JP4672966B2 - 分析物の結合体プローブおよび光学的検出 - Google Patents
分析物の結合体プローブおよび光学的検出 Download PDFInfo
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Description
本出願は、特許協力条約第8条によって、2001年3月9日に出願された、米国仮特許出願60/274,177号の優先権の利益を主張し、この出願は、その全体を参考として本明細書中で援用される。
本発明は、分析物の結合体プローブおよび光学的検出に関する。より具体的には、本発明は、センサーのアレイまたは分析物の光学的検出を使用するシステムを形成するために使用される結合体プローブに関する。
生体分子および他の分析物は、標的の分析物を結合する、選択的または特異的なプローブのアレイまたはマイクロアレイを使用して、検出され得る。バイオセンサーアレイ技術において、基材またはバイオチップ上にプローブスポットを配置するためのスキームが、開発されてきた。アレイは、遺伝子配列を検出および発見するために、薬物分子候補を選択および試験するために、毒物学的作用または薬理学的作用を調査するために、および他の使用のために使用される。標的は、種々の相互作用のプローブのアレイに結合し得、その相互作用としては、核酸塩基対形成またはハイブリダイゼーション、タンパク質−タンパク質相互作用、タンパク質−リガンド相互作用、酵素−基質相互作用、レセプター−リガンド相互作用、および他の化学反応が挙げられる。
1つの局面において、本発明は、ポリマーに結合する化学物質または生体分子を含む結合体に関する。1つの実施形態において、このポリマーは、ジオール含有ポリマーである。別の実施形態において、このポリマーは、直鎖ポリサッカリドまたは分枝鎖ポリサッカリドである。このポリマーはまた、直鎖ポリヌクレオチドまたは分枝鎖ポリヌクレオチドであり得る。この化学物質または生体分子は、例えば、オリゴヌクレオチド、タンパク質核酸、タンパク質、抗体または抗体フラグメントであり得る。1つの実施形態において、結合体は、ポリサッカリドを、酸化剤、またはカルボニル化剤、もしくは、例えば、3−マレイミドプロピオン酸(3−maleimidopropionic acid)と反応する工程を含む方法によって調製される。ポリマーへの化学物質または生体分子のカップリングは、光反応性架橋剤、またはヘテロ二官能性光反応性架橋剤を用いてなされ得る。
「アレイ」または「マイクロアレイ」は、直線状または2次元のマトリクスまたは不連続領域のアレイであり、各々は、規定された領域を有し、固体支持体の表面上に形成される。この不連続領域は、重なり合っても、重なり合わなくても良い。マイクロアレイ上の不連続領域の密度は、単一の固相支持体の表面上で検出される標的分子(例えば、ポリヌクレオチド)の総数によって決定される。2つ以上の領域がアレイを形成し得るが、不連続領域の代表的な密度は、少なくとも約50/cm2であり、しばしば少なくとも約100/cm2であり、よりしばしば少なくとも約500/cm2であり、そして時々少なくとも約1,000/cm2である。本明細書中で使用される場合、DNAマイクロアレイは、標的ポリヌクレオチドを増幅またはクローニングするために使用される、チップまたは他の表面上に位置づけられるオリゴヌクレオチドプライマーのアレイである。アレイ中のプライマーの各特定の群の位置が既知であるので、標的ポリヌクレオチドの同一性は、マイクロアレイ上の特定の位置へのそれらの結合に基づいて決定され得る。
一般的に、アレイのプローブは、検出される標的分析物を捕捉し、結合する。プローブ捕捉部分または標的結合部分としては、核酸、ポリヌクレオチド、タンパク質、ペプチド核酸、低分子、および広範囲の生体分子が挙げられる。標的結合プローブ部分としては、抗体のエピトープ結合ドメインが挙げられる。
本発明の1つの局面において、バイオセンサーシステムを用いる分析物の検出は、そのアレイに結合した分析物からのシグナルを、より少ないバックグラウンドで、相応して、もっと高いシグナル対ノイズ比で読み出すために使用され得る、デジタル画像または「機械視覚」感知技術を使用することによって、増強される。1つの実施形態において、そのバイオセンサーシステムは、デジタル画像感知技術を使用し、この技術は、ドーターボード上のデジタル画像センサー、結合体プローブのアレイ、そのセンサーに熱冷却を提供し得るそのセンサーのエンクロージャー、ならびに分析物シグナルを積分する方法を含む。
結合体プローブに結合した分析物の光学的検出としては、蛍光法、化学発光法、生物発光法、比色法、吸収法、および量子ドット法による検出が挙げられる。標識種またはシグナル分子は、ポリマーに、結合体プローブのプローブに、または標的混合物中の分析物に付着される。標識種またはシグナル分子の例としては、放射性同位元素、蛍光剤、化学発光剤、ケミルミノフォア(chemiluminophore)、生物発光剤、酵素、抗体、および粒子(例えば、磁気粒子および量子ドット)が挙げられる。短いアミン誘導性オリゴヌクレオチドに付着した蛍光色素分子が、標識種として使用され得、ここで、アミン基は、結合体のポリマーに結合される。分析物検出に使用されるシグナル分子としては、放射標識、蛍光色素(例えば、Cy3、Cy5、Alexa Flour 488、フルオレセイン、ローダミン、Texas red、ローズベンガル(rose bengal)、ダンシルクロリド、エチジウムブロミド、アミノナフタレン、ピレン、およびポルフィリン)、化学発光系(例えば、ルミノール、ジオキシエタン(dioxetane)、アクリジニウムフェニルエステル、およびルテニウム塩)、発色団および比色プローブ(例えば、コロイド金、アゾ色素、キノリン色素およびシアニン色素)が挙げられる。
1つの実施形態において、図3に示されるように、バイオセンサーシステムは、微光(low−light)エンクロージャー100内のデジタル画像センサー600、ハイデータスループット読取り位置400、および汎用コンピューター700を備える。読取り位置400は、CMOSデジタル画像センサー600を含む低光量エンクロージャー100を挿入するための少なくとも1つのソケットを備え、これによって、センサーエンクロージャーを読取り位置に電気的および機械的に接続する。読取り位置は、例えば、ユニバーサルシリアルバス(USB)454を介して、または異なるパラレルポートインターフェースデバイス452によってコンピューターに接続され得る。必要に応じて、読取り位置は、Ethernet(登録商標)インターフェース456を介してコンピューターに接続され得る。このシステムにおいて使用され得る他の接続としては、限定しないが、Firewire、SCSI、およびPCMCIAが挙げられる。代替の実施形態において、コンピューターおよび読取り位置は、ハンドヘルドコンピューター、またはパーソナルデジタルアシスタント(例えば、Palm Pilot)によって置換され得る。
線状ポリサッカリドデキストラン(Sigma)を、脱イオン水中に、1%の最終濃度まで溶解し、次いでオートクレーブした。0.40mlデキストラン溶液のアリコートを、ロッキングプラットフォームにおいて、室温暗所で、一晩、44μlの0.5M過ヨウ素酸ナトリウムで酸化した。次いで、酸化されたデキストランを0.3M NaOACおよび2×VolのEtOHで2回沈殿させることによって洗浄した。ペレットを空気乾燥し、そして0.4mlの5mM NaPO4緩衝液(pH7.2)中に再溶解した。
高分子量分枝ポリサッカリド、グリコーゲン(Sigma)およびアミロペクチン(Sigma)を使用して、アミン誘導体化オリゴヌクレオチドに結合した。これらのポリマーのジオール基を、NaIO4を用いる酸化によってアルデヒド基に変換した。過ヨウ素酸ナトリウムを、それぞれ、グリコーゲンについて25mMの最終濃度、そしてアミロペクチンについて20mMの最終濃度まで、0.4mlの1%ポリサッカリド溶液(H2O中)に添加した。酸化を、ロッキングプラットフォームにおいて、室温暗所で、一晩続けた。次いで、酸化されたポリサッカリドを、0.3M NaOAcおよび2×Vol のEtOHで2回沈殿させて、過剰のNaIO4を除去した。空気乾燥後、ペレットを0.4mlの5mM NaPO4緩衝液(pH7.2)に溶解した。アミン誘導体化オリゴヌクレオチドのカップリングおよび結合した生成物のゲル−分析を、デキストランについて実施例1に記載される通りに実施した。
10グルコースモノマー当たり1つのオリゴヌクレオチドの結合密度で各グリコーゲン分子に結合される、平均して約1000オリゴヌクレオチド分子を有する、実施例2の結合体プローブを調製する。
5’末端においてアミンで誘導体化され、そして3’末端において、蛍光色素Cy5で誘導体化された、シグナル伝達分子5’−ACTGCT−3’(BP001)、および5’末端においてアミン基で誘導体化された、認識プローブ分子オリゴヌクレオチドAKH108
オリゴヌクレオチド
カルボジイミダゾール(CDI)活性化について、4μlの0.5M CDI(DMSO中)を、DMSO中の10μlの0.5%ポリサッカリドおよび6μlのDMSOに添加した。反応を、室温で3時間、ときどきボルテックスしながらインキュベートした。次いで、1mlのn−ブタノールを各チューブに添加し、ボルテックスにより完全に混合し、そして微量遠心管中で遠心分離した。ブタノールの除去後、ペレットを空気乾燥し、そして10μlのDMSO中に溶解した。オリゴヌクレオチドカップリングのために、2.2nmolのアミン誘導体化オリゴヌクレオチドを、5μlの0.5%CDI−活性化ポリサッカリドと、20μlの最終容量で混合した。反応物を、室温で5日間、ときどきボルテックスしながらインキュベートした。反応の最後に、1μlの10%エタノールアミンを添加し、そしてさらに30分間インキュベートし、次いで、EtOHで沈殿させた。次いで、生成物を10μlのTE緩衝液中に溶解し、そしてゲル電気泳動によって分析した。
CMOS画像センサーを、ハイブリダイゼーションシグナルの直接的なオンチップ(on−chip)検出のために使用した。
最大での利得(Gain)(レジスター53、および43〜46);1フレームあたり0.1秒での積分時間(レジスター9および10);最大でのアナログマイナスオフセット(Analogue negative offset)(レジスター32および57);74での増幅段利得(Gainstage)(レジスター62)。レジスターの静止(rest)を、デフォルト設定で残した。
Claims (3)
- 光学的画像センサーを用いて分析物を検出するためのアレイであって、該アレイは結合体を含み、
該結合体は、ジオール含有ポリマー、直鎖状ポリサッカリド、または分枝状ポリサッカリドに共有結合によって結合された化学物質または生体分子を含み、ここで、該結合体は該光学的画像センサーの表面に結合されており、かつ
該結合体は、該光学的画像センサーの表面上にスポットされている、
ことを特徴とする上記アレイ。 - 該結合体は、直鎖状ポリサッカリドに共有結合によって結合された化学物質または生体分子を含む、請求項1に記載のアレイ。
- 該分枝状ポリサッカリドは、アミロース、アミロペクチン、グリコゲン、デキストラン、セルロース、キチン、キトサン、ペプチドグリカン、グリコサミノグリカンおよびそれらの混合物からなる群より選択される、請求項1に記載のアレイ。
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Publication number | Publication date |
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AU2002255699A1 (en) | 2002-09-24 |
ES2593683T3 (es) | 2016-12-12 |
EP2295971A3 (en) | 2011-06-08 |
US20090082214A1 (en) | 2009-03-26 |
EP2295971A2 (en) | 2011-03-16 |
HK1155515A1 (zh) | 2012-05-18 |
JP2009092675A (ja) | 2009-04-30 |
US20120202714A1 (en) | 2012-08-09 |
ES2441412T3 (es) | 2014-02-04 |
EP2295971B1 (en) | 2016-09-07 |
JP2004532397A (ja) | 2004-10-21 |
EP1373573A2 (en) | 2004-01-02 |
EP1373573A4 (en) | 2006-11-29 |
EP1373573B1 (en) | 2013-10-02 |
CA2440656A1 (en) | 2002-09-19 |
WO2002073158A3 (en) | 2003-02-20 |
DK1373573T3 (da) | 2014-01-20 |
WO2002073158A2 (en) | 2002-09-19 |
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