugates. 270. The method of claim 269, wherein the nucleic acid is contacted and hybridized with the oligonucleotide on the nanoparticles of the first type of conjugate prior to contacting with the second type of conjugate. 288. The method of claim 288, wherein the first type of conjugate is attached to the substrate. 269. The method of claim 269, wherein the nucleic acid is double stranded and hybridization to an oligonucleotide on the nanoparticle results in a triple stranded complex. A method for detecting a nucleic acid having at least two parts, comprising:
252. The step of providing at least one type of nanoparticle according to any of claims 243-252, wherein the recognition oligonucleotide on each nanoparticle has at least two portions of a nucleic acid. Having a sequence complementary to the sequence;
Contacting the nucleic acid and the nanoparticle under conditions that allow the oligonucleotide on the nanoparticle to effectively hybridize to the two or more portions of the nucleic acid; and the detectable change caused by hybridization of the nucleic acid and the oligonucleotide on the nanoparticle Observing. A method for detecting a nucleic acid having at least two parts, comprising:
The step of contacting a nucleic acid with at least two types of nanoparticles according to any one of claims 243 to 250 attached with a recognition oligonucleotide, wherein the recognition oligonucleotide on the first type of nanoparticles is The recognition oligonucleotide on the second type of nanoparticles has a sequence complementary to a first portion of the sequence of the nucleic acid, and the recognition oligonucleotide on the second type of nanoparticles has a sequence complementary to the second portion of the sequence of the nucleic acid. Occurring under conditions that allow the above recognition oligonucleotide to effectively hybridize to the nucleic acid;
Observing a detectable change caused by the hybridization of the nucleic acid to the recognition oligonucleotide on the nanoparticle. 293. The method of claim 292, wherein said contacting conditions include freezing and thawing. 292. The method of claim 292, wherein said contacting conditions include heating. 292. The method of claim 292, wherein the detectable change is observed on a solid surface. 292. The method of claim 292, wherein the detectable change is a color change observable to the naked eye. 297. The method of claim 296, wherein a change in color is observed on a solid surface. 292. The method of claim 292, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 298. The method of claim 298, wherein the nanoparticles are formed from gold. The end of the recognition oligonucleotide attached to the nanoparticle that is not attached to the nanoparticle is labeled with a molecule that produces a detectable change based on the hybridization of the oligonucleotide on the nanoparticle with the nucleic acid. Item 292. The method according to Item 292. 300. The method of claim 300, wherein the nanoparticles are metal or semiconductor nanoparticles and the oligonucleotide attached to the nanoparticles is labeled with a fluorescent molecule. The nucleic acid has a third portion located between the first and second portions, and the sequence of the oligonucleotide on the nanoparticle has no sequence complementary to the third portion of the nucleic acid;
293. The nucleic acid of claim 292, wherein the nucleic acid is further contacted with a filler oligonucleotide having a sequence complementary to the third portion of the nucleic acid, wherein the contacting occurs under conditions that effectively hybridize the filler oligonucleotide with the nucleic acid. Method. 292. The method of claim 292, wherein said nucleic acid is viral RNA or DNA. 292. The method of claim 292, wherein the nucleic acid is a disease associated gene. 293. The method of claim 292, wherein said nucleic acid is bacterial DNA. 293. The method of claim 292, wherein said nucleic acid is fungal DNA. 292. The method of claim 292, wherein the nucleic acid is a synthetic DNA, a synthetic RNA, a structurally modified natural or synthetic RNA, or a structurally modified natural or synthetic DNA. 293. The method of claim 292, wherein the nucleic acid is of a biological source. 293. The method of claim 292, wherein the nucleic acid is a product of a polymerase chain reaction amplification method. 292. The method of claim 292, wherein the nucleic acid is contacted simultaneously with the first and second types of nanoparticles. 292. The method of claim 292, wherein the nucleic acid is contacted and hybridized with an oligonucleotide on the first type of nanoparticle before contacting with the second type of nanoparticle. 311. The method of claim 311, wherein the first type of nanoparticles is applied to a substrate. 292. The method of claim 292, wherein the nucleic acid is double stranded and hybridization to an oligonucleotide on the nanoparticle results in a triple stranded complex. A method for detecting a nucleic acid having at least two parts, comprising:
265. The step of providing certain types of nanoparticles according to any of claims 253-265, wherein the recognition oligonucleotides on each nanoparticle have a sequence of at least two portions of the nucleic acid. Having a sequence complementary to
Contacting the nucleic acid with the nanoparticle under conditions that allow the recognition oligonucleotide on the nanoparticle to effectively hybridize to two or more portions of the nucleic acid;
Observing a detectable change caused by the hybridization of the nucleic acid to the recognition oligonucleotide on the nanoparticle. A method for detecting a nucleic acid having at least two parts, comprising:
The step of contacting at least two types of nanoparticles according to any one of claims 253-263 with a recognition oligonucleotide, wherein the recognition oligonucleotide on the first type of nanoparticles is a nucleic acid. The recognition oligonucleotide on the second type of nanoparticle has a sequence complementary to the first portion of the sequence, the recognition oligonucleotide on the second type of nanoparticle has a sequence complementary to the second portion of the sequence of the nucleic acid, and the contacting occurs on the nanoparticle. Occurring under conditions that allow the recognition oligonucleotide to effectively hybridize to the nucleic acid;
Observing a detectable change caused by the hybridization of the nucleic acid to the recognition oligonucleotide on the nanoparticle. 315. The method of claim 315, wherein said contact conditions include freezing and thawing. 315. The method of claim 315, wherein the contacting conditions include heating. 315. The method of claim 315, wherein the detectable change is observed on a solid surface. 315. The method of claim 315, wherein the detectable change is a color change observable to the naked eye. 320. The method of claim 319, wherein the color change is observed on a solid surface. 315. The method of claim 315, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 322. The method of claim 321, wherein the nanoparticles are formed from gold. The end of the recognition oligonucleotide attached to the nanoparticle that is not attached to the nanoparticle is labeled with a molecule that produces a detectable change based on the hybridization of the recognition oligonucleotide on the nanoparticle with the nucleic acid. 315. The method of claim 315. 334. The method of claim 323, wherein the nanoparticles are metal or semiconductor nanoparticles and the oligonucleotide attached to the nanoparticles is labeled with a fluorescent molecule. The nucleic acid has a third portion located between the first and second portions, and the sequence of the oligonucleotide on the nanoparticle has no sequence complementary to the third portion of the nucleic acid;
315. The nucleic acid of claim 315, wherein the nucleic acid is further contacted with a filler oligonucleotide having a sequence complementary to the third portion of the nucleic acid, wherein the contacting occurs under conditions that effectively hybridize the filler oligonucleotide with the nucleic acid. Method. 315. The method of claim 315, wherein the nucleic acid is a viral RNA or DNA. 315. The method of claim 315, wherein the nucleic acid is a disease associated gene. 315. The method of claim 315, wherein the nucleic acid is bacterial DNA. 315. The method of claim 315, wherein the nucleic acid is fungal DNA. 315. The method of claim 315, wherein the nucleic acid is a synthetic DNA, a synthetic RNA, a structurally modified natural or synthetic RNA, or a structurally modified natural or synthetic DNA. 315. The method of claim 315, wherein the nucleic acid is of a biological source. 315. The method of claim 315, wherein the nucleic acid is a product of a polymerase chain reaction amplification. 315. The method of claim 315, wherein the nucleic acid is contacted simultaneously with the first and second types of conjugates. 315. The method of claim 315, wherein the nucleic acid is contacted and hybridized with a recognition oligonucleotide on the first type of nanoparticle before contacting with the second type of nanoparticle. 334. The method of claim 334, wherein the first type of nanoparticles is applied to a substrate. 315. The method of claim 315, wherein the nucleic acid is double stranded and hybridization with an oligonucleotide on the nanoparticle results in a triple stranded complex. A method for detecting a nucleic acid having at least two parts, comprising:
(A) contacting the oligonucleotide-attached substrate with a nucleic acid, the oligonucleotide having a sequence complementary to a first portion of the nucleic acid sequence, wherein the contacting comprises causing the oligonucleotide on the substrate to A step occurring under conditions that effectively hybridize with the nucleic acid;
(B) contacting the first kind of nanoparticle-oligonucleotide conjugate according to any one of claims 237 to 240 with the nucleic acid bound to a substrate, wherein the conjugate is attached to the conjugated nanoparticles. At least one of the oligonucleotides having a sequence complementary to the second portion of the nucleic acid sequence, wherein the contacting is performed under conditions that allow the oligonucleotide attached to the conjugated nanoparticles to effectively hybridize to the nucleic acid. The steps that take place,
(C) observing a detectable change. (D) contacting the first type of nanoparticle-oligonucleotide conjugate bound to the substrate with the second type of nanoparticle-oligonucleotide conjugate according to any one of claims 237 to 240. Wherein at least one of the oligonucleotides attached to the nanoparticles of the second type of conjugate has a sequence complementary to at least one sequence of the oligonucleotides attached to the nanoparticles of the first type of conjugate When,
337. The method of claim 337, further comprising: (e) observing a detectable change. At least one of the oligonucleotides on the first type of conjugated nanoparticles has a sequence complementary to at least one type of oligonucleotide on the second type of conjugated nanoparticles;
(F) contacting a second type of conjugate bound to the substrate with a first type of conjugate, wherein the contacting comprises contacting the oligonucleotides on the nanoparticles of the first and second types of conjugates; Steps that occur under conditions that allow effective hybridization;
338. The method of claim 338, further comprising: (g) observing a detectable change. 339. The method of claim 339, wherein step (d) or steps (d) and (f) are repeated one or more times to observe a detectable change. (D) providing a type of binding oligonucleotide having a sequence comprising at least two portions, the first portion comprising at least one type of oligonucleotide attached to a first type of conjugated nanoparticle; Steps complementary to
(E) contacting the conjugated oligonucleotide with a first type of conjugate attached to the substrate, wherein the contacting effectively hybridizes the conjugated oligonucleotide with an oligonucleotide on a nanoparticle of the first type of conjugate. Steps that take place under soy conditions;
(F) providing a second type of nanoparticle-oligonucleotide conjugate according to any one of claims 237 to 240, wherein the oligonucleotide is attached to the second type of conjugated nanoparticle. At least one having a sequence complementary to the second portion of the sequence of the binding oligonucleotide;
(G) contacting the binding oligonucleotide bound to the substrate with a second type of conjugate, wherein the contacting effectively causes the oligonucleotide attached to the second type of conjugate nanoparticle to bind to the binding oligonucleotide. Steps that occur under hybridizing conditions;
337. The method of claim 337, further comprising: (h) observing a detectable change. (I) contacting a second type of conjugate bound to the substrate with the binding oligonucleotide, wherein the contacting effectively hybridizes the oligonucleotide on the nanoparticle of the second type of conjugate with the binding oligonucleotide; Steps that take place under soy conditions;
j) contacting the binding oligonucleotide bound to the substrate with the first type of conjugate, wherein said contacting effectively hybridizes the oligonucleotide on the nanoparticles of the first type of conjugate with the binding oligonucleotide. Steps that take place under soy conditions;
341. The method of claim 341, further comprising: (k) observing a detectable change. 345. The method of claim 342, wherein steps (e) and (g) or steps (e), (g), (i) and (j) are repeated one or more times and a detectable change is observed. 337. The method of claim 337, wherein the substrate is a transparent substrate or an opaque white substrate. 344. The method of claim 344, wherein the detectable change is the formation of a dark region on the substrate. 337. The method of claim 337, wherein the conjugated nanoparticles are metal nanoparticles or semiconductor nanoparticles. 347. The method of claim 346, wherein the conjugated nanoparticles are formed from gold or silver. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 337. The method of claim 337. 337. The method of claim 337, wherein the substrate is contacted with a silver dye to produce a detectable change. 348. The method of claim 348, wherein the substrate is contacted with a silver dye to produce a detectable change. 337. The method of claim 337, wherein the detectable change is observed with an optical scanner. 352. The method of claim 351, wherein the optical scanner is a flatbed scanner. 358. The method of claim 351, wherein the scanner is coupled to a computer equipped with software capable of calculating grayscale measurements and calculates the grayscale measurements to provide a quantitative measurement of the detected nucleic acid. 337. The method of claim 337, wherein the oligonucleotide attached to the substrate is disposed between the two electrodes, the conjugated nanoparticles are formed from a conductive material, and the detectable change is a change in conductivity. 354. The method of claim 354, wherein the electrodes are formed from gold and the nanoparticles are formed from gold. 354. The method of claim 354, wherein the substrate is contacted with a silver dye to cause a change in conductivity. 348. The method of claim 348, wherein each of the plurality of oligonucleotides attached to the substrate in an array is disposed between the two electrodes, the nanoparticles are formed from a conductive material, and the detectable change is a change in conductivity. The method described in. 358. The method of claim 357, wherein the electrodes are formed from gold and the nanoparticles are formed from gold. 358. The method of claim 357, wherein the substrate is contacted with a silver dye to cause a change in conductivity. A method for detecting a nucleic acid having at least two parts, comprising:
(A) contacting a nucleic acid with a substrate having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to a first portion of the sequence of the nucleic acid, wherein the contacting comprises: And a step that occurs under conditions that effectively hybridize the nucleic acid,
(B) contacting the first type of nanoparticles according to any one of claims 243-250 with one or more types of recognition oligonucleotides attached thereto, with the nucleic acid bound to a substrate; At least one of the one or more recognition oligonucleotides has a sequence complementary to a second portion of the sequence of the nucleic acid, and the contacting effectively hybridizes the oligonucleotide on the nanoparticle with the nucleic acid. Steps that take place under soy conditions;
(C) observing a detectable change. (D) contacting the second type of nanoparticles according to any one of claims 243-250 with a recognition oligonucleotide attached to the first type of nanoparticles bound to the substrate, At least one of the recognition oligonucleotides on the two types of nanoparticles has a sequence that is complementary to at least one type of sequence of the oligonucleotides on the first type of nanoparticles, and wherein the contacting occurs with the first and second types of nanoparticles. Occurring under conditions that effectively hybridize the oligonucleotide on the type of nanoparticles;
360. The method of claim 360, further comprising: (e) observing a detectable change. At least one of the recognition oligonucleotides on the first type of nanoparticles has a sequence complementary to at least one sequence of the oligonucleotides on the second type of nanoparticles;
(F) contacting the second type of nanoparticles associated with the substrate with the first type of nanoparticles, wherein the contacting effectively hybridizes the oligonucleotides on the first and second types of nanoparticles. Steps that occur under conditions that cause
360. The method of claim 360, further comprising: (g) observing a detectable change. 372. The method of claim 362, wherein step (d) or steps (d) and (f) are repeated one or more times to observe a detectable change. (D) providing a type of binding oligonucleotide having a sequence comprising at least two portions, wherein the first portion is complementary to at least one of the oligonucleotides on the first type of nanoparticles. Steps and
(E) contacting the binding oligonucleotide with the first type of nanoparticle bound to the substrate, wherein the contacting condition effectively hybridizes the binding oligonucleotide with the oligonucleotide on the first type of nanoparticle. The steps that take place below,
(F) providing a second type of nanoparticles according to any one of claims 243-250, wherein a recognition oligonucleotide is attached, wherein at least one of the recognition oligonucleotides on the second type of nanoparticles is provided. One type has a sequence complementary to a second portion of the sequence of the binding oligonucleotide;
(G) contacting the binding oligonucleotide bound to the substrate with the second type of nanoparticles, wherein said contacting effectively hybridizes the oligonucleotide on the second type of nanoparticles with the binding oligonucleotide. The steps that take place below,
360. The method of claim 360, further comprising: (h) observing a detectable change. (I) contacting the second type of nanoparticles attached to the substrate with the binding oligonucleotide, wherein said contacting effectively hybridizes the oligonucleotide on the second type of nanoparticles with the binding oligonucleotide; The steps that take place below,
(J) contacting the binding oligonucleotide bound to the substrate with the first type of nanoparticles, wherein said contacting effectively hybridizes the binding oligonucleotide with the oligonucleotide on the first type of nanoparticles. The steps that take place below,
364. The method of claim 364, further comprising: (k) observing a detectable change. 370. The method of claim 365, wherein steps (e) and (g) or steps (e), (g), (i), and (j) are repeated one or more times and a detectable change is observed. . 360. The method according to claim 360, wherein the substrate is a transparent substrate or an opaque white substrate. 367. The method of claim 367, wherein the detectable change is the formation of a dark region on the substrate. 360. The method of claim 360, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 369. The method of claim 369, wherein the nanoparticles are formed from gold or silver. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 360. The method according to 360. 360. The method of claim 360, wherein the substrate is contacted with a silver dye to produce a detectable change. 372. The method of claim 371, wherein the substrate is contacted with a silver dye to produce a detectable change. 360. The method of claim 360, wherein the detectable change is observed with an optical scanner. 375. The method of claim 375, wherein the optical scanner is a flatbed scanner. 375. The method of claim 375, wherein the scanner is coupled to a computer equipped with software capable of calculating grayscale measurements and calculates the grayscale measurements to provide a quantitative measurement of the detected nucleic acid. 360. The method of claim 360, wherein the oligonucleotide attached to the substrate is disposed between the two electrodes, the conjugated nanoparticles are formed from a conductive material, and the detectable change is a change in conductivity. 400. The method of claim 378, wherein the electrodes are formed from gold and the nanoparticles are formed from gold. 400. The method of claim 378, wherein the substrate is contacted with a silver dye to cause a change in conductivity. 372. The method of claim 371, wherein each of the plurality of oligonucleotides attached to the substrate in an array is disposed between two electrodes, the nanoparticles are formed from a conductive material, and the detectable change is a change in conductivity. The method described in. 381. The method of claim 381, wherein the electrodes are formed from gold and the nanoparticles are formed from gold. 381. The method of claim 381, wherein the substrate is contacted with a silver dye to cause a change in conductivity. A method for detecting a nucleic acid having at least two parts, comprising:
(A) contacting a nucleic acid with a substrate having an oligonucleotide attached thereto, wherein the oligonucleotide has a sequence complementary to a first portion of the sequence of the nucleic acid, wherein the contacting comprises: And a step that occurs under conditions that effectively hybridize the nucleic acid,
(B) contacting the first kind of nanoparticles according to any one of claims 253-263 with one or more kinds of recognition oligonucleotides attached thereto, with the nucleic acid bound to a substrate; At least one of the one or more types of recognition oligonucleotides has a sequence complementary to a second portion of the sequence of the nucleic acid, wherein the contacting effectively causes the recognition oligonucleotide on the nanoparticle to interact with the nucleic acid. Steps that occur under hybridizing conditions;
(C) observing a detectable change. (D) contacting the first type of nanoparticle bound to the substrate with the second type of nanoparticle according to any one of claims 253-263 to which a recognition oligonucleotide is attached, At least one of the recognition oligonucleotides on the first type of nanoparticles has a sequence complementary to at least one sequence of the oligonucleotides on the first type of nanoparticles, and wherein the contacting comprises: Occurring under conditions that allow the oligonucleotides on the nanoparticles to effectively hybridize;
384. The method of claim 384, further comprising: (e) observing a detectable change. At least one of the recognition oligonucleotides on the first type of nanoparticles has a sequence complementary to at least one sequence of the oligonucleotides on the second type of nanoparticles;
(F) contacting the second type of nanoparticles associated with the substrate with the first type of nanoparticles, wherein the contacting effectively hybridizes the oligonucleotides on the first and second types of nanoparticles. Steps that take place under soy conditions;
385. The method of claim 385, further comprising: (g) observing a detectable change. 386. The method of claim 386, wherein step (d) or steps (d) and (f) are repeated one or more times to observe a detectable change. (D) providing a type of binding oligonucleotide having a sequence comprising at least two portions, wherein the first portion is complementary to at least one of the oligonucleotides on the first type of nanoparticles. Steps and
(E) contacting the binding oligonucleotide with a first type of nanoparticles attached to the substrate, said contact effectively hybridizing the binding oligonucleotide with the oligonucleotide on the first type of nanoparticles. Steps that occur under conditions;
(F) providing a second type of nanoparticles according to any one of claims 253-263, wherein the recognition oligonucleotides are attached, wherein at least one of the recognition oligonucleotides on the second type of nanoparticles is present. A species having a sequence complementary to a second portion of the sequence of the binding oligonucleotide;
(G) contacting the binding oligonucleotide bound to the substrate with the second type of nanoparticles, wherein said step is a condition for effectively hybridizing the oligonucleotide on the second type of nanoparticles with the binding oligonucleotide. The steps that take place below,
386. The method of claim 386, further comprising: (h) observing a detectable change. (I) contacting the second type of nanoparticles attached to the substrate with the binding oligonucleotide, wherein said contacting effectively hybridizes the binding oligonucleotide with the oligonucleotide on the second type of nanoparticles; The steps that take place below,
(J) contacting the binding oligonucleotide bound to the substrate with the first type of nanoparticles, wherein said contacting effectively hybridizes the oligonucleotide on the first type of nanoparticles with the binding oligonucleotide; The steps that take place below,
388. The method of claim 388, further comprising: (k) observing a detectable change. 390. The method of claim 389, wherein steps (e) and (g) or steps (e), (g), (i), and (j) are repeated one or more times and a detectable change is observed. . 394. The method of claim 384, wherein the substrate is a transparent substrate or an opaque white substrate. 391. The method of claim 391, wherein the detectable change is the formation of a dark region on the substrate. 383. The method of claim 384, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 403. The method of claim 393, wherein the nanoparticles are formed from gold or silver. The substrate has a plurality of types of oligonucleotides attached thereto in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or both of them. 384. The method of claim 384. 386. The method of claim 384, wherein the substrate is contacted with a silver dye to produce a detectable change. 398. The method of claim 395, wherein the substrate is contacted with a silver dye to produce a detectable change. 386. The method of claim 384, wherein the detectable change is observed with an optical scanner. 398. The method of claim 398, wherein the optical scanner is a flatbed scanner. 398. The method of claim 398, wherein the scanner is coupled to a computer equipped with software capable of calculating grayscale measurements, and calculates the grayscale measurements to provide a quantitative measurement of the detected nucleic acid. 394. The method of claim 384, wherein the oligonucleotide attached to the substrate is disposed between the two electrodes, the conjugated nanoparticles are formed from a conductive material, and the detectable change is a change in conductivity. 404. The method of claim 401, wherein the electrodes are formed from gold and the nanoparticles are formed from gold. 404. The method of claim 401, wherein the substrate is contacted with a silver dye to cause a change in conductivity. 397. Each of the plurality of oligonucleotides attached to the substrate in an array is disposed between two electrodes, the nanoparticles are formed from a conductive material, and the detectable change is a change in conductivity. The method described in. 404. The method of claim 404, wherein the electrodes are formed from gold and the nanoparticles are formed from gold. 404. The method of claim 404, wherein the substrate is contacted with a silver dye to cause a change in conductivity. A method for detecting a nucleic acid having at least two parts, comprising:
(A) contacting a nucleic acid with a substrate having an oligonucleotide attached thereto, wherein the oligonucleotide is disposed between a pair of electrodes, and the oligonucleotide is complementary to a first portion of the sequence of the nucleic acid. Having the following sequence, wherein the contacting occurs under conditions that allow the oligonucleotide on the substrate to effectively hybridize to the nucleic acid;
(B) contacting the nucleic acid bound to a substrate with a first type of nanoparticles, wherein the nanoparticles are formed of a material capable of conducting electricity and the nanoparticles have one or more At least one of the oligonucleotides has a sequence complementary to a second portion of the sequence of the nucleic acid, and the contacting causes the oligonucleotide on the nanoparticle to be effective with the nucleic acid. A step that occurs under conditions that hybridize to
(C) detecting a change in conductivity. On the substrate, a plurality of pairs of electrodes are arranged in an array so as to enable detection of a plurality of portions in one nucleic acid, detection of a plurality of different nucleic acids, or detection of both of them. 407. The method of claim 407, wherein each has some type of oligonucleotide attached to the substrate between the pair. 407. The method of claim 407, wherein the nanoparticles are formed from a metal. 407. The method of claim 407, wherein the nanoparticles are formed from gold or silver. 407. The method of claim 407, wherein the substrate is contacted with a silver dye to cause a change in conductivity. (D) contacting the first type of nanoparticles associated with the substrate with the second type of nanoparticles, wherein the nanoparticles are formed of a material capable of conducting electricity, and Has one or more types of oligonucleotides attached thereto, and at least one of the oligonucleotides on the second type of nanoparticles has at least one of the one or more types of oligonucleotides on the first type of nanoparticles Having a sequence that is complementary to one type of sequence, wherein said contacting occurs under conditions that effectively hybridize the oligonucleotides on the first and second types of nanoparticles;
407. The method of claim 407, further comprising: (e) detecting a change in conductivity. At least one of the oligonucleotides on the first type of nanoparticles has a sequence complementary to at least one sequence of the oligonucleotides on the second type of nanoparticles;
(F) contacting the second type of nanoparticles associated with the substrate with the first type of nanoparticles, wherein the contacting effectively hybridizes the oligonucleotides on the first and second types of nanoparticles. Steps that occur under conditions that cause
412. The method of claim 412, further comprising: (g) detecting a change in conductivity. 414. The method of claim 413, wherein step (d) or steps (d) and (f) are repeated one or more times to detect a change in conductivity. (D) contacting the first type of nanoparticles associated with the substrate with an aggregate probe to which the oligonucleotide is attached, wherein the nanoparticles of the aggregate probe are formed of a material capable of conducting electricity; At least one of the oligonucleotides on the aggregate has a sequence that is complementary to a sequence of one of the one or more types of oligonucleotides on the first type of nanoparticles, and wherein the contacting comprises: Occurring under conditions that effectively hybridize the oligonucleotide on the probe and the oligonucleotide on the first type of nanoparticles;
407. The method of claim 407, further comprising: (e) detecting a change in conductivity. A method for detecting a nucleic acid having at least two parts, comprising:
(A) contacting a substrate having an oligonucleotide attached thereto with a nucleic acid, wherein the oligonucleotide is disposed between a pair of electrodes, and the oligonucleotide is complementary to a first portion of the sequence of the nucleic acid. Having the following sequence, wherein the contacting occurs under conditions that allow the oligonucleotide on the substrate to effectively hybridize to the nucleic acid;
(B) contacting the nucleic acid attached to the substrate with an aggregate probe having an oligonucleotide attached thereto, wherein at least one of the oligonucleotides on the aggregate probe has a sequence in the second portion of the nucleic acid Having a complementary sequence, the nanoparticles of the aggregate probe are formed of a material capable of conducting electricity, and the conditions are such that the contacting effectively hybridizes the oligonucleotide on the aggregate probe with the nucleic acid. The steps that take place,
(C) detecting a change in conductivity. A method for detecting nucleic acids, performed on a substrate, comprising detecting the presence, amount, or both, of the nucleic acids with an optical scanner. 418. The method of claim 417, wherein the optical scanner is a flatbed scanner. 418. The method of claim 417, wherein the scanner is coupled to a computer equipped with software capable of calculating grayscale measurements, and calculates the grayscale measurements to provide a quantitative measurement of the detected nucleic acid. 418. The method of claim 417, wherein the scanner is coupled to a computer equipped with software capable of calculating grayscale measurements and qualitatively determines the presence of nucleic acids, the amount of nucleic acids, or both. A kit comprising a container containing the nanoparticle-oligonucleotide conjugate according to any one of claims 237-242. A kit comprising a container containing the nanoparticles of any one of claims 243-265. A kit comprising a substrate having at least one pair of electrodes attached thereto, wherein an oligonucleotide attached to the substrate is present between the electrodes. 423. The substrate of claim 423, wherein the substrate has a plurality of pairs of electrodes attached thereto in an array to enable detection of multiple portions of a single nucleic acid, detection of multiple different nucleic acids, or both. The kit as described. A method of nanofabrication,
Providing at least one type of linking oligonucleotide having a selected sequence, wherein the sequence of each type of linking oligonucleotide has at least two portions;
247. A step of providing one or more types of nanoparticle-oligonucleotide conjugates according to any one of claims 237-242, wherein the one or more types of conjugates are attached to each nanoparticle. The oligonucleotide having a sequence complementary to a sequence of a portion of the linking oligonucleotide;
The oligonucleotides attached to the conjugated nanoparticles are effectively hybridized to the linked oligonucleotides, resulting in the formation of the desired nanomaterial or nanostructure where the conjugated nanoparticles are held together by the oligonucleotide connector. Contacting the conjugate with the ligating oligonucleotide under conditions. A method of nanofabrication,
Providing at least one type of linking oligonucleotide having a selected sequence, wherein the sequence of each type of linking oligonucleotide has at least two portions;
265. The method of providing one or more types of nanoparticles according to any one of claims 243-265, wherein the recognition oligonucleotide on each of the one or more types of nanoparticles is a linking oligonucleotide. Having a sequence complementary to the partial sequence;
Under conditions where the oligonucleotides on the nanoparticles are effectively hybridized to the tethered oligonucleotide, such that the nanoparticles form the desired nanomaterial or nanostructure that is held together by the oligonucleotide connector, the tethered oligonucleotide And contacting with the nanoparticles. A method of nanofabrication,
Providing at least two types of nanoparticle-oligonucleotide conjugates according to any one of claims 237-242,
The oligonucleotide attached to the nanoparticles of the first type of conjugate has a sequence complementary to the sequence of the oligonucleotide attached to the nanoparticles of the second type of conjugate,
An oligonucleotide attached to the second type of conjugated nanoparticles having a sequence complementary to the sequence of the oligonucleotides attached to the first type of conjugated nanoparticles;
Contacting the first and second types of conjugates under conditions where the oligonucleotides on the nanoparticles of the conjugate effectively hybridize to each other, thereby forming the desired nanomaterial or nanostructure. , Consisting of. A method of nanofabrication,
Providing at least two types of nanoparticles according to any one of claims 243-265,
The recognition oligonucleotide on the first type of nanoparticles has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticles,
The recognition oligonucleotide on the second type of nanoparticles has a sequence complementary to the sequence of the oligonucleotide on the first type of nanoparticles,
Contacting the first and second types of nanoparticles under conditions in which the oligonucleotides on the nanoparticles effectively hybridize to each other, resulting in the formation of the desired nanomaterial or nanostructure. Method. 246. A nanomaterial or nanostructure composed of the nanoparticle-oligonucleotide conjugate according to any one of claims 237-242, wherein the nanoparticles are held together by an oligonucleotide connector. Construction. 265. A nanomaterial or nanostructure composed of the nanoparticles of any of claims 243-265, wherein the nanoparticles are held together by an oligonucleotide connector. A method for separating a selected nucleic acid having at least two portions from other nucleic acids, comprising:
247. A step of providing two or more nanoparticle-oligonucleotide conjugates according to any one of claims 237-242, wherein the oligonucleotides attached to each nanoparticle of the two or more conjugates. Has a sequence that is complementary to the sequence of one of the at least two portions of the selected nucleic acid;
Contacting the nucleic acid and the conjugate such that the oligonucleotides on the conjugated nanoparticles hybridize with the selected nucleic acid under effective conditions, such that the conjugate hybridized to the selected nucleic acid aggregates and precipitates. And a method consisting of: A method for separating a selected nucleic acid having at least two portions from other nucleic acids, comprising:
265. The method of providing two or more nanoparticles according to any one of claims 243-265, wherein the oligonucleotide on each of the two or more nanoparticles comprises at least two of the selected nucleic acids. Having a sequence complementary to the sequence of one of the portions;
Contacting the nucleic acid with the nanoparticle such that the oligonucleotide on the nanoparticle is hybridized with the selected nucleic acid under effective conditions, so that the nanoparticle hybridized to the selected nucleic acid aggregates and precipitates. How to become. A nanoparticle-oligonucleotide conjugate that is a nanoparticle having an oligonucleotide attached thereto, wherein the oligonucleotide has a covalently linked cyclic disulfide functional group capable of binding to the nanoparticle. A nanoparticle-oligonucleotide conjugate, which is a nanoparticle having an oligonucleotide attached thereto, wherein the oligonucleotide has a covalently bound polythiol functional group capable of binding to the nanoparticle. A nanoparticle-oligonucleotide conjugate, which is a nanoparticle to which an oligonucleotide is attached, wherein the oligonucleotide has a covalently bonded disulfide functional group capable of binding to the nanoparticle, at least a portion of the oligonucleotide being A conjugate having a sequence complementary to at least a portion of the sequence of a nucleic acid or another oligonucleotide. A nanoparticle-oligonucleotide conjugate, which is a nanoparticle to which an oligonucleotide is attached, wherein the oligonucleotide has a covalently bound polythiol functional group capable of binding to the nanoparticle, at least a portion of the oligonucleotide being A conjugate having a sequence complementary to at least a portion of the sequence of a nucleic acid or another oligonucleotide. 438. The conjugate of claim 435 or 436, wherein said oligonucleotide is present at a surface density sufficient to stabilize the conjugate. 438. The conjugate of claim 437, wherein the oligonucleotide is present on the nanoparticle surface at a surface density of at least 10 picomoles / cm. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 15 picomoles / cm ^2, conjugate according to claim 438. Present on the nanoparticle surfaces with a surface density of oligonucleotides of about 15 picomoles / cm ² ~ about 40 picomoles / cm ^2, conjugate according to claim 439. The conjugate according to claim 435 or 436, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 441. The conjugate of claim 441, wherein the nanoparticles are gold nanoparticles. 439. The conjugate of claim 435 or 436, wherein the oligonucleotide comprises at least one recognition oligonucleotide, wherein the recognition moiety has a sequence that is complementary to at least a portion of the sequence of a nucleic acid or another oligonucleotide. 443. The conjugate of claim 443, wherein each recognition oligonucleotide has a spacer moiety and a recognition moiety, wherein the spacer moiety is designed to be capable of binding to a nanoparticle. 445. The conjugate of claim 444, wherein the spacer moiety is covalently linked to a moiety having a cyclic disulfide functionality that links the spacer moiety to the nanoparticle. 445. The conjugate of claim 444, wherein the spacer moiety is covalently linked to a moiety having a polythiol functional group that links the spacer moiety to the nanoparticle. 457. The conjugate of claim 442, wherein the spacer moiety comprises at least about 10 nucleotides. The conjugate of claim 447, wherein the spacer moiety comprises up to about 10 to about 30 nucleotides. 449. The conjugate of claim 448, wherein the bases of the spacer nucleotides are all adenine, all thymine, all cytosine, all uracil, or all guanine. 439. The conjugate of claim 435 or 436, further comprising a type of diluent oligonucleotide. 460. The nanoparticle of claim 450, wherein the diluent oligonucleotide comprises about the same number of nucleotides as contained in the spacer portion of the recognition oligonucleotide. 451. The nanoparticle of claim 451, wherein the sequence of the diluent oligonucleotide is the same as the sequence of the spacer portion of the recognition oligonucleotide. A method of attaching an oligonucleotide to a nanoparticle to produce a nanoparticle-oligonucleotide conjugate, comprising:
Providing an oligonucleotide having a covalently attached cyclic sulfide functionality capable of binding to the nanoparticles;
Contacting the oligonucleotide with the nanoparticle under conditions effective to bind at least a portion of the oligonucleotide to the nanoparticle to form a nanoparticle-oligonucleotide conjugate. A method of attaching an oligonucleotide to a nanoparticle to produce a nanoparticle-oligonucleotide conjugate, comprising:
Providing an oligonucleotide having a polythiol functional group capable of binding to the nanoparticle covalently bound;
Contacting the oligonucleotide with the nanoparticle under conditions effective to bind at least a portion of the oligonucleotide to the nanoparticle to form a nanoparticle-oligonucleotide conjugate. 455. The method of claim 454 or 455, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 455. The method of claim 455, wherein the nanoparticles are gold nanoparticles. The oligonucleotide comprises at least one kind of recognition oligonucleotide, each recognition oligonucleotide has a spacer portion and a recognition portion, and a portion having a functional group capable of binding to the nanoparticle is covalently linked to the spacer portion. 453. The method of claim 453 or 454, wherein 457. The method of claim 457, wherein the spacer moiety comprises at least about 10 nucleotides. 458. The method of claim 458, wherein the spacer moiety comprises about 10 to about 30 nucleotides. 460. The method of claim 459, wherein the bases of the nucleotides of the spacer are all adenines, all thymines, all cytosines, all uracils, or all guanines. The oligonucleotide further comprises a type of diluent oligonucleotide, wherein the oligonucleotide and the nanoparticles are combined with at least a portion of each type of oligonucleotide of the oligonucleotide with a nanoparticle to form a nanoparticle-oligo. 457. The method of claim 457, wherein the contacting is under conditions effective to form a nucleotide conjugate. 462. The method of claim 461, wherein the diluent oligonucleotide comprises approximately the same number of nucleotides as included in the spacer portion of the recognition oligonucleotide. 462. The method of claim 462, wherein the sequence of the diluent oligonucleotide is the same as the sequence of the spacer portion of the recognition oligonucleotide. 457. The method of claim 457, wherein the oligonucleotide comprises at least two recognition oligonucleotides. A method of attaching an oligonucleotide to a charged nanoparticle to produce a nanoparticle-oligonucleotide conjugate, comprising:
Providing an oligonucleotide having a covalently attached cyclic disulfide functional group capable of binding to the nanoparticle, wherein the oligonucleotide comprises:
A certain type of recognition oligonucleotide,
And a type of diluent oligonucleotide.
Contacting the oligonucleotide with the nanoparticle in water for a period of time sufficient for at least a portion of each type of oligonucleotide of the oligonucleotide to bind to the nanoparticle;
Adding at least one salt to the water to form a salt solution, wherein the ionic strength of the salt solution is determined by the electrostatic attraction or repulsion of the oligonucleotides to the nanoparticles, and the electrostatic attraction of the oligonucleotides to each other. Steps that are strong enough to at least partially overcome the repulsion;
The additional oligonucleotides of each type of oligonucleotides of the oligonucleotides are combined with the nanoparticles in a salt solution for an additional period sufficient to form a nanoparticle-oligonucleotide conjugate. Contacting. A method of attaching a binding oligonucleotide to a charged nanoparticle to produce a nanoparticle-oligonucleotide conjugate, comprising:
Providing an oligonucleotide having covalently linked polythiol functional groups capable of binding to the nanoparticles, wherein the oligonucleotide comprises:
Certain types of recognition oligonucleotides,
And a type of diluent oligonucleotide.
Contacting the oligonucleotide with the nanoparticle in water for a period of time sufficient for at least a portion of each type of oligonucleotide of the oligonucleotide to bind to the nanoparticle;
Adding at least one salt to the water to form a salt solution, wherein the ionic strength of the salt solution is determined by the electrostatic attraction or repulsion of the oligonucleotides to the nanoparticles, and the electrostatic attraction of the oligonucleotides to each other. Steps that are strong enough to at least partially overcome the repulsion;
The additional oligonucleotides of each type of oligonucleotides of the oligonucleotides are combined with the nanoparticles in a salt solution for an additional period sufficient to form a nanoparticle-oligonucleotide conjugate. Contacting. 465. The method of claim 465 or 466, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles. 468. The method of claim 467, wherein the nanoparticles are gold nanoparticles. 465. The method of claim 465 or 466, wherein all the salt is added to the water in one addition. 465. The method of claim 465 or 466, wherein the salt is added gradually over time. The salt comprises sodium chloride, magnesium chloride, potassium chloride, ammonia, chloride, sodium, acetate, ammonium acetate, a combination of two or more of these salts, one of these salts in a phosphate buffer, and 465. The method of claim 465 or 466, wherein the method is selected from the group consisting of a combination of two or more of these salts in a phosphate buffer. 472. The method of claim 471, wherein said salt is sodium chloride in a phosphate buffer. Nanoparticles having oligonucleotides present nanoparticle surfaces with a surface density of at least 10 picomoles / cm ² - oligonucleotide conjugates are produced, the method according to claim 465 or 466. Oligonucleotide is present on the surface of the nanoparticles at the surface density of at least 15 picomoles / cm ^2, The method of claim 473. The method of claim 474 oligonucleotide is present on the surface of the nanoparticles at the surface density of about 15 picomoles / cm 2 ^~ about 40 picomoles / cm ^2, is present on the oligonucleotide. 465. The method of claim 465, wherein each recognition oligonucleotide has a spacer moiety and a recognition moiety, wherein the spacer moiety has attached thereto a moiety having a cyclic disulfide functional group capable of binding to the nanoparticles. 466. The method of claim 466, wherein each recognition oligonucleotide has a spacer moiety and a recognition moiety, wherein the spacer moiety has attached thereto a moiety having a polythiol functional group capable of binding to the nanoparticles. 475. The method of claim 476 or 477, wherein the spacer portion comprises at least about 10 nucleotides. 498. The method of claim 478, wherein the method comprises about 10 to about 30 nucleotides of the spacer moiety. 475. The method of claim 476 or 477, wherein the bases of the spacer nucleotides are all adenine, all thymine, all cytosine, all uracil, or all guanine. 475. The method of claim 476 or 477, wherein the diluent oligonucleotide comprises about the same number of nucleotides as contained in the spacer portion of the recognition oligonucleotide. 481. The method of claim 481, wherein the sequence of the diluent oligonucleotide is the same as the sequence of the spacer portion of the recognition oligonucleotide. 475. The method of claim 476 or 477, wherein the oligonucleotide comprises at least two recognition oligonucleotides. Oligonucleotides having a covalently linked cyclic disulfide function capable of binding to the nanoparticles. Oligonucleotides having covalently linked polythiol functional groups capable of binding to nanoparticles. 493. The composition of claims 433, 435, 445, 446, 453, 465, and 484, wherein a large hydrophobic group is located between the oligonucleotide and the cyclic disulfide function. A method for detecting an analyte in a sample, comprising:
Providing a type of nanoparticle conjugate having the oligonucleotide attached thereto, wherein at least a portion of the oligonucleotide attached to the nanoparticle is bound to a specific binding complement of the analyte as a result of hybridization. Binding to two oligonucleotides;
Contacting the analyte with the nanoparticle conjugate under conditions effective to permit a specific binding interaction between the analyte and a specific binding complement associated with the nanoparticle conjugate;
Observing a detectable change caused by a specific binding interaction between the analyte and a specific binding complement of the analyte. A method for detecting a sample, comprising:
Providing a type of nanoparticle conjugate having the oligonucleotide attached thereto, wherein at least a portion of the oligonucleotide attached to the nanoparticle binds to the first portion of the linked oligonucleotide as a result of hybridization, and The oligonucleotide having a second portion that binds to the oligonucleotide that has bound the specific binding complement of the analyte as a result of hybridization;
Contacting the analyte with the nanoparticle conjugate under conditions effective to permit a specific binding interaction between the analyte and the specific binding complement associated with the nanoparticle conjugate;
Observing a detectable change caused by a specific binding interaction between the analyte and a specific binding complement of the analyte. A method for detecting a sample, comprising:
(I) an analyte associated with the oligonucleotide, (ii) a first type of nanoparticle associated with an oligonucleotide having a sequence complementary to the sequence of the oligonucleotide associated with the analyte, and (iii) an oligonucleotide associated. Providing a second type of nanoparticles, wherein the portion of the oligonucleotide associated with the second type of nanoparticles binds to the oligonucleotide associated with the specific binding complement of the analyte as a result of hybridization. Providing
Oligonucleotide bound to the analyte under conditions effective to hybridize the oligonucleotide attached to the analyte and the oligonucleotide attached to the first type of nanoparticles to form a nanoparticle-analyte conjugate Contacting with a first type of nanoparticles;
The nanoparticle-analyte conjugate is combined with the second type of nanoparticle conjugate under conditions effective to permit a specific binding interaction between the analyte and the specific binding complement of the second type of nanoparticle. Contacting;
Observing a detectable change caused by a specific binding interaction between the analyte and a specific binding complement of the analyte. A method for detecting a sample, comprising:
(Ii) a linking oligonucleotide having at least two portions, (iii) a first type of nano-attached oligonucleotide having a portion of which is complementary to a second portion of the linking oligonucleotide. A particle, (i) an analyte associated with an oligonucleotide having a sequence complementary to the first portion of the linking oligonucleotide, and (iv) an oligonucleotide, wherein a portion of the oligonucleotide is specific to the analyte as a result of hybridization. Providing a second type of nanoparticle associated oligonucleotide that binds to the oligonucleotide associated with the binding complement;
Contacting the linking oligonucleotide with the first type of nanoparticles under conditions effective to permit hybridization between the oligonucleotide attached to the first type of nanoparticles and the first portion of the linking oligonucleotide. Steps and
Contacting the linking oligonucleotide with the oligonucleotide linked to the sample under conditions effective to permit hybridization between the oligonucleotide linked to the sample and the second portion of the linking oligonucleotide;
Under conditions effective to permit a specific binding interaction between an analyte associated with the first type of nanoparticles and a specific binding complement associated with the second type of nanoparticles, the first type of nanoparticles Contacting an analyte associated with the nanoparticles with a second type of nanoparticles;
Observing a detectable change in the analyte caused by specific binding of the analyte to specific binding complement of the analyte. 490. The method of any one of claims 487-490, wherein the analyte is multivalent and binds to more than one nanoparticle conjugate. 490. The method of any one of claims 487-490, wherein the analyte is multivalent and specifically binds to more than one nanoparticle conjugate. 490. The method of any one of claims 487-490, wherein said contacting conditions include freezing and thawing. 490. The method of any one of claims 487-490, wherein said contacting conditions include heating. 490. The method of any one of claims 487-490, wherein the detectable change is observed on a solid surface. 490. The method of any one of claims 487-490, wherein the detectable change is a color change observable to the naked eye. 490. The method of any one of claims 487-490, wherein a color change is observed on a solid surface. 490. The method of any one of claims 487-490, wherein the nanoparticles are formed from gold. A method for detecting a sample, comprising:
(I) a support that binds the analyte, and (ii) an oligonucleotide that partially binds to a second oligonucleotide having a specific binding complement of the analyte as a result of hybridization. Providing a nanoparticle conjugate,
Contacting a support-bound analyte with a nanoparticle conjugate under conditions effective to permit a specific binding interaction between the analyte and a specific binding complement bound to the nanoparticle conjugate The step of causing
Observing a detectable change based on the specific binding of the analyte to the specific binding complement of the analyte. A method for detecting a sample, comprising:
(I) a support to which the oligonucleotide is bound, (ii) a specimen to which an oligonucleotide having a sequence complementary to the oligonucleotide bound to the support is bound, and (iii) the oligonucleotide, and a part thereof. Providing, as a result of the hybridization, a type of nanoparticle conjugate associated oligonucleotide that binds to a second oligonucleotide associated with the specific binding complement of the analyte;
The oligonucleotide attached to the analyte under conditions effective to allow hybridization of the oligonucleotide attached to the support to the oligonucleotide attached to the support and the oligonucleotide attached to the analyte. Contacting with
The support-bound analyte can be loaded onto the nanoparticle under conditions effective to permit specific binding complement between the support-bound analyte and the specific binding complement bound to the nanoparticle. Contacting with a conjugate;
Observing a detectable change based on the specific binding of the analyte to the specific binding complement of the analyte. A method for detecting an analyte in a sample, comprising:
Providing (i) a support having the oligonucleotide attached thereto, (ii) a linked oligonucleotide, (ii) a specimen having the oligonucleotide attached thereto, and (iii) a kind of nanoparticle conjugate having the oligonucleotide attached thereto. Wherein at least a portion of the oligonucleotide bound to the nanoparticle conjugate binds to the oligonucleotide bound to the specific binding complement of the analyte as a result of the hybridization, and the sequence of the linking oligonucleotide comprises at least two portions. Wherein the oligonucleotide bound to the support has a sequence complementary to the first portion of the linking oligonucleotide, and the oligonucleotide bound to the analyte has a sequence complementary to the second portion of the linking oligonucleotide. A step having
The linking oligonucleotide is contacted with the support-linked oligonucleotide under conditions effective to permit hybridization between the support-linked oligonucleotide and the first portion of the linking oligonucleotide. Steps and
Contacting the linking oligonucleotide with the oligonucleotide associated with the sample under conditions effective to permit hybridization between the oligonucleotide linked to the analyte and the second portion of the linking oligonucleotide;
The support-bound analyte is subjected to conditions effective to permit a specific binding interaction between the support-bound analyte and the specific binding complement bound to the nanoparticle conjugate, Contacting with a nanoparticle conjugate;
Observing a detectable change based on the specific binding of the analyte to the specific binding complement of the analyte. A method for detecting a sample, comprising:
(I) a support having an oligonucleotide attached thereto, (ii) a specimen having an oligonucleotide which is complementary to the sequence of the oligonucleotide attached to the support attached, and (iii) a kind of nanoparticles having an oligonucleotide attached thereto. Wherein a portion of the oligonucleotide attached to the nanoparticle binds to the first portion of the linking oligonucleotide as a result of the hybridization, and the second portion of the linking oligonucleotide further comprises a Being bound to an oligonucleotide having the oligonucleotide bound to a specific binding complement of the analyte;
Contacting the support-bound oligonucleotide with the analyte-bound oligonucleotide under conditions effective to permit hybridization between the support-bound oligonucleotide and the analyte-bound oligonucleotide. When,
The support-bound analyte is combined with the nanoparticle conjugate under conditions effective to permit a specific binding interaction between the support-bound analyte and the specific binding complement bound to the nanoparticle. Contacting;
Observing a detectable change based on the specific binding of the analyte to the specific binding complement of the analyte. A method for detecting a sample, comprising:
Providing an aggregate probe comprising (i) a support associated with the analyte, and (ii) at least two types of nanoparticles associated with the oligonucleotide, wherein the nanoparticles of the aggregate probe are attached thereto. Some of the oligonucleotides that have been bound to each other as a result of the hybridization, and at least one of the at least two nanoparticles of the aggregate probe has bound the specific binding complement of the analyte as a result of the hybridization. Binding to two oligonucleotides;
Contacting the support with the aggregate probe under conditions effective to permit a specific binding interaction between the analyte bound to the support and the specific binding complement bound to the aggregate probe. When,
Observing a detectable change based on the specific binding of the analyte to the specific binding complement of the analyte. A method for detecting a sample, comprising:
(I) a support having an oligonucleotide attached thereto, (ii) an analyte having an oligonucleotide complementary to the oligonucleotide attached to the support attached, and (iii) a coagulation comprising at least two types of nanoparticles having the oligonucleotide attached thereto. Providing an aggregated probe, wherein the nanoparticles of the aggregated probe bind to each other as a result of a portion of the oligonucleotides attached to each being hybridized, and wherein at least two of the nanoparticles of the aggregated probe Providing at least one species that binds to the associated second oligonucleotide as a result of hybridization, the specific binding complement of the analyte;
Contacting the oligonucleotide-bound support with the oligonucleotide-bound sample, wherein the contacting is performed under conditions where hybridization between the sample-bound oligonucleotide and the support-bound oligonucleotide is effective. Steps that take place in
The support-bound analyte is subjected to coagulation under conditions effective to permit the specific binding interaction between the support-bound analyte and the specific binding complement attached to the aggregate probe. Contacting with a collecting probe;
Observing a detectable change based on the specific binding of the analyte to the specific binding complement of the analyte. A method for detecting a sample, comprising:
(I) a support having attached oligonucleotides, (ii) a linked oligonucleotide having at least two parts, (iii) an analyte having attached oligonucleotides, and (iv) at least two kinds of nanoparticles having attached oligonucleotides. Providing an aggregate probe, wherein the nanoparticles of the aggregate probe bind to each other as a result of hybridization of a portion of the oligonucleotide attached to each, and at least two of the nanoparticles of the aggregate probe Binds to the second oligonucleotide associated with the specific binding complement of the analyte as a result of hybridization, and the oligonucleotide associated with the support is complementary to the first portion of the linked oligonucleotide. Has a unique sequence and is associated with the sample. Go nucleotides and steps have a sequence complementary to the second portion of the linking oligonucleotide,
Contacting the tethered oligonucleotide with the support-tethered oligonucleotide under conditions effective to permit hybridization between the support-tethered oligonucleotide and the first portion of the tethered oligonucleotide; ,
The linking oligonucleotide is coupled to the analyte under conditions effective to permit hybridization between the second portion of the linking oligonucleotide bound to the support and the oligonucleotide bound to the analyte. Contacting with
The support-bound analyte is subjected to coagulation under conditions effective to permit a specific binding interaction between the support-bound analyte and the specific binding complement attached to the aggregate probe. Contacting with a collecting probe;
Observing a detectable change based on the specific binding of the analyte to the specific binding complement of the analyte. A method for detecting a sample, comprising:
(I) a support to which oligonucleotides are linked, (ii) a sample to which oligonucleotides having a sequence complementary to the sequence of oligonucleotides to which the support is linked, and (iii) at least two kinds of samples to which oligonucleotides are linked. Providing an aggregate probe comprising nanoparticles, wherein the nanoparticles of the aggregate probe bind to each other as a result of a portion of the oligonucleotides attached to each being hybridized, and provide at least two types of aggregate probes. At least one of the nanoparticles has an oligonucleotide that binds to the first portion of the linking oligonucleotide as a result of hybridization, and the second portion of the linking oligonucleotide binds to the analyte as a result of hybridization. Oligonus with bound complement And a step that binds to Reochido,
The oligonucleotide-bound support is contacted with the sample-bound oligonucleotide under conditions effective to permit hybridization of the sample-bound oligonucleotide with the support-bound oligonucleotide. Steps and
The support-bound analyte is allowed to aggregate under conditions effective to permit a specific binding interaction between the support-bound analyte and the specific binding complement bound to the aggregation probe. Contacting with a probe;
Observing a detectable change based on the specific binding of the analyte to the specific binding complement of the analyte. A method for detecting a sample, comprising:
Providing (i) a support to which the analyte is bound, and (ii) nanoparticles to which the oligonucleotide is bound, wherein a portion of the oligonucleotide attached to the nanoparticle is characterized as a result of the hybridization. Binding to a second oligonucleotide associated with the specific binding complement;
The support associated with the analyte is subjected to conditions effective to permit a specific binding interaction between the analyte bound to the support and the specific binding complement bound to the nanoparticle conjugate, Contacting with a nanoparticle conjugate;
Contacting the nanoparticle conjugate bound to the support with a silver dye to produce a detectable change;
Observing a detectable change based on specific binding of the analyte to the analyte with specific binding complement. A method for detecting a multivalent sample, comprising:
Providing a nanoparticle probe having the oligonucleotide attached thereto, wherein at least a portion of the oligonucleotide attached to the nanoparticle binds to a first portion of the reporter oligonucleotide as a result of hybridization, and A second part, as a result of the hybridization, binding to the oligonucleotide that bound the specific binding complement of the analyte;
Contacting the multivalent analyte with the nanoparticle probe under conditions effective to permit a specific binding interaction between the analyte and the nanoparticle probe and form an aggregated complex;
Separating the aggregated complex;
Subjecting the aggregated complex to conditions effective to dehybridize the aggregated complex, release the reporter oligonucleotide and the reporter oligonucleotide,
Detecting the presence of the reporter oligonucleotide. A method for detecting a nucleic acid, comprising:
(I) one or more types of nanoparticles having an oligonucleotide having a sequence complementary to the first portion of the nucleic acid, and (ii) two or more biotin molecules each having an oligonucleotide associated therewith. Providing a complex comprising streptavidin or avidin linked by a specific binding interaction;
Nucleic acids, nanoparticles, and complexes are allowed to hybridize with a first portion of the nucleic acid and an oligonucleotide associated with the nanoparticle and with a second portion of the nucleic acid and an oligonucleotide associated with the complex. Contacting under effective conditions;
Observing a detectable change resulting from the hybridization of the nanoparticles, the complex and the nucleic acid. A method for detecting a nucleic acid, comprising:
(I) one or more types of nanoparticles conjugated to an oligonucleotide having a sequence complementary to the first portion of the nucleic acid; (ii) conjugated to biotin having a sequence complementary to the second portion of the nucleic acid. Providing an oligonucleotide, and (iii) streptavidin or avidin;
Permits hybridization of the nucleic acid with a first portion of the nucleic acid and the oligonucleotide attached to the nanoparticle, and between the second portion of the nucleic acid and the oligonucleotide attached to biotin, thereby forming a complex. Contacting with the oligonucleotide conjugated to the nanoparticle conjugate and biotin under conditions effective to
Contacting the conjugate with streptavidin or avidin under conditions effective to permit a specific binding interaction between biotin associated with the conjugate and streptavidin or avidin;
Observing a detectable change caused by specific binding of streptavidin or avidin to biotin associated with the complex. A method for detecting a nucleic acid, comprising:
A first type of nanoparticle conjugate having an oligonucleotide attached thereto, wherein at least a portion of the oligonucleotide attached to the nanoparticle has a sequence complementary to a first portion of a nucleic acid Providing a
Contacting the nucleic acid with the first type of nanoparticle under conditions effective to permit hybridization between the oligonucleotide attached to the nanoparticle and the first portion of the nucleic acid;
providing an oligonucleotide attached to the sbp member, wherein the oligonucleotide attached to the sbp member has a sequence complementary to a second portion of the sequence of the nucleic acid;
contacting the nucleic acid linked to the sbp member under conditions effective to permit hybridization between the oligonucleotide linked to the sbp member and a second portion of the nucleic acid;
Providing a second type of nanoparticle conjugate associated with the oligonucleotide, wherein at least a portion of the oligonucleotide associated with the second type of nanoparticle has a specific binding of the analyte as a result of hybridization. Binding complement to the associated oligonucleotide;
The nucleic acid associated with the first type of nanoparticles and the oligonucleotide associated with the sbp member are combined with the sbp member associated with the first type of nanoparticles and the sbp complement associated with the second type of nanoparticles. Contacting with an oligonucleotide associated with the second type of nanoparticles under conditions effective to permit a specific binding interaction between the two;
Observing a detectable change resulting from specific binding to streptavidin or avidin. The method of claim 511, wherein the sbp member is biotin. The method of claim 511, wherein the sb complement is streptavidin or avidin. A method for detecting a nucleic acid, comprising:
Providing a support attached oligonucleotide having a sequence complementary to the first portion of the nucleic acid;
Contacting the nucleic acid with the oligonucleotide-attached support under conditions effective to permit hybridization between the oligonucleotide associated with the support and the first portion of the nucleic acid;
providing an oligonucleotide attached to the sbp member, wherein the oligonucleotide attached to the sbp member has a sequence complementary to a second portion of the nucleic acid;
Contacting the nucleic acid bound to the support with the oligonucleotide bound to the sbp member under conditions effective to permit hybridization between the nucleic acid bound to the sbp member and a second portion of the nucleic acid Steps and
Providing a type of nanoparticle conjugate to which the oligonucleotide is attached, wherein at least a portion of the oligonucleotide attached to the nanoparticle conjugate, upon hybridization, results in specific binding complement of an sbp member. Binding to the oligonucleotide associated with
The sbp member bound to the support can be combined with the nanoparticle under conditions effective to permit a specific binding interaction between the sbp member bound to the support and a specific binding complement bound to the nanoparticle. Contacting with a particle conjugate;
Under conditions effective to permit a specific binding interaction between the support-bound sbp member and the specific binding complement bound to the nanoparticle, the support-bound sbp member is Contacting with a nanoparticle conjugate;
observing a detectable change based on the specific binding between the sbp member and the specific binding complement. 515. The method of claim 514, wherein the sbp member is streptavidin or avidin. 515. The method of claim 514, wherein the sb complement is biotin. A method for detecting a nucleic acid, comprising:
Providing a nanoparticle conjugate attached to the oligonucleotide, wherein at least a portion of the oligonucleotide attached to the nanoparticle has a sequence complementary to the first portion of the nucleic acid;
Contacting the nucleic acid with the oligonucleotide attached to the nanoparticle under conditions effective to permit hybridization of the oligonucleotide attached to the nanoparticle with the first portion of the nucleic acid;
providing an oligonucleotide attached to the sbp member, wherein the oligonucleotide attached to the sbp member has a sequence complementary to the second portion of the nucleic acid;
Contacting the nucleic acid with an oligonucleotide having an sbp member under conditions effective to permit hybridization between the oligonucleotide having the sbp member and the nucleic acid;
providing a support associated with the specific binding complement of the sbp member;
Contacting the support with a sbp member associated with the nanoparticle under conditions effective for a specific binding interaction to occur between the sbp member and the sb complement associated with the support;
observing a detectable event based on the specific binding of the sbp member to the complement of the sbp member. The method of claim 517, wherein the sbp member is biotin. The method of claim 517, wherein the sb complement is streptavidin or avidin. A method for detecting a nucleic acid, comprising:
Providing a support having the oligonucleotide attached thereto, wherein the oligonucleotide attached to the support has a sequence complementary to the first portion of the nucleic acid;
Contacting the nucleic acid with the support under conditions effective to permit hybridization between the oligonucleotide attached to the support and the first portion of the nucleic acid;
providing an oligonucleotide attached to the sbp member, wherein the oligonucleotide attached to the sbp member has a sequence complementary to the second portion of the nucleic acid;
The support-bound nucleic acid and the sbp member are bound under conditions effective to permit hybridization between the second portion of the support-bound nucleic acid and the oligonucleotide bound to the sbp member. Contacting the oligonucleotide thus obtained,
Providing an aggregate probe comprising at least two types of nanoparticles associated oligonucleotides, wherein the nanoparticles of the aggregate probes bind to each other as a result of a portion of the oligonucleotides attached thereto being hybridized. And at least one of the at least two nanoparticles of the aggregate probe is hybridized so that it is bound to an oligonucleotide that binds the specific binding complement of the sbp member;
Under conditions effective to permit a specific binding interaction between the support-bound sbp member and the specific binding complement bound to the aggregate probe, the support-bound sbp member is removed. Contacting with an aggregate probe;
observing a detectable change based on the specific binding of the sbp member and the sbp complement of the sbp member. The method of claim 520, wherein the sbp member is biotin. The method of claim 520, wherein the sb complement is streptavidin or avidin. A method for detecting a nucleic acid, comprising:
(I) an aggregate probe comprising at least two types of nanoparticles associated oligonucleotides, wherein the nanoparticles of the aggregate probe bind to each other as a result of a portion of the oligonucleotides attached to them hybridizing; At least one of the at least two nanoparticles of the aggregate probe has attached thereto some oligonucleotides complementary to the first portion of the nucleic acid; and (ii) the second probe of the nucleic acid. Providing an oligonucleotide having an sbp member having a sequence complementary to the portion;
The oligonucleotide attached to the aggregate probe and the first portion of the nucleic acid under conditions effective to permit hybridization between the portion of the oligonucleotide attached to the aggregate probe and the first portion of the nucleic acid. Contacting the nucleic acid with an aggregate probe under conditions effective to permit hybridization between
Contacting the nucleic acid with an oligonucleotide having an sbp member, wherein the oligonucleotide having the sbp member attached thereto is capable of allowing hybridization between the oligonucleotide having the sbp member attached and the second portion of the nucleic acid. Having, under effective conditions, a sequence complementary to the second portion of the nucleic acid;
providing a support that has bound the specific binding complement of the sbp member;
The specific binding complement bound to the aggregate probe under conditions effective to permit a specific binding interaction to occur between the sbp member bound to the aggregate probe and the sb complement bound to the support. Contacting the body with an sbp member associated with the aggregate;
observing a detectable event based on the specific binding of the sbp member and sb complement. The method of claim 523, wherein the sbp member is streptavidin or avidin. The method of claim 523, wherein the sb complement is biotin. The substrate has a plurality of types of nanoparticles attached thereto in an array to enable detection of multiple portions of a single nucleic acid, detection of multiple different nucleic acids, or both. The method according to any one of claims 499-525. 525. The method of any one of claims 499-525, wherein the substrate is a transparent substrate or an opaque white substrate. 525. The method of any one of claims 499-525, wherein the detectable change is the formation of a dark region on the substrate. 525. The method of any one of claims 499-525, wherein the nanoparticles are formed from gold. 529. The method of any one of claims 498-523, wherein the substrate is contacted with a silver dye to produce a detectable change. 529. The method of any one of claims 498-523, wherein the detectable change is observed with an optical scanner. A nanoparticle conjugate for detecting an analyte,
(I) nanoparticles having oligonucleotides attached thereto;
(Ii) an oligonucleotide that binds the specific binding complement of the analyte, the oligonucleotide having a sequence complementary to at least a portion of the oligonucleotide that is bound to the nanoparticle, and that has been bound to the nanoparticle as a result of hybridization. A oligonucleotide conjugated to at least a portion of the oligonucleotide. A nanoparticle conjugate for detecting an analyte,
(I) nanoparticles having oligonucleotides attached thereto;
(Ii) an oligonucleotide that binds a specific binding complement of a sample member;
(Iii) a linking oligonucleotide having at least two portions, wherein the first portion of the linking oligonucleotide binds to the oligonucleotide associated with the nanoparticle as a result of hybridization, and the second portion of the linking oligonucleotide comprises , As a result of the hybridization, a linking oligonucleotide that binds to the oligonucleotide associated with the specific binding complement of the sample,
A nanoparticle conjugate comprising: An aggregate probe for detecting a sample,
(I) at least two types of nanoparticles that bind oligonucleotides, wherein the nanoparticles attached to each other bind as a result of hybridization of a portion of the oligonucleotides;
(Ii) an oligonucleotide to which a specific binding complement of the analyte is bound, wherein the oligonucleotide binds to at least one of the at least two nanoparticles of the aggregate probe as a result of hybridization. ,
An aggregate probe comprising: An aggregate probe for detecting a sample,
(I) at least two types of nanoparticles that bind oligonucleotides, wherein the nanoparticles attached to each other bind as a result of hybridization of a portion of the oligonucleotides;
(Ii) an oligonucleotide binding the specific binding complement of the sample;
(Iii) a linking oligonucleotide having at least two portions, wherein the first portion of the linking oligonucleotide is a hybrid of at least one of the at least two nanoparticles of the aggregate probe as a result of hybridization. A second portion of the linking oligonucleotide that binds to the oligonucleotide, wherein, as a result of the hybridization, a linking oligonucleotide that binds to the oligonucleotide that bound the specific binding complement of the analyte;
An aggregate probe comprising: A method for preparing a nanoprobe conjugate for detecting an analyte,
(I) a nanoparticle conjugate associated with the oligonucleotide, and (ii) an oligonucleotide associated with the specific binding complement of the analyte, wherein at least a portion of the oligonucleotide associated with the nanoparticle has a specific binding complement. Providing an oligonucleotide having a name sequence complementary to the sequence of the oligonucleotide tied to the body;
The oligonucleotide attached to the nanoparticle conjugate can be combined with the nanoparticle under conditions effective to permit hybridization between the oligonucleotide attached to the nanoparticle and the oligonucleotide attached to the specific binding complement. Contacting with the oligonucleotide attached to the conjugate. A kit for detecting a specimen,
(A) at least one container holding a nanoparticle conjugate comprising the following (i) to (iii):
(I) a nanoparticle associated with the oligonucleotide; (ii) an oligonucleotide associated with a specific binding complement of the analyte, wherein the oligonucleotide having a specific binding pair member comprises an oligonucleotide associated with the nanoparticle. An oligonucleotide that binds and, as a result of hybridization, binds to at least a portion of the oligonucleotide associated with the nanoparticle;
(B) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding a nanoparticle conjugate comprising the following (i) to (iii):
(I) a nanoparticle associated oligonucleotide (ii) an oligonucleotide associated specific binding complement of an analyte (iii) a linking oligonucleotide having at least two portions, wherein the first portion of the linking oligonucleotide comprises: As a result of the hybridization, the second portion of the linking oligonucleotide binds to the oligonucleotide attached to the nanoparticle, and the second portion of the linking oligonucleotide binds to the oligonucleotide that binds the specific binding complement of the analyte as a result of hybridization. b) an optional support for observing a detectable change. A kit for detecting a specimen,
(B) at least one container holding an aggregate probe comprising the following (i) to (iii):
(I) at least two types of nanoparticles that bind oligonucleotides, wherein the nanoparticles attached to each other bind as a result of hybridization of a portion of the oligonucleotides;
(Ii) an oligonucleotide to which a specific binding complement of an analyte is bound, and as a result of hybridization, binds to an oligonucleotide of at least one of the at least two nanoparticles of the aggregate probe; Oligonucleotide (b) an optional support for observing the detectable change. A kit for detecting a specimen,
(A) at least one container holding an aggregate probe comprising the following (i) to (iii):
(I) at least two types of nanoparticles that bind oligonucleotides, wherein the nanoparticles attached to each other bind as a result of hybridization of a portion of the oligonucleotides;
(Ii) an oligonucleotide binding the specific binding complement of the sample;
(Iii) a linking oligonucleotide having at least two portions, wherein the first portion of the linking oligonucleotide is a hybrid of at least one of the at least two nanoparticles of the aggregate probe as a result of hybridization. A container comprising a linking oligonucleotide that binds to the oligonucleotide and binds to the oligonucleotide that bound the specific binding complement of the analyte as a result of hybridization;
(B) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding certain types of nanoparticles, including nanoparticles with associated oligonucleotides;
(B) the container holding the oligonucleotide binding the specific binding complement of the analyte, and the oligonucleotide binding the specific binding pair member, wherein the oligonucleotide has a sequence complementary to at least a portion of the oligonucleotide bound to the nanoparticle. Having
(C) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding a nanoparticle conjugate associated oligonucleotide;
(B) a container for holding an oligonucleotide linked to a specific binding complement of a sample member;
(C) a container holding a linking oligonucleotide having at least two portions, and a first portion of the linking oligonucleotide is complementary to at least a portion of the oligonucleotide associated with the nanoparticle and a second portion of the linking oligonucleotide. The portion is complementary to the oligonucleotide that bound the specific binding complement of the analyte; and
(D) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, and the aggregate probe being separated from each other as a result of a portion of the oligonucleotide attached thereto being hybridized; Being connected,
(B) a container holding an oligonucleotide to which the specific binding complement of the sample is bound, and the oligonucleotide to which the specific binding complement is bound, at least one of at least two types of nanoparticles of the aggregate probe. Being complementary to one type of nanoparticle oligonucleotide;
(C) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, and the aggregate probe being separated from each other as a result of a portion of the oligonucleotide attached thereto being hybridized; Being connected,
(B) a container for holding the oligonucleotide to which the specific binding complement of the sample is bound;
(C) a container holding a linking oligonucleotide having at least two portions, wherein the first portion of the linking oligonucleotide is an oligonucleotide of at least one of the at least two nanoparticles of the aggregate probe; Having a complementary sequence, wherein the second portion of the linking oligonucleotide has a sequence complementary to the oligonucleotide that bound the specific binding complement of the analyte;
(D) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding a type of nanoparticle associated oligonucleotide;
(B) a container holding an oligonucleotide to which a functional group for binding to a specific binding complement of a sample is bound by a covalent bond, and an oligonucleotide bound to the functional group is an oligonucleotide bound to a nanoparticle. Having a sequence complementary to at least a portion of the nucleotides;
(C) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding nanoparticles having associated oligonucleotides;
(B) a container for holding an oligonucleotide to which a functional group for binding to a specific binding complement of a sample is covalently bound,
(C) a container holding a linking oligonucleotide having at least two portions, and a first portion of the linking oligonucleotide is complementary to at least a portion of the oligonucleotide associated with the nanoparticle and a second portion of the linking oligonucleotide. The moiety is complementary to the oligonucleotide having a functional group; and
(D) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding an aggregation probe containing at least two types of nanoparticles linked to oligonucleotides, wherein the nanoparticles of the aggregated probe are partially hybridized to oligonucleotides attached thereto; As a result of
(B) a container for holding an oligonucleotide to which a functional group for binding to a specific binding complement of a specimen is covalently bound, and an oligonucleotide bound to the functional group comprises at least two types of aggregates. Having a sequence complementary to the oligonucleotide of at least one of the nanoparticles;
(C) an optional support for observing a detectable change. A kit for detecting a specimen,
(A) at least one container holding an aggregation probe containing at least two types of nanoparticles linked to oligonucleotides, wherein the nanoparticles of the aggregated probe are partially hybridized to oligonucleotides attached thereto; As a result of
(B) a container for holding an oligonucleotide to which a functional group for binding to a specific binding complement of a sample is covalently bound,
(C) a container holding a linking oligonucleotide having at least two portions, wherein the first portion of the linking oligonucleotide is complementary to an oligonucleotide of at least one of the at least two nanoparticles of the aggregation probe. Wherein the second portion of the linking oligonucleotide is complementary to the oligonucleotide to which the functional group is attached;
(D) an optional support for observing a detectable change. A kit for detecting a specimen,
A substrate having an oligonucleotide attached thereto,
An oligonucleotide having a functional group for binding to a specific binding complement of an analyte bound by a covalent bond, and the oligonucleotide bound to the functional group has a sequence complementary to the oligonucleotide bound to the substrate. That
(I) a nanoparticle associated with the oligonucleotide, (ii) an oligonucleotide associated with the specific binding complement of the analyte, wherein the oligonucleotide has a sequence complementary to at least a portion of the oligonucleotide associated with the nanoparticle. A kit comprising: an oligonucleotide that binds to at least a portion of the oligonucleotide associated with the nanoparticle as a result of the hybridization; and a nanoparticle conjugate comprising the oligonucleotide. A kit for detecting a specimen,
A substrate having an oligonucleotide attached thereto,
An oligonucleotide having a functional group for binding to a specific binding complement of an analyte bound by a covalent bond, and the oligonucleotide bound to the functional group has a sequence complementary to the oligonucleotide bound to the substrate. That
(I) an oligonucleotide associated oligonucleotide, (ii) an oligonucleotide associated specific binding complement of an sbp member, and (iii) a linking oligonucleotide having at least two portions, wherein the linking oligonucleotide comprises One portion binds to the oligonucleotide attached to the nanoparticle as a result of hybridization, and the second portion of the linking oligonucleotide binds to the oligonucleotide attached to the specific binding complement of the analyte as a result of hybridization. A conjugate comprising a linking oligonucleotide and a nanoparticle conjugate. A kit for detecting a specimen,
A substrate having the oligonucleotide attached thereto, and the oligonucleotide attached to the substrate having a sequence complementary to the first portion of the nucleic acid;
an oligonucleotide associated with the sbp member, the oligonucleotide having a sequence complementary to the second portion of the nucleic acid;
(I) a nanoparticle associated with the oligonucleotide, (ii) an oligonucleotide associated with the specific binding complement of the sbp member, wherein the oligonucleotide has a sequence complementary to at least a portion of the oligonucleotide associated with the nanoparticle. A nanoparticle conjugate comprising: an oligonucleotide that binds to at least a portion of the oligonucleotide associated with the nanoparticle as a result of hybridization. The kit of claim 551, wherein the sbp member is biotin. 552. The kit of claim 551, wherein the specific binding complement of the sbp member is streptavidin or avidin. A kit for detecting a nucleic acid,
A substrate having the oligonucleotide attached thereto, and the oligonucleotide attached to the substrate having a sequence complementary to the first portion of the nucleic acid;
an oligonucleotide associated with the sbp member, the oligonucleotide having a sequence complementary to the second portion of the nucleic acid;
(I) an oligonucleotide associated oligonucleotide, (ii) an oligonucleotide associated specific binding complement of an sbp member, and (iii) a linking oligonucleotide having at least two portions, wherein the linking oligonucleotide comprises One portion binds to the oligonucleotide attached to the nanoparticle as a result of hybridization, and the second portion of the linking oligonucleotide binds to the oligonucleotide attached to the specific binding complement of the analyte as a result of hybridization. A conjugate comprising a linking oligonucleotide and a nanoparticle conjugate. The kit of claim 554, wherein the sbp member is biotin. 555. The kit of claim 554, wherein the specific binding complement of the sbp member is streptavidin or avidin. A kit for detecting a specimen,
A substrate having an oligonucleotide attached thereto,
An oligonucleotide having a functional group for binding to an analyte bound by a covalent bond, wherein the oligonucleotide bound to the functional group has an oligonucleotide complementary to at least a portion of the oligonucleotide bound to the substrate. Nucleotides;
An aggregate probe comprising the following (i) and (ii):
(I) at least two types of nanoparticles associated with the oligonucleotide, wherein the nanoparticles attached to each bind to each other as a result of the hybridization of a part of the oligonucleotide; (ii) associated with the specific binding complement of the specimen; A kit comprising: an oligonucleotide, wherein the oligonucleotide of at least one of the at least two nanoparticles of the aggregate probe further binds as a result of the hybridization. A kit for detecting a specimen,
A substrate having an oligonucleotide attached thereto,
An oligonucleotide having a functional group for binding to an analyte bound by a covalent bond, wherein the oligonucleotide bound to the functional group has an oligonucleotide complementary to at least a portion of the oligonucleotide bound to the substrate. Nucleotides;
An aggregate probe comprising the following (i) to (iii):
(I) at least two types of nanoparticles that bind oligonucleotides, each of which binds to each other as a result of hybridization of a part of the attached oligonucleotide; (ii) binding of specific binding complement of an analyte; A linked oligonucleotide having at least two portions, wherein the first portion of the linked oligonucleotides has at least one of the at least two nanoparticles of the aggregate probe as a result of hybridization. A kit comprising a linking oligonucleotide that binds to an oligonucleotide associated with the nanoparticle of the type and the second portion of the linking oligonucleotide binds to the oligonucleotide that bound the specific binding complement of the analyte as a result of hybridization. . A kit for detecting a nucleic acid,
A substrate having the oligonucleotide attached thereto, and the oligonucleotide attached to the substrate having a sequence complementary to the first portion of the nucleic acid;
An aggregate probe comprising the following (i) and (ii):
(I) at least two types of nanoparticles having oligonucleotides attached thereto, wherein the nanoparticles bind to each other as a result of hybridization of a part of the oligonucleotide attached to each of them (ii) specific binding complement of sbp member A kit comprising an associated oligonucleotide that binds to an oligonucleotide associated with at least one of said at least two nanoparticles of said aggregate probe as a result of hybridization. The kit of claim 559, wherein the sbp member is biotin. The kit of claim 559, wherein the specific binding complement of the sbp member is streptavidin or avidin. A kit for detecting a nucleic acid,
A substrate having the oligonucleotide attached thereto, and the oligonucleotide attached to the substrate having a sequence complementary to the first portion of the nucleic acid;
an oligonucleotide associated with the sbp member, the oligonucleotide having a sequence complementary to the second portion of the nucleic acid;
An aggregate probe comprising the following (i) to (iii):
(I) at least two types of nanoparticles that bind oligonucleotides, each of which binds to each other as a result of hybridization of a part of the attached oligonucleotide; (ii) binding of specific binding complement of an analyte; A linked oligonucleotide having at least two portions, wherein the first portion of the linked oligonucleotides has at least one of the at least two nanoparticles of the aggregate probe as a result of hybridization. A second portion of the linking oligonucleotide comprising a linking oligonucleotide that binds to the oligonucleotide that bound the specific binding complement of the sbp member as a result of hybridization. kit. 562. The kit of claim 562, wherein the sbp member is biotin. 561. The kit of claim 562, wherein the specific binding complement of the sbp member is streptavidin or avidin. A method of nanofabrication,
Providing a linking oligonucleotide having a selected sequence, wherein each type of sequence of the linking oligonucleotide has at least two portions;
Providing one or more types of nanoparticles attached to the oligonucleotides, wherein the oligonucleotides on each type of nanoparticles have a sequence complementary to a second portion of the sequence of the linked oligonucleotides;
Providing a complex composed of streptavidin or avidin conjugated to two or more biotin molecules, wherein each molecule is associated with an oligonucleotide, wherein the oligonucleotide is a member of the sequence of the linking oligonucleotide. Having a sequence complementary to the two parts;
Allow the linking oligonucleotides, complexes and nanoparticles to hybridize the oligonucleotides on the nanoparticles and the first oligonucleotide conjugated to biotin to the linking oligonucleotide to form the desired nanomaterial or nanostructure. Contacting under effective conditions. A method of nanofabrication,
(I) at least one type of linking oligonucleotide in which the sequence of each type of linking oligonucleotide has at least two portions, (ii) at least two types of nanoparticles to which the oligonucleotide is attached, and The oligonucleotide on the nanoparticle has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticle and a sequence complementary to the first portion of the sequence of the linked oligonucleotide, The oligonucleotide on the particle comprises a nanoparticle having a sequence complementary to the sequence of the oligonucleotide on the first type of nanoparticle-oligonucleotide conjugate and a sequence complementary to the first portion of the sequence of the linking oligonucleotide. (Iii) a complex comprising streptavidin or avidin conjugated to two or more biotin molecules A is, in each molecule bound oligonucleotide, the method comprising providing an oligonucleotide that is attached to the biotin, the composite having a sequence complementary to a second portion of the sequence of the linking oligonucleotide, a,
Hybridization of the first and second types of nanoparticles, linking oligonucleotides, and the conjugates to the oligonucleotides on the nanoparticles and to each other and to the linking oligonucleotides to form the desired nanomaterial or nanostructure. Contacting under conditions effective to permit hybridization of the oligonucleotide of the complex to the nucleotide. A method of nanofabrication,
(A) at least one type of linking oligonucleotide in which the sequence of each type of linking oligonucleotide has at least two parts, (b) one or more nanoparticles to which the oligonucleotides are attached, wherein The oligonucleotide on the particle is a nanoparticle having a sequence complementary to the first portion of the sequence of the linking oligonucleotide, (c) the biotin to which the oligonucleotide is attached, wherein the oligonucleotide to which the biotin molecule is attached is: Providing biotin, (d) streptavidin or avidin, having a sequence complementary to the second portion of the sequence of the linking oligonucleotide;
The linking oligonucleotide, the biotin linked to the oligonucleotide, and the nanoparticles are used to hybridize the oligonucleotide on the nanoparticle and the oligonucleotide linked to biotin to the linking oligonucleotide to form a complex. Contacting under sufficient conditions;
Contacting the complex with streptavidin or avidin under conditions effective to permit a specific binding interaction between biotin and streptavidin or avidin to form a desired nanomaterial or nanostructure. And a method consisting of: A nanomaterial,
(I) at least one type of linking oligonucleotide in which the sequence of each type of linking oligonucleotide has at least two portions, (ii) at least two types of nanoparticles to which the oligonucleotide is attached, and The oligonucleotide on the nanoparticle has a sequence complementary to the sequence of the oligonucleotide on the second type of nanoparticle and a sequence complementary to the first portion of the sequence of the linked oligonucleotide, The oligonucleotide on the particle comprises a nanoparticle having a sequence complementary to the sequence of the oligonucleotide on the first type of nanoparticle-oligonucleotide conjugate and a sequence complementary to the first portion of the sequence of the linking oligonucleotide. (Iii) a complex comprising streptavidin or avidin conjugated to two or more biotin molecules A is, in each molecule bound oligonucleotide, the method comprising providing an oligonucleotide that is attached to the biotin, the composite having a sequence complementary to a second portion of the sequence of the linking oligonucleotide, a,
Hybridization of the first and second types of nanoparticles, linking oligonucleotides, and the conjugates to the oligonucleotides on the nanoparticles and to each other and to the linking oligonucleotides to form the desired nanomaterial or nanostructure. Contacting under conditions effective to permit hybridization of the oligonucleotide of the complex to nucleotides. A nanomaterial,
(A) at least one type of linking oligonucleotide in which the sequence of each type of linking oligonucleotide has at least two parts, (b) one or more nanoparticles to which the oligonucleotides are attached, wherein The oligonucleotide on the particle is a nanoparticle having a sequence complementary to the first portion of the sequence of the linking oligonucleotide, (c) the biotin to which the oligonucleotide is attached, wherein the oligonucleotide to which the biotin molecule is attached is: Providing biotin, (d) streptavidin or avidin, having a sequence complementary to the second portion of the sequence of the linking oligonucleotide;
The linking oligonucleotide, the biotin linked to the oligonucleotide, and the nanoparticles are used to hybridize the oligonucleotide on the nanoparticle and the oligonucleotide linked to biotin to the linking oligonucleotide to form a complex. Contacting under sufficient conditions;
Contacting the complex with streptavidin or avidin under conditions effective to permit a specific binding interaction between biotin and streptavidin or avidin to form a desired nanomaterial or nanostructure. And a nanomaterial produced by a method comprising: A method for separating a selected target nucleic acid having at least two portions from other nucleic acids, comprising:
(A) one or more types of nanoparticles attached with oligonucleotides, wherein the oligonucleotide on each type of nanoparticle has a sequence complementary to the sequence of the first portion of the selected nucleic acid; and (b) each molecule A composite comprising streptavidin or avidin conjugated to two or more biotin molecules, wherein the first oligonucleotide is conjugated and the first oligonucleotide has a sequence complementary to the sequence of the second portion of the selected nucleic acid Providing a body,
Effective for allowing selected nucleic acids and other nucleic acids to permit hybridization of the oligonucleotide on the nanoparticle and the first oligonucleotide of the complex with the selected nucleic acid, and subsequently to form an aggregate. Contacting the nanoparticles under various conditions;
Fractionating aggregates containing the selected nucleic acid. A method for separating a selected target nucleic acid having at least two portions from other nucleic acids, comprising:
(A) one or more types of nanoparticles attached with oligonucleotides, wherein the oligonucleotide on each type of nanoparticle has a sequence complementary to the sequence of the first portion of the selected nucleic acid; Providing biotin having an oligonucleotide having a sequence complementary to the two-part sequence, and (c) streptavidin or avidin;
Under conditions effective to permit hybridization of the selected nucleic acid and other nucleic acids with the selected nucleic acid of the oligonucleotide on the nanoparticle and the oligonucleotide of the biotin construct with the selected nucleic acid, Contacting the oligonucleotide with the associated nanoparticles and biotin;
Contacting the conjugate with streptavidin or avidin under conditions effective to permit a specific binding interaction between biotin and streptavidin or avidin, followed by formation of an aggregate. When,
Fractionating aggregates containing the selected nucleic acid. A method for accelerating the movement of nanoparticles to an electrode surface,
Providing at least one nanoparticle associated with a charged first member of the specific binding pair and an electrode surface having a second member of the specific binding pair;
Contacting the nanoparticles and the electrode surface under conditions effective to allow binding between the first and second members of the specific binding pair;
Subjecting the nanoparticles to an electric field to accelerate migration of the nanoparticles to the electrode surface and promote binding between the first and second members of the binding pair. 572. The method of claim 572, wherein the specific binding pair is an antibody / antigen. 572. The method of claim 572, wherein the specific binding pair is a receptor / ligand. A method for detecting a nucleic acid having one or more moieties bound to an electrode surface,
Providing one or more types of nanoparticles having oligonucleotides attached thereto, wherein the oligonucleotide on each of the one or more types of nanoparticles is one of at least one or more portions of the nucleic acid. Having a sequence complementary to the two sequences;
Contacting the nucleic acid and the nanoparticle under conditions that allow the oligonucleotide on the nanoparticle to effectively hybridize to the nucleic acid;
Subjecting the nanoparticles to an electric field to accelerate the movement of the nanoparticles to the electrode surface;
Observing a detectable change. 575. The method of claim 575, wherein the nucleic acid has at least two portions. A method for detecting a nucleic acid having one or more moieties attached to a surface, comprising:
Contacting the nucleic acid with at least two types of nanoparticles having the oligonucleotide attached thereto, wherein the oligonucleotide on the first type of nanoparticles has a sequence complementary to the sequence of the first portion of the nucleic acid; The oligonucleotides on the two nanoparticles have a sequence complementary to the sequence of the second portion of the nucleic acid, and contacting occurs under conditions that effectively hybridize the oligonucleotide on the nanoparticle with the nucleic acid;
Subjecting the nanoparticles to an electric field to accelerate their movement to the surface;
Observing a detectable change caused by hybridization of the oligonucleotide and the nucleic acid on the nanoparticle. The method of claim 577, wherein the nucleic acid has at least two parts. The method of claim 577, wherein the contact conditions include freezing and thawing. 580. The method of claim 577, wherein said contacting conditions include heating. The method of claim 577, wherein the detectable change is observed on a solid surface. 580. The method of claim 577, wherein the detectable change is a color change observable to the naked eye. The method of claim 582, wherein the change in color is observed on a solid surface. The method of claim 577, wherein the nanoparticles are formed from gold. 580. The method of claim 577, wherein the nanoparticles are metal or semiconductor nanoparticles, and the oligonucleotide attached to the nanoparticles is labeled with a fluorescent molecule at the end that is not attached to the nanoparticles. The method of claim 577, wherein the nucleic acid has at least two parts. The nucleic acid has a third portion located between the first and second portions, and the sequence of the oligonucleotide on the nanoparticle has no sequence complementary to the third portion of the nucleic acid;
589. The nucleic acid of claim 586, wherein the nucleic acid is further contacted with a filler oligonucleotide having a sequence complementary to the third portion of the nucleic acid, wherein the contacting occurs under conditions that effectively hybridize the filler oligonucleotide with the nucleic acid. Method. The method of claim 577, wherein the nucleic acid is viral RNA or DNA. The method of claim 577, wherein the nucleic acid is a disease-related gene. The method of claim 577, wherein the nucleic acid is bacterial DNA. The method of claim 577, wherein the nucleic acid is fungal DNA. 580. The method of claim 577, wherein the nucleic acid is synthetic DNA, synthetic RNA, structurally modified natural or synthetic RNA, or structurally modified natural or synthetic DNA. The method of claim 577, wherein the nucleic acid is of a biological source. The method of claim 577, wherein the nucleic acid is a product of a polymerase chain reaction amplification method. The method of claim 577, wherein the nucleic acid is a fragment obtained by cleavage of the DNA with a restriction enzyme. 580. The method of claim 577, wherein the nucleic acid is contacted simultaneously with the first and second types of nanoparticles. 580. The method of claim 577, wherein the nucleic acid is contacted and hybridized with an oligonucleotide on the first type of nanoparticle before contacting with the second type of nanoparticle. 598. The method of claim 597, wherein the first type of nanoparticles is attached to a substrate. 589. The method of claim 575 or 577, wherein the detectable change is caused by hybridization of an oligonucleotide on the nanoparticle to a nucleic acid.

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