JP2004512497A - Interaction of colloid-fixed species with species on non-colloidal structures - Google Patents

Abstract

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Description

【００７２】
本明細書で使用する“分子ワイヤ”は、ＳＡＭで被覆した電極と接触している流体が電極と電気的に連絡する能力を高めるワイヤを意味する。これには、先に記載し、以下でさらに完全に例示する伝導性分子、または流体が電極と接触するようにＳＡＭ中に欠陥を生じることができる分子が含まれる。付加的な分子ワイヤの非限定的なリストには、２−メルカプトピリジン、２−メルカプトベンゾチアゾール、ジチオスレイトール、１，２−ベンゼンジチオール、１，２−ベンゼンジメタンチオール、ベンゼン‐エタンチオール、および２−メルカプトエチルエーテルが挙げられる。また、単分子層の伝導性は電極の平面で伝導性を促進する分子の添加により高めることが可能である。伝導性ＳＡＭは、以下のものからなるが、それらに限定されない：１）硫黄で停止するポリ（エチニルフェニル）鎖；２）ベンゼン環で停止するアルキルチオール；３）ＤＮＡ塩基で停止するアルキルチオール；４）単分子層中に不完全に詰まる、いずれかの硫黄停止種；５）上記の全てに非特異的吸着を阻害するエチレングリコール単位またはメチル基のいずれかで停止するアルキルチオールスペーサー分子を足したものまたは引いたもの。チオールはＳＡＭの即座の形成において、それらの金への親和性のために記載される。米国特許第５，６２０，８２０号および他の参考文献に基づいて、チオールを当技術分野で既知の他の分子と置換してもよい。
【００７３】
シグナル伝達コロイド上の既知のリガンドがそれらのコグネイト細胞由来タンパク質と相互作用するとき、薬物候補を溶液に添加し、異なる作用をモニターしてもよい。細胞に直接的に、または間接的に結合する頭基で停止する分子（たとえば、メチル基、ポリＫ，陽性電荷、ＲＧＤ配列、Ｋｒｉｎｇｌｅモチーフ、インテグリン、およびそれらのペプチド模倣物）も提示する伝導性ＳＡＭにより被覆された電極に細胞が補充されてもよい。あるいは異なる相互作用は、伝導性ＳＡＭ上に提示されたコグネイトリガンドと相互作用している、ＳＡＭに結合した細胞表面タンパク質を有することにより定量することができる。この場合、リガンドが構成的に発現された受容体に結合した後、電子的または電気化学的シグナルが生成される。流動力下では、電子的または電気化学的シグナルはＳＡＭ固定化リガンドと相互作用する細胞表面受容体の数に比例するだろう。流速の関数としての細胞運動性の速度は、表面の細胞接着分子に対する受容体の密度を示す。相互に作用するＳＡＭの下流は、電荷読みとり装置（たとえば、電流計、電荷計数装置）、または光学式読みとり機（たとえば、表面プラズモン共鳴検出器、または蛍光読みとり機）である。さらに、これらの方法は、関与する特異的な受容体‐接着分子相互作用の予備知識なしに、浸潤または転移が特徴であることが既知の細胞表現型を阻害する薬物のスクリーニングに使用することもできる。また、生理学的に適切な相互作用を模倣する薬物のスクリーニングはこの系を使用して以下のいずれかにより行うことができる：溶液中に遊離で薬物候補を添加し、検出シグナルの損失を探索するか、または固体支持体上の薬物候補と共に溶液中で遊離した既知のリガンドを添加し、検出シグナルの出現を探索する。
【００７４】
わずかに陽性のバイアスをＡＣ電位ランプ（ｒａｍｐ）に適用すると細胞は陰性荷電されるという事実から、感知電極に引き寄せられる。リガンドが連結するデンドリマーまたはポリマーに付加的な陰性に荷電した基を添加すると感知電極への補充がさらに促進される。当業者は、デンドリマーまたはポリマーに複数のシグナル伝達実体を容易に固定することができる。あるいは、金属結合タグにより認識リガンドを結合することは、アルキルチオールＮＴＡへのそれらの結合（および結果として関連した細胞の結合）を促進する。金被覆磁気ビーズ上にフェロセンのような金属含有化合物を有するＳＡＭｓは、感知表面電極に複合体を補充するために電磁場、または固定磁石の使用を許容する。また、リガンドを包含する感知電極は、細胞接着分子によるもののような特異的相互作用または非特異的相互作用により、直接的に、またはヒスチジンタグ標識により細胞を引き寄せることができる。
【００７５】
しかし、結合パートナーの一つが分析溶液より比重が大きいため、複合体が重力により沈殿する場合には、検出系への補充も単に重力を用いて行うことができる。早すぎる沈殿を避けるためにインキュベーション段階で機械的混合が行われてもよい。図３２Ａおよび３２Ｂは、表面への結合相互作用を示すシグナル伝達実体および対照、を補充するための重力の使用を示す実験のＡＣＶプロットを示す。ＧＳＴは金属結合タグ／金属／キレート結合（ＮＴＡ‐ＳＡＭ）によりコロイドに結合した。またコロイドは電子的または電気化学的シグナル伝達のためのフェロセン誘導体を提示した。陰性対照としてコロイドを測定電極に溶液中で提示し、図３２Ａのプロットを得た。コロイドの１セットは、グルタチオンで被覆されたポリマービーズ（アガロース）に暴露した。コロイドはグルタチオン／ＧＳＴ結合によりビーズに固定され、ビーズは重力により電極上に固定された。電極に近いフェロセンの電気活性検出を示す図３２ＢのＡＣＶプロットが得られた。
【００７６】
細胞由来タンパク質生成装置として電極を使用する方法において、細胞を溶解させる電位スパイクを発生させるために電極パッド上の点を使用することができる。次に細胞の内容物をシグナル伝達リガンドとインキュベートし、相互作用を検出する。
【００７７】
さらに細胞結合分子は、伝導性分子とともにチャネルを並べ、系の検出能力を高めるために、ＳＡＭを使用してフローチャネルの底に沿って指定のトポロジーの形状にされてもよい。したがって、ＳＡＭは感知能力（光学的または電子的もしくは電気化学的）と細胞結合能力を交互にする形状で、結合部分を提示してもよい。例としては、メチル停止アルキルチオールの縞はＳＡＭ中で伝導性のポリピロール基と交互になる。また、ＳＡＭのトポロジーは細胞を溝中に置き、その結果細胞の側面に沿った受容体を評価できるように操作することができる。交互官能基性ＳＡＭの縞の形成は、共に本明細書に援用される米国特許第５，５１２，１３１号および国際特許公開公報第ＷＯ９６／２９６２９号に記載のように行うことができる。これらの複合体は電子的または電気化学的に検出することができる。１つの検出方法は、交流ボルタンメトリー（ＡＣＶ）と呼ばれる電気化学的技術である。この検出方法は、より高次な調和解析のような付加的な分析技術の使用により補充することができる。これらの複合体は、ＳＰＲのような光学的手段によって検出してもよい。後者の技術を使用すると、感知表面への複合体の補充により、光学的性質の大きなシフトが引き起こされる。
【００７８】
一態様において、以下のように細胞表面タンパク質を検出し、定量することができる：細胞表面タンパク質を認識するヒスチジンタグ標識リガンドをＮＴＡ（Ｈｉｓ‐タグ標識タンパク質を捕捉するため）およびフェロセン部分（電子的または電気化学的シグナル伝達のため）の両方を提示するＳＡＭを持つコロイドに、連結させる。次に、これらの生物特異的、電子的または電気化学的シグナル伝達コロイドを、標的受容体を提示する細胞と共にインキュベートする。細胞はその後、ＳＡＭ被覆電極上に沈殿させ、接着させ、または引き寄せられ、ＡＣＶにより分析される（図３）。シグナル伝達コロイドに固定化されたリガンドが細胞表面上のそれらのコグネイト受容体に結合した場合、フェロセン部分の特徴的な酸化電位で電流ピークが生じるであろう。細胞表面受容体を認識する抗体は、初めにＨｉｓタグ標識プロテインＡまたはＧが結合しているＮＴＡ‐フェロセンを持つコロイドに結合してもよい。あるいは、抗体は金属結合タグ／金属／キレート結合によりコロイドに直接結合してもよく、ここで金属結合タグは抗体に結合さえる。ヒスチジンタグを抗体に結合するための技術は、“Ｃｏｎｓｔｒｕｃｔｉｏｎ　ｏｆ　ｔｈｅ　ｓｉｎｇｌｅ−ｃｈａｉｎ　Ｆｖ　ｆｒｏｍ　１９６−１４　ａｎｔｉｂｏｄｙ　ｔｏｗａｒｄ　ｏｖａｒｉａｎ　ｃａｎｃｅｒ−ａｓｓｏｃｏｉａｔｅｄ　ａｎｔｉｇｅｎ　ＣＡ１２５”Ｈａｓｈｉｍｏｔｏ，Ｙ．，Ｔａｎｉｇａｗａ，Ｋ．，Ｎａｋａｓｈｉｍａ，Ｍ．，Ｓｏｎｏｄａ，Ｋ．，Ｕｅｄａ，Ｔ．，Ｗａｔａｎａｂｅ，Ｔ．，ａｎｄ　Ｉｍｏｔｏ，Ｔ．：１９９９，Ｂｉｏｌｏｇｉｃａｌ　ａｎｄ　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｂｕｌｌｅｔｉｎ，Ｖｏｌ　２２：（１０）１０６８−１０７２．；“Ｈｕｍａｎ　ａｎｔｉｂｏｄｉｅｓ　ｗｉｔｈ　ｓｕｂｎａｎｏｍｏｌａｒ　ａｆｆｉｎｉｔｉｅｓ　ｉｓｏｌａｔｅｄ　ｆｒｏｍ　ａ　ｌａｒｇｅ　ｎｏｎ−ｉｍｍｕｎｉｚｅｄ　ｐｈａｇｅ　ｄｉｓｐｌａｙ　ｌｉｂｒａｒｙ”，Ｖａｕｇｈａｎ，Ｔ．Ｊ．，Ｗｉｌｌｉａｍｓ，Ａ．Ｊ．，Ｐｒｉｔｃｈａｒｄ，Ｋ．，Ｏｓｂｏｕｒｎ，Ｊ．Ｋ．，Ｐｏｐｅ，Ａ．Ｒ．，Ｅａｒｎｓｈａｗ，Ｊ．Ｃ．ｅｔ　ａｌ．，１９９６，Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｖｏｌ　１４（３）ｐ．２６７．；“Ｅｘｐｒｅｓｓｉｏｎ　ａｎｄ　ｐｕｒｉｆｉｃａｔｉｏｎ　ｏｆ　ｓｉｎｇｌｅ　ｃｈａｉｎ　ａｎｔｉ−ＨＢｘ　ａｎｔｉｂｏｄｙ　ｉｎ　Ｅ．ｃｏｌｉ”Ｚｏｕｈ　Ｇ，ｌｕｉ　ＫＤ，Ｓｕｎ　Ｈ．Ｃ．，Ｃｈｅｎ　Ｙ．Ｈ．，Ｔａｎｇ　Ｚ．Ｙ．，ａｎｄ　Ｓｃｈｒｏｄｅｒ　Ｃ．Ｈ．，１９９７，ｖｏｌ　１２３（１１−１２）ｐｇｓ６０９−１３に見出すことができる。
【００７９】
本発明の技術の一例として、図４では、細胞へのコロイド粒子の固定を含む有用な技術の例が記載される。腫瘍マーカー、ＭＵＣ−１は腫瘍細胞上に異常に発現される。ＡＴＣＣから入手できるヒト組織培養乳癌細胞系統ＭＣＦ−７は、ＭＵＣ−１を過剰発現する。Ｄａｎａ−Ｆａｒｂｅｒ　Ｃａｎｃｅｒ　Ｉｎｓｔｉｔｕｔｅから入手できる抗体５０，ＤＦ３またはおよびＤＦ３−ｐは、ヒスチジンタグ標識プロテインＧ　５６により電子的または電気化学的シグナル伝達コロイド５２（ＮＴＡ‐ＳＡＭｓ５４を持つ）に結合する。標的細胞５８は、抗体を持つシグナル伝達コロイド５２と共にインキュベートし、分子ワイヤ含有ＳＡＭ５８で被覆した電極に電気泳動し、ＡＣＶにより分析する。電極４０上のＳＡＭは、より慣用の、堅く詰まったＳＡＭ‐形成種と混合した分子ワイヤ５８を含む。すべての図において説明を簡単にするために、分子ワイヤ５８だけを図式的に示す。抗体を持つシグナル伝達コロイドをＭＵＣ−１を持つ細胞とインキュベートすると、電流ピークが得られる。あるいは、ＭＵＣ−１の推定コグネイトリガンド、Ｉ−ＣＡＭをＨｉｓタグ標識し、ＮＴＡ基をも持つシグナル伝達コロイドに結合してもよい。
【００８０】
別のアッセイを図５に示す。薬物ライブラリーは、ＭＵＣ−１／Ｉ−ＣＡＭ相互作用のような、細胞由来タンパク質との特異的相互作用を阻害するそれらの能力に対してスクリーニングしてもよい。Ｉ−ＣＡＭ　６４は先に記載のようにシグナル伝達コロイド６２に結合し、その後ＭＵＣ−１（６６）を提示する細胞７０および対照細胞と共にインキュベートする。薬物候補６８は、細胞およびコロイドが懸濁している溶液内に添加し、その後細胞は電極に接着し、ＡＣＶにより分析する。シグナルの損失は、薬物候補との相互作用を示す。図６は、使い捨てマイクロウェル７２のアレイを微小電極７４の接続可能なアレイと共に使用することにより、何千もの薬物候補を同時にスクリーニングするためにこのスキームがどのようにして容易に多重化されるかを説明する。それぞれのウェルは、図５に示されるアッセイ、または別のアッセイを含む。
【００８１】
次に図７では、シグナル増加アッセイのために、またはコグネイトリガンドが未知の細胞表面受容体８０に結合する薬物をスクリーニングするために、小分子薬物ライブリーを、電子的または電気化学的シグナル伝達部分３４をも持つコロイドまたは粒子７８上で合成するか、またはそこに共有結合することができる手順が示される。シグナル伝達粒子に結合した薬物候補７６は、関心のある受容体８０を提示する細胞８２、または対照細胞８４とインキュベートすることができる。本アッセイにおける薬物‐標的相互作用により、シグナルが増加する（図７）。
【００８２】
細胞表面受容体、αＶβ３は細胞接着分子ビトロネクチンとの相互作用により血管新生を促進することが示唆されている。αＶβ３細胞表面受容体を提示するヒト臍静脈内皮細胞（ＨＵＶＥＣ）は、Ｃｌｏｎｅｔｉｃｓから入手できる。αＶβ３の作用を阻害する薬物のスクリーニングのために、ビトロネクチン由来のＲＧＤ‐含有配列を提示するＨｉｓタグ標識ペプチドを、ＮＴＡ基およびフェロセン部分を共に提示するＳＡＭｓを持つコロイドに連結させる。その後、生物特異的シグナル伝達コロイドはＨＵＶＥＣ細胞および薬物候補と共にインキュベートする。ＨＵＶＥＣ細胞は電極上で増殖させることができる。しかし、細胞が溶液または懸濁液中にある場合、それらを電気泳動させるか、または感知電極に磁気的に結合させて、ＡＣＶにより分析することができる。生物特異的コロイドは、対照細胞よりむしろ、ＨＵＶＥＣ細胞と共にインキュベートするとき、電流ピークが生じる。薬物候補がαＶβ３‐ＲＧＤ配列相互作用を妨害する場合、結果としてシグナル損失がおきる。既知の血管新生阻害剤エンドスタチンでαＶβ３‐ＲＧＤ相互作用を阻害することにより、このアッセイの実行可能性が示された。アッセイは、説明したように電子的に、または標準プラスチック皿中で細胞を増殖させてアッセイを行い、細胞を４０倍の倍率で観察することにより、視覚的に行うことができる。
【００８３】
あるいは、薬物候補は、ビーズ、コロイド、または電子的または電気化学的シグナル伝達部分を同様に提示する支持体上で合成されるか、それらに連結してもよい。これらの“粒子”または表面を標的細胞とインキュベートし、感知電極に引き寄せ、ＡＣＶにより分析する。吸引場を逆にし、そしてＲＧＤ配列を含むペプチドを溶液中で滴定する。細胞は再び感知電極に電気泳動され、再分析される。シグナルの損失は、薬物‐細胞相互作用がαＶβ３受容体に特異的であることを示し、ＲＧＤペプチドのＩＣ_５０を薬物への結合親和性と関連付けられる。
【００８４】
あるいは、同様にαＶβ３に結合する４９アミノ酸ペプチド、エチスタチンは、先に記載のアッセイにおいてＲＧＤ‐含有ペプチドと交換するためにＨｉｓタグ標識されてもよい。
【００８５】
メタロセンは、以下の理由からシグナル伝達実体としてとりわけ有用である。１００ｍＶから８００ｍＶ間の特有の電位でそれぞれ酸化されるように各種フェロセン誘導体を選択してもよい。それぞれの酸化電位は特有の標識を表し、その結果複数の細胞表面受容体を同時に探索することができる。細胞表面受容体とリガンドを固定化したコロイド間で生物学的に適切な相互作用が起こる場合、細胞は電子的または電気化学的シグナル伝達粒子で修飾され、その結果電流ピークが生じる。電流ピークの大きさは、シグナル伝達コロイドにより認識された細胞表面受容体の数に比例するだろう。
【００８６】
細胞表面分子は懸濁液中、または図８に示すように、組織試料中に埋め込まれた細胞上で検出することができる。凍結組織標本８６は、冷凍で切片にし、ＲＧＤ‐含有ペプチドまたはメチル停止基のような、細胞結合基９０で誘導体化されている柔軟で半透過性の膜支持体８８上に直接置かれる。その後、関心のある細胞表面受容体に対するリガンド９４をも提示する電子的または電気化学的シグナル伝達コロイド９２と共に試料をインキュベートする。非結合コロイドは、インキュベーション時間の後に洗い流される。その後支持体膜を、細胞サイズに匹敵する電極の面積を有する、微小電極アレイ９６と物理的に接触させて、ＡＣＶにより分析する。組織試料のそれぞれの部分は、タンパク質量および発現レベルを分析し、組織病理学的に関連付ける。この能力は単一細胞分析法の妥当性を保証する、なぜなら、その方法により研究者は癌細胞に特に関連したタンパク質の型を同定し、任意の異常なタンパク質発現を排除することができるからである。懸濁液中の細胞は同様に支持体膜に結合することができる。
【００８７】
この技術は、健康な組織または細胞に対して病気を罹ったそれらにおいて異なって発現される受容体またはタンパク質のような、細胞由来分子を同定するために使用することができる。この異なる発現が、健康な組織または細胞に対して病気を罹ったそれらにおける異なる発現のレベル、および／または容易に同定することができる、組織または細胞における発現の異なる型と関連してもよい。この技術が疾患状態の測定のための診断用アッセイを容易にする。たとえば、特定の疾患であると推定される患者に関連して、細胞、具体的には、生検由来の細胞、血液試料などのような疾患の指標に関連する細胞が患者から採取されてもよく、そしてこれらの細胞は、疾患を示す発現レベルまたは型を測定するために健康な細胞に対して分析することができる。また、各種病理学的状態に関与する細胞由来タンパク質のアップレギュレーションを阻害する薬物をスクリーニングするためにこの系を使用することができる。薬物スクリーニングのための２つの技術の例として以下のものが挙げられる：（１）疾患と推定されるか、またはその徴候を示す患者に候補薬物を投与し、そして疾患の治療における薬物の効力を測定するために先に記載の患者の細胞を含む生物学的試料をモニターすること；（２）疾患を有すると推定されるまたは有する患者由来の細胞を含む生物学的試料を採取し、生物学的試料または細胞の成分を候補薬物に暴露し、そして先に記載の技術を使用して、発現レベルおよび／または型をモニターすること。一度結合パートナー（薬物、抗体、または細胞由来タンパク質のタンパク質／ペプチドリガンドを含んでいてもよい）が同定されると、結合パートナーは疾患状態または治療に反応した細胞由来タンパク質の発現レベルを定量するために検出部分に結合させることができる。これは細胞表面および細胞内コンパートメントの両方への細胞由来タンパク質の発現および移動に対する薬物候補の間接的影響を試験する、いずれのアッセイであってもよい。
【００８８】
また本発明は、個々の細胞表面上および／または組織試料中に埋め込まれた細胞上の細胞表面受容体発現の型を視覚的に研究するための能力を提供する。これは、疾患状態に関連付けることができる細胞表面受容体発現の型を示すことができる。また、これらは診断または薬物スクリーニング方法に使用することができる。具体的なアッセイにおいて、細胞表面受容体に結合するリガンドを持つコロイド粒子は個々の細胞または埋め込まれた細胞に暴露され、そして個々の細胞に関するそれらの結合の位置は、視覚的に測定可能で、細胞表面受容体発現の型を示す。たとえば、ＭＵＣ−１は乳癌に関連する細胞表面受容体である。正常にはＭＵＣ−１は、各種細胞型の表面に均一に発現される。各種癌に関連する形質転換細胞において、受容体は過剰発現され、細胞の先端部分に濃縮される。これは、記載された技術を使用して測定することができる。薬物スクリーニングにおいて、形質転換細胞の培養物が提供され、薬物候補により処理されてもよい。先端での型発現の損失が検討される。この態様において、視覚的同定はヒト裸眼、顕微鏡、吸光光度法、電子顕微鏡検査法、蛍光検出などによる観察のような本明細書に記載の任意の技術を含んでいてもよい。
【００８９】
先に記載の電子的または電気化学的検出を含む技術では、発現された種のレベルは、個々の細胞または他の非常に少量のものを含む試料をそれぞれ含む試料間で比較することが可能であり、そして型は組織試料を含むより大きな試料上で測定することができる。先に記載の視覚的検出態様では、発現された種のレベルは、大きな試料および単細胞試料を含む小さい試料の両方に発現された種の型と同様に測定することができる。電子的または電気化学的検出に有用なシグナル伝達実体は、フェロセンのような酸化還元活性分子を含む、電子的または電気化学的検出のために本明細書に記載されたシグナル伝達実体を含む。先に記載の視覚的検出では、コロイドを含む本明細書に記載のいずれかのシグナル伝達実体は単独で、または蛍光もしくは視覚的に同定可能な実体のような補助的なシグナル伝達実体を有しているものとして、使用することができる。複数のシグナル伝達実体が使用されてもよい（すなわち、結合事象ごとに複数のシグナル伝達）。電子的もしくは電気化学的または視覚的シグナル伝達ではともに、異なるアッセイに関して異なるシグナル伝達実体を使用してもよい。たとえば、第１の受容体またはタンパク質を標的にするように選択された第１のリガンドは、第１のシグナル伝達実体に関して固定化されてもよく、一方第２の受容体またはタンパク質を標的にするように選択された第２のリガンドは、第２のシグナル伝達実体に関して固定化されてもよい。電子的または電気化学的シグナル伝達では、異なるシグナル伝達実体が異なる酸化還元電位を含んでいてもよく、それらの間の差は電子的に区別可能であり、そして視覚的同定では、異なるシグナル伝達実体が異なる色の放射性または吸収性実体であってもよい。そのような場合、タンパク質または受容体の発現レベルおよび型が測定されるだけでなく、型は一つの受容体またはタンパク質の発現の位置と他のそれを区別することができる。
【００９０】
また、先に記載の生物特異的コロイドはｉｎ　ｖｉｖｏ画像診断を容易にするために使用することができる。使用されるコロイドは金であり、それは比較的不活性である。腫瘍マーカーに対する抗体または他のリガンドを提示するように誘導体化されている金コロイドは、内部に取り込ませるか、または注入することができる。コグネイト腫瘍マーカーを提示する腫瘍塊はコロイドで覆われ、濃縮装置として作用するであろう。次に腫瘍はＸ線、およびＸ線コンピュータトモグラフィー（ＣＴ）のような画像診断技術により検出することができる、なぜなら、有効な結果は金属中に包まれた腫瘍塊であってもよいからである。溶液中の個々のコロイドは検出に対して見えないであろう。また、生物特異的コロイドでそのように標識されている腫瘍は、ＭＲＩ（磁気共鳴イメージング）により検出することができる。画像診断技術は、常磁性金属および他の対比物質、たとえばＦｅ、Ｇｄ、またはＣｒを取り込むことにより高めることができる。対比物質は、コロイド上に形成されたＳＡＭｓに金属キレートチオールを取り入れることによりコロイドに結合させることができる。あるいは、腫瘍上に発現される標的タンパク質のコグネイトリガンドだけを提示するＳＡＭｓは、対比物質材料からなる粒子上で形成されてもよい。鉄または磁鉄鉱コアを有するコロイドは、金で被覆され、生物特異的ＳＡＭｓで誘導体化されてもよい。あるいは、リポソーム様粒子がキレート基で停止された脂質鎖から形成され、その結果金属対比物質が“コア”を形成し、生物特異的リガンドが溶媒に暴露されてもよい。あるいは、コロイドが生物特異的リガンドに加え、特異的分光分析的特徴を有する部分を持っていてもよく、その部分はＲａｍａｎ分光法を含むエネルギー吸収、または散乱技術により検出することができる。画像診断装置は本体の外側に含まれるか、または観察機械の本体もしくは光ファイバーに導入されてもよい。あるいは、腫瘍塊上で濃縮された場合、高周波を伝える部分を持つコロイドが検出可能なシグナルを生成してもよい。個々のコロイド由来のシグナルは、腫瘍塊に結合したコロイドの集合物から生成されるシグナルに比べ、無視できるか、または検出不可能でなければならない。
【００９１】
図９では、２つのタンパク質がお互いに相互作用するか否かを測定するために、第１のタンパク質１０４をＮＴＡ　５４およびフェロセンリガンド３４を共に提示するコロイド１０６に結合し、第２のタンパク質１００をＮＴＡリガンド５４のみを提示するＳＡＭ‐磁気ビーズ１０２に結合させるであろう。２つの粒子型は一緒に混合され、その後磁石１０８が下にある感知電極４０に磁気的に補充される。磁気ビーズが２つのタンパク質種の相互作用によりシグナル伝達コロイドに結合した場合だけ、電子的または電気化学的シグナルが、生成されるであろう。また、非ヒスチジンタグ標識タンパク質も、シグナル伝達または磁気（補充可能）粒子の上に、それらは荷電され、そしてカップリング化学に関与しない暴露基で停止したチオール（たとえばＮＴＡチオール）とともにカルボキシで停止するチオールをを組み込んだＳＡＭｓを形成することにより、それらに連結できる。次に、第１級アミンを提示するいずれかの分子を結合させるために標準ＥＤＣ／ＮＨＳカップリング化学を使用することができる。電子的または電気化学的標識工程はタンパク質製造工程と分かれているため、一般的な電子的または電気化学的シグナル伝達コロイドおよび磁気粒子を使用して系を効率的に多重化してもよい。これは、研究者が癌の分子的特徴を明確にするのに役立つタンパク質相互作用データベースの構築を容易にする。
【００９２】
相互作用するタンパク質パートナーは、小分子薬物ライブラリーとともにインキュベートすることができ、および薬物リードはシグナルの損失を検出することにより識別した。あるいは、小分子薬物ライブラリーは既知の標的タンパク質に結合する薬物候補を直接スクリーニングするための磁気ビーズ上のものを購入してもよい。
【００９３】
また、本明細書に記載の基礎的な技術は、酵素活性を調節する薬物をスクリーニングするために使用することができる。ある酵素は、進行性疾患に重要なものであると同定されている。ある場合には、分子は活性になるためにその特異的部位で開裂されなければならず、これを行う酵素の活性を阻害する薬物のスクリーニングが望まれるであろう。本明細書に記載の技術を、この目的のために以下で記載のように使用することができる：
以下に記載したものはこの技術の実行に有用である：Ｅｄｅｌｓｔｅｉｎ　ａｎｄ　Ｄｉｓｔｅｆａｎｏは、光励起性交差結合部分で修飾されたファルネシルピロフォスフェート基が、酵母ＦＰＴによりＲＡＳ由来のペプチドモチーフに添加できることを報告している（Ｅｄｅｌｓｔｅｉｎ　Ｒ．ａｎｄ　Ｄｉｓｔｅｆａｎｏ　Ｍ．（１９９７）．Ｐｈｏｔｏａｆｆｉｎｉｔｙ　ｌａｂｅｌｉｎｇ　ｏｆ　ｙｅａｓｔ　ｆａｒｎｅｓｙｌ　ｐｒｏｔｅｉｎ　ｔｒａｎｓｆｅｒａｓｅ　ａｎｄ　ｅｎｚｙｍａｔｉｃ　ｓｙｓｔｈｅｓｉｓ　ｏｆ　ａ　ＲＡＳ　ｐｒｏｔｅｉｎ　ｉｎｃｏｒｐｏｒａｔｉｎｇ　ａ　ｐｈｏｔｏａｃｔｉｖｅ　ｉｓｏｐｒｅｎｏｉｄ．Ｂｉｏｃｈｅｍ．ａｎｄ　Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍ．２３５，３７７−３８２）。彼らの目的は、このアッセイを使用して、２つを交差結合することにより、ファルネシル誘導体がＦＰＴにより認識されることを示すことであった。この発見は、酵素活性または特異性を妨害せずにピロフォスフェートから離れた末端で、ファルネシルまたはゲラニル部分をビオチンで修飾することができるという概念と一致する。
【００９４】
図１０では、既知の結合パートナー（ＢＰ１）がある分子１２２に、酵素開裂部位（ＥＣＳ）１１０が１つの末端１１２で連結することができる。他の末端１１４では、それがＳＡＭ（ＨＳ−Ｒ−Ｘ）に取り込まれてもよい分子１１６に連結していてもよく、ここでＳは硫黄であり、ＲはＳＡＭに取り込まれることができる分子種であり、そしてＸはリンカーである。得られた分子は、電子的シグナルを送達できる基１１８（ＨＳ−Ｒ−ＸＭ）（または電極に近くに引き寄せられたとき、電子的または電気化学的シグナルを変換することができる他の基）と共にコロイド１１６上でＳＡＭｓに組み込まれてもよく、ここでＭは酸化還元‐活性金属もしくは遷移金属である。磁気ビーズまたは粒子１２０は、相互標的分子（ＢＰ３）に同時に結合することにより直接的、または間接的のいずれかにより、ＢＰ１　１２２に結合する分子１２４（たとえばストレプトアビジン）（ＢＰ２）を提示するように誘導体化されてもよい。
【００９５】
たとえば、電子的または電気化学的シグナル伝達コロイド１１６は、酵素開裂部位（ＥＣＳ）を持ち、およびビオチンで停止したチオールにより誘導体化されてもよい。磁気ビーズはストレプトアビジンにより誘導体化されてもよい。磁気ビーズおよびシグナル伝達コロイドは関心のある酵素および薬物候補と共にインキュベートしてもよい。薬物候補が酵素作用を調節するか、または阻害する場合、（たとえば、タンパク質またはペプチドの）部位は開裂されず、そしてシグナル伝達コロイドはビオチン／ストレプトアビジン相互作用により磁気ビーズに連結するであろう。複合体が磁気的に感知電極に連結するとき、結果として電流ピークが発生し、アッセイはシグナルの増加検出アッセイになる。すなわち、タンパク質は１つの末端でのＳＡＭ‐形成種、およびビーズ１２０上に固定化された結合パートナー２４に結合可能な結合パートナー１２２を介してコロイド１１６およびビーズ１２０の両方に結合するように適合してもよく、そして開裂またはその調節がモニターされてもよい。タンパク質は記載されたものを含む各種の方法でコロイドおよびビーズに結合してもよいか、または、さらにコロイドが金属を配位結合するキレートを提示してもよく、タンパク質が金属結合タグにより提供されていてもよい。本発明はさらにいっそう一般化され、そこにおいてコロイド粒子１１６およびいずれかの非コロイド構造、たとえばビーズ１２０の両方への結合に適合したいずれかの実体が、実体を開裂する能力を有する酵素、および酵素活性を低下させる候補薬物の両方の存在下で、コロイドおよび非コロイド構造に提示され、そしてそこに結合してもよい。非コロイド構造は図解したようにビーズであってもよく、または電極自体であってもよい。
【００９６】
ＢＰ１はＢＰ２および／またはＢＰ３と同じであってもよい。たとえば、ＥＣＳおよびＢＰ１（ビオチン）を持つチオールは、遊離ストレプトアビジン（ＢＰ３）、次に磁気ビーズ上のビオチン（ＢＰ２）に結合してもよい。あるいは、標的分子に分子の基を添加する酵素を阻害または促進しようとしてもよい、すなわちコロイド粒子および非コロイド構造を、非コロイド構造への結合に適合した酵素の基質、粒子への結合に適合した酵素活性により基質に結合可能な分子種、および基質に対する酵素に暴露してもよい。このことはさらに、酵素の活性調節のための候補薬物の存在下で行ってもよく、非コロイド構造は固定化された電気活性実体を持つコロイド粒子を持つ磁気ビーズであってもよい。あるいは、非コロイド構造は電極表面であってもよい。
【００９７】
具体的には、図１１では、そのような場合、電子的または電気化学的シグナル伝達コロイド１２６は、キレート／金属／金属結合タグ結合によって、基が添加される分子１２８により誘導体化される。添加される小片、添加分子１３０は第１の結合パートナー（ＢＰ１）１３２（ビオチンであってもよい）により停止する。磁気ビーズ１２は、ＢＰ１の結合パートナー（ＢＰ２）１３４（ストレプトアビジンであってもよい）を提示する。コロイド、磁気ビーズ、添加分子、関心のある酵素および薬物候補をインキュベートし、その後分析のために電極に引き寄せる。対照に比較したシグナルの損失は、薬物候補が酵素活性を阻害することを示し、一方シグナルの増加は薬物により亢進した酵素の活性を示す。
【００９８】
本発明に従って、電極、コロイド粒子などのようなアーティクルの表面で結合パートナー、シグナル伝達実体などのような所望する種を提示するために、各種のＳＡＭｓをさまざまな種類の表面上で使用することができる。当業者は、さまざまな種類の表面、官能基、スペーサー部分などの中から選択することができる。例示的な説明は、米国特許第５，６２０，８５０号に見出すことができる。また、この米国特許はニトリロトリ酢酸、２，２′−ビス（サリシリデンアミノ）−６，６′−デメチルジフェニル、および１，８−ビス（ａ−ピリジル）−３，６−ジチアオクタンなどを含む、使用することができる各種金属結合タグについて説明する。
【００９９】
ビーズを含む各種非コロイド構造は先に記載される。ビーズはポリマー材料、アガロース、テンタゲル、および／または磁気材料を含むことができる。ポリスチレンビーズは非常に有用である。本発明のこれらおよび他の態様の効用および利点は、以下の実施例により十分に理解されるであろう。以下の実施例は本発明の利点を説明することを意図するが、本発明の完全な範囲を例証しない。
【０１００】
以下の実施例および実験は、本発明の具体的な態様を説明し、本発明がいずれか特定の態様に限定されると解釈すべきではない。
以下に記載の態様のいくつかにおいて、実施例はＳＡＭ形成、コラーゲン被覆、細胞増殖、コロイド形成、および交流ボルタンメトリー（ＡＣＶ）を含む。ＳＡＭ形成のために、顕微鏡用ガラススライドはＴｉの層、次にＡｕの層で覆った。それぞれの電極はＲＴで０．５時間、１０％メチル停止チオール（ＨＳ−（ＣＨ２）１５ＣＨ３）、４０％トリ−エチレングリコ−ル停止チオール、ＨＳ（ＣＨ_２）_１１（ＣＨ_２ＣＨ_２）_３ＯＨ、（式）および５０％ＭＦ−１を含有する、３００μｌのＤＭＦ溶液でインキュベートした。その後、チップを含むシンチレーションバイアルに４００μＭ　トリ−エチレングリコ−ル停止チオール２ｍｌを添加し、バイアルは以下の様に水浴中で熱サイクルにかけた：２分間＠５５℃；２分間＠３７℃；１分間＠５５℃；２分間＠３７℃続いてＲＴで１０分間。その後電極はＥｔＯＨに浸し、続いて滅菌ＰＢＳで洗浄した。それらは、生物学的安全性キャビネット中でＬＴＶ殺菌灯下に１時間置き、滅菌を確実にした。
【０１０１】
コラーゲン被覆のために、ＰＢＳ中の０．００５ｍｇ／ｍｌ　コラーゲンの２００μＬ液滴をそれぞれの電極に添加し、４℃で２時間インキュベートした。
細胞増殖のために、電極を細胞増殖フラスコに入れ、培養培地および特定の細胞表面受容体αＶβ３を提示するヒト内皮細胞（ＨＵＶＥＣｓ）の溶液を添加した。電極および細胞含有溶液はＣＯ_２インキュベータ内で２４時間、３７℃でインキュベートした。１００倍の倍率による肉眼検査は、細胞が電極に接着し、増殖の表現型であるウェブ様の広がりを示したことを示した。
【０１０２】
コロイド製造のために、市販の金コロイド（Ａｕｒｏ　Ｄｙｅ）１．５ｍｌをで微少遠心管中で高速で１０分間遠心分離し、ペレットにした。ペレットは保存用バッファー（クエン酸ナトリウムおよびｔｗｅｅｎ‐２０）１００μＬに再懸濁した。９０μＭ　ニトリロトリ酢酸（ＮＴＡ）‐チオール、９０μＭ　フェロセン‐チオール、および５００μＭカルボキシ停止チオールを含有するジメチルホルムアミド（ＤＭＦ）溶液１００μＬ。そのチオール溶液中で３時間インキュベーション後、コロイドをペレットにし、上清を捨てた。それらをＤＭＦ中の４００μＭ　トリ−エチレングリコ−ル停止チオール１００μＬ中で、２分間、５５℃；２分間、３７℃；１分間、５５℃；２分間、３７℃、続いて室温で１０分間インキュベートした。コロイドはその後ペレットにし、リン酸緩衝生理食塩水（ＰＢＳ）１００μＬを添加した。コロイドは次にコロイド保存用緩衝液中の１８０μＭ　ＮｉＳＯ４で１：１に希釈した。ＰＢＳ中の１００μＭのＨｉｓ‐タグ標識ペプチド　１００μＬを、コロイドを提示するＮＴＡ‐Ｎｉ（ＩＩ）１００μＬに添加し、０．５時間インキュベートした。遊離の非結合ペプチドを取り除くために、その後コロイドをペレットにし、上清を捨てた。コロイドペレットは１００μＬ　ＰＢＳに再懸濁した。コロイドは以下のいずれかに結合した：ａ）αＶβ３受容体に結合するように設計されたペプチド、ＨＨＨＨＨＨ（Ｓ_４Ｇ_１）_３ＧＲＧＤＳＧＲＧＤＳ；またはｂ）不適切なペプチド、ＨＨＨＨＨＨ‐グルタチオンＳ‐トランスフェラーゼ（ＧＳＴ）。ＲＧＤモチーフを含むペプチドは内皮細胞上のαＶβ３受容体に結合することが示されている。ビトロネクチン（αＶβ３の天然のリガンド）中のＲＧＤモチーフが相互作用に関与すると考えられている。
【０１０３】
ＡＣＶ分析は、ＣＨ　Ｉｎｓｔｒｕｍｅｎｔｓ　ｅｌｅｃｔｒｏｃｈｅｍｉｃａｌ　ａｎａｌｙｚｅｒを使用して行われた。３電極系が使用された。銀対塩化銀参照電極を白金補助電極とともに使用した。誘導体化された金被覆チップを作用電極として使用した。２５ｍＶの過電位が１０Ｈｚの周波数で電極に適用された。
【０１０４】
【実施例】
実施例１
この実施例はファルネシルタンパク質トランスフェラーゼ（ＦＲＴ）およびゲラニルゲラニルタンパク質トランスフェラーゼ（ＧＧＰＴ）の阻害剤のスクリーニングのためのアッセイを記載する。
【０１０５】
本発明者らは以下のことを容易に多重化できるアッセイを考案している：１）初期スクリーニング工程に野生型標的タンパク質および変異体を組み入れる；２）関心のある酵素によるファルネシル／ゲラニル基およびそれらの類似体の標的タンパク質への添加を直接検出し、定量する；および３）酵素活性に対する薬物候補の異なる作用を検出できる。本発明者らの研究方法は以下のとおりである：１）ＮＴＡ／Ｎｉ（ＩＩ）基（ヒスチジンタグ標識タンパク質を捕捉するため）および電子的または電気化学的シグナル伝達のためにフェロセンのような遷移金属の両方を提示するＳＡＭｓを持つコロイドにヒスチジンタグ標識ＲＡＳタンパク質（または各種変異体由来のペプチドモチーフもしくはフラグメント）を結合させる；２）ビオチンでファルネシルピロフォスフェート誘導体を修飾する（図１３）；３）酵素を添加する；４）候補阻害剤を添加する；５）ストレプトアビジンをもつ磁気ビーズを添加する；６）感知電極に磁気的に引き寄せ、電子的に分析する。酵素ＦＰＴは、シグナル伝達コロイド上に固定化されたＲＡＳにビオチン化ファルネシル（またはゲラニル）部分を添加するだろう。ストレプトアビジンをもつ磁気ビーズはビオチンに結合し、その複合体は補充も検出も可能であろう。アッセイは、微小電極アレイと接続したマイクロウェル中で、シグナル伝達コロイド上に固定化されたＲＡＳ変異体および薬物候補を変えて、並行して行われるであろう（図１４および１５）。微小電極アレイの全体は、特定の標的タンパク質のための酵素活性に対する薬物候補の異なる作用を測定するために電子的に分析されるであろう。薬物候補が標的変異体タンパク質にファルネシル基を添加する酵素を妨害する場合、結果として電子的または電気化学的シグナルが減少するであろう。
【０１０６】
この研究方法は酵素の活性をモニターするか、またはその活性を調節する薬物のスクリーニングに使用することができる。また、非ヒスチジンタグ標識タンパク質は標準カップリング化学によりシグナル伝達コロイドに結合することができる。あるいは、ファルネシル誘導体は、磁気ビーズ上に固定化された基の結合パートナーである、ビオチン以外の認識基により修飾されてもよい。
【０１０７】
ＥｄｅｌｓｔｅｉｎおよびＤｉｓｔｅｆａｎｏは、光励起性交差結合部分で修飾されたファルネシルピロフォスフェート基が、酵母ＦＰＴによりＲＡＳ由来のペプチドモチーフに添加できることを報告している（Ｅｄｅｌｓｔｅｉｎ　Ｒ．ａｎｄ　Ｄｉｓｔｅｆａｎｏ　Ｍ．（１９９７）．Ｐｈｏｔｏａｆｆｉｎｉｔｙ　ｌａｂｅｌｉｎｇ　ｏｆ　ｙｅａｓｔ　ｆａｒｎｅｓｙｌ　ｐｒｏｔｅｉｎ　ｔｒａｎｓｆｅｒａｓｅ　ａｎｄ　ｅｎｚｙｍａｔｉｃ　ｓｙｎｔｈｅｓｉｓ　ｏｆ　ａ　ＲＡＳ　ｐｒｏｔｅｉｎ　ｉｎｃｏｒｐｏｒａｔｉｎｇ　ａ　ｐｈｏｔｏａｃｔｉｖｅ　ｉｓｏｐｒｅｎｏｉｄ．Ｂｉｏｃｈｅｍ．ａｎｄ　Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍ．２３５，３７７−３８２）。彼らの目的は、ファルネシル誘導体がＦＰＴと交差結合することにより、ＦＰＴにより認識されることを示すためにこのアッセイを使用することであった。この発見は、酵素活性または特異性を妨害することなしに、ピロフォスフェートから離れた末端でファルネシルまたはゲラニル部分がビオチンで修飾できるという概念と一致する。
【０１０８】
実施例２
この実施例では、細胞をＳＡＭｓにより誘導体化された金被覆電極に連結させた。電極に連結したままの細胞は次に、金コロイドを含む溶液と共にインキュベートし、それらのコロイドは、細胞表面上の受容体に特異的なリガンドおよび電子的または電気化学的シグナルを電極に送達することができる酸化還元‐活性金属を共に提示するように誘導体化されていた。多少のインキュベーション時間の後、電極は交流ボルタンメトリー（ＡＣＶ）により読みとった。コロイド結合リガンドおよび細胞表面受容体間の正の相互作用が、同様にコロイド上にある酸化還元‐活性金属を電極の十分近くに引き寄せ、電子的または電気化学的シグナルを変換するであろう。
【０１０９】
より具体的には、電極への電子の流れを容易にするために、電極は５０％ビス（エチルフェニルチオール）（すなわち、Ｃ_１６Ｈ_１０Ｓ）のバックグラウンド中で１０％メチル頭基を提示するようにＳＡＭｓにより誘導体化された。４０％トリエチレングリコール停止チオール（ＨＳ（ＣＨ_２）_１１（ＣＨ_２ＣＨ_２）_３ＯＨ）が単分子層の充填を促進するために包含された。細胞増殖がコラーゲンで被覆されたＨＳＣ１５‐メチル停止ＳＡＭｓ上で維持できることは以前に示されていた。ＨＳＣＨ_２Ｃ_１５ＣＨ_３およびコラーゲンは共に絶縁分子であり、電極への電子の流れを阻害することができる。このため、本実施例では、飽和炭素鎖‐コラーゲン被覆度を低下させ、より伝導性の分子ワイヤに隣接した増殖細胞の島状構造を生じた。細胞表面受容体αＶβ３を提示する、血管新生に重要なヒト内皮細胞（ＨＵＶＥＣｓ）は電極上で増殖した。受容体のリガンドおよび電子的または電気化学的シグナル伝達のためのフェロセン部分を持つＳＡＭ‐被覆金コロイドは細胞提示電極としばらくインキュベートし、その後ＡＣＶにより分析した。
【０１１０】
ＡＣＶ分析のために、１ｍｌ容量のシリコンガスケットを細胞提示電極上に固定した。Ｈｉｓタグ標識ＲＧＤモチーフペプチドを前もって結合させたＮＴＡ‐Ｎｉコロイド　１００μｌおよび１００μｌ　ＰＢＳを細胞提示電極と共にインキュベーションするためにガスケットに添加した。１５分後、初めのＡＣＶスキャンが記録された。１５分間隔で２つの連続したスキャンが記録された。電流出力は電圧に対してプロットされた。初めのスキャンはＰＢＳバッファーに特徴的な広幅の電流バルジを生じた。第２および第３のスキャンは、特徴的なフェロセン電位（７８０ｍＶｏｌｔｓ）においてそれぞれ０．５μＡｍｐｓおよび１．６μＡｍｐｓの異なる電流ピークを発生した、図１６を参照されたい。Ｈｉｓタグ標識ＧＳＴが予め結合したＮＴＡ‐Ｎｉ（ＩＩ）コロイド　１００μＬと共に同じ細胞を提示する同一の電極をインキュベートした陰性対照は、ＲＧＤ‐提示コロイドと共にインキュベートした電極に類似した第１のスキャンを生じた。しかし広幅のバルジは、第２および第３のスキャンで減衰し、同一であった（図１７）。陽性対照の１．６μＡｍｐｓのピークと明らかに異なり、陰性対照では鮮明なピークは見られなかった。
【０１１１】
実施例３：伝導性表面上の細胞増殖
この実施例では、コラーゲンで被覆されていない“伝導性”表面上での細胞増殖の電子的検出について記載する。細胞は、いくつかの場合には単分子層に構築されるが、コラーゲンでは被覆されなかった硫黄含有分子で修飾された金電極上で増殖させた。１００％候補薬物と共にインキュベートした電極がトリ‐エチレングリコール停止チオール中で循環加熱されないことを除いて、電極修飾は実施例２の電極製造部分に記載したように行われた。いくつかの電極を同じ細胞増殖フラスコ中に集め、ＨＵＶＥＣ細胞を含む培地を添加した。電極および細胞はＣＯ_２インキュベータ中で２４時間インキュベートした。表面は１００倍の倍率を使用して視覚的に分析した。表１は、表面修飾およびその後の細胞接着／増殖の特徴を記載する。表面は低い非特異的結合を示した。しかし、ひとたび細胞が結合すると、細胞増殖は十分であった。ＳＡＭを介して金属を配位結合する金属キレートを提示しタンパク質上の金属結合タグによってＳＡＭにタンパク質を結合し、タンパク質が細胞を引き寄せることにより、細胞をＳＡＭ上に容易に固定化することができる。結果を実証するために写真を撮影した（示していない）。細胞は、フェロセン部分および細胞表面のαＶβ３受容体に特異的な、ペプチド、ＨＨＨＨＨＨ（Ｓ_４Ｇ_１）_３ＧＲＧＤＳＧＲＧＤＳ；または陰性対照としての、不適切なペプチド、ＨＨＨＨＨＨ‐グルタチオンＳ‐トランスフェラーゼ（ＧＳＴ）を提示するコロイド（先に記載）と共にインキュベートした。図１８は、１００％　エチニルフェニルチオール（ＭＦ１）ＳＡＭ‐被覆電極上で増殖した細胞を、αＶβ３受容体に特異的なリガンドを持つコロイドと共にインキュベートしたとき（図１８、実線）だけ電流ピークを生じ、不適切なペプチドＧＳＴにより誘導体化されたコロイドと共にインキュベートしたとき（図１８、点線）は生じなかったということを示す。
【０１１２】
図１９は、絶縁トリ‐エチレングリコール停止チオールのバックグラウンド中で２５％　２−メルカプトエチルエーテルにより誘導体化された電極の電気化学的スキャンを示す。ＲＧＤ配列ペプチドを提示するコロイドで誘導体化された細胞は小ピークを生じ（図１９、実線）、一方ＧＳＴペプチドを提示するコロイドと共にインキュベートした細胞はピークを生じなかった（図１９、点線）。
【０１１３】
【表１】

【０１１４】
実施例４：グルタチオン−Ｓ−トランスフェラーゼ／グルタチオン結合を介したコロイド粒子によるポリマービーズの修飾
図２０および２１を参照されたい。以下の実施例は、標的タンパク質の結合パートナーである小分子、薬物候補、ペプチド、タンパク質、核酸、またはそれらの組合せを提示する非コロイド粒子上に、標的タンパク質を提示するコロイドがどのように凝集するかを説明する。多くのコンビナトリアル薬物ライブラリーは非コロイド粒子またはビーズ上で合成され、医学的に適切な標的種を表示するコロイド粒子と混合することができる。標的種の結合パートナーである薬物を表示するビーズは容易に同定することができる、なぜなら、コロイド粒子がそのビーズ上に凝集し、それを赤く着色するからである。
【０１１５】
標的タンパク質、グルタチオン−Ｓ−トランスフェラーゼ（ＧＳＴ）はヒスチジンタグ標識され、そしてＮＴＡ‐Ｎｉ（ヒスチジンタグ標識はＮＴＡ‐Ｎｉに結合する）を提示するＳＡＭ‐被覆コロイド上に固定化された。表面上に４０μＭ　ＮＴＡ‐Ｎｉを提示するコロイド　３０μｌを２１．５μＭ　ＧＳＴ　６５μｌに添加し、溶液中のＧＳＴ最終濃度を１４μＭとした。ＧＳＴを結合する小分子グルタチオンは、アガロースビーズに結合したものをＳｉｇｍａ‐Ａｌｄｒｉｃｈから入手できる。グルタチオン被覆ビーズは、ＧＳＴ‐結合コロイドの溶液とともにインキュベートした。数分位内に、ＧＳＴはグルタチオンビーズに結合し、着色コロイドを溶液から除去し、ビーズを赤く修飾した（図２０）。ＧＳＴに結合しない、小分子を表示するビーズは、ＧＳＴ結合コロイドとインキュベートしたとき、無色のままであった（図２１）。第２の陰性対照（その中でグルタチオン被覆ビーズをＧＳＴ非存在下でＮＴＡ‐Ｎｉコロイド　３０μｌとインキュベートした）は、ＮＴＡ‐Ｎｉコロイドがビーズ表面に、またはグルタチオンに非特異的に結合しないことを示した。
【０１１６】
実施例５：ＳＡＭの電子透過性の制御の実例
この実施例は、促進された電子的連絡を含むＳＡＭを形成する能力を示す。ＳＡＭは、第１の堅く詰まる種および、電子的連絡に対するＳＡＭの透過性を高める、異なる分子構造の第２の種の混合物を含んで表面上で形成される。表面が暴露された流体が表面と電気的に連絡できるように、欠陥、または開口部がＳＡＭ中に形成される。具体的には、全体としてＳＡＭに関して破壊的な構造を有する硫黄含有小分子が安定してＳＡＭに取り込まれ、電子に対する透過性亢進が示された。この実施例は、表面を電気的に比較的伝導性にし、およびその後、細胞増殖を支持できることを示す。
【０１１７】
水溶性フェロセン誘導体は電解質溶液：５００μＭ　ＮａＣｌＯ_４中のフェロセンジカルボン酸の１００ｍＭ溶液、に溶解した。作用電極は、異なる量の２−単位分子ワイヤ（ＭＦ１）を含むＳＡＭにより誘導体化された金被覆電極であった。特徴的なフェロセン電位でのピークの高さを分子ワイヤ密度の関数としてプロットした。陰性対照として、金被覆電極は１００％　トリ‐エチレングリコール停止チオールからなる絶縁ＳＡＭで誘導体化した。図２２は、ＳＡＭの“伝導性”、または溶液中の電子がＳＡＭに侵入する能力が、ＳＡＭに統合された２−単位分子ワイヤの密度の関数であることを示す。この系は、一連の硫黄含有化合物で修飾された電極の伝導性を試験するために使用された。５０％　候補薬物および５０％　トリ‐エチレングリコール停止チオールにおいて、化合物をＤＭＦ中に溶解した。電極は実施例１に記載のように誘導体化した。表２は、ＡＣＶにより分析したときの、候補薬物および生じた電流ピークの高さを表にする。図２２は、ポリ（エチニルフェニルチオール）による単分子層破壊の関数としての、単分子層の伝導性試験の２つの実験結果を示す。
【０１１８】
ＳＡＭｓは、ＤＭＦ中の５００μＭ　トリエチレングリコール停止チオールおよび５００μＭのメルカプトベンゾチアゾールまたは２−メルカプトエチルエーテルのいずれかから、金チップ上で形成された。チップは平らな表面と１ｍｌ　容量のシリコンガスケット間に固定された。フェロセンジカルボン酸溶液は、５００μＭのＮａＣｌＯ４中に溶解し、Ａｇ／ＡｇＣｌ参照電極およびＰｔ補助電極とともにシリコンガスケット中に入れた。金チップは作用電極に結合した。系はＡＣＶにより分析した。溶液中のフェロセンが電極と電気的に連絡することにより得られた電流ピークの大きさは、電極と連絡するＳＡＭ内に欠陥を作製することにより、ＳＡＭの電子流への透過性を高める試験化合物の能力の指標であった。図２３は、単に絶縁種トリ‐エチレングリコール停止（ＣＨ２）１１−ＳＨ（＋＋＋線）および、報告によれば伝導性が亢進した、ポリ‐エチニルフェニルチオール種（ＭＦ１）（実線）を単独で包含するＳＡＭのオーバーレイである。伝導性ポリ‐エチニルフェニルチオール種はより伝導性であると予想される、なぜなら、同一の分子で２つの付加的な反復単位を持つものは伝導性であると報告されているからである（Ｓｃｉｅｎｃｅ　１９９７　Ｂｕｍｍ　ｅｔ　ａｌ．，）。予測したように、アルキルチオールＳＡＭは、フェロセンジカルボン酸溶液中で電流ピークを生じないが、５０％　ポリ‐エチニルフェニルチオールからなるＳＡＭはそれを生じる。図２４は、２−メルカプトエチルエーテル（実線）およびメルカプトベンゾチアゾール（＋＋＋線）を含む伝導性が亢進したＳＡＭｓに対する図２３のプロット（点はＴＥＧ停止チオール、星印はポリ‐エチニルフェニルチオールである）を示し、ポリ‐エチニルフェニルＳＡＭ由来の電流ピークは、５０％　２−メルカプトエチルエーテルを包含するＳＡＭｓ、または５０％　メルカプトベンゾチアゾールを包含するＳＡＭにより発生するピークより桁違いに小さいことを示し、ＳＡＭに伝導性種を取り込むことによるか、またはＳＡＭに“欠陥”または“開口”分子を挿入することにより、ＳＡＭｓが電子流に透過性になるという概念に一致する。
【０１１９】
表２．電極は、トリ‐エチレングリコール停止チオールバックグラウンド中で以下の化合物により誘導体化された。電極製造は、実施例１に記載のように行われた。表面の“伝導性”または透過性は、電解質溶液（０．５Ｍ　ＮａＣｌＯ４）中のフェロセンジカルボン酸の酸化／還元により生じた電流ピークの大きさを測定することによりアッセイした。非特異的結合に抵抗するそれぞれの化合物の能力は、測定前にＢＳＡ（ウシ血清アルブミン）にそれぞれの表面を浸すことによりアッセイした。非特異的に吸着されたタンパク質は、単分子層から電極への電子の伝導を妨げるであろう。
【０１２０】
【表２】

【０１２１】
実施例６：タンパク質‐タンパク質相互作用の検出
この実施例は、固定化されたシグナル伝達実体および固定化されたタンパク質を有するコロイド粒子の有用性を示す（図２７参照）。
【０１２２】
ヒスチジンタグ標識グルタチオン−Ｓ−トランスフェラーゼ（ＧＳＴ‐Ｈｉｓ）は、４０μＭ　ＮＴＡ‐Ｎｉおよび１００μＭフェロセンチオールを表示する、ＮＴＡ‐ＳＡＭ‐被覆コロイドに連結させた。プロテインＡを提示する市販の磁気ビーズは、抗‐ＧＳＴ抗体により１／１０の結合容量に被覆し、１：５の比でＧＳＴ‐コロイドに添加し、そして磁石の頂部に置いた５０％　ＭＦ‐１　ＳＡＭ被覆電極上で測定した。磁石は電極表面に磁気ビーズを引き寄せ、厚い、可視の沈殿物を形成した。ＧＳＴ‐コロイドは磁気ビーズ上のＧＳＴ抗体と相互作用し、電極表面に引き寄せられ、約２８０ｍＶにおいて電流ピークを生じた。２つの陰性対照、ＧＳＴがコロイド表面に結合しない対照、およびＧＳＴ抗体が磁気ビーズに結合しない他の一つの対照を使用した。いずれの陰性対照も電流ピークを生じなかった。図２５はこの実例の結果をプロットする。実線は、ＧＳＴ‐Ｈｉｓ‐提示コロイドおよび磁気ビーズ上の抗‐ＧＳＴ／Ａｂ間の相互作用を表す。○はＧＳＴを提示しないコロイドとインキュベートした抗体を提示する磁気ビーズを表す。●はＧＳＴを提示するコロイドとインキュベートした抗体を提示しないビーズを表す。
【０１２３】
実施例７：細胞検出
この実施例は、ＳＡＭを横切る電気的連絡を高める分子種を含む混合物を含み、ＳＡＭに欠陥を形成することにより、表面が暴露された流体が表面と電気的に連絡できる表面上のＳＡＭを形成する利点、および固定化されたシグナル伝達実体を持つコロイドのタンパク質への連結の有用性を共に説明する。タンパク質は次にはＳＡＭを提示する電極の表面に連結した細胞に固定化される。この場合の欠陥は、フェニル環を含むＳＡＭに組み込まれた分子のバルクにより生じる。
【０１２４】
ＨＵＶＥＣ細胞は培地に懸濁し、金表面上を被覆したＳＡＭとフラスコに入れた。ＳＡＭは、５０％　直鎖チオール、および５０％　２−単位ポリ（エチニルフェニル）チオール（ＭＦ１）を含んでいた。８．４ｍＭ　ＲＧＤ‐Ｈｉｓペプチド溶液　５μｌを培地に添加し、細胞を一晩３７度でインキュベートして電極表面に接着させた。約１６時間後、ＲＧＤ‐Ｈｉｓペプチドを捕捉するためのＮＴＡおよびシグナル伝達のためのフェロセンを提示するＳＡＭ‐被覆コロイド　１００μｌを細胞に添加し、室温で２０分間インキュベートした。次に電極をバッファーで洗浄し、どのような非結合コロイドも洗い流して、測定した。電流ピークは２００〜２５０ｍＶで記録した。陰性対照は、細胞に結合できない不適切なタンパク質、Ｈｉｓ‐ＧＳＴと共にインキュベートした細胞であった。コロイドを陰性対照に添加し、電極をバッファーで洗浄し、測定を行った。陰性対照ではピークは全く見られなかった。図２６は、フェロセンシグナル伝達実体および細胞表面受容体に対するｈｉｓタグ標識リガンドを提示するコロイドが、細胞／表面相互作用により電極表面に運ばれたときに、発生したピーク（実線）を示す。菱形は陰性対照を表し、コロイドは細胞表面受容体に結合しないように選択された不適切なタンパク質を提示した。
【０１２５】
実施例８：非修飾リガンドに対するリガンド‐受容体相互作用の検出
この実施例は、タンパク質またはリガンドのいずれも標識せずに、ＳＰＲの非存在下で、タンパク質／リガンド相互作用を測定するための能力を示す。具体的には、電極表面上にリガンドおよびフェロセンを含むＳＡＭを形成することにより、リガンドと相互作用すると推定されるタンパク質にリガンドを暴露すること、ここでリガンドは、その電気活性シグナルがタンパク質への接近度に依存する電気活性実体、すなわちフェロセンと一定の接近関係にある。
【０１２６】
本発明者らは、フェロセン誘導体の特徴的な酸化電位はフェロセン誘導体の局所環境の化学的性質に基づいてシフトされてもよいことを見出している。タンパク質はフェロセン誘導体の近くに運ばれるため、変化した電位において第２の電流ピークが現れる。この変化したピークの大きさは系のタンパク質の密度に比例する。
【０１２７】
一連の金‐被覆電極は、トリ‐エチレングリコール停止チオールバックグラウンド中で一定密度の伝導性分子ワイヤチオールおよび可変密度のメチル停止チオールを含む、異種のＳＡＭにより誘導体化された。以下のことは以前に示されている：１）トリ‐エチレングリコール停止ＳＡＭｓはタンパク質の非特異的吸着に抵抗する；および２）メチル停止ＳＡＭｓはタンパク質、特にコラーゲンに結合する。したがって、電極を溶液中のコラーゲンと共にインキュベートする場合、非特異的に吸着するコラーゲンの密度はメチル停止チオールの密度が増大するにつれて増大するであろうと考えた。２％、４％、１０％および１５％メチル停止チオールを含むＳＡＭｓが金被覆電極上に形成された。電極の半分はＰＢＳ中の０．００５ｍｇ／ｍｌ　コラーゲンと共に４℃で２時間インキュベートし、他の半分はＰＢＳのみとインキュベートした。電極は、電解質溶液（０．５Ｍ　ＮａＣｌＯ４）に溶解した水溶性フェロセン誘導体、フェロセンジカルボン酸と共に交流ボルタンメトリー（ＡＣＶ）により分析した。特徴的な電位におけるフェロセンの交互の酸化／還元が電流ピークを生み出す。この電流ピークの大きさおよびそれが発生する電位が記録された。低密度メチル停止チオールでは、電流ピークは約４５０ｍＶで発生した；その大きさおよび位置はタンパク質、コラーゲンの存在により影響されないと考えられた。１０％以上の高密度のメチル停止チオールでは、タンパク質コラーゲンの存在下でのみ、第２の電流ピークが約３００ｍＶで生じる。図２８は、フェロセンジカルボン酸の存在下における高密度メチル停止チオール電極を示す。実線は、メチルチオールに結合したコラーゲンを持つ電極を表す。電極表面上のコラーゲンにより生み出された疎水性の環境は、フェロセンの酸化電位をシフトさせ、２つのピークが見られる。黒丸はコラーゲンに結合していない高密度メチル停止チオール電極を表し、したがってフェロセンの酸化に影響を与えない。
【０１２８】
この研究方法は、非修飾タンパク質、ペプチド、細胞の存在を検出するために以下のように使用することができる：混合ＳＡＭはアルキルチオールに連結したフェロセンジカルボン酸ＳＡＭを電子流について透過性にするチオールおよび標的種の結合パートナー（結合パートナーは、ヒスチジンタグ標識され、ＳＡＭ中のＮＴＡ‐Ｎｉ部分への結合により電極に結合していてもよい）で停止するチオールから形成される。標的種が試料溶液中に存在する場合、それはＳＡＭ上に提示されているその結合パートナーに結合する。ＳＡＭ表面の近くのタンパク質の存在は、フェロセンジカルボン酸のまわりの化学的環境を変化させ、その酸化電位をより低い電位側にシフトさせる。固定化された酸化還元‐活性金属の特徴的な酸化電位のシフトが標的種の存在を示す。
【０１２９】
これらの実験が環上に極性置換基を持たないフェロセン誘導体により行われると、作用は全く観察されなかった。したがって、この研究方法が機能するには、固定化された酸化還元‐活性金属が極性環境中にあり、固定化された結合パートナー（チオールによりＳＡＭに結合された）ができるだけ小さいことが極めて重要である。
【０１３０】
実施例９：ＥＬＩＳＡ
分子または細胞生物学の技術者によく知られた技術は酵素結合免役吸着検定法（ＥＬＩＳＡ）（Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｖｏｌｕｍｅ　２，Ｉｍｍｕｎｏｌｏｇｙ　１１．２，１９９６，著作権所有者Ｗｉｌｅｙ　ａｎｄ　Ｓｏｎｓ　Ｉｎｃ．１９９４−１９９８）である。ＥＬＩＳＡに伴う重要な問題は感度である。酵素シグナル伝達は１：１の比で起こる；１抗体に結合した酵素は、１抗原の存在をシグナル伝達する。したがって、抗原のマイクログラム量が効果的なＥＬＩＳＡに必要であるが、すべての場合に妥当ではない。このことから、低レベルで発現されるか、または提示される細胞表面上の抗原の検出にはＥＬＩＳＡは適さない。ＥＬＩＳＡに伴う別の問題は、低い抗原濃度では、アッセイに非常に時間がかかることである。酵素加水分解の量は、加水分解の時間に直接比例する。一般にＥＬＩＳＡを行う場合、標的種はプラスチック支持層に直接的にか、または間接的に連結させる。次に固定化された種の存在は、同様にシグナル伝達能力を有する“第２”の抗体がそれに結合することにより検出される；第２の抗体は、典型的にはホースラディッシュペルオキシダーゼ（ＨＲＰＯ）、アルカリホスファターゼ（ＡＰ）といった酵素、または添加された基質上で結果として色が変化する反応（分光光度計により検出される）を行うことができる蛍光タグ、もしくは蛍光計により検出できる蛍光標識タグに通常は結合されている（たとえば、“Ｌｏｃａｌｉｚａｔｉｏｎ　ｏｆ　ａ　ｐａｓｓｉｖｅｌｙ　ｔｒａｎｓｆｅｒｒｅｄ　ｈｕｍａｎ　ｒｅｃｏｍｂｉｎａｎｔ　ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ　ｔｏ　ｈｅｒｐｅｓ　ｓｉｍｐｌｅｘ　ｖｉｒｕｓ　ｇｌｙｃｏｐｒｏｔｅｉｎ　Ｄ　ｔｏ　ｉｎｆｅｃｔｅｄ　ｎｅｒｖｅ　ｆｉｂｅｒｓ　ａｎｄ　ｓｅｎｓｏｒｙ　ｎｅｕｒｏｎｓ　ｉｎ　ｖｉｖｏ”，Ｓａｎｎａ　ＰＰ，Ｄｅｅｒｉｎｃｋ　ＴＪ，ａｎｄ　Ｅｌｌｉｓｍａｎ　ＭＨ，１９９９，Ｊｏｕｒｎａｌ　ｏｆ　Ｖｉｒｏｌｏｇｙ　Ｏｃｔ．Ｖｏｌ　７３（１０８８１７−２３）を参照されたい）。多くの場合、便宜上すべての抗体が酵素に結合する必要がないように、マウス抗体を特異的認識抗体として使用し、その後酵素結合ウサギ‐抗マウス抗体を添加する。
【０１３１】
本明細書に記載の技術を使用して、ＥＬＩＳＡの感度を非常に高め、そしてプローブ分子として天然のリガンド（タンパク質もしくはペプチド）または薬物候補を使用して固定化された標的種の存在を検出することができる。固定化された種の存在は、複数のホースラディッシュペルオキシダーゼ（ＨＲＰＯ）またはアルカリホスファターゼ（ＡＰ）をも提示するコロイドに結合したリガンドをそれに結合することにより検出する。酵素は、酵素に直接連結したヒスチジンタグを含む各種の手段により、またはヒスチジンタグ標識プロテインＧを介してコロイドに結合するヤギ抗体に、マウス‐抗ヤギ酵素結合抗体を結合することにより、コロイドに好都合に結合することができる（Ａｋｅｒｓｔｏｍ，Ｂ．，Ｎｉｅｌｓｏｎ，Ｅ．，Ｂｊｏｒｃｋ，Ｌ．Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ，１９８７　Ｏｃｔ．５　Ｖｏｌ．２６２（２８）；ｐｇｓ．１３３８８−９１　ａｎｄ　Ｆａｈｎｅｓｔｏｃｋ，Ｓ．Ｒ．，Ａｌｅｘａｎｄｅｒ，Ｐ．，Ｎａｇｌｅ，Ｊ．ａｎｄ　Ｆｉｌｐｕｌａ，Ｄ．（１９８６）Ｊｏｕｒｎａｌ　ｏｆ　Ｂａｃｔｅｒｉｏｌｏｇｙ　Ｖｏｌ．１６７，８７０−８８０）。複数の酵素と共にコロイドに共固定化されたリガンドを第２抗体の代わりに標的種に結合することにより、結合事象に対するシグナル伝達分子の比は桁が増加する。ＥＬＩＳＡプレート上に提示された抗原へのコロイド上の１抗体またはリガンドの結合の結果、間接的に何千または何百万の酵素が結合する。あるいは、既知の種を意図的に９６‐ウェル上プレートのウェルに結合させ、それぞれがシグナル伝達酵素と共に異なる薬物候補を提示することをコロイドにより調べることができる。今のところ、現在のＥＬＩＳＡ技術ではこれを行うことは不可能である。なぜなら、それぞれの薬物候補は酵素に結合することができないからである。あるいは、固定化された標的に対する天然のリガンドが、シグナル伝達酵素と共にコロイド上に提示されてもよく、および薬物候補が相互作用を破壊するためにプレートのそれぞれのウェルに添加されてもよい。非結合コロイド、およびしたがってそれらのシグナルは洗浄工程で失われる。抗体‐またはリガンド提示コロイドの標的は、ＥＬＩＳＡプレートに直接的または間接的に（別の抗体またはリガンドにより）結合した細胞またはタンパク質であってもよい。ＥＬＩＳＡのこの修正の利点は感度だけでなく、効率にもある。数百のシグナル伝達酵素がコロイドにより１抗原に結合したままでいるため、基質加水分解がより速く起き、より少ない時間で適切な解釈が行われるであろう。
【０１３２】
実施例１０：細胞表面受容体とコロイド固定化リガンドの相互作用およびその阻害の視覚的検出
この実施例は、以下のこと除いては実施例３に記載の方法で行われた。細胞は複数ウェルプレート上で増殖させた。相互作用後、肉眼検査は、受容体が発現される細胞上の位置での、コロイドによる細胞の選択的な修飾を示した。図２９Ａでは、全く結合が起きない対照が示される。任意の配列のペプチドが使用された。図２９Ｂは、タンパク質が発現される細胞上の位置におけるコロイドによる選択的な細胞の修飾を示す。
【０１３３】
実施例１１：非特異的に固体支持体に結合した一結合パートナーとコロイドに結合した他のパートナーとの間の結合相互作用の検出
タンパク質、具体的にはストレプトアビジン（Ｐｒｏｍｅｇａ）は、非特異的に固体支持体、具体的にはＱ−セファロースに連結した。その結合パートナー、ビオチンは、コロイド粒子上の自己組織化単分子層に組み込まれたチオールに共有結合的に連結した。コロイド粒子をビーズに暴露すると、ビオチンとストレプトアビジン間の結合により固定化され、ビーズを赤く着色した。図３０Ａは、結合相互作用により修飾されたビーズ（本来、赤色）のコンピュータデジタル化顕微鏡画像の写真複写である。図３０Ｂ（対照）は、修飾されなかったビーズの同様の画像である；同様のビーズ（ストレプトアビジン‐結合）は不適切なタンパク質を提示するコロイドに暴露された。
【０１３４】
また、この実施例は、重要なことには、コロイド粒子上にビオチンを含む自己組織化単分子層について記載する。ビオチンＳＡＭｓは、以下のようにコロイド上に形成された。ＤＭＦ中に５８０μＭ　ＣＯＯＨ停止チオールおよび２０μＭビオチン停止チオールを含む溶液が作製された。チオールはＣ１１であった。コロイド製造は先に記載のように行い、記載された２０μＭビオチンおよび５８０μＭ　ＣＯＯＨチオールのＤＭＦ溶液はコロイドとともにインキュベートした。他の工程は先に記載したものと同様であった。
【０１３５】
実施例１２：単一結合事象の複数のシグナル伝達によるＥＬＩＳＡ
この実施例は以下の例外／詳細を伴って、典型的なＥＬＩＳＡと同様に行われた：洗剤および界面活性剤は洗浄工程から除かれた。ＧＳＴを、マルチウェルプレート上に固定化した。マウス抗‐ＧＳＴ抗体をウェルに添加し、ＧＳＴに結合させた。一般的な洗浄工程後、ウェルの第１セット（実験用）にヒスチジンタグ標識プロテインＧを提示するコロイドを添加し、抗‐ＧＳＴに結合させた。ウェルの第２のセット（対照）にはプロテインＧ固定化コロイドを暴露しなかった。別の一般的な洗浄工程後、すべてのウェルにホースラディッシュペルオキシダーゼ結合ヤギ抗‐マウス抗体を暴露した。次にすべてにホースラディッシュペルオキシダーゼ基質および硫酸を暴露した。反応は、通常の方法で進行させた。プレートはマルチウェル分光光度計リーダーにより分析した。
【０１３６】
この実施例の結果は、ホースラディッシュペルオキシダーゼがコロイド粒子上の複数のＧ位置に結合するに従い、実験用ウェルが対照ウェルに比べ６桁大きいシグナル伝達をすることを示す。図３１は、この６桁の増大を示す棒グラフである。この実施例は、複数のシグナル伝達実体による、第１の生物学的または化学的作用物質の単一の結合の、第２の生物学的または化学的作用物質へのシグナル伝達を示す。複数の実体のシグナル伝達は、それらがコロイド粒子に結合するのと同時に起こる。
【０１３７】
実施例１３：コロイド表面上に提示された自己組織化単分子層への非修飾化学的または生物学的分子の連結、具体的には、コロイドへのストレプトアビジンの連結
この実施例は、カルボン酸またはその塩を提示することができる表面へのいずれかの本質的にアミンを含有する実体の連結のための技術を提供する。技術は、コロイド表面上に形成された自己組織化単分子層への連結により、コロイドに実体を連結するために使用することができる。自己組織化単分子層に連結される分子は、結合前に親和性タグなどのような、いずれかの特定の化学的官能基を含む必要がない。ただ一つの要件は、多くのアミノ酸に見出されるように、少なくとも一つの第１級アミンを含むということである。したがって技術は、表面にカルボン酸を提示するコロイドに第１級アミノ基を持つタンパク質またはペプチドを共有結合するためにとりわけ適する。
【０１３８】
コロイド上に自己組織化単分子層を形成するための技術は先に記載されている。これらの技術には、以下の具体的な詳細な説明および但し書きが付け加えられた。
【０１３９】
タンパク質はコロイド表面上のカルボキシレート（ＳＡＭによって結合）とペプチドのアミン残基間にアミド結合を形成することにより結合した。アミド結合は、修正されたＥＤＣ／ＮＨＳカップリングプロトコルの適用により形成された。カップリングの成否は、ストレプトアビジンを提示するコロイドとビオチン基を提示するコロイド（ビオチンＳＡＭを介して、先述）を混合することにより試験した。ストレプトアビジンが首尾よく結合した場合、２つの型のコロイドは凝集し、懸濁液が色を変え、最後にコロイドが沈殿して透明な溶液が残るであろう。
【０１４０】
カップリング手順に使用されるコロイドは、他に記載されたように製造し、５４０μＭ（マイクロモラー）ＣＯＯＨ停止チオール、６０μＭ　ＮＴＡ‐停止チオールを含有するチオール溶液中でのインキュベーション、およびその後の４００μＭ　エチレングリコール停止チオール中での循環加熱により形成された。ＰＢＳ中の５０μｌ　コロイドは、水中の５０μｌの１０ｍＭ　ＥＤＣ、４０ｍＭ　ＮＨＳで処理された。７分後、コロイドを遠心分離して沈殿させ、液体を除去し、その後ｐＨ６．４のリン酸バッファー中の０．１ｍｇ／ｍｌ　ストレプトアビジン　５００μｌ（マイクロリットル）に再懸濁した。１．５時間後、コロイドの未反応部位をエタノールアミンを添加することによりブロックし、そしてコロイドを遠心分離して沈殿させ、その後ｐＨ６．４のリン酸バッファー　１００μＬ中に再懸濁し、過剰な、結合しなかったストレプトアビジンを洗い流した。コロイドはその後再び遠心分離して沈殿させ、５０μｌ　ＰＢＳに再懸濁した。２０μＬ　リン酸バッファーおよび１５μｌ　ビオチン提示コロイドの混合物に、先に製造されたストレプトアビジン‐提示コロイド　１５μｌを添加した。この懸濁液を３５μｌ　リン酸バッファーおよび１５μｌ　ビオチン提示コロイドの混合物と比較した。ストレプトアビジン‐提示コロイドの添加は、赤色から青色への速やかな色の変化を引き起こした。〜１０分後、ビオチンコロイドおよびストレプトアビジンコロイドの混合物は透明になり、底に沈殿物が見られた。ビオチンコロイドを含まない混合物は視覚的な変化がなく、赤色のままであった。
【０１４１】
先に記載のプロトコルは、コロイド上の自己組織化単分子層への、アミンを含む本質的にどんな化学的または生物学的分子の結合にも適用できる。結果として凝集を起こす複数のコロイドへの結合よりむしろ、ただ一つのコロイドへの１分子の結合を確実にするために、以下の修正を適宜行ってもよい。望ましく結合した分子の濃度を維持し、そしてＥＤＣ／ＮＨＳ反応物質の濃度を維持する一方で、溶液中のコロイドの濃度を低下させてもよい。各種分子に対して当業者は、他のわずかな修正を行ってもよい。アミン‐含有分子は、当業者により選択されてもよく、そして限定はしないが、タンパク質、合成分子、ペプチド、誘導体化核酸、および他のアミンを含む誘導体化された、または天然に存在する生物学的分子を含んでいてもよい。当業者は、本発明のこの態様に従ってコロイドに連結可能にするために、多様な種がアミンまたはアミン‐含有種とともに合成されるか、またはそれらに連結してもよいことは理解するだろう。
【０１４２】
当業者は、本明細書に記載のすべてのパラメータは例示的なものであって、実際のパラメータは本発明が使用される方法および装置についての具体的な用途に依存することを容易に理解するであろう。したがって、先の態様は実施例のためだけに提示されること、および添付の請求項およびそれらと同等のものの範囲内で、具体的な記載とは異なって本発明が実行されてもよいということを理解するべきである。
【図面の簡単な説明】
【図１】
図１は無傷細胞上のリガンドのコグネイト細胞表面受容体と相互作用するデンドリマーまたはポリマーを介して、フェロセンのような金属含有化合物に連結したリガンドを表す。
【図２】
図２は無傷細胞上のリガンドのコグネイトの受容体と相互作用する粒子に結合したリガンド、フェロセンのような金属含有化合物を表す。
【図３】
図３は、コロイド上の自己組織化単分子層に練得ｋつしたタンパク質を示し、それらは電子的または電気化学的シグナル伝達のための酸化還元‐活性金属も提示する。標的受容体を持つ細胞は、関心のある受容体に対するコグネイトリガンドを持つコロイドとインキュベートし、続いて感知電極に電気泳動的に、または磁気的に引き寄せられる。図は一定の比率に拡大したものではなく、そしてコロイドは２０の金原子から直径数百ナノメーターまでに及ぶ、各種サイズで作られてもよい。
【図４】
図４は、腫瘍マーカーＭＵＣ−１特異的抗体、ＤＦ−３を持つ電子的シグナル伝達コロイドとインキュベートされるＭＵＣ−１を有する細胞を表す。抗体は、コロイド上の自己組織化単分子層に取り込まれたＮＴＡ／Ｎｉ（ＩＩ）基に結合するＨｉｓタグ標識プロテインＧを介して、コロイドに結合する。
【図５】
図５は、薬物ライブラリーの個々のメンバーの細胞表面受容体との相互作用を阻害する能力をスクリーニングするために使用することができる、アッセイの一態様を示す。
【図６】
図６は、マイクロウェル（使い捨てであってもよい）と微小電極アレイおよびロボットをつなぎ合わせることにより、薬物スクリーニングアッセイを容易に多重化できることを示す。電極アレイの分析は、多数の電極の同時分析が可能な、修正した電気化学的アナライザーを使用して行うことができる。電極はマイクロウェルに挿入されるように（矢印を参照されたい）設計することができる。
【図７】
図７は、電気活性シグナル伝達部分をも持つ粒子上で合成されるか、またはそれらに共有結合的に連結することができる薬物候補を示す。シグナル伝達粒子に連結した薬物候補は、関心のある受容体を提示する細胞、および関心のある受容体を発現しない対照細胞と共にインキュベートすることができる。このアッセイにおける薬物−標的相互作用の結果、シグナルが増加するであろう。
【図８】
図８は、ＲＧＤモチーフを含むペプチドとの相互作用により、柔軟な、半透過性支持体に連結するする組織試料を示す。次に、試料は関心のある細胞表面受容体に対するリガンドを持つ、電気活性シグナル伝達コロイドとインキュベートする。試料を洗浄し、次に微小電極アレイにつなげる。電極アレイは電極の面積が細胞の面積に相当するように作製することができる。試料はさらにＡＣＶにより特徴付け、その後組織病理学に関連付けることができる。
【図９】
図９は一般的な補充およびシグナル伝達粒子に基づいた、容易に多重化可能なタンパク質‐タンパク質相互作用アッセイを示す。Ｈｉｓタグ標識タンパク質を選択的に捕捉するＮＴＡ／Ｎｉ（ＩＩ）リガンドを持つ自己組織化単分子層は、シグナル伝達コロイドおよび金被覆磁気ビーズ上に形成される。次に、それぞれの粒子型を、異なるＨｉｓタグ標識タンパク質と手短にインキュベートする。次に磁場が磁気ビーズを感知電極に引き寄せる。第１タンパク質と第２タンパク質間の相互作用を通してコロイド上の電気活性標識（酸化還元活性部分であってもよい）が電極に運ばれるときだけ、シグナルが変換される。非Ｈｉｓタグ標識タンパク質もカルボキシ停止チオールを提示する、自己組織化単分子層を持つ一般的な粒子に結合できることに注意されたい。
【図１０】
図１０は、酵素開裂部位（ＥＣＳ）を持ち、ビオチンで停止したチオールにより誘導体化された電気活性シグナル伝達コロイドを示す。磁気ビーズはストレプトアビジンにより誘導体化し、次に磁気ビーズとシグナル伝達コロイドを関心のある酵素および薬物候補と共にインキュベートする。もし薬物候補が関心のある酵素の作用を阻害すると、その部位は開裂されず、そしてシグナル伝達コロイドはビオチンストレプトアビジン相互作用により磁気ビーズに結合したままであろう。得られた複合体が磁気的に感知電極に結合するとき、電流のピークが得られ、アッセイがシグナル増加検出アッセイになるであろう。
【図１１】
図１１は、酵素活性を調節する薬物のスクリーニングのための研究方法を示す。このスキームにおいて、酵素修飾の標的であるタンパク質は、やはり電気活性部分を持つコロイドに連結する。酵素が標的タンパク質に添加する化学基はビオチンで標識されている。酵素機能が適切であるとき、ビオチン標識化合物はシグナル伝達コロイド上のタンパク質に添加される。このようにして得られた複合体は、さらにストレプトアビジン被覆磁気ビーズに結合する。複合体はさらに感知電極に電磁気的に引き寄せられる。薬物候補はアッセイに添加されてもよい。対照に比較したシグナルの損失は、薬物候補が酵素の活性を阻害したことを示し、一方シグナルの増加は薬物が酵素の活性を高めたことを示すであろう。
【図１２】
図１２は、ファルネシル基、ファルネシルピロフォスフェート、およびゲラニル−ゲラニルピロフォスフェートの化学構造を示す。
【図１３】
図１３は、ビオチンのような認識基によるファルネシル−またはゲラニル−様部分の修飾を示す。
【図１４】
ビオチン部分を持つファルネシルピロフォスフェート誘導体を示す。ファルネシルタンパク質トランスフェラーゼ（ＦＰＴ）酵素は、ＲＡＳタンパク質変異体へのファルネシル誘導体の添加を触媒する。複合体を感知電極に補充するためにストレプトアビジンを持つ磁気ビーズを使用するとき、酵素の活性、または薬物候補が酵素活性を修飾する能力は電流出力を測定することにより定量することができる。
【図１５】
図１５は、薬物候補がそれらの酵素活性を調節できる能力に対して評価されてもよいことを示す。空間的にアドレスできるマイクロウェルはＲＡＳタンパク質（野生型または変異体）、ＦＰＴ酵素、ビオチン修飾付加基（ファルネシル誘導体）、ストレプトアビジンを持つ磁気ビーズ、およびプラスまたはマイナス薬物候補を含む。電極は、さらに交流ボルタンメトリー（ＡＣＶ）を含む、各種電子的および電気化学的技術により分析することができる。
【図１６】
図１６は、ＲＧＤ‐含有リガンドとコラーゲン被覆電極上で増殖する細胞との相互作用のＡＣＶ分析のプロットを示す。
【図１７】
図１７は、（陰性）対照リガンドとコラーゲン被覆電極上で増殖する細胞との相互作用のＡＣＶ分析のプロットを示す。
【図１８】
図１８は、伝導性分子の単一の型のみを含有する金属支持体上で増殖する細胞のＡＣＶ分析のプロットを示す。
【図１９】
図１９は、ＲＧＤ‐含有リガンドと、絶縁および伝導性分子の不均一単分子層を含む金属支持体上で増殖する細胞との相互作用のＡＣＶ分析のプロットを示す。
【図２０】
図２０は、コロイドで修飾されたビーズ、具体的にはタンパク質／タンパク質相互作用によりビーズに結合したコロイドの写真の写真複写である。
【図２１】
図２１は、図２０で示された実験の陰性対照の写真の写真複写である。
【図２２】
図２２は、（絶縁種の芝生中の）伝導性分子の割合の増大に従った単分子層の伝導性増大を表すプロットを示す。
【図２３】
図２３は、フェロセン溶液とともに測定した、非伝導性自己組織化単分子層の陰性対照に対する２単位分子ワイヤ、ポリ（エチニルフェニル）チオール、ＭＦ１を含有する伝導性自己組織化単分子層の単分子層の伝導性のＡＣＶ分析のプロットを示す。
【図２４】
図２４は、フェロセン溶液とともに測定した、より高い伝導性単分子層に対して測定した、図２３の２つの自己組織化単分子層の単分子層の伝導性のＡＣＶ分析のプロットを示す。
【図２５】
磁気ビーズへのコロイドの結合により測定した、タンパク質／タンパク質相互作用のＡＣＶ分析を示す。
【図２６】
図２６は、対照に対する自己組織化単分子層を横切る増大した電子的連絡、および細胞表面へのタンパク質固定化の酸化還元シグナル伝達のＡＣＶ表示を示す。
【図２７】
図２７は、図によるアッセイの説明であり、磁気ビーズへのコロイドの結合により測定したタンパク質／タンパク質相互作用のプロットは、図２５に示す。
【図２８】
図２８は、表面‐結合タンパク質を含む電極、およびフェロセン酸化電位に対するそれらの作用（実線）および陰性対照（点線）のＡＣＶ分析のプロットを示す。
【図２９】
図２９Ａおよび２９Ｂは、受容体が発現される部位で選択的にコロイドにより修飾される細胞（２９Ｂ）および対照（２９Ａ）の顕微鏡写真（倍率４０倍）の写真複写である。
【図３０】
図３０Ａおよび３０Ｂは、自己組織化単分子層上で、タンパク質の結合パートナーを持つコロイド粒子により修飾された非特異的に結合するタンパク質を持つビーズ（３０Ａ）および対照（３０Ｂ）のコンピュータデジタル化顕微鏡画像の写真複写である。
【図３１】
図３１は、複数のシグナル伝達実体により単一結合事象をシグナル伝達することに起因する、６桁改善されたシグナル伝達を示す棒グラフである。
【図３２】
図３２Ａおよび３２Ｂは、表面への結合相互作用を示すシグナル伝達実体を補充するための重力の使用を表示する実験、および対照のＡＣＶプロットを示す。[0001]
Technical field to which the invention belongs
The present invention relates generally to chemical and biochemical detection methods, and more particularly to techniques in which colloids bind to non-colloidal structures such as electrodes, beads, or cells, and techniques in which a composite signal represents a single binding event. . Techniques involving drug screening are facilitated by the present invention.
[0002]
Background of the Invention
Drug discovery is facilitated by screening a large number of candidate compounds that interact with the target receptor or protein under physiological conditions. Of particular interest are cell surface receptors or proteins. Many of the biomolecular interactions that promote tumorigenesis are related to cell surface proteins that mediate both intracellular and intercellular signaling. “Tumor markers” are molecules on the cell surface that are exclusively expressed or overexpressed as a result of transformation into a neoplastic state. Some of these markers are associated with the ability of tumors to invade tissue and exhibit invasive growth processes characterized by metastases (these tumors generally lead to poor prognosis for patients). For example, the interaction between the cell surface receptor αVβ3 and the cell adhesion molecule vitronectin has been linked to angiogenesis. J. Varner et al. , "Integrins and Cancer," Curr Opin Cell Biol. 8: 724 (1996); Vailhe @ et @ al. , “In vitro angiogenesisｇis modulated the mechanical properties of fibrin gels and is related to a αVβ3 integrin localizationａｔｉｏｎ, Integral. , 33: 763 (1997); See Horton, "The @ .alpha.V.beta.3 @ integrin @ vitronectin @ receptor", "Int @ J Biochem @ Cell @ Biol, 29: 721 (1997). In fact, increased concentrations of this receptor on melanoma cells have been associated with poor prognosis. Another example of a cell surface receptor that promotes tumorigenesis and / or angiogenesis is the MUC-1 antigen; this antigen is overexpressed on breast, prostate, lung and ovarian cancer.
[0003]
The search for drugs that bind to and inhibit cell surface receptors involved in tumorigenesis has been a technological challenge, since few assays are available for intact cells. The ability to detect interactions between ligands and target receptors on the surface of living intact cells will allow the screening of candidate compounds that inhibit these interactions. Screening compound libraries of drugs that inhibit the action of cell surface receptors is highly dependent on the receptors being in a native conformation and multimerization state throughout the drug screening process. With current technology, it is difficult or impossible to detect the interaction of cell surface receptors with their natural ligands.
[0004]
Current techniques have limited their ability to assay ligand interactions on cells. Fluoresceable cell sorting (FACS) is one of the few technologies that allows the detection of cell surface receptors. One obstacle in using this technology to screen for drugs that inhibit cell surface receptors is: 1) the technology cannot be easily multiplexed to facilitate sequential and large number of drug screens; And 2) the technique is only suitable for detecting antibodies that bind to the receptor; the antigen-antibody reaction is generally a high-affinity interaction that cannot be inhibited by the drug. With current technology, it is difficult or impossible to detect cell surface receptors and their natural ligands. Screening compound libraries of drugs that inhibit the action of cell surface receptors is highly dependent on the receptors being in a native conformation and multimerization state throughout the drug screening process.
[0005]
Another technique that can be used to test for cell surface receptors is an ELISA assay. In this technique, cells are attached to a 96-well plastic plate. Cognate antibodies are allowed to bind to the cell surface receptor of interest, and unbound antibodies are washed away. Receptor availability is estimated by detecting the presence of cognate antibodies. The presence of the bound antibody is detected by introducing a detectable level of a second antibody that is active against the species of the cognate antibody. There are several inherent problems that limit the effectiveness of this technique for assessing the availability of cell surface proteins, or for screening for drugs that inhibit them: 1) resulting from non-specific adsorption of antibodies to plastic False positives hamper the technique; 2) ligand-receptor interactions are often inhibited by many washing steps; and most importantly, 3) use antibodies rather than natural ligands in this assay They typically have high-affinity interactions, so that drugs that bind to the receptor cannot be easily inhibited; and, unlike natural ligands, antibodies are responsible for the normal function of the receptor. May bind to the target receptor at a site that does not inhibit.
[0006]
What is needed is an approach to monitor or control binding events between chemical or biological species, which can increase the versatility and sensitivity of a wide range of specific interactions. For example, approaches for screening for drugs that inhibit the specific interaction of cell receptors with their ligands, including but not limited to the interaction of the αVβ3 receptor, would be advantageous. In the latter case, the assay should be flexible, thus allowing screening for a wide range of tumor types, and especially for invasive tumors. Importantly, the screening method allows for rapid screening of a large number of candidate drugs.
[0007]
Summary of the Invention
The present invention provides a variety of novel methods, compositions, and species, including techniques useful for drug screening, and articles for monitoring (detecting) interactions between chemical or biological species. The main aspects of the present invention include the following. Means for proteomic research, including protein chips and particles for signaling interactions, and multiparticulate systems such as two-particle systems. In multiparticulate systems, one particle may be a replenishable particle, another particle may have a binding partner for the agent presented by the replenishable particle, and may be a signaling entity. Or ancillary signaling entities. Other major parts include cell research, especially techniques involving interactions between ligands and cell surface proteins and receptors. Findings and treatments for drugs that can influence these interactions are also described with particular emphasis on drug therapies related to angiogenesis. Specifically, cell receptor / ligand interactions that can inhibit or promote angiogenesis are described. Other parts include detecting the protein either in solution or on the surface of intact cells for diagnosis.
[0008]
In one aspect, the invention provides a method of imparting the ability of a colloid particle to be anchored in a non-colloidal structure, and determining whether the colloidal particle is anchored in a non-colloidal structure. This aspect deals with a wide range of interactions involving colloidal particles or, preferably, many colloidal particles having various structures. Non-colloidal structures, including beads, such as magnetic beads, can interact with colloidal particles as an aid in measuring the interaction between the species immobilized on the beads and the species immobilized on the colloidal particles. Colloidal particles can "modify" the beads so that they can be identified visually as a measure of unique species / species interactions. Alternatively, magnetic beads may be used to replenish the surface with colloidal particles, where the identification of the particles can be performed electronically or electrochemically, or by other means. Non-colloidal structures, such as electrodes, can work with colloidal particles or species immobilized on colloidal particles to indicate specific assay results. Non-colloidal structures, such as cells, can interact with the colloidal structure to indicate binding of the cells to the species possessed by the colloid particles. The advantages of these and other technologies, including colloidal particles and non-colloidal structures, will become even more apparent from the following description.
[0009]
In another aspect, the invention provides techniques for attaching a chemical or biological agent to an article, such as a bead, colloid particle, or the like, via a metal binding tag / metal chelate bond. Articles thus bound to chemical or biological species are encompassed by the present invention.
[0010]
Another aspect of the invention includes techniques for the detection of ligand / protein interactions involving: providing ligands in fixed proximity to an electroactive entity; By monitoring the interaction of a ligand with a protein by monitoring the electroactive signal provided by the entity, the signal is dependent on the proximity of the electroactive entity to the protein. This technique has a first protein immobilized on a first surface, and a second surface, which may be a particle, with an electroactive entity also immobilized on a second surface. It may be facilitated by an article of the invention having a putative ligand that interacts with the protein of interest. This aspect of the invention facilitates another feature of the invention that is a method of measuring protein / ligand interactions in the absence of surface plasmon resonance without labeling either the protein or the ligand.
[0011]
The present invention relates to techniques including methods for signaling a single bond of a first biological or chemical agent to a second biological or chemical agent having multiple signaling entities, and Ingredients are also provided. The article provided by the present invention facilitates this technique, and it is capable of biologically or chemically binding a second agent, which is bound to a plurality of signaling entities, Has chemical agent. The plurality of signaling entities may be bound to the agent as a polymer or dendrimer with the plurality of signaling entities, and are compatible with binding to the agent. Also, the technique may be facilitated by immobilizing the agent on the colloid particles, for example, on the surface of the colloid particles where a plurality of signaling entities are also immobilized. In a preferred embodiment, three or more signaling entities simultaneously signal a single chemical or biological binding event. More preferably, at least 10, even more preferably at least 50, and even more preferably at least 1000 or 10,000 signaling entities simultaneously signal a single chemical or biological binding event according to this aspect of the invention.
[0012]
Another aspect of the invention is illustrated by a colloid particle having both a signaling entity and a protein immobilized thereon. Techniques for the use of this component are also encompassed by the present invention.
[0013]
The present invention also provides a technique for controlling electron permeability across a self-assembled monolayer at a surface. In such a technique, a self-assembled monolayer is formed on a surface and is described by a mixture of at least a first species and a second species. The first molecular species has a structure that promotes self-assembly at the surface in a tightly packed manner that prevents electrical communication between the surface and the fluid to which the surface is exposed. That is, the first species is a relatively tightly packed self-assembled monolayer (SAM) that seals the surface to a fluid for SAMs (self-assembled monolayers) formed only from the first species. ) Is formed. A SAM according to this aspect of the invention has a first species and a different molecular structure, wherein the molecular structure is formed from a second species that results in the disruption of a tightly packed self-assembled structure; This reveals defects in the tightly packed structure that allow the fluid to which the surface is exposed to be in electronic communication with the surface. In such a situation, the fluid makes electrical contact with the surface, either by contacting the surface with which it can be in electrical communication, such as by tunneling, or by being sufficiently close to the surface. As will be appreciated from the following description, this aspect of the invention will be very useful when combined with other techniques of the invention.
[0014]
As noted above, one aspect of the present invention is to provide a range of components and techniques for drug screening. The method provides: 1) a modular system for binding of natural ligands to universal signaling elements; 2) increased detection sensitivity due to the binding of multiple signaling elements to each ligand. 3) simpler format (no need for washing steps, enzymatic cleavage and toxic substrates); 4) convenient electronic output; and 5) the ability to multiplex.
[0015]
In one embodiment of the assay, the present invention contemplates immobilizing live, intact growing cells, or cell-derived molecules on a conductive solid support. Such cells or cell-derived molecules, having both a surface molecule (eg, a receptor) and an intracellular signaling protein, bind to a solid support, which may be substantially surface or particle-like. Ligands (or “binding partners”) for these cells or cell-derived molecules are known and unknown, as well as putative drug candidates (not bound to other solid supports or bound to surface or particle-like structures) Which interact with other cell-derived molecules in such a way that binding between the two binding partners occurs and may be detected. The binding partner or one of its binding supports may be additionally derivatized with an agent that can be recognized and quantified by a detection device. This complex (via interaction) is then transported to a detection device using the properties of the bound complex, which can be distinguished from the unbound binding partner.
[0016]
In one embodiment, cell growth is performed on a metal support (eg, gold) that has been treated (eg, coated with a molecule compatible with cell growth) to maintain conductivity of the metal support. In one embodiment, a homogeneous conductive layer of molecules is attached to a metal surface, where it conducts electrons at a faster rate than a metal uniformly coated with an insulating species of molecules (eg, a saturated alkyl thiolate). In another aspect, a layer of foreign molecules comprising a mixture of first and second species with which a fluid exposed to a surface as described above can be in electrical communication. In another embodiment, an extraneous conductive molecular layer is attached to the metal surface; the extraneous layer is interrupted by conductive or highly conductive molecules (or lawn "defects"), low or non-conductive. It can be seen as a lawn of a species (eg, one such species or many such species).
[0017]
In a preferred embodiment of a heterogeneous conductive layer, or a heterogeneous layer in which defects promote fluid / surface electrical communication, two types of molecules are attached to the metal surface: 1) to form a lawn; The alkylthiol spacer molecule used (stopped at ethylene glycol units or methyl groups to inhibit non-specific adsorption) and 2) conductive or defect-forming species. Conducting species can be easily identified by the experiments described below. In one aspect, the conductive species comprises at least one cyclic structure (eg, a benzene ring, a pyrimidine ring, etc.). In another embodiment, the conductive species does not include any cyclic structures, but includes -O-, -OH-, -NH₂And a group selected from SH.
[0018]
In one embodiment of a homogeneous conductive layer, only one type of molecule, ie, only the conductive species, is attached to the metal surface. Other embodiments using multiple conductive species are also contemplated. Another description of defect-forming molecules, and other aspects of this procedure, are described below.
[0019]
As noted above, the present invention contemplates both methods and compositions. In one aspect, the invention relates to a method wherein the first molecule and one or more signaling elements (ie, molecules or clusters of molecules) attached to a solid support are altered in light transmission, in solution color, in charge, Providing a signal that can be detected by any means, including but not limited to detection by alteration, wherein the first molecule is a ligand and the protein or Can interact with cell surface receptors. In another aspect, the invention contemplates a composition comprising a first molecule, a second molecule, and a third molecule attached to a solid support, wherein the first molecule interacts with a protein or cell surface receptor. Encompasses a ligand capable of acting, wherein the second molecule forms a monolayer on the solid support, and the third molecule is electroactive.
[0020]
The present invention is not intended to be limited by the nature of the solid support. In one aspect, the solid support is a colloid (eg, colloidal gold). It is not intended to be limited by the nature of the binding of the ligand to the solid support. In one embodiment, the ligand is covalently linked to the solid support (either directly or via another ligand or by a binding moiety). In another embodiment, the ligands are linked non-covalently or by electrostatic or ionic interactions.
[0021]
The present invention contemplates a variety of signaling elements, including but not limited to fluorescent molecules and enzymes capable of acting on a chromogenic substrate. In a preferred embodiment, the present invention provides an electroactive molecule, ie, a molecule having an oxidation or reduction potential that can be measured electronically or electrochemically in the vicinity of a working electrode in a suitable and conventional electrical configuration as a signaling element. Intend. The preferred electroactive molecule is a metallocene. Metallocenes that can act as electroactive signaling elements according to the present invention are known. Examples of particularly preferred electroactive molecules include ferrocene or ferrocene derivative groups, or ferrocenyl thiol (C₃₅H₂₄Organic compounds of other transition metals are also contemplated as signaling elements, including those containing derivatives such as FeS).
[0022]
It is not intended that the invention be limited by the nature of the chemical or biological agent. For example, protein / protein, protein / peptide, antibody / antigen, antibody / hapten, enzyme / substrate, enzyme / inhibitor, enzyme / cofactor, binding protein / substrate, carrier protein / substrate, lectin / carbohydrate, receptor / hormone A wide range of agents and their binding partners such as receptors / effectors, complementary strands of nucleic acids, proteins / nucleic acids, repressors / inducers, ligands / cell surface receptors, viruses / ligands, etc. It can be used for the binding interaction of the present invention. In one embodiment, the agent is a ligand, especially a peptide. In a preferred embodiment, the peptide is derivatized with a moiety capable of binding a metal chelate (eg, a histidine tag). In this embodiment, it is advantageous for the solid support to comprise a metal chelate and for the earlier peptide to be linked to the earlier solid support by attachment of the earlier moiety to the earlier metal chelate.
[0023]
In some embodiments, the cell-derived molecule comprising both a cell surface receptor and an intracellular signaling protein is on or on a solid support, which may be either substantially surface or particle-like. Connected. The binding partners of these cell-derived proteins may include both known and unknown ligands, as well as putative drug candidates, which are linked to surface and / or particle-like structures, and Interact with cell-derived proteins such that binding of One of the binding partners or its linking support may be additionally derivatized with a detectable agent. Interacting complexes are identified utilizing the properties of the binding complex that distinguish the bound complex from the unbound binding partner. The presence of, or a change in, a detectable moiety that is immobilized with one of the binding partners on a common solid support or directly linked to one of the binding partners is detected. Molecules that inhibit the relevant interaction can be identified by detecting loss of this signal. Interacting partners manipulate the properties of the bound complex, leaving one of the binding partners in the sensing moiety and recruiting it to other binding partners, or distinguishing bound from unbound binding partners. Or coupled to the replenishable element to one of the binding partners or its coupled solid support.
[0024]
For example, a chemical or biological binding partner may be bound to the surface of a solid support, which may be an electrode, a substantially planar article such as a common biotechnology chip, or the like. Good. The binding partner of the chemical or biological agent may bind to the colloid, and exposing the colloid to the surface of the article binds the binding partner, thus immobilizing the colloid on the surface. This can be measured by measuring the colloid at the surface, specifically at a particular part of the surface. The colloid particles may alternatively include ancillary signaling entities that facilitate this measurement (eg, electroactive signaling entities or visible signaling entities such as fluorescent tags).
[0025]
Specifically, cell-derived molecules such as proteins link to surfaces and ligands, likewise putative drug candidates link to particles, and interact with cell-derived proteins such that binding between two binding partners occurs. I do. One of the binding partners or its linking support may be derivatized with a detectable substance. By retaining one of the binding partners in the sensing moiety and replenishing it with the other binding partner, the interacting partner is carried to the sensing device. Alternatively, one of the binding partners may be linked to a replenishable particle, which may or may not display a signaling entity. One aspect of the invention involves recruiting electronic signaling entities to the electrodes using magnetic materials. This aspect may find use in many assays and other techniques of the present invention. Typically, the method provides the signaling entity with the ability to be immobilized in association with a magnetic material, which may be a magnetic bead. The magnetic material and the signaling entity may be immobilized relative to each other by various chemical and / or physical associations described herein. For example, a first species may be immobilized, or immobilized in relation to a magnetic material, and a second species may be immobilized or immobilized with respect to a signaling entity, or the first and second species. May be combined with each other. The first and second species are essentially as described herein, or may be any species known in the art for conjugation, and in one aspect are proteins. In a preferred embodiment, the protein is not an antibody, but for example, a ligand and a cognate receptor. The signaling entity may be anchored to an intermediate entity, such as a colloid particle, to which one of the proteins acting as a binding partner is also anchored. Also, the signaling entity may be supplemented to the electrodes without using magnetic materials. In this procedure, the signaling entity may be immobilized with respect to the binding partner of the species for the electrode, and the binding partners may bind to each other. In essence, binding partner interactions described herein or known in the art can facilitate this technology.
[0026]
Another aspect of the invention provides a biotin-containing self-assembled monolayer on a surface. In one aspect, the surface is the surface of a colloid particle.
Other advantages, novel aspects, and objects of the present invention, as determined in accordance with the accompanying drawings, which are diagrammatic and are not intended to be drawn to scale, are set forth below. It will be clear from the description. In the drawings, each identical or nearly identical component that is described in various figures is represented by a single numeral. For the sake of clarity, if no explanation is necessary for a person skilled in the art to understand the invention, not all components of all figures are shown and not all components of each aspect of the invention.
[0027]
DETAILED DESCRIPTION OF THE INVENTION
Bamdad et al., Entitled "Rapid and Sensitive Detection of Aberrant Protein Protein Aggregation in Neurogenerative Diseases". International Patent Application No. PCT / US00 / 01997 (published on 07/27/00 as WO / 00/43991), filed on 01/25/00, "Interaction \ of \ Colloid-Immobilized \ Specifics \ With \ Specialties \ Non \ Non-patented. -Colloid {Structures "" in Bamdad et al. International Patent Application No. PCT / US00 / 01504 (published on 07/27/00 as WO / 00/34783), filed on 01/21/00, "Interaction \ of \ Colloid-Immobilized \ Specifics \ With \ Specialties \ Non \ Non-patented. -Colloidal Structures "in Bamdad et al. Commonly-owned, co-pending U.S. Patent Application Serial No. 09 / 602,778, filed 06/23/00, and "Rapid \ and \ Sensitive \ Detection \ of \ Protein \ Aggregation". Bamdad @ et @ al. Commonly-owned, co-pending U.S. patent application Ser. No. 09 / 631,818, filed Aug. 08, 2008, which is hereby incorporated by reference. .
[0028]
Definition:
As used herein, "small molecule" means a molecule of less than 5 kilodaltons, and more specifically, less than 1 kilodalton. "Small molecule" as used herein excludes proteins.
[0029]
As used herein, the term “candidate drug” refers to any medicament used in humans, animals, or plants. Included within this definition are compound analogs, naturally occurring substances, synthetic and recombinant pharmaceuticals, hormones, antibacterials, neurotransmitters, and the like. This includes treating any neurodegenerative disease, or other disease characterized by abnormal aggregation, or any agent or precursor (naturally occurring) that is to be evaluated for use as a drug for its prevention. Present, either synthetic or recombinant). Evaluation is performed by activity in an assay, such as a screening assay of the invention.
[0030]
Various types of particle types are used in the present invention. For example, "fluid suspendable particles" can themselves remain in suspension in the fluid (generally an aqueous solution) used for the present invention, or can be magnetic, electromagnetic, or stirring, Particles that can be maintained in solution by application of shaking, shaking, sonication, centrifugation, agitation such as vortexing, and the like. A “magnetically suspendable” particle is one that can be maintained as a suspension in a fluid by application of a magnetic field. An electromagnetically suspendable particle is one that can be maintained as a suspension in a fluid by the application of an electromagnetic field (eg, a charged particle or a particle modified to have a charge). A “self-suspendable particle” is of sufficiently small size and / or mass that it remains in suspension in the fluid (usually an aqueous solution) used for at least one hour without the aid of a magnetic field, for example. . Other self-suspendable particles will remain in suspension according to the invention for 5 hours, 1 day, 1 week, or even 1 month without assistance.
[0031]
"Proteins" and "peptides" are known in the art and are not clearly defined in the art as to the number of amino acids each comprises. These terms as used herein are given their ordinary meaning in the art. Generally, peptides are amino acid sequences that are less than 100 amino acids in length, but can include sequences up to 300 amino acids. A protein is generally considered to be a molecule of at least 100 amino acids.
[0032]
As used herein, “metal binding tag” refers to a group of molecules that may be immobilized on a metal coordinated to a chelate. Suitable groups of such molecules include amino acid sequences (polyamino acid tags), including but not limited to histidine and cysteine. Metal binding tags include histidine tags, defined below.
[0033]
As used herein, "chelate coordinating a metal" or a metal coordinated by a chelate refers to a metal coordinated by a chelator, wherein the chelator is any metal on the metal. Does not fill the available coordination sites, leaving some coordination sites available for binding by metal binding tags.
[0034]
As used herein, "metal-binding tag / metal / chelate bond" defines a bond between a first and second species, wherein the first species is immobilized with respect to the metal-binding tag, and The second species is immobilized with respect to the chelate, where the chelate coordinates the metal, where the metal binding tag also coordinates. Bamdad {et} al., Incorporated herein by reference. U.S. Patent No. 5,620,850 describes an exemplary combination.
[0035]
"Signaling entity" means an entity whose presence can be indicated in a particular sample or at a particular site. The signaling entities of the present invention are those that are identifiable by the naked human eye, may be invisible when separated, but can be detected by the naked human eye if there is a sufficient amount (eg, colloidal particles), An entity that absorbs or emits electromagnetic radiation within a level or wavelength range that is readily detectable spectroscopically (by the naked eye or a microscope, including an electron microscope, etc.), and that is characteristic upon exposure to the appropriate activation energy. It may be an entity that can be detected electronically or electrochemically (“electronic signaling entity”), such as a redox-active molecule that exhibits an oxidation / reduction pattern. Examples include dyes, dyes, electroactive molecules such as redox-active molecules, fluorescent moieties (including phosphorescent moieties by definition), up-regulating phosphors, chemiluminescent entities, electroluminescent Chemiluminescent entities, or enzyme-linked signaling moieties, including horseradish peroxidase and alkaline phosphatase. A "precursor of a signaling entity" may not itself be capable of transmitting signals, but is capable of interacting chemically, electrochemically, electrically, magnetically, or physically with another species. An entity that becomes a signaling entity. Examples include chromophores that have the ability to emit within a specific, detectable wavelength only when they chemically interact with other molecules. A precursor of a signaling entity can be distinguished from "signaling entity" as used herein, but is included within its definition.
[0036]
As used herein, a species is "immobilized or adapted to be immobilized" with respect to the surface of another species or article, when the species is covalently linked, specific biological binding ( (Eg, biotin / streptavidin), or chemically or biologically linked, such as by coordination bonds such as chelate / metal bonds. For example, in this context, “immobilized” include binding species such as peptides synthesized on polystyrene beads, antibodies specific for proteins such as protein A covalently linked to the beads. Biologically bound binding species, binding species that form (by genetic design) parts of molecules such as GST and Phase, which are also specific for binding partners covalently immobilized on the surface A plurality of chemical bonds, including but not limited to those chemically and biologically bonded (for example, glutathione in the case of GST), and a plurality of chemical / biological bonds. As another example, a moiety that covalently binds to thiol is adapted to be immobilized on the gold surface because thiol is covalently bound to gold. Similarly, a species with a metal binding tag is adapted to be immobilized on a surface with molecules that covalently bind to the surface (thiol / gold bonds) and also have a chelate that binds the metal coordinatively . If the surface has a particular nucleotide sequence and the species contains a complementary nucleotide sequence, the species is adapted to be immobilized on the surface as well.
[0037]
"Covalently immobilized" means immobilized by one or more covalent bonds. For example, a species covalently attached by EDC / NHS chemistry to a carboxylate-presenting alkylthiol that is also immobilized on a gold surface is covalently immobilized on that surface.
[0038]
As used herein, a component "immobilized with respect to" another component is immobilized on the other component or, for example, immobilized on a third component to which the other component is similarly immobilized. Thereby, it is either indirectly fixed to other components, or is otherwise translationally associated with other components. For example, if the signaling entity is fixed with respect to the binding species, the binding species is fixed to the colloid particles to which it is fixed, the binding species is fixed to the dendrimer or polymer to which the binding species is fixed, etc. Is fixed with respect to The species immobilized on the surface of the first colloid particle binds to the entity, and the species on the surface of the second colloid particle binds to the same entity, where the entity is a single entity, a complex of multiple species. If it can be an entity, a cell, another particle, etc., the colloid particles are immobilized with respect to the other colloid particles. Any entity that can be fixed or adapted to be fixed to another entity of the present invention is also fixed or adapted to be fixed to another entity. Is possible, and vice versa.
[0039]
“Specifically immobilized” or “adapted to be specifically immobilized” is defined as a species as described above with respect to the definition of “fixed or adapted to be immobilized”. Binds chemically or biochemically to another sample or surface, but excludes all non-specific binding.
[0040]
As used herein, "non-specific binding" is given its ordinary meaning in the field of biochemistry.
As used herein, “colloid” refers to nanoparticles, ie, very small, self-suspendable particles, including inorganic, polymeric, and metallic particles. Typically, the colloidal particles have a cross-section in all dimensions of less than 250 nm, more typically less than 100 nm in all dimensions, and preferably 10-30 nm, and include metallic, non-metallic, crystalline It may be crystalline or amorphous. As used herein, the term includes definitions commonly used in the field of biochemistry, and typically means colloidal gold particles.
[0041]
As used herein, "a moiety capable of coordinating to a metal" is any that can occupy at least two coordination binding sites on a metal atom, such as a metal binding tag or chelate. Means the molecule.
[0042]
"Diverse biological species" means mice and hamsters, mice and goats and the like.
The term "sample" includes, but is not limited to, a biological source ("biological sample") or any other biological or biological source that can be conveniently assessed according to the present invention. Represents any cell, tissue, or fluid from a non-biological medium: a biological sample taken from a human patient, a sample taken from an animal, a sample taken from human food for consumption, or a livestock feed. Samples containing food for consumption of such animals, milk, donated organ samples, blood samples for blood supply, samples for water supply, etc. One example of a sample is a sample taken from a human or animal that has been administered a drug to determine the drag of the drug.
[0043]
"Sample presumed to contain" a particular component means a sample in which the amount of the component is unknown. For example, a fluid sample from a human that is presumed to have a disease such as a neurodegenerative disease or a non-neurodegenerative disease but is not known to have the disease is presumed to contain neurodegenerative disease aggregate-forming species. Define the sample to be used. Related "samples" include physiological samples from humans or animals, samples from food, naturally occurring samples such as livestock feed, as well as "predetermined samples" Alternatively, it is defined herein to mean a sample used in an assay to test whether a biological sequence or structure is associated with a specific process, such as a neurodegenerative disease. For example, the "sample whose structure has been previously determined" includes a peptide sequence, an arbitrary peptide sequence in a phage display library, and the like. Representative samples taken from humans or other animals include cells, blood, urine, ocular fluid, saliva, cerebrospinal fluid, or tonsils, lymph nodes, fluids from needle biopsies or other samples, etc. Can be
[0044]
As used herein, “metal binding tag” refers to a group of molecules that can bind to a metal coordinated to a chelate. A suitable group of such molecules typically comprises an amino acid sequence of from about 2 to about 10 amino acid residues. These include, but are not limited to, histidine and cysteine ("polyamino acid tag"). Such binding tags, when they contain histidine, may be referred to as "poly-histidine tracts" or "histidine tags" or "HIS-tags", either at the amino- or carboxy-terminus, or at the peptide or protein level. Alternatively, it may be present in any exposed region of the nucleic acid. Poly-histidine tracts of up to 6 to 10 residues are preferred for use in the present invention. Also, poly-histidine tracts are functionally defined as being a large number of contiguous histidine residues added to the protein of interest, which can be used for affinity purification of the resulting protein on a metal chelating column, or other methods. Interaction with a molecule (eg, an antibody that reacts with the HIS-tag) allows identification of protein termini.
[0045]
"Affinity tag" is given its ordinary meaning in the art. Affinity tags include, for example, metal binding tags, GST (in GST / glutathione binding clips), and streptavidin (in biotin / streptavidin binding). Various portions of the specification describe specific affinity tags in connection with binding interactions. In any of the embodiments using an affinity tag, it should be understood that the present invention includes a series of individual embodiments, each including a selection from any of the affinity tags described herein. .
[0046]
As used herein, "molecular wire" means a wire that enhances the ability of a fluid in contact with an electrode coated with a SAM to be in electrical communication with the electrode. This includes conductive molecules or molecules that can create defects in SAMs for communicating with electrodes, as described above and exemplified in more detail below. A non-limiting list of additional molecular wires includes 2-mercaptopyridine, 2-mercaptobenzothiazole, dithiothreitol, 1,2-benzenedithiol, 1,2-benzenedimethanethiol, benzene-ethanethiol, And 2-mercaptoethyl ether. Also, the conductivity of the monolayer can be increased by the addition of molecules that promote conductivity in the plane of the electrode. Conductive SAMs can consist of, but are not limited to: 1) a poly (ethynylphenyl) chain terminated by sulfur; 2) an alkylthiol terminated by a benzene ring; 3) an alkylthiol terminated by a DNA base. 4) any sulfur-terminated species that clogs poorly in the monolayer; 5) alkylthiol spacer molecules that terminate at either the ethylene glycol unit or the methyl group, which inhibit non-specific adsorption to all of the above. Addition or subtraction. Thiols are described in the easy formation of SAMs because of their affinity for gold. Based on U.S. Patent No. 5,620,820 and other references, thiols may be substituted with other molecules known in the art. Molecular wires typically create defects in otherwise tightly packed SAMs, due to their bulk and other shapes, preventing the SAM from tightly sealing the surface to the fluid to which it is exposed. . Molecular wires cause the destruction of tightly packed self-assembled structures, thereby defining defects where the fluid to which the surface is exposed can be in electrical communication with the surface. In this context, the fluid is in electrical communication with the surface by contacting the surface or sufficiently close to the vicinity of the surface that electrical communication may occur, such as by tunneling.
[0047]
The term “biological binding” refers to an interaction between corresponding pairs of molecules, typically including biochemical, physical, and / or drug interactions, which describes the affinity or binding ability of each other. Target or non-specific binding or interaction. Biological binding defines the type of interaction that occurs between pairs of molecules, including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, and the like. Specific examples include antibody / antigen, antibody / hapten, enzyme / substrate, enzyme / inhibitor, enzyme / cofactor, binding protein / substrate, carrier protein / substrate, lectin / carbohydrate, receptor / hormone, receptor / Effector, complementary strand of nucleic acid, protein / nucleic acid, repressor / inducer, ligand / cell surface receptor, virus / ligand and the like.
[0048]
The term "binding partner" refers to a molecule that can bind to a particular molecule. Biological binding partners are an example. For example, Protein A is a binding partner for the biological molecule IgG and vice versa.
[0049]
The term "determining" refers to a quantitative or qualitative analysis of a species, for example, by spectroscopy, ellipsometry, piezometry, immunoassay, electrochemical measurement, and the like. "Measure" also means detecting or quantifying an interaction between species, such as detecting binding between two species.
[0050]
The term “self-assembled monolayer” (SAM) refers to a relatively ordered set of molecules that are spontaneously chemisorbed to a surface, where the molecules are approximately parallel to each other and approximately perpendicular to the surface. Is done. Each of the molecules includes a functional group that adheres to the surface and a portion that interacts with adjacent molecules to form a relatively ordered array in the monolayer. Laibinis, P .; E. FIG. Hickman, J .; Wrighton, M .; S. Whitesides, G .; M.Science{245,845 (1989); Eval, J .; Whitesides, G .; M.J. Am. Chem. Soc.111, 7155-7164 (1989); Whitesides, G .; M.J. Am. Chem. Soc.111, 7164-7175 (1989), each of which is incorporated herein by reference.
[0051]
The term "self-assembled mixed monolayer" refers to a heterogeneous self-assembled monolayer, i.e., a monolayer made up of a relatively ordered collection of at least two different molecules.
[0052]
In one aspect, the invention contemplates interactions between chemical or biological agents for analysis, drug screening, and the like. The present invention involves analyzing and / or inhibiting ligand interactions including, but not limited to, ligands on intact cells (growing on electrodes or in solution or suspension). But not limited to them. The present invention contemplates various embodiments including the use of drug candidates, known or putative ligands, and small molecule drug libraries.
[0053]
In one aspect, the cells grow on electrodes that may or may not be derivatized by self-assembled monolayers (SAMs). A putative ligand (eg, of a particular cell surface receptor) is immobilized on a solid support (eg, colloidal gold) along with a signaling element (eg, an electroactive complex). These derivatized solid supports are incubated with cells immobilized on a sensing electrode (eg, a metal support). Interaction between the target receptor and a ligand on the solid support (eg, a ligand bound to a colloid) tethers the co-immobilized signaling element near the sensing electrode. Without being limited to any particular mechanism, it is believed that when a potential is applied to the electrodes, nearby redox-active metal complexes receive their characteristic oxidation potential and emit electrons. When the oscillating component is added to the top of the voltage lamp, more electrons are emitted by each metal composite and can be detected as a current output. This type of electrochemical analysis is called alternating current voltammetry (ACV). As known to those skilled in the art, "cell surface receptor" as used herein is a generic term that also includes cell surface proteins.
[0054]
Certain embodiments of the present invention use self-assembled monolayers (SAMs) on surfaces, such as the surface of colloidal particles, and articles, such as colloidal particles, having surfaces coated with SAMs. In one set of preferred embodiments, the SAMs formed entirely of synthetic molecules completely cover the surface or area of the surface, eg, completely cover the surface of the colloid particles. As used herein, a "synthetic molecule" refers to a molecule that does not occur in nature, rather, is one that has been designed by a human, or designed by a human, or synthesized under the control of a human. "Completely cover" herein means that there is no portion or region of the surface that directly contacts proteins, antibodies, or other species, which would prevent complete and direct coverage by the SAM. That is, in a preferred embodiment, the surface or region comprises a SAM consisting entirely of a non-naturally occurring molecule (ie, a synthetic molecule). SAMs are SAM-forming species that form SAMs that are tightly packed at the surface, or these species in combination with molecular wires, or other species that can facilitate electrical communication through the SAM (defect-promoting species that can participate in SAM) Or other species capable of participating in the SAM, and any combination thereof. Preferably, all of the species involved in the SAM contain functional groups that bind to the surface, and possibly covalently, such as thiols that bind covalently to the gold surface. The self-assembled monolayer on the surface of the present invention is a species that can essentially present (expose) any chemical or biological functionality (eg, a thiol species when gold is the surface). Or a mixture of For example, they are tri-ethylene glycol-terminated species to inhibit non-specific adsorption (eg, tri-ethylene glycol terminated thiols), and when complexed with, for example, a nickel atom, the histidine-tag labeled binding species Includes other species that terminate at the binding partner of the affinity tag (eg, thiols), such as those that terminate at chelates that can coordinate the metal, such as nitrilotriacetic acid, which captures the metal-binding tag-labeled species. You can go out. The present invention provides a method for closely controlling the concentration of essentially any chemical or biological species presented on a colloid surface, or any other surface. In many embodiments of the present invention, a self-assembled monolayer is formed on a colloidal gold surface. The present invention provides a method for strictly controlling the concentration of a histidine-tagged peptide displayed on a colloid surface. Without strict control of the peptide density on each colloid surface, the co-immobilized peptides easily aggregate with each other to form microhydrophobic regions, and the non-aggregate-forming species present in the sample It will catalyze colloid-colloid aggregation in the presence. This is an advantage of the present invention over existing colloid aggregation assays.
[0055]
The method according to the invention produces a self-assembled monolayer on a colloid that resists non-specific adsorption without a protein blocking step, such as blocking with BSA. Also, the method according to the present invention is stable in biologically relevant fluids and requires a surfactant (for stability; keeping the colloid in suspension) that interferes with the binding reaction. This produces derivatized colloids that are not needed. This allows a sensitive binding assay to be performed in solution. This eliminates the need to have a binding partner that adheres to the adsorption surface, which is common in existing colloid aggregation assays. As explained below, surfactants can be conveniently used for SAM formation on colloids. In this case, the surfactant may be removed after SAM formation, preferably removed and no longer present on the colloid, in the SAM, or elsewhere during the binding interaction, or other use of the colloid.
[0056]
The present invention is not intended to be limited to embodiments in which cells bind directly to a metal support (eg, an electrode). Embodiments contemplate that the cells bind indirectly due to the interaction of the ligand with the cells bound to the metal support. That is, cells can be recruited to the surface by coating the surface with molecules that bind directly or indirectly to the cell by specific or non-specific interactions. For example, methyl-terminated self-assembled monolayers (SAMs) bind non-specifically to collagen and then non-specifically to cells. Alternatively, peptides containing an arginine, glycine, aspartate (RGD) motif mimic vitronectin and bind to specific cell types such as endothelial cells. Similarly, polylysine, positively charged, Kringle domains, integrins, and peptides, or molecular mimetics thereof, can be attached to a surface to connect cells. These ligands may be displayed on the surface by incorporation into SAMs. The ligand itself need not be directly incorporated into the SAM. A SAM that presents a binding partner for an affinity tag linked to a ligand may be used. For example, peptides modified with a histidine tag can readily bind to SAMs containing nitrilotriacetic acid (NTA) -nickel thiol. Alternatively, the glutathione S-transferase fusion protein may be linked to a SAM that incorporates glutathione or a derivative thereof. One advantage of using metal electrodes is that they can provide many known functional groups at the ends of molecules that form SAMs on the surface.
[0057]
For example, cells in solution can be linked to a detection electrode by electrophoresis. Specifically, the cell-derived molecule may bind to a colloid-linked ligand that also presents an electroactive molecule, such as a ferrocene derivative, to assist in detecting the binding complex. However, the detection element used may be an electroactive species or fluorescent tag, either charged, which is easily detectable, or the colloid itself.
[0058]
The signaling entity is used in various assays and procedures of the present invention. Any variety, including dyes, dyes, electroactive molecules, fluorescent moieties, up-regulating fluorophores, enzyme-linked signaling moieties including horseradish peroxidase and alkaline phosphatase, chemiluminescent moieties, electrochemiluminescent moieties, etc. It should be understood that the signaling entity may be selected in each case. Knight, "Trends in Analytical Chemistry", vol. 18, 1999, pg. 47; Knight, et al. , Analyst, vol. 119, 1994, page 879; Stults, et al. , "Use \ Recombinant \ Biotinylated \ Aequorin \ in \ Microtiter \ and \ Membrane-Based \ Assays: Purification \ of \ Recombinant \ ecoe, economy.com, e. , "A Microplate Assay for Analysis, Analysis for Solution-Phase, Glycosyltransferase, Reactions: Determination of Of Kinetic, Constants, see, for example, see, for example, Analytical, 1931, and 28. If desired, the various signaling entities may be immobilized on the surface of the particle, such as a bead or a colloid particle. The signaling entity displayed on any of these may be a fluorescent molecule. Fluorophore-bound antibodies and other fluorophore fusion proteins, including green fluorescent protein, are widely used in biomedical research and testing. These fluorescent proteins and molecules may be based on affinity tags, EDC / NHS chemistry, or by binding to His-tagged protein A or G presented on NTA-SAM-coated colloids according to the present invention. Either way, the putative binding partner can be easily linked to the presenting gold colloid. Also, signaling entities such as fluorescent moieties may be co-immobilized on the colloid with a biotin stopping ligand, or may be immobilized with a chelate / metal / metal binding tag linkage. Alternatively, the fluorescent moiety may be immobilized by using a chelate / metal / metal binding tag with His-Protein G to bind it to the antibody and bind to the antibody. The part can then be detected directly.
[0059]
The ligand-derivatized colloid may be anchored near the electrode, which may or may not be modified by a self-assembled monolayer (SAM), and will bind the bound cells by perturbations in the electrode current induced by binding. Can be detected. Cell-derived proteins can also bind to ligands linked to magnetic beads. Alternatively, the cell-derived protein can bind to a ligand on the electroactive dendrimer. In addition, cell-derived proteins can bind ligands linked to polymers that also display an electroactive or redox-active complex. Cell-derived proteins can also bind ligands linked to conductive polymers that present an electroactive or redox-active complex.
[0060]
In an alternative scheme, the described system can use two different ligands. One ligand that binds to a constitutively expressed cell surface receptor can be immobilized on magnetic beads and used to recruit cells to the sensing electrode. The second ligand, which recognizes the cell surface receptor of interest, binds to a signaling colloid.
[0061]
In another alternative scheme, the described system can use two distinguishable electroactive complexes, such as two ferrocene derivatives that oxidize at different potentials. The first ferrocene derivative can be directly or indirectly linked to the ligand of the cell surface receptor of interest. The second ferrocene derivative is directly or indirectly linked to a second ligand that binds to a constitutively expressed cell surface receptor. In this means, the ratio of the two signals can be used to calibrate the level of induction of cell-derived molecules (eg, proteins) induced by physiological, environmental, or disease-related changes in expression. it can. This comparison of expression ratios can be performed for those having both ligands with the same metal complex, but incubating different aliquots of the sample on different electrode pads.
[0062]
The present invention provides for the attachment of ligands (including, but not limited to putative drug candidates) to a surface that may be particle-like, and to attach them to a support or leave them on cells. It is also contemplated that cell-derived proteins, such as, for example, cell surface proteins, are allowed to interact for identification of binding partners, determination of their presence or absence, and quantification of their levels. Specifically, an intact cell presenting a cell surface receptor can be used as the first binding partner. Known or putative ligands that bind directly or indirectly to the signaling entity act as second binding partners. Alternatively, the cell-derived protein can bind to a surface and their binding partner, such as a ligand, or a signaling capable particle, such as a colloid or derivatized colloid. Drug candidates can be added to facilitate drug screening by inhibiting the interaction. This method is also very useful for diagnosis. The protein or antibody may be immobilized on a surface, and a sample suspected of containing the binding partner of the protein may be incubated with the sample to facilitate binding, and the remainder of the sample may be washed away. it can. A ligand for the binding partner and particles with signaling capabilities may be added. If the sample contains a binding partner, the particles will bind indirectly to the surface and provide a signal. The surface may be refillable particles. The connection may be direct or indirect, that is, the connection may involve two entities that are immobilized on each other. The particles present an immobilized antibody, such as a histidine-tagged protein G to which the antibody binds, and the antibody may recognize a common ligand or may contain proteins that recognize each other May be.
[0063]
One general technique of the present invention is to use a magnetic field to recruit an electronic or electrochemical signaling entity to the surface of an electrode that exhibits the capture of a binding moiety (biological or chemical agent) by a binding partner. It involves using beads. In FIG. 27, a first species 30 is linked to a colloid particle 32 that also has signaling capabilities, such as metallocene-ferrocene,. A second species 36, presumed to be the 30 binding partner, couples to the magnetic particles 38, which are unable to signal but can be magnetically attracted to the electrode 40. In one particularly useful technique, species 30 and second species 36 are proteins, so the present invention finds particular use in the field of proteomics. The above technique is an example of an aspect of the invention that involves allowing the colloidal particles 32 to be immobilized on a non-colloidal structure, magnetic beads, 38. Measurement of the fixation of the colloidal particles to the non-colloidal structure is performed by attracting the magnetic beads to the electrode and measuring whether the colloidal particles are also near or unconnected to the electrode. Specifically, the two components are incubated together in a solution and the resulting complex is magnetically attracted to the electrode. The electrodes are then analyzed by alternating current voltammetry (ACV) (Laviron E: J Electro Anal Chem., 1979, 105: 35). The current is plotted in real time as a function of the potential. If the electronic or electrochemical signaling moiety on the first component is very close to the electrode, a distinct current peak will occur at a characteristic potential. If the two proteins interact with each other and the magnetic particles are attracted to the sensing electrode, it will also have colloidal particles with signaling capabilities. Because the first component is a small colloidal gold, the interaction between the first species on the colloidal particles and the second species bound to the magnetic particles causes the interaction in the suspension unless specifically recruited to the sensing electrode. Will remain. An electronic or electrochemical signal will only be generated if the signaling particle is bound to the magnetic beads by an interaction between the first species and the second species. In this embodiment, the colloid particles include ancillary signaling entities, as exemplified by ferrocene. In other embodiments described below, the colloid particles are signaling entities themselves and do not require auxiliary entities.
[0064]
This approach may be used for rapid screening of protein-protein interactions in an array format. When searching for potential therapeutic targets, researchers often need to identify binding partners for particular proteins. Known proteins can be bound to magnetic beads. This can be achieved by direct chemical coupling of known proteins to commercially available magnetic beads displaying chemical functional groups, by coupling to protein A-coated magnetic beads via cognate antibodies, or by derivatization with NTA-SAM. (See, eg, US Pat. No. 5,620,850, which is incorporated herein by reference). . Potential binding partners may be separately bound to NTA-SAM-coated colloids that are expressed as a histidine-tagged protein and then also present a ferrocene moiety. Each well of the multi-well array connected to the electrode array will contain known proteins on magnetic beads. Separate putative binding partners linked to signaling colloids may be added separately to each well. The resulting complex may then be magnetically attracted to the electrodes to determine which candidate proteins have bound to the known protein. Known species need not be proteins. When a binding partner is identified, a drug candidate may be added to the assay to inhibit the interaction.
[0065]
Alternatively, biological or chemical agent 30 is a drug candidate and its binding partner (potential binding partner) 36 is the target of the drug candidate, or vice versa. Specifically, drug candidates may be linked to magnetic particles, and known proteins may be linked to signaling colloids to identify drug candidates that bind to a medically relevant target species. Alternatively, species 30 and 36 may be a target and a ligand for the target, respectively, and the technique brings colloid 32 and magnetic beads 38 closer together in the presence of a candidate drug that inhibits binding of the ligand to the target. 36. As will be seen below, this technique can be applied to the interaction of colloids with various non-colloidal structures.
[0066]
The advantages of this technique over existing methods such as ELISA, fluorescent labeling and SPR include: In this system, there is no need for protein labeling; proteins bind to labeled components. Gold colloids can be pre-labeled with both: a) a signaling moiety; and b) functional groups for protein linkage. NTA / Ni for capturing histidine-tagged proteins²⁺Self-assembled monolayers, which together present ferrocene derivatives for electronic and electrochemical signaling, may be formed on the colloid. SAMs containing carboxylic acid groups for chemical coupling of unmodified proteins (standard EDC / NHS chemistry) can also be used. The technology is modular. Virtually any biological species can be co-immobilized on a colloid having a signaling entity. The technique allows cost-effective multiplexing because it can be easily multiplexed on a microelectrode array.
[0067]
Liposomes can also take up the first binding partner 30. As described above, the signaling itself may be incorporated into the liposome, or lipids may be incorporated into the liposome to capture the His-tagged soluble receptor after liposome formation. Liposomes can contain UV-activated, cross-linking, photo-excitable groups after liposome formation to increase stability.
[0068]
Identification of drug candidates can also be performed using competitive inhibition assays. Specifically, free drug candidates in solution can be separately incubated with the composition. Competitive inhibition of the target cell-derived protein caused by the drug bound to the receptor or ligand can be expressed as a time or dose dependent loss of the detection signal.
[0069]
To do this requires a method of directly or indirectly binding a signaling moiety to a known ligand, and then provides a method of linking or recruiting a complex binding partner to the sensing surface. The former is described above and the latter is described below.
[0070]
The sensing surface used can be of various shapes. However, for purposes of illustration, a method for recruiting the binding partner complex using an electrode modified with a conductive SAM is described. A conductive SAM is a layer of molecules bound to a metal surface, which conducts electrons at a faster rate than a metal uniformly coated with an insulating species such as a saturated alkyl thiolate. A preferred route for electron conduction can be provided by a monolayer incorporating molecular wires (polymers of aromatic ring compounds). The poly (ethynylphenylthiol) (ie, C₁₆H₁₀A variety of molecules, including but not limited to S), can be used for this purpose:
[0071]
Embedded image

[0072]
As used herein, "molecular wire" means a wire that enhances the ability of a fluid in contact with a SAM-coated electrode to be in electrical communication with the electrode. This includes conductive molecules, as described above and more fully illustrated below, or molecules that can cause defects in the SAM such that the fluid contacts the electrodes. A non-limiting list of additional molecular wires includes 2-mercaptopyridine, 2-mercaptobenzothiazole, dithiothreitol, 1,2-benzenedithiol, 1,2-benzenedimethanethiol, benzene-ethanethiol, And 2-mercaptoethyl ether. Also, the conductivity of the monolayer can be increased by the addition of molecules that promote conductivity in the plane of the electrode. Conductive SAMs include, but are not limited to: 1) a poly (ethynylphenyl) chain terminated by sulfur; 2) an alkylthiol terminated by a benzene ring; 3) an alkylthiol terminated by a DNA base; 4) any of the sulfur-terminated species that incompletely clog in the monolayer; 5) any of the above with alkylthiol spacer molecules terminated with either ethylene glycol units or methyl groups that inhibit nonspecific adsorption. What was done or subtracted. Thiols are described in their immediate formation of SAMs due to their affinity for gold. Based on U.S. Patent No. 5,620,820 and other references, thiols may be substituted with other molecules known in the art.
[0073]
As known ligands on signaling colloids interact with their cognate cell-derived proteins, drug candidates may be added to the solution to monitor different effects. Conductivity that also presents molecules that terminate at head groups that bind directly or indirectly to cells (eg, methyl groups, polyK, positive charge, RGD sequences, Kringle motifs, integrins, and their peptidomimetics) Cells may be recruited to the electrodes coated with the SAM. Alternatively, different interactions can be quantified by having a cell surface protein bound to the SAM interacting with a cognate ligand presented on a conductive SAM. In this case, an electronic or electrochemical signal is generated after the ligand binds to the constitutively expressed receptor. Under flow force, the electronic or electrochemical signal will be proportional to the number of cell surface receptors that interact with the SAM-immobilized ligand. The rate of cell motility as a function of flow rate indicates the density of receptors for surface cell adhesion molecules. Downstream of the interacting SAM is a charge reader (eg, ammeter, charge counter), or an optical reader (eg, surface plasmon resonance detector, or fluorescence reader). In addition, these methods can also be used to screen for drugs that inhibit cellular phenotypes known to be characterized by invasion or metastasis without prior knowledge of the specific receptor-adhesion molecule interactions involved. it can. Screening for drugs that mimic physiologically relevant interactions can also be performed using this system by either: adding drug candidates free in solution and looking for loss of detection signal Alternatively, the known ligand released in solution with the drug candidate on the solid support is added and the appearance of the detection signal is explored.
[0074]
Applying a slightly positive bias to the AC potential ramp attracts the sensing electrode due to the fact that the cells are negatively charged. Addition of additional negatively charged groups to the dendrimer or polymer to which the ligand is linked further enhances recruitment to the sensing electrode. One of skill in the art can readily immobilize multiple signaling entities on a dendrimer or polymer. Alternatively, binding the recognition ligands with metal binding tags facilitates their binding to alkylthiol NTA (and consequently the associated cells). SAMs with metal-containing compounds such as ferrocene on gold-coated magnetic beads allow the use of electromagnetic fields, or fixed magnets, to replenish the complex to the sensing surface electrode. Also, sensing electrodes that include ligands can attract cells directly, through specific or non-specific interactions, such as with cell adhesion molecules, or through histidine tag labeling.
[0075]
However, since one of the binding partners has a higher specific gravity than the analysis solution, if the complex precipitates due to gravity, replenishment to the detection system can be performed simply using gravity. Mechanical mixing may be performed during the incubation step to avoid premature precipitation. FIGS. 32A and 32B show ACV plots of experiments showing the use of gravity to recruit signaling entities and controls showing binding interactions to the surface. GST was bound to the colloid by a metal binding tag / metal / chelate linkage (NTA-SAM). Colloids have also presented ferrocene derivatives for electronic or electrochemical signaling. The colloid was presented in solution to the measurement electrode as a negative control, yielding the plot of FIG. 32A. One set of colloids was exposed to glutathione-coated polymer beads (agarose). The colloid was immobilized on the beads by glutathione / GST binding, and the beads were immobilized on the electrodes by gravity. The ACV plot of FIG. 32B was obtained showing the electroactivity detection of ferrocene close to the electrode.
[0076]
In methods using an electrode as a cell-derived protein generator, a point on the electrode pad can be used to generate a potential spike that lyses the cells. The contents of the cells are then incubated with the signaling ligand and the interaction is detected.
[0077]
In addition, cell binding molecules may be channeled with conductive molecules and shaped into a designated topology along the bottom of the flow channel using SAM to enhance the detection capabilities of the system. Thus, the SAM may present the binding moiety in a form that alternates between sensing (optical or electronic or electrochemical) and cell binding capabilities. As an example, the methyl-terminated alkylthiol stripes alternate with conductive polypyrrole groups in the SAM. Also, the topology of the SAM can be manipulated so that the cells are placed in the furrows, so that receptors along the sides of the cells can be evaluated. The formation of alternating functional SAM stripes can be performed as described in US Pat. No. 5,512,131 and International Patent Publication No. WO 96/29629, both of which are incorporated herein by reference. These complexes can be detected electronically or electrochemically. One detection method is an electrochemical technique called alternating current voltammetry (ACV). This detection method can be supplemented by the use of additional analytical techniques, such as higher order harmonic analysis. These complexes may be detected by optical means such as SPR. Using the latter technique, recruitment of the complex to the sensing surface causes a large shift in optical properties.
[0078]
In one embodiment, a cell surface protein can be detected and quantified as follows: a histidine-tagged ligand that recognizes the cell surface protein is labeled with NTA (to capture the His-tagged protein) and a ferrocene moiety (electronically). (Or for electrochemical signaling) to a colloid with a SAM that presents both. These biospecific, electronic or electrochemical signaling colloids are then incubated with cells presenting the target receptor. Cells are then sedimented, adhered or attracted onto SAM-coated electrodes and analyzed by ACV (FIG. 3). When ligands immobilized on signaling colloids bind to their cognate receptors on the cell surface, a current peak will occur at the characteristic oxidation potential of the ferrocene moiety. An antibody that recognizes a cell surface receptor may first bind to a colloid with NTA-ferrocene to which His-tagged protein A or G is bound. Alternatively, the antibody may be directly bound to the colloid via a metal binding tag / metal / chelate bond, wherein the metal binding tag is bound to the antibody. Techniques for attaching a histidine tag to an antibody are described in “Construction of the single-chain Fv from 196-14 antibody toward ovarian cancer-associated antigen CA125” Hashi. Tanigawa, K .; , Nakashima, M .; , Sonoda, K .; Ueda, T .; Watanabe, T .; , And @Imoto, T .; : 1999, Biological and Pharmaceutical Bulletin, Vol. 22: (10) 1068-1072. "Human \ antibodies \ with \ subnanomolar \ affinities \ isolated \ from \ a \ large \ non-immunized \ phage \ display \ library", Vaughan, T .; J. , Williams, A .; J. , Pritchard, K .; , Osbourn, J .; K. Pope, A .; R. , Earnshaw, J .; C. et @ al. , 1996, Nature Biotechnology Vol 14 (3) p. 267. "Expression and purification, of single-chain, anti-HBx antibody in E. coli" Zouh G, lui KD, Sun H .; C. , Chen @ Y. H. , Tang @ Z. Y. , And {Schroder} C. H. , 1997, vol @ 123 (11-12) pgs609-13.
[0079]
As an example of the technique of the present invention, FIG. 4 describes an example of a useful technique that includes immobilizing colloidal particles on cells. The tumor marker, MUC-1, is abnormally expressed on tumor cells. The human tissue-cultured breast cancer cell line MCF-7, available from the ATCC, overexpresses MUC-1. Antibodies 50, DF3 or DF3-p, available from Dana-Farber Cancer Institute, bind to electronic or electrochemical signaling colloid 52 (with NTA-SAMs 54) by histidine-tagged protein G56. Target cells 58 are incubated with signaling colloids 52 with antibodies, electrophoresed on electrodes coated with SAMs 58 containing molecular wires, and analyzed by ACV. The SAM on the electrode 40 includes a more conventional, tightly packed SAM-forming species mixed molecular wire 58. For simplicity in all figures, only the molecular wires 58 are shown schematically. Incubation of signaling colloids with antibodies with cells with MUC-1 gives current peaks. Alternatively, a putative cognate ligand of MUC-1, I-CAM, may be His-tagged and bound to a signaling colloid also having an NTA group.
[0080]
Another assay is shown in FIG. Drug libraries may be screened for their ability to inhibit specific interactions with cell-derived proteins, such as the MUC-1 / I-CAM interaction. I-CAM # 64 binds to signaling colloid 62 as described above and is then incubated with MUC-1 (66) presenting cells 70 and control cells. Drug candidate 68 is added into the solution in which the cells and colloid are suspended, after which the cells adhere to the electrodes and are analyzed by ACV. Loss of signal indicates an interaction with the drug candidate. FIG. 6 shows how this scheme is easily multiplexed to screen thousands of drug candidates simultaneously by using an array of disposable microwells 72 with a connectable array of microelectrodes 74. Will be described. Each well contains the assay shown in FIG. 5, or another assay.
[0081]
Next, in FIG. 7, small molecule drug libraries are used to generate electronic or electrochemical signaling for signal increase assays or to screen for drugs where the cognate ligand binds to an unknown cell surface receptor 80. A procedure is shown that can be synthesized on, or covalently bonded to, a colloid or particle 78 that also has a portion 34. The drug candidate 76 bound to the signaling particles can be incubated with cells 82 presenting the receptor 80 of interest, or control cells 84. The signal is increased by the drug-target interaction in this assay (FIG. 7).
[0082]
It has been suggested that αVβ3, a cell surface receptor, promotes angiogenesis by interacting with the cell adhesion molecule vitronectin. Human umbilical vein endothelial cells (HUVEC) displaying the αVβ3 cell surface receptor are available from Clonetics. For screening for drugs that inhibit the action of αVβ3, a His-tagged peptide displaying an RGD-containing sequence from vitronectin is ligated to a colloid with SAMs presenting both an NTA group and a ferrocene moiety. Subsequently, the biospecific signaling colloid is incubated with the HUVEC cells and the drug candidate. HUVEC cells can be grown on electrodes. However, if the cells are in solution or suspension, they can be electrophoresed or magnetically coupled to the sensing electrode and analyzed by ACV. Biospecific colloids produce a current peak when incubated with HUVEC cells, rather than control cells. If a drug candidate interferes with the αVβ3-RGD sequence interaction, signal loss results. Inhibiting the αVβ3-RGD interaction with the known angiogenesis inhibitor endostatin demonstrated the feasibility of this assay. The assay can be performed electronically as described, or visually by growing the cells in a standard plastic dish and performing the assay and viewing the cells at 40 × magnification.
[0083]
Alternatively, the drug candidate may be synthesized or linked to a bead, colloid, or support that also presents an electronic or electrochemical signaling moiety. These "particles" or surfaces are incubated with the target cells, attracted to the sensing electrodes and analyzed by ACV. The suction field is reversed and the peptide containing the RGD sequence is titrated in solution. The cells are again electrophoresed on the sensing electrode and re-analyzed. Loss of signal indicates that the drug-cell interaction is specific for the αVβ3 receptor and the IC of the RGD peptide₅₀Is associated with the binding affinity for the drug.
[0084]
Alternatively, echistatin, a 49 amino acid peptide that also binds αVβ3, may be His-tagged for exchange with an RGD-containing peptide in the assay described above.
[0085]
Metallocenes are particularly useful as signaling entities for the following reasons. Various ferrocene derivatives may be selected such that they are each oxidized at a specific potential between 100 mV and 800 mV. Each oxidation potential represents a unique label so that multiple cell surface receptors can be probed simultaneously. When a biologically relevant interaction occurs between the cell surface receptor and the ligand-immobilized colloid, the cell is modified with electronic or electrochemical signaling particles, resulting in a current peak. The magnitude of the current peak will be proportional to the number of cell surface receptors recognized by the signaling colloid.
[0086]
Cell surface molecules can be detected in suspension or on cells embedded in a tissue sample, as shown in FIG. Frozen tissue specimens 86 are sectioned frozen and placed directly on a flexible, semi-permeable membrane support 88 that has been derivatized with a cell binding group 90, such as an RGD-containing peptide or a methyl terminating group. The sample is then incubated with an electronic or electrochemical signaling colloid 92 that also presents a ligand 94 for the cell surface receptor of interest. Unbound colloid is washed away after the incubation time. The support membrane is then physically contacted with a microelectrode array 96 having an electrode area comparable to the cell size and analyzed by ACV. Each portion of the tissue sample is analyzed for protein content and expression level and correlated histopathologically. This ability guarantees the validity of the single-cell assay, as it allows researchers to identify protein types that are particularly relevant to cancer cells and eliminate any abnormal protein expression. is there. Cells in suspension can be bound to a support membrane as well.
[0087]
This technique can be used to identify cell-derived molecules, such as receptors or proteins that are differentially expressed in healthy tissues or cells that have been diseased. This different expression may be associated with different levels of expression in those diseased relative to healthy tissues or cells, and / or different types of expression in tissues or cells that can be easily identified. This technique facilitates diagnostic assays for measuring disease states. For example, in connection with a patient presumed to have a particular disease, cells may be taken from the patient, particularly cells associated with an indicator of the disease, such as cells from a biopsy, a blood sample, etc. Well, these cells can be analyzed against healthy cells to determine expression levels or types indicative of disease. The system can also be used to screen for drugs that inhibit the up-regulation of cell-derived proteins involved in various pathological conditions. Examples of two techniques for drug screening include: (1) administering a candidate drug to a patient who is presumed to be or exhibiting a disease, and assessing the efficacy of the drug in treating the disease. Monitoring a biological sample comprising cells of a patient as described above for measuring; (2) collecting a biological sample comprising cells from a patient presumed to have or having a disease; Exposure of a target sample or cell components to a candidate drug and monitoring expression levels and / or types using the techniques described above. Once a binding partner (which may include a drug, antibody, or protein / peptide ligand of a cell-derived protein) is identified, the binding partner is used to quantify the level of expression of the cell-derived protein in response to a disease state or treatment. To the detection moiety. This may be any assay that tests the indirect effect of a drug candidate on the expression and transfer of cell-derived proteins to both cell surface and intracellular compartments.
[0088]
The invention also provides the ability to visually study the type of cell surface receptor expression on individual cell surfaces and / or on cells embedded in tissue samples. This can indicate the type of cell surface receptor expression that can be linked to the disease state. They can also be used in diagnostic or drug screening methods. In a specific assay, colloid particles with ligands that bind to cell surface receptors are exposed to individual or embedded cells, and the location of their binding with respect to individual cells is visually measurable, 1 shows the type of cell surface receptor expression. For example, MUC-1 is a cell surface receptor associated with breast cancer. Normally, MUC-1 is uniformly expressed on the surface of various cell types. In transformed cells associated with various cancers, the receptor is overexpressed and concentrated at the cell tip. This can be measured using the described technique. In drug screening, a culture of transformed cells may be provided and treated with a drug candidate. The loss of mold expression at the tip is considered. In this embodiment, visual identification may include any of the techniques described herein, such as observation by the naked human eye, microscopy, absorption spectroscopy, electron microscopy, fluorescence detection, and the like.
[0089]
In techniques involving electronic or electrochemical detection as described above, the level of the expressed species can be compared between samples, each containing individual cells or other samples containing very small amounts. Yes, and the mold can be measured on larger samples, including tissue samples. In the visual detection embodiment described above, the level of the expressed species can be measured as well as the type of species expressed in both large and small samples, including single cell samples. Signaling entities useful for electronic or electrochemical detection include the signaling entities described herein for electronic or electrochemical detection, including redox-active molecules such as ferrocene. For the visual detection described above, any of the signaling entities described herein, including colloids, alone or have an auxiliary signaling entity, such as a fluorescent or visually identifiable entity. Can be used as is. More than one signaling entity may be used (ie, more than one signaling per binding event). Both electronic or electrochemical or visual signaling may use different signaling entities for different assays. For example, a first ligand selected to target a first receptor or protein may be immobilized with respect to a first signaling entity, while targeting a second receptor or protein. The second ligand so selected may be immobilized with respect to the second signaling entity. In electronic or electrochemical signaling, different signaling entities may contain different redox potentials, the differences between them are electronically distinguishable, and in visual identification, different signaling entities May be radioactive or absorbing entities of different colors. In such cases, not only is the expression level and type of the protein or receptor measured, but the type can distinguish the location of expression of one receptor or protein from the others.
[0090]
The biospecific colloids described above can also be used to facilitate in vivo imaging. The colloid used is gold, which is relatively inert. Colloidal gold, which has been derivatized to present antibodies or other ligands for tumor markers, can be incorporated or injected. The tumor mass presenting a cognate tumor marker will be covered with colloid and will act as a concentrator. The tumor can then be detected by x-rays and diagnostic imaging techniques such as x-ray computed tomography (CT), since the useful result may be a tumor mass encapsulated in metal . Individual colloids in solution will not be visible for detection. Tumors so labeled with biospecific colloids can also be detected by MRI (magnetic resonance imaging). Diagnostic techniques can be enhanced by incorporating paramagnetic metals and other contrasting materials, such as Fe, Gd, or Cr. The contrast material can be attached to the colloid by incorporating the metal chelate thiol into the SAMs formed on the colloid. Alternatively, SAMs that present only the cognate ligand of the target protein expressed on the tumor may be formed on particles made of a contrast material. Colloids with iron or magnetite cores may be coated with gold and derivatized with biospecific SAMs. Alternatively, liposome-like particles may be formed from lipid chains terminated by chelating groups, such that the metal counterpart forms a "core" and the biospecific ligand is exposed to a solvent. Alternatively, the colloid may have, in addition to the biospecific ligand, a moiety having specific spectroscopic characteristics, which moiety can be detected by energy absorption or scattering techniques, including Raman spectroscopy. The diagnostic imaging device may be included outside the body or may be introduced into the body or optical fiber of the viewing machine. Alternatively, when enriched on a tumor mass, a colloid with a radio frequency transmitting moiety may produce a detectable signal. The signal from each colloid must be negligible or undetectable compared to the signal generated from the collection of colloids bound to the tumor mass.
[0091]
In FIG. 9, to determine whether the two proteins interact with each other, the first protein 104 is bound to a colloid 106 that presents both NTA # 54 and ferrocene ligand 34, and the second protein 100 is Will bind SAM-magnetic beads 102 that present only NTA ligand 54. The two particle types are mixed together, after which a magnet 108 is magnetically replenished to the underlying sensing electrode 40. Only when the magnetic beads bind to the signaling colloid by the interaction of the two protein species will an electronic or electrochemical signal be generated. Also, non-histidine-tagged proteins, on signaling or magnetic (replenishable) particles, are charged and carboxy terminated with thiols terminated with exposed groups that do not participate in coupling chemistry (eg, NTA thiols). By forming SAMs incorporating thiols, they can be linked to them. Next, standard EDC / NHS coupling chemistry can be used to attach any molecule that presents a primary amine. Because the electronic or electrochemical labeling step is separate from the protein production step, common electronic or electrochemical signaling colloids and magnetic particles may be used to efficiently multiplex the system. This facilitates the construction of a protein interaction database that helps researchers define the molecular characteristics of cancer.
[0092]
Interacting protein partners can be incubated with a small molecule drug library, and drug leads were identified by detecting loss of signal. Alternatively, small molecule drug libraries may be purchased on magnetic beads for direct screening of drug candidates that bind to a known target protein.
[0093]
Also, the basic techniques described herein can be used to screen for drugs that modulate enzyme activity. Certain enzymes have been identified as important in progressive disease. In some cases, a molecule must be cleaved at its specific site to become active, and it may be desirable to screen for drugs that inhibit the activity of the enzyme that does this. The techniques described herein can be used for this purpose as described below:
What is described below is useful in practicing this technique: Edelstein {and} Distefano reports that farnesyl pyrophosphate groups modified with photoexcitable crosslinkable moieties can be added to peptide motifs derived from RAS by yeast FPT. and that (Edelstein R.and Distefano M. (1997) .Photoaffinity labeling of yeast farnesyl protein transferase and enzymatic systhesis of a RAS protein incorporating a photoactive isoprenoid.Biochem.and Biophys.Res.Comm.235,377 382). Their purpose was to use this assay to show that by cross-linking the two, the farnesyl derivative would be recognized by the FPT. This finding is consistent with the notion that farnesyl or geranyl moieties can be modified with biotin at the end remote from pyrophosphate without interfering with enzyme activity or specificity.
[0094]
In FIG. 10, an enzyme cleavage site (ECS) 110 can be linked at one end 112 to a molecule 122 with a known binding partner (BP1). At the other end 114, it may be linked to a molecule 116 that may be incorporated into a SAM (HS-RX), where S is sulfur and R is a molecule that can be incorporated into a SAM. Is a species and X is a linker. The resulting molecule is coupled with a group 118 (HS-R-XM) that can deliver an electronic signal (or other group that can convert an electronic or electrochemical signal when attracted close to an electrode). It may be incorporated into SAMs on colloid 116, where M is a redox-active metal or transition metal. The magnetic beads or particles 120 may present a molecule 124 (eg, streptavidin) (BP2) that binds to BP1 に よ り 122, either directly or indirectly by simultaneously binding to a mutual target molecule (BP3). It may be derivatized.
[0095]
For example, the electronic or electrochemical signaling colloid 116 may have an enzyme cleavage site (ECS) and be derivatized with a thiol terminated with biotin. Magnetic beads may be derivatized with streptavidin. Magnetic beads and signaling colloids may be incubated with enzymes and drug candidates of interest. If a drug candidate modulates or inhibits the action of an enzyme, the site (eg, of a protein or peptide) will not be cleaved, and the signaling colloid will link to the magnetic beads via a biotin / streptavidin interaction. When the complex magnetically couples to the sensing electrode, a current peak results, and the assay becomes an increased signal detection assay. That is, the protein is adapted to bind to both colloid 116 and bead 120 via a SAM-forming species at one end and a binding partner 122 capable of binding to a binding partner 24 immobilized on bead 120. And its cleavage or its regulation may be monitored. The protein may be bound to the colloids and beads in various ways, including those described, or the colloid may also present a chelate that coordinates the metal, and the protein may be provided by a metal binding tag. May be. The invention is even more generalized, in which the colloid particles 116 and any entity that is adapted to bind to both non-colloidal structures, such as beads 120, have the ability to cleave the entity, and enzymes It may be presented in and bound to colloidal and non-colloidal structures in the presence of both candidate drugs that reduce activity. The non-colloidal structure may be a bead, as illustrated, or the electrode itself.
[0096]
BP1 may be the same as BP2 and / or BP3. For example, a thiol with ECS and BP1 (biotin) may bind free streptavidin (BP3) and then biotin (BP2) on magnetic beads. Alternatively, one may try to inhibit or promote the enzyme that adds the group of the molecule to the target molecule, i.e. the colloidal particles and the non-colloidal structure are adapted for binding to the non-colloidal structure, the substrate of the enzyme adapted for binding to the non-colloidal structure. It may be exposed to molecular species capable of binding to the substrate by enzymatic activity and to the enzyme for the substrate. This may further be performed in the presence of a candidate drug for regulating the activity of the enzyme, and the non-colloidal structure may be magnetic beads with colloidal particles having immobilized electroactive entities. Alternatively, the non-colloidal structure may be an electrode surface.
[0097]
Specifically, in FIG. 11, in such a case, the electronic or electrochemical signaling colloid 126 is derivatized by the molecule 128 to which the group is added by a chelate / metal / metal bond tag bond. The added particles, the added molecules 130, are stopped by the first binding partner (BP1) 132 (which may be biotin). Magnetic beads 12 present a binding partner for BP1 (BP2) 134, which may be streptavidin. The colloids, magnetic beads, added molecules, enzymes of interest and drug candidates are incubated and then attracted to the electrodes for analysis. Loss of signal as compared to control indicates that the drug candidate inhibits enzyme activity, while an increase in signal indicates activity of the enzyme enhanced by the drug.
[0098]
In accordance with the present invention, various SAMs can be used on various types of surfaces to present desired species, such as binding partners, signaling entities, etc., on the surface of articles, such as electrodes, colloid particles, etc. it can. One of skill in the art can select from various types of surfaces, functional groups, spacer moieties, and the like. An exemplary description can be found in US Pat. No. 5,620,850. This U.S. patent also discloses nitrilotriacetic acid, 2,2'-bis (salicylideneamino) -6,6'-demethyldiphenyl, and 1,8-bis (a-pyridyl) -3,6-dithiaoctane. Various metal binding tags that can be used are described.
[0099]
Various non-colloidal structures, including beads, are described above. Beads can include polymeric materials, agarose, tentagels, and / or magnetic materials. Polystyrene beads are very useful. The utility and advantages of these and other aspects of the present invention will be better understood with reference to the following examples. The following examples are intended to illustrate the advantages of the present invention, but do not exemplify the full scope of the invention.
[0100]
The following examples and experiments illustrate specific embodiments of the invention and should not be construed as limiting the invention to any particular embodiment.
In some of the embodiments described below, examples include SAM formation, collagen coating, cell proliferation, colloid formation, and alternating current voltammetry (ACV). For SAM formation, the microscope glass slide was covered with a layer of Ti followed by a layer of Au. Each electrode was 0.5 h at RT, 10% methyl terminated thiol (HS- (CH2) 15CH3), 40% tri-ethylene glycol terminated thiol, HS (CH₂)₁₁(CH₂CH₂)₃Incubated with 300 μl DMF solution containing OH, (Formula) and 50% MF-1. Thereafter, 2 ml of 400 μM tri-ethyleneglycol-stopped thiol was added to the scintillation vial containing the tip, and the vial was heat cycled in a water bath as follows: 2 minutes @ 55 ° C; 2 minutes @ 37 ° C; 1 minute @ 55 ° C; 2 minutes @ 37 ° C followed by 10 minutes at RT. The electrodes were then immersed in EtOH and subsequently washed with sterile PBS. They were placed in a biological safety cabinet under an LTV germicidal lamp for 1 hour to ensure sterility.
[0101]
For collagen coating, 200 μL drops of 0.005 mg / ml collagen in PBS were added to each electrode and incubated at 4 ° C. for 2 hours.
For cell growth, the electrodes were placed in cell growth flasks and a culture medium and a solution of human endothelial cells (HUVECs) displaying the specific cell surface receptor αVβ3 were added. The electrode and cell containing solution are CO₂Incubated at 37 ° C. for 24 hours in an incubator. Visual inspection at 100 × magnification showed that the cells adhered to the electrodes and exhibited a web-like spread, a phenotype of proliferation.
[0102]
For colloid production, 1.5 ml of commercially available gold colloid (Auro @ Dye) was centrifuged at high speed in a microfuge for 10 minutes to pellet. The pellet was resuspended in 100 μL of storage buffer (sodium citrate and tween-20). 100 μL of a dimethylformamide (DMF) solution containing 90 μM @ nitrilotriacetic acid (NTA) -thiol, 90 μM @ ferrocene-thiol, and 500 μM carboxy-stopped thiol. After incubation in the thiol solution for 3 hours, the colloid was pelleted and the supernatant was discarded. They were incubated in 100 μL of 400 μM tri-ethyleneglycol-stopped thiol in DMF for 2 minutes, 55 ° C .; 2 minutes, 37 ° C .; 1 minute, 55 ° C .; 2 minutes, 37 ° C., followed by 10 minutes at room temperature. . The colloid was then pelleted and 100 μL of phosphate buffered saline (PBS) was added. The colloid was then diluted 1: 1 with 180 μM @ NiSO4 in colloid storage buffer. 100 μL of 100 μM His-tagged peptide in PBS was added to 100 μL of colloid-presenting NTA-Ni (II) and incubated for 0.5 hours. To remove free unbound peptide, the colloid was then pelleted and the supernatant discarded. The colloid pellet was resuspended in 100 μL PBS. The colloid bound to any of the following: a) a peptide designed to bind to the αVβ3 receptor, HHHHHH (S₄G₁)₃GRGDSGRGDS; or b) an unsuitable peptide, HHHHHH-glutathione S-transferase (GST). Peptides containing the RGD motif have been shown to bind to the αVβ3 receptor on endothelial cells. It is believed that the RGD motif in vitronectin (a natural ligand of αVβ3) is involved in the interaction.
[0103]
ACV analysis was performed using a CH Instruments Instruments electrochemical analyzer. A three electrode system was used. A silver to silver chloride reference electrode was used with a platinum auxiliary electrode. Derivatized gold-coated tips were used as working electrodes. An overpotential of 25 mV was applied to the electrodes at a frequency of 10 Hz.
[0104]
【Example】
Example 1
This example describes an assay for screening for inhibitors of farnesyl protein transferase (FRT) and geranylgeranyl protein transferase (GGPT).
[0105]
We have devised an assay that can easily multiplex the following: 1) Incorporate wild-type target proteins and variants in the initial screening step; 2) Farnesyl / geranyl groups and enzymes by enzymes of interest Can directly detect and quantify the addition of analogs to the target protein; and 3) detect the different effects of drug candidates on enzyme activity. Our method of study is as follows: 1) NTA / Ni (II) group (to capture histidine-tagged proteins) and ferrocene-like transitions for electronic or electrochemical signaling. Histidine-tagged RAS protein (or peptide motifs or fragments from various mutants) is bound to colloids with SAMs that present both metals; 2) Modification of the farnesyl pyrophosphate derivative with biotin (FIG. 13); 3 4) Add the candidate inhibitor; 5) Add magnetic beads with streptavidin; 6) Attract magnetically to the sensing electrode and analyze electronically. The enzyme FPT will add a biotinylated farnesyl (or geranyl) moiety to the RAS immobilized on the signaling colloid. Magnetic beads with streptavidin bind to biotin, and the complex will be recruitable and detectable. The assay will be performed in parallel, with the RAS variant and drug candidate immobilized on the signaling colloid in microwells connected to a microelectrode array (FIGS. 14 and 15). The entire microelectrode array will be analyzed electronically to determine the different effects of drug candidates on enzyme activity for a particular target protein. If the drug candidate interferes with the enzyme that adds a farnesyl group to the target mutant protein, the electronic or electrochemical signal will decrease.
[0106]
This approach can be used to monitor the activity of the enzyme or to screen for drugs that modulate that activity. Also, non-histidine-tagged proteins can be bound to signal transducing colloids by standard coupling chemistry. Alternatively, the farnesyl derivative may be modified with a recognition group other than biotin, which is a binding partner of the group immobilized on the magnetic beads.
[0107]
Edelstein and Disefano report that a farnesyl pyrophosphate group modified with a photoexcitable cross-linking moiety can be added to peptide motifs derived from RAS by yeast FPT (Edelstein @ R. And @ Distefano @ M. (1997). Photoaffinity). labeling of yeast farnesyl protein transferase and enzymatic synthesis of a RAS protein incorporating a photoactive isoprenoid.Boy. Their purpose was to use this assay to show that the farnesyl derivative is recognized by FPT by cross-linking with FPT. This finding is consistent with the notion that farnesyl or geranyl moieties can be modified with biotin at the end remote from pyrophosphate without interfering with enzymatic activity or specificity.
[0108]
Example 2
In this example, cells were connected to a SAMs-derivatized gold-coated electrode. The cells remaining connected to the electrodes are then incubated with a solution containing colloidal gold, which colloids deliver receptor-specific ligands on the cell surface and electronic or electrochemical signals to the electrodes. Have been derivatized to present a redox-active metal together. After some incubation time, the electrodes were read by alternating current voltammetry (ACV). Positive interactions between the colloid-bound ligand and the cell surface receptor will also attract redox-active metals sufficiently on the colloid to close to the electrode and convert the electronic or electrochemical signal.
[0109]
More specifically, to facilitate the flow of electrons to the electrode, the electrode is 50% bis (ethylphenylthiol) (ie, C₁₆H₁₀Derivatized with SAMs to present 10% methyl head group in background of S). 40% triethylene glycol terminated thiol (HS (CH₂)₁₁(CH₂CH₂)₃OH) was included to facilitate the packing of the monolayer. It has previously been shown that cell growth can be maintained on HSC15-methyl arrested SAMs coated with collagen. HSCH₂C_FifteenCH₃And collagen are both insulating molecules and can inhibit the flow of electrons to the electrodes. For this reason, in this example, the degree of saturated carbon chain-collagen coverage was reduced, resulting in an island-like structure of proliferating cells adjacent to the more conductive molecular wire. Human endothelial cells (HUVECs), which are important for angiogenesis, presenting the cell surface receptor αVβ3, grew on the electrodes. SAM-coated gold colloids with receptor ligands and ferrocene moieties for electronic or electrochemical signaling were incubated with cell-presenting electrodes for some time and then analyzed by ACV.
[0110]
For ACV analysis, a 1 ml silicon gasket was immobilized on the cell presentation electrode. NTA-Ni colloid {100 μl and 100 μl} PBS pre-coupled with His-tagged RGD motif peptide were added to the gasket for incubation with the cell display electrode. After 15 minutes, the first ACV scan was recorded. Two consecutive scans were recorded at 15 minute intervals. Current output was plotted against voltage. The first scan produced a broad current bulge characteristic of the PBS buffer. The second and third scans generated different current peaks of 0.5 μAmps and 1.6 μAmps, respectively, at the characteristic ferrocene potential (780 mVolts), see FIG. Negative controls incubated with the same electrode presenting the same cells with 100 μL of NTA-Ni (II) colloid pre-bound with His-tagged GST resulted in a first scan similar to the electrode incubated with RGD-presented colloid . However, the broad bulge attenuated in the second and third scans and was identical (FIG. 17). The peak was clearly different from the 1.6 μAmps peak of the positive control, and no sharp peak was observed in the negative control.
[0111]
Example 3: Cell growth on conductive surfaces
This example describes the electronic detection of cell growth on a "conductive" surface that is not coated with collagen. Cells were grown on gold electrodes modified with sulfur-containing molecules, which in some cases were built up in a monolayer, but were not coated with collagen. Electrode modification was performed as described in the electrode fabrication section of Example 2, except that the electrode incubated with 100% candidate drug was not circulated in tri-ethylene glycol terminated thiol. Several electrodes were collected in the same cell growth flask and medium containing HUVEC cells was added. Electrodes and cells are CO₂Incubated for 24 hours in incubator. The surface was analyzed visually using 100 × magnification. Table 1 describes the characteristics of surface modification and subsequent cell attachment / proliferation. The surface showed low non-specific binding. However, once the cells were attached, cell growth was sufficient. A metal chelate that coordinates and binds a metal via SAM is displayed, the protein is bound to SAM by a metal binding tag on the protein, and the protein is attracted to the cell, whereby the cell can be easily immobilized on the SAM. . Pictures were taken to verify the results (not shown). Cells are treated with a peptide, HHHHHH (S), specific for the ferrocene moiety and the αVβ3 receptor₄G₁)₃GRGDSGRGDS; or as a negative control, incubated with a colloid presenting an inappropriate peptide, HHHHHH-glutathione S-transferase (GST) (described above). FIG. 18 shows that when cells grown on a 100% @ethynylphenylthiol (MF1) SAM-coated electrode were incubated with a colloid having a ligand specific for αVβ3 receptor (FIG. 18, solid line), a current peak occurred only. This shows that no incubation occurred (FIG. 18, dotted line) when incubated with colloids derivatized with the inappropriate peptide GST.
[0112]
FIG. 19 shows an electrochemical scan of an electrode derivatized with 25% @ 2-mercaptoethyl ether in a background of insulating tri-ethylene glycol terminated thiol. Cells derivatized with colloid displaying the RGD sequence peptide produced a small peak (FIG. 19, solid line), while cells incubated with colloid displaying the GST peptide did not produce a peak (FIG. 19, dotted line).
[0113]
[Table 1]

[0114]
Example 4: Modification of polymer beads with colloid particles via glutathione-S-transferase / glutathione linkage
See FIGS. 20 and 21. The following example demonstrates how a colloid that presents a target protein aggregates on non-colloidal particles that present a small molecule, drug candidate, peptide, protein, nucleic acid, or combination thereof that is a binding partner for the target protein. Is explained. Many combinatorial drug libraries are synthesized on non-colloidal particles or beads and can be mixed with colloidal particles displaying a medically relevant target species. Beads displaying a drug that is a binding partner of the target species can be easily identified because the colloid particles aggregate on the beads and color it red.
[0115]
The target protein, glutathione-S-transferase (GST), was histidine-tagged and immobilized on a SAM-coated colloid displaying NTA-Ni (the histidine tag-label binds to NTA-Ni). 30 μl of colloid presenting 40 μM NTA-Ni on the surface was added to 65 μl of 21.5 μM GST to give a final GST concentration of 14 μM in the solution. A small molecule glutathione that binds GST is available from Sigma-Aldrich, bound to agarose beads. Glutathione-coated beads were incubated with a solution of GST-bound colloid. Within a few minutes, GST bound to the glutathione beads, removing the colored colloid from solution and modifying the beads to red (FIG. 20). Beads displaying small molecules that did not bind to GST remained colorless when incubated with GST-bound colloids (FIG. 21). A second negative control, in which glutathione-coated beads were incubated with 30 μl of NTA-Ni colloid in the absence of GST, showed that the NTA-Ni colloid did not bind nonspecifically to the bead surface or to glutathione. Was.
[0116]
Example 5: Example of control of electron permeability of SAM
This example demonstrates the ability to form a SAM that includes enhanced electronic communication. The SAM is formed on a surface including a first tightly packed species and a mixture of a second species of different molecular structure that enhances the permeability of the SAM to electronic communication. Defects or openings are formed in the SAM so that the fluid to which the surface is exposed can be in electrical communication with the surface. Specifically, the sulfur-containing small molecule having a destructive structure with respect to the SAM as a whole was stably incorporated into the SAM, and enhanced permeability to electrons was shown. This example shows that the surface can be made relatively electrically conductive and can subsequently support cell growth.
[0117]
The water-soluble ferrocene derivative is an electrolyte solution: 500 μM NaClO₄In a 100 mM solution of ferrocenedicarboxylic acid. The working electrode was a SAM-derivatized gold-coated electrode containing different amounts of the 2-unit molecular wire (MF1). The peak height at the characteristic ferrocene potential was plotted as a function of molecular wire density. As a negative control, the gold-coated electrode was derivatized with an insulating SAM consisting of 100% tri-ethylene glycol terminated thiol. FIG. 22 shows that the "conductivity" of the SAM, or the ability of electrons in solution to penetrate the SAM, is a function of the density of the two-unit molecular wire integrated into the SAM. This system was used to test the conductivity of electrodes modified with a series of sulfur-containing compounds. Compounds were dissolved in DMF at 50% {candidate drug and 50%} tri-ethylene glycol terminated thiol. The electrodes were derivatized as described in Example 1. Table 2 tabulates the candidate drug and the height of the resulting current peak when analyzed by ACV. FIG. 22 shows two experimental results of a monolayer conductivity test as a function of monolayer destruction by poly (ethynylphenylthiol).
[0118]
SAMs were formed on gold chips from 500 μM triethylene glycol terminated thiol in DMF and either 500 μM mercaptobenzothiazole or 2-mercaptoethyl ether. The chip was fixed between a flat surface and a 1 ml volume silicon gasket. The ferrocene dicarboxylic acid solution was dissolved in 500 μM NaClO 4 and placed in a silicon gasket with an Ag / AgCl reference electrode and a Pt auxiliary electrode. The gold tip was bonded to the working electrode. The system was analyzed by ACV. The magnitude of the current peak obtained when the ferrocene in solution is in electrical communication with the electrode is a test compound that enhances the permeability of the SAM to the electron flow by creating defects in the SAM that communicates with the electrode. Was an indicator of their ability. FIG. 23 solely includes the insulating species tri-ethylene glycol terminated (CH2) 11-SH (++++ line) and the reportedly enhanced conductivity, poly-ethynylphenylthiol species (MF1) (solid line) alone. SAM overlay. Conducting poly-ethynylphenylthiol species are expected to be more conductive, since the same molecule with two additional repeating units has been reported to be conductive (Science 1997 {Bumm et al.,). As expected, alkylthiol SAMs do not produce a current peak in ferrocenedicarboxylic acid solutions, whereas SAMs consisting of 50% poly-ethynylphenylthiol do. FIG. 24 is a plot of FIG. 23 for SAMs with enhanced conductivity containing 2-mercaptoethyl ether (solid line) and mercaptobenzothiazole (++++ line) (dots indicate TEG-stopped thiol, asterisk indicates poly-ethynylphenylthiol). ), Indicating that the current peaks from the poly-ethynylphenyl SAM are orders of magnitude smaller than the peaks generated by SAMs containing 50% {2-mercaptoethyl ether, or SAMs containing 50%} mercaptobenzothiazole; Consistent with the notion that SAMs become permeable to electron flow, either by incorporating conductive species into the SAM, or by inserting "defect" or "open" molecules into the SAM.
[0119]
Table 2. The electrodes were derivatized with the following compounds in a tri-ethylene glycol terminated thiol background. Electrode fabrication was performed as described in Example 1. The “conductivity” or permeability of the surface was assayed by measuring the magnitude of the current peak resulting from the oxidation / reduction of ferrocenedicarboxylic acid in an electrolyte solution (0.5 M NaClO 4). The ability of each compound to resist non-specific binding was assayed by immersing each surface in BSA (bovine serum albumin) prior to measurement. Non-specifically adsorbed proteins will prevent conduction of electrons from the monolayer to the electrodes.
[0120]
[Table 2]

[0121]
Example 6: Detection of protein-protein interaction
This example demonstrates the utility of colloid particles with immobilized signaling entities and immobilized proteins (see FIG. 27).
[0122]
Histidine-tagged glutathione-S-transferase (GST-His) was linked to an NTA-SAM-coated colloid displaying 40 μM ΔNTA-Ni and 100 μM ferrocentiol. Commercial magnetic beads displaying protein A were coated with anti-GST antibody to a binding volume of 1/10, added to GST-colloid in a ratio of 1: 5, and placed on top of a magnet with 50% ΔMF- Measured on 1 @ SAM coated electrodes. The magnet attracted the magnetic beads to the electrode surface, forming a thick, visible precipitate. The GST-colloid interacted with the GST antibody on the magnetic beads and was attracted to the electrode surface, producing a current peak at about 280 mV. Two negative controls were used, one in which GST did not bind to the colloid surface, and another one in which the GST antibody did not bind to the magnetic beads. None of the negative controls produced a current peak. FIG. 25 plots the results of this example. The solid line represents the interaction between GST-His-presenting colloids and anti-GST / Abs on magnetic beads. ○ represents magnetic beads displaying antibodies incubated with colloids not displaying GST. ● represents beads that do not display antibodies incubated with colloids that display GST.
[0123]
Example 7: Cell detection
This example includes a mixture that includes a molecular species that enhances electrical communication across the SAM, and forms a defect in the SAM to form a SAM on the surface where the fluid to which the surface is exposed can be in electrical communication with the surface. Together with the benefits of linking a colloid with an immobilized signaling entity to a protein. The protein is then immobilized on cells connected to the surface of the SAM-presenting electrode. The defect in this case is caused by the bulk of the molecule incorporated in the SAM containing the phenyl ring.
[0124]
HUVEC cells were suspended in medium and placed in a flask with SAM coated on a gold surface. The SAM contained 50% linear thiol, and 50% 2-unit poly (ethynylphenyl) thiol (MF1). 5 μl of 8.4 mM {RGD-His peptide solution} was added to the medium, and the cells were incubated overnight at 37 ° C. to adhere to the electrode surface. Approximately 16 hours later, 100 μl of SAM-coated colloid presenting NTA for capturing the RGD-His peptide and ferrocene for signaling was added to the cells and incubated at room temperature for 20 minutes. The electrodes were then washed with buffer and any unbound colloid was washed off and measured. Current peaks were recorded between 200 and 250 mV. The negative control was cells incubated with His-GST, an inappropriate protein that could not bind to the cells. Colloids were added to the negative control, the electrodes were washed with buffer and measurements were taken. No peak was seen in the negative control. FIG. 26 shows the peak generated (solid line) when a colloid presenting a his-tagged ligand for a ferrocene signaling entity and a cell surface receptor was carried to the electrode surface by cell / surface interaction. Diamonds represent negative controls, colloids displayed inappropriate proteins selected to not bind to cell surface receptors.
[0125]
Example 8: Detection of ligand-receptor interaction for unmodified ligand
This example demonstrates the ability to measure protein / ligand interactions in the absence of SPR without labeling either protein or ligand. Specifically, exposing the ligand to a protein that is presumed to interact with the ligand by forming a SAM containing the ligand and ferrocene on the electrode surface, where the ligand has an electrical activity signal that is transmitted to the protein. There is a certain close relationship with the electroactive entity, ferrocene, which depends on the degree of proximity.
[0126]
The inventors have found that the characteristic oxidation potential of a ferrocene derivative may be shifted based on the local environment chemistry of the ferrocene derivative. As the protein is transported near the ferrocene derivative, a second current peak appears at the altered potential. The size of this changed peak is proportional to the density of the protein in the system.
[0127]
A series of gold-coated electrodes were derivatized with a heterogeneous SAM containing a constant density of conductive molecular wire thiols and a variable density of methyl terminated thiols in a tri-ethylene glycol terminated thiol background. The following has been previously shown: 1) tri-ethylene glycol terminated SAMs resist non-specific adsorption of proteins; and 2) methyl terminated SAMs bind proteins, especially collagen. Thus, when the electrodes were incubated with collagen in solution, it was thought that the density of nonspecifically adsorbed collagen would increase as the density of methyl-terminated thiols increased. SAMs containing 2%, 4%, 10% and 15% methyl terminated thiols were formed on gold coated electrodes. One half of the electrode was incubated with 0.005 mg / ml collagen in PBS at 4 ° C. for 2 hours, and the other half was incubated with PBS only. The electrodes were analyzed by alternating current voltammetry (ACV) together with a water-soluble ferrocene derivative and ferrocene dicarboxylic acid dissolved in an electrolyte solution (0.5 M NaClO 4). Alternating oxidation / reduction of ferrocene at a characteristic potential produces a current peak. The magnitude of this current peak and the potential at which it occurred were recorded. For the low-density methyl-terminated thiol, the current peak occurred at about 450 mV; its size and location did not appear to be affected by the presence of the protein, collagen. At high densities of methyl-terminated thiols of 10% or more, a second current peak occurs at about 300 mV only in the presence of protein collagen. FIG. 28 shows a high density methyl terminated thiol electrode in the presence of ferrocene dicarboxylic acid. The solid line represents an electrode with collagen bound to methyl thiol. The hydrophobic environment created by collagen on the electrode surface shifts the oxidation potential of ferrocene, and two peaks are seen. Closed circles represent high density methyl-terminated thiol electrodes that are not bound to collagen and therefore do not affect ferrocene oxidation.
[0128]
This approach can be used to detect the presence of unmodified proteins, peptides, cells as follows: Mixed SAMs make ferrocenedicarboxylic acid SAMs linked to alkyl thiols permeable to electron flow. And a thiol that terminates at the binding partner of the target species (the binding partner may be histidine-tagged and bound to the electrode by binding to the NTA-Ni moiety in the SAM). If the target species is present in the sample solution, it will bind to its binding partner presented on the SAM. The presence of a protein near the SAM surface alters the chemical environment around ferrocenedicarboxylic acid, shifting its oxidation potential to a lower potential. The characteristic oxidation potential shift of the immobilized redox-active metal indicates the presence of the target species.
[0129]
No effect was observed when these experiments were performed with ferrocene derivatives without polar substituents on the ring. Therefore, for this approach to work, it is crucial that the immobilized redox-active metal is in a polar environment and that the immobilized binding partner (bound to the SAM by a thiol) is as small as possible. is there.
[0130]
Example 9: ELISA
A technique well known to those skilled in molecular or cell biology is the enzyme-linked immunosorbent assay (ELISA) (Current Protocols in Molecular Biology, Volume 2, Immunology 11.2, 1996, copyright owner Wiley and Sons Inc.). 1994-1998). An important issue with ELISA is sensitivity. Enzyme signaling occurs in a 1: 1 ratio; an enzyme linked to one antibody signals the presence of one antigen. Thus, microgram quantities of antigen are required for effective ELISA, but are not valid in all cases. This makes ELISA unsuitable for detecting antigens on cell surfaces that are expressed or displayed at low levels. Another problem with ELISAs is that at low antigen concentrations, the assay is very time consuming. The amount of enzymatic hydrolysis is directly proportional to the time of hydrolysis. In general, when performing an ELISA, the target species is directly or indirectly linked to the plastic support layer. The presence of the immobilized species is then detected by binding to a "second" antibody that also has signaling capabilities; the second antibody is typically horseradish peroxidase (HRPO). , An enzyme such as alkaline phosphatase (AP), or a fluorescent tag capable of performing a resulting color-changing reaction (detected by a spectrophotometer) on an added substrate, or a fluorescent labeled tag detectable by a fluorometer Usually coupled (e.g., "Localization of a, passive, transfer, human, recombinant, monoclonal, antibody, to, herpes, simplex, virus, Glycoprotein" nerve fibers and sensory neurons in vivo ", Sanna PP, Deerinck TJ, and Ellisman MH, 1999, see the Journal of Virology Oct.Vol 73 (108817-23)). In many cases, a mouse antibody is used as the specific recognition antibody so that not all antibodies need to be conjugated to the enzyme for convenience, followed by addition of an enzyme-conjugated rabbit-anti-mouse antibody.
[0131]
Using the techniques described herein greatly enhance the sensitivity of the ELISA and detect the presence of a target species immobilized using a natural ligand (protein or peptide) or drug candidate as a probe molecule. be able to. The presence of the immobilized species is detected by binding to a colloid-bound ligand that also presents multiple horseradish peroxidases (HRPO) or alkaline phosphatase (AP). Enzymes may be favored for colloids by various means, including a histidine tag directly linked to the enzyme, or by coupling a mouse-anti-goat enzyme-conjugated antibody to a goat antibody that binds to the colloid via histidine-tagged protein G. (Akerstom, B., Nielson, E., Bjorck, L. Journal of Biological Chemistry, 1987 Oct. 5 Vol. 262 (28); pgs. 13388-91 and Fahnes. Alexander, P., Nagle, J. and Filfila, D. (1986) Journal of Bacteriology Vol. 167, 870-880). By binding the ligand co-immobilized to the colloid with multiple enzymes to the target species instead of the second antibody, the ratio of signaling molecule to binding event is increased by orders of magnitude. Binding of one antibody or ligand on the colloid to the antigen displayed on the ELISA plate results in indirect binding of thousands or millions of enzymes. Alternatively, known species can be intentionally bound to the wells of the 96-well upper plate, and colloidally examined, each presenting a different drug candidate with a signaling enzyme. At present, this is not possible with current ELISA technology. This is because each drug candidate cannot bind to the enzyme. Alternatively, a natural ligand for the immobilized target may be presented on the colloid with the signaling enzyme, and drug candidates may be added to each well of the plate to disrupt the interaction. Unbound colloids, and thus their signals, are lost in the washing step. The target of the antibody- or ligand-presenting colloid may be a cell or protein bound directly or indirectly (by another antibody or ligand) to the ELISA plate. The advantage of this modification of the ELISA is not only sensitivity but also efficiency. Since hundreds of signaling enzymes remain bound to one antigen by colloids, substrate hydrolysis will occur faster and a proper interpretation will take less time.
[0132]
Example 10: Visual Detection of Interaction of Cell Surface Receptor with Colloid-Immobilized Ligand and Its Inhibition
This example was performed in the manner described in Example 3, except for the following. Cells were grown on multi-well plates. After the interaction, macroscopic examination showed selective modification of the cells by colloids at the location on the cells where the receptor was expressed. FIG. 29A shows a control in which no binding occurs. Peptides of any sequence were used. FIG. 29B shows the selective modification of cells by colloids at the location on the cell where the protein is expressed.
[0133]
Example 11: Detection of binding interaction between one binding partner non-specifically bound to a solid support and another partner bound to a colloid
The protein, specifically streptavidin (Promega), was non-specifically linked to a solid support, specifically Q-Sepharose. Its binding partner, biotin, was covalently linked to a thiol incorporated into a self-assembled monolayer on colloidal particles. When the colloid particles were exposed to the beads, they were immobilized by the binding between biotin and streptavidin, and the beads were colored red. FIG. 30A is a photographic copy of a computer digitized microscopic image of a bead modified by binding interaction (red in nature). FIG. 30B (control) is a similar image of unmodified beads; similar beads (streptavidin-bound) were exposed to colloids displaying inappropriate proteins.
[0134]
This example also importantly describes a self-assembled monolayer containing biotin on colloidal particles. Biotin SAMs were formed on colloids as follows. A solution containing 580 μM @ COOH-stopped thiol and 20 μM biotin-stopped thiol in DMF was made. The thiol was C11. Colloid production was performed as previously described, and the described 20 μM biotin and 580 μM @COOH thiol DMF solution was incubated with the colloid. Other steps were similar to those described above.
[0135]
Example 12: ELISA with multiple signaling of a single binding event
This example was performed similarly to a typical ELISA, with the following exceptions / details: detergents and surfactants were omitted from the washing step. GST was immobilized on a multiwell plate. Mouse anti-GST antibody was added to the wells and allowed to bind to GST. After a general washing step, a colloid displaying histidine-tagged protein G was added to the first set of wells (for experiments) and allowed to bind to anti-GST. A second set of wells (control) was not exposed to the protein G immobilized colloid. After another general washing step, all wells were exposed to horseradish peroxidase-conjugated goat anti-mouse antibody. All were then exposed to horseradish peroxidase substrate and sulfuric acid. The reaction proceeded in the usual way. Plates were analyzed on a multiwell spectrophotometer reader.
[0136]
The results of this example show that as horseradish peroxidase binds to multiple G positions on the colloid particles, the experimental wells signal six orders of magnitude greater than the control wells. FIG. 31 is a bar graph showing this six digit increase. This example illustrates the signaling of a single binding of a first biological or chemical agent to a second biological or chemical agent by multiple signaling entities. Signaling of multiple entities occurs simultaneously as they bind to the colloid particles.
[0137]
Example 13: Linking unmodified chemical or biological molecules to a self-assembled monolayer presented on a colloid surface, specifically linking streptavidin to a colloid
This example provides a technique for attachment of any essentially amine-containing entity to a surface that can present a carboxylic acid or salt thereof. The technique can be used to link entities to colloids by linking to a self-assembled monolayer formed on the colloid surface. Molecules linked to the self-assembled monolayer need not include any specific chemical functional groups, such as affinity tags, prior to binding. The only requirement is that it contain at least one primary amine, as found in many amino acids. Thus, the technique is particularly suitable for covalently attaching proteins or peptides having primary amino groups to a colloid presenting a carboxylic acid on the surface.
[0138]
Techniques for forming self-assembled monolayers on colloids have been described previously. The following specific details and proviso are added to these techniques.
[0139]
The protein was bound by forming an amide bond between the carboxylate (bound by SAM) on the colloid surface and the amine residue of the peptide. The amide bond was formed by applying a modified EDC / NHS coupling protocol. Successful coupling was tested by mixing a colloid presenting streptavidin with a colloid presenting a biotin group (via biotin SAM, described above). If streptavidin binds successfully, the two types of colloid will aggregate, the suspension will change color, and finally the colloid will precipitate, leaving a clear solution.
[0140]
The colloids used in the coupling procedure were prepared as described elsewhere and incubated in a thiol solution containing 540 μM (micromolar) COOH-stopped thiol, 60 μM {NTA-stopped thiol, and then 400 μM} ethylene glycol Formed by circulating heating in a stopped thiol. 50 μl colloid in PBS was treated with 50 μl 10 mM EDC, 40 mM NHS in water. After 7 minutes, the colloids were spun down by centrifugation, the liquid was removed and then resuspended in 500 μl (microliter) of 0.1 mg / ml {streptavidin} in phosphate buffer at pH 6.4. After 1.5 hours, the unreacted sites of the colloid were blocked by adding ethanolamine and the colloid was centrifuged to precipitate, then resuspended in 100 μL of pH 6.4 phosphate buffer and the excess, Unbound streptavidin was washed away. The colloid was then centrifuged again and precipitated, and resuspended in 50 μl PBS. To a mixture of 20 μL of phosphate buffer and 15 μl of biotin-presenting colloid, 15 μl of the previously prepared streptavidin-presenting colloid was added. This suspension was compared to a mixture of 35 μl phosphate buffer and 15 μl biotin presenting colloid. Addition of streptavidin-presenting colloid caused a rapid color change from red to blue. After 〜１０10 minutes, the mixture of biotin colloid and streptavidin colloid became clear and a precipitate was seen at the bottom. The mixture without biotin colloid had no visual change and remained red.
[0141]
The protocol described above is applicable to the attachment of essentially any chemical or biological molecule, including amines, to a self-assembled monolayer on a colloid. The following modifications may be made as appropriate to ensure binding of one molecule to a single colloid, rather than binding to multiple colloids that result in aggregation. The concentration of the colloid in solution may be reduced while maintaining the concentration of the desired bound molecule and the concentration of the EDC / NHS reactant. One skilled in the art may make other minor modifications to the various molecules. Amine-containing molecules may be selected by one of skill in the art and include, but are not limited to, proteins, synthetic molecules, peptides, derivatized nucleic acids, and derivatized or naturally occurring biology, including other amines. Target molecules. One skilled in the art will appreciate that a variety of species may be synthesized with or linked to an amine or amine-containing species to enable linking to a colloid according to this aspect of the invention.
[0142]
Those skilled in the art will readily appreciate that all parameters described herein are exemplary and that the actual parameters will depend on the particular application for the method and apparatus in which the invention is used. Will. Therefore, the foregoing embodiments are presented by way of example only, and that the invention may be practiced differently from the specific description within the scope of the appended claims and their equivalents. You should understand.
[Brief description of the drawings]
FIG.
FIG. 1 depicts a ligand linked to a metal-containing compound, such as ferrocene, via a dendrimer or polymer that interacts with the cognate cell surface receptor for the ligand on intact cells.
FIG. 2
FIG. 2 depicts a metal-containing compound, such as ferrocene, bound to a particle that interacts with the receptor for the cognate ligand of the ligand on intact cells.
FIG. 3
FIG. 3 shows proteins assembled into self-assembled monolayers on colloids, which also present redox-active metals for electronic or electrochemical signaling. Cells bearing the target receptor are incubated with a colloid bearing a cognate ligand for the receptor of interest and subsequently attracted electrophoretically or magnetically to the sensing electrode. The figures are not to scale, and the colloids may be made in various sizes, ranging from 20 gold atoms to several hundred nanometers in diameter.
FIG. 4
FIG. 4 depicts cells with MUC-1 incubated with an electronic signaling colloid bearing the tumor marker MUC-1 specific antibody, DF-3. The antibody binds to the colloid via a His-tagged protein G that binds to NTA / Ni (II) groups incorporated into a self-assembled monolayer on the colloid.
FIG. 5
FIG. 5 shows one embodiment of an assay that can be used to screen the ability of individual members of a drug library to inhibit interaction with cell surface receptors.
FIG. 6
FIG. 6 shows that by tying microwells (which may be disposable) to microelectrode arrays and robots, drug screening assays can be easily multiplexed. Analysis of the electrode array can be performed using a modified electrochemical analyzer that allows simultaneous analysis of multiple electrodes. The electrodes can be designed to be inserted into microwells (see arrows).
FIG. 7
FIG. 7 shows drug candidates that can be synthesized on particles that also have an electroactive signaling moiety or that can be covalently linked to them. Drug candidates linked to the signaling particles can be incubated with cells displaying the receptor of interest and control cells that do not express the receptor of interest. The signal will increase as a result of the drug-target interaction in this assay.
FIG. 8
FIG. 8 shows a tissue sample linked to a flexible, semi-permeable support by interaction with a peptide containing an RGD motif. The sample is then incubated with an electroactive signaling colloid having a ligand for the cell surface receptor of interest. The sample is washed and then connected to the microelectrode array. The electrode array can be manufactured such that the area of the electrode corresponds to the area of the cell. The sample can be further characterized by ACV and subsequently linked to histopathology.
FIG. 9
FIG. 9 shows an easily multiplexable protein-protein interaction assay based on generic recruitment and signaling particles. Self-assembled monolayers with NTA / Ni (II) ligands that selectively capture His-tagged proteins are formed on signaling colloids and gold-coated magnetic beads. Next, each particle type is briefly incubated with a different His-tagged protein. The magnetic field then attracts the magnetic beads to the sensing electrode. Only when the electroactive label on the colloid (which may be a redox active moiety) is carried to the electrode through the interaction between the first and second proteins, the signal is converted. Note that non-His-tagged proteins can also bind to common particles with self-assembled monolayers that display carboxy-terminated thiols.
FIG. 10
FIG. 10 shows a biotin terminated thiol-derivatized electroactive signaling colloid with an enzyme cleavage site (ECS). The magnetic beads are derivatized with streptavidin, and the magnetic beads and signaling colloid are then incubated with the enzyme and drug candidate of interest. If the drug candidate inhibits the action of the enzyme of interest, the site will not be cleaved and the signaling colloid will remain bound to the magnetic beads by biotin-streptavidin interaction. When the resulting complex magnetically binds to the sensing electrode, a current peak is obtained and the assay will be a signal increase detection assay.
FIG. 11
FIG. 11 shows a research method for screening drugs that modulate enzyme activity. In this scheme, the protein that is the target of the enzyme modification is linked to a colloid that also has an electroactive moiety. The chemical groups that the enzyme adds to the target protein are labeled with biotin. When the enzyme function is appropriate, the biotin-labeled compound is added to the protein on the signaling colloid. The complex thus obtained is further bound to streptavidin-coated magnetic beads. The complex is further electromagnetically attracted to the sensing electrode. Drug candidates may be added to the assay. Loss of signal compared to control would indicate that the drug candidate inhibited the activity of the enzyme, while an increase in signal would indicate that the drug increased the activity of the enzyme.
FIG.
FIG. 12 shows the chemical structures of the farnesyl group, farnesyl pyrophosphate, and geranyl-geranyl pyrophosphate.
FIG. 13
FIG. 13 shows the modification of a farnesyl- or geranyl-like moiety with a recognition group such as biotin.
FIG. 14
3 shows a farnesyl pyrophosphate derivative having a biotin moiety. Farnesyl protein transferase (FPT) enzymes catalyze the addition of farnesyl derivatives to RAS protein variants. When using magnetic beads with streptavidin to recruit the complex to the sensing electrode, the activity of the enzyme, or the ability of the drug candidate to modify the enzyme activity, can be quantified by measuring the current output.
FIG.
FIG. 15 shows that drug candidates may be evaluated for their ability to modulate enzyme activity. The spatially addressable microwell contains RAS protein (wild-type or mutant), FPT enzyme, biotin-modified addition group (farnesyl derivative), magnetic beads with streptavidin, and plus or minus drug candidates. The electrodes can be further analyzed by various electronic and electrochemical techniques, including alternating current voltammetry (ACV).
FIG.
FIG. 16 shows a plot of an ACV analysis of the interaction of an RGD-containing ligand with cells growing on a collagen-coated electrode.
FIG.
FIG. 17 shows a plot of an ACV analysis of the interaction of a (negative) control ligand with cells growing on a collagen-coated electrode.
FIG.
FIG. 18 shows a plot of an ACV analysis of cells growing on a metal support containing only a single type of conductive molecule.
FIG.
FIG. 19 shows a plot of an ACV analysis of the interaction of an RGD-containing ligand with cells growing on a metal support containing a heterogeneous monolayer of insulating and conducting molecules.
FIG.
FIG. 20 is a photocopy of a photograph of a colloid-modified bead, specifically a colloid bound to the bead by a protein / protein interaction.
FIG. 21
FIG. 21 is a photocopy of a photograph of the negative control of the experiment shown in FIG.
FIG. 22
FIG. 22 shows a plot representing the increase in conductivity of the monolayer with increasing proportion of conductive molecules (in the grass of insulating species).
FIG. 23
FIG. 23 shows a 2 unit molecular wire, a monomolecule of a conductive self-assembled monolayer containing poly (ethynylphenyl) thiol, MF1 for a negative control of a non-conductive self-assembled monolayer measured with a ferrocene solution. 3 shows an ACV analysis plot of the conductivity of the layer.
FIG. 24
FIG. 24 shows a plot of an ACV analysis of the conductivity of the monolayer of the two self-assembled monolayers of FIG. 23, measured against a higher conductivity monolayer, measured with the ferrocene solution.
FIG. 25
Figure 3 shows ACV analysis of protein / protein interaction as measured by binding of colloids to magnetic beads.
FIG. 26
FIG. 26 shows an ACV representation of redox signaling of protein immobilization to the cell surface and increased electronic communication across the self-assembled monolayer relative to controls.
FIG. 27
FIG. 27 is a graphical description of the assay, and a plot of protein / protein interaction as measured by colloid binding to magnetic beads is shown in FIG.
FIG. 28
FIG. 28 shows an ACV analysis plot of electrodes containing surface-bound proteins and their effect on ferrocene oxidation potential (solid line) and negative control (dotted line).
FIG. 29
FIGS. 29A and 29B are photocopy of photomicrographs (40 × magnification) of cells (29B) and control (29A) selectively colloidally modified at the site where the receptor is expressed.
FIG. 30
FIGS. 30A and 30B show computer digitized microscopy of nonspecifically binding protein-containing beads (30A) and control (30B) modified by colloidal particles with protein binding partners on self-assembled monolayers. This is a photo copy of the image.
FIG. 31
FIG. 31 is a bar graph showing six orders of magnitude improved signaling due to signaling a single binding event by multiple signaling entities.
FIG. 32
Figures 32A and 32B show an ACV plot of an experiment displaying the use of gravity to recruit signaling entities showing binding interactions to the surface, and controls.

Claims (204)

A method comprising providing the colloid particles with the ability to be immobilized with respect to the non-colloid structure; and measuring the immobilization of the colloid particles to the non-colloid structure. 2. The method of claim 1, wherein the colloidal particles include an auxiliary signaling entity. The auxiliary signaling entity is a dye, dye, electroactive molecule, chemiluminescent moiety, electrochemiluminescent moiety, fluorescent moiety, upregulating fluorophore, or enzyme-linked signal including horseradish peroxidase and alkaline phosphatase 3. The method of claim 2, comprising a transmitting portion. 4. The method of claim 3, wherein the signaling entity comprises a metallocene. 5. The method of claim 4, wherein the signaling entity comprises ferrocene or a ferrocene derivative. 2. The method of claim 1, comprising binding the plurality of colloidal particles to the non-colloidal structure and measuring the binding of the plurality of particles to the non-colloidal structure. 7. The method of claim 6, wherein the plurality of colloid particles include an auxiliary signaling entity. The auxiliary signaling entity is a dye, dye, electroactive molecule, chemiluminescent moiety, electrochemiluminescent moiety, fluorescent moiety, up-regulating fluorophore, or enzyme-linked signaling, including horseradish peroxidase and alkaline phosphatase The method of claim 7, comprising a portion. A biological or chemical agent that binds or is adapted to bind to a non-colloidal structure, and a binding partner of a biological or chemical agent that binds or is adapted to bind to a particle. The method of claim 1, further comprising providing, wherein the providing comprises binding the particles to the non-colloidal structure by the agent and the binding partner. 2. The method of claim 1, wherein the non-colloidal structure is a bead. The method of claim 10, wherein the beads comprise a polymeric material, agarose, tentagel, and / or a magnetic material. The method according to claim 11, wherein the beads are polystyrene beads. 10. The method of claim 9, comprising binding the agent to the non-colloidal structure, binding the binding partner to the particle, and binding the agent and the binding partner to each other. 14. The method of claim 13, comprising binding the agent and the binding partner biologically to each other. 10. The method of claim 9, wherein the biological or chemical agent is a drug candidate and the binding partner is a target of the drug candidate. 16. The method of claim 15, wherein the non-colloidal structure is a bead. 16. The method of claim 15, wherein the non-colloidal structure is an essentially planar substrate or chip surface. 10. The method according to claim 9, wherein the biological or chemical agent is a nucleic acid sequence. 10. The method according to claim 9, wherein the biological or chemical agent is a peptide and the binding partner is a binding partner of the peptide. 10. The method according to claim 9, wherein the biological or chemical agent is a protein and the binding partner is a binding partner of the protein. The method of claim 1, wherein the colloidal particles have an immobilized ligand and the non-colloidal structure has a binding partner for the ligand, wherein the non-colloidal structure has a non-colloidal structure in the presence of a candidate drug for blocking ligand binding to a target. Such a method, comprising providing the colloid particles with the ability to bind to a colloid structure. The non-colloidal structure is a bead, a plurality of beads, a plurality of biological or chemical agents adapted for binding to the beads, a plurality of particles, and a biological or chemical agent adapted for binding to the particles. 2. The method of claim 1, further comprising providing a plurality of binding partners, wherein at least some of the agents and the binding partners are presumed to have the ability to bind to each other. Exposing at least some of the beads to at least some of the particles, and measuring the immobilization of the particles on the beads. 23. The method of claim 22, wherein the biological or chemical agent is a drug candidate, and the binding partner is a target of the drug candidate, wherein the two have different immobilized drug candidates. Providing at least first and second beads at different locations, exposing the plurality of particles to at least two beads, and distinguishing binding of the particles to the first and second beads. Said method. 24. The method of claim 23, wherein the at least two beads are separately located in at least two different wells of a multi-well plate. 23. The method of claim 22, wherein the biological or chemical agent is a drug candidate, and the binding partner is a target of the drug candidate, wherein at least two beads having the drug candidate at two different locations. Providing, exposing the first bead to a first set of colloid particles having a first target of the drug candidate, and exposing the second bead to a second set of colloid particles having a second target of the drug candidate. Exposing the second set of particles to the first bead, and discriminating the binding of the second set of particles to the second bead relative to the binding of the first set of particles to the first bead. . 10. The method of claim 9, comprising measuring the immobilization of the particles in the non-colloidal structure by measuring a change in the spectrum of electromagnetic radiation that is absorbed or transmitted in interaction with the particles. 10. The method of claim 9, comprising measuring the immobilization of the particles in the non-colloidal structure by visual inspection. Providing a plurality of beads and an agent bound to the beads, a plurality of particles and a binding partner bound to the particles, and exposing the particles to the beads, and measuring immobilization of the particles on the beads, 23. The method according to claim 22. 10. The method of claim 9, wherein at least one of the agent or binding partner is each adapted for binding to a non-colloidal structure or particle by affinity tag / binding partner binding. 10. The method of claim 9, wherein at least one of the agent or binding partner is adapted for binding to a non-colloidal structure or particle by a metal binding tag / metal / chelate linkage, respectively. 31. The method of claim 30, wherein at least one of the agent or binding partner has a chelate to coordinate a metal immobilized thereon, and at least one of the agent or binding partner is derivatized with a polyamino acid tag. . 10. The method of claim 9, wherein at least one of the agent or binding partner is adapted for binding to a non-colloidal structure or particle, respectively, by a self-assembled monolayer. 10. The method of claim 9, wherein at least one of the agent or binding partner is adapted for binding to a bead or particle, respectively, by a complementary nucleic acid sequence pair. 10. The method of claim 9, wherein the binding partner is adapted for binding to the particle by a glutathione / glutathione-s-transferase ligand interaction. Providing at least a first and a second non-colloidal structure comprising a polymer bead and at least a first and a second agent respectively associated with the first and the second bead;
Providing a plurality of colloid particles each having a putative binding partner of the first and / or second agent immobilized on the colloid particles;
10. The method of claim 9, comprising exposing the beads to the particles; and distinguishing binding of the particles to the second bead relative to the first bead. The first and second agents bound to the first and second polymer beads are presumed to undergo a biological or chemical interaction with the binding partner, and the step of distinguishing between the first agent and the binding partner. 36. The method of claim 35, comprising distinguishing a biological interaction between the second agent and the binding partner. 10. The method of claim 9, comprising:
Providing a non-colloidal structure, including a plurality of beads, each with an immobilized agent;
Providing a first set and a second set of colloidal particles, wherein the first set has a first putative binding partner of the agent immobilized thereon, respectively, and the second set has immobilized thereon. Each having a second putative binding partner of the identified agent;
Exposing at least a first bead to a first set of particles and at least a second bead to a second set of particles;
Distinguishing the binding of a second set of particles to a bead relative to the binding of a first set of particles to a first bead. The first and second putative binding partners are presumed to have a biological or chemical interaction with the agent, and the step of distinguishing is between the agent and the first putative binding partner. 38. The method of claim 37, comprising identifying a biological interaction between the putative binding partners of the. 2. The method according to claim 1, wherein the non-colloidal structure is a cell. Further comprising providing a ligand for a cell surface receptor or protein adapted for binding to the particle, wherein the providing step comprises binding the particle to the cell with a ligand that interacts with the receptor or protein. Item 39. The method according to Item 39. 40. The method of claim 39, wherein the ligand is adapted to bind to the particle via a self-assembled monolayer. 41. The method of claim 40, wherein the ligand is a peptide, protein, antibody, enzyme, or small molecule. 41. The method of claim 40, wherein the ligand is adapted to bind to the particle by affinity tag / binding partner binding. 41. The method of claim 40, wherein the ligand is adapted to bind to the particle via a metal binding tag / metal / chelate bond. 43. The method of claim 42, wherein the ligand has a polyamino acid tag and the particle has an immobilized chelate that coordinates the metal. 46. The method of claim 45, wherein the chelate is nitrilotriacetic acid. 46. The method of claim 45, wherein the particles have a self-assembled monolayer comprising nitrilotriacetic acid. 46. The method of claim 45, wherein the particles have a self-assembled monolayer that includes an immobilized chelate. 41. The method of claim 40, comprising exposing the ligand and the particle to a cell, binding the ligand to the particle, and measuring binding of the ligand to a receptor or protein. 40. The method of claim 39, comprising measuring the immobilization of the particle on the cell by measuring a change in the spectrum of the electromagnetic radiation that is absorbed or transmitted by interacting with the particle. 40. The method of claim 39, comprising measuring the immobilization of the particles on the cells electronically. 52. The method of claim 51, comprising measuring immobilization of the particles on the cells by alternating current voltammetry. 51. The method of claim 50, comprising measuring the immobilization of the particles on the cells by visual inspection. 40. The method of claim 39, comprising:
Providing cells that present receptors or proteins on their surface;
Providing colloidal particles; and binding the colloidal particles to a receptor or protein. 55. The method of claim 54, wherein the colloid particles include an auxiliary signaling entity. A non-colloidal structure is a cell whose surface exposes a receptor or protein, providing a ligand for the receptor or protein that is adapted for binding to the particle, and hindering the interaction between the ligand and the receptor or protein 2. The method of claim 1, further comprising exposing the ligand and the particle to the cell in the presence of a candidate drug for; and measuring the binding of the particle to the cell. 57. The method of claim 56, wherein the measuring step comprises measuring the inhibition of binding of the particle to the cell, indicating the effectiveness of the candidate drug in interfering with the receptor or protein / target protein interaction. Method. Providing at least the first and second cells at different locations;
Ligand and colloid particles for a cell receptor or protein of the first cell, ligand adapted for binding to the particle, and first candidate for disrupting the interaction between the ligand and the receptor or protein in the first cell Exposing to a drug; and adding a ligand and a colloid to a second cell, and a second candidate drug to disrupt an interaction between the ligand and a receptor or protein; Distinguishing the binding of the particles to a second cell;
57. The method of claim 56, comprising: The method of claim 1, wherein the non-colloidal structure is a cell that exposes a receptor or protein on its surface, further comprising:
Providing at least two cells at different locations;
Exposing each cell to a different target protein for a cell receptor or protein and a colloidal particle adapted for binding to the respective target protein; and Measuring the binding of the particles. 10. The method of claim 9, wherein the non-colloidal structure is a magnetic bead and the colloidal particles include ancillary signaling entities. 61. The method of claim 60, wherein the signaling entity is a metallocene. 61. The method of claim 60, wherein the signaling entity is ferrocene or a ferrocene derivative. 61. The method of claim 60, comprising exposing the particles to the beads in the presence of the agent and the binding partner, and further in the presence of an enzyme capable of cleaving the agent and the binding partner. 64. The method of claim 63, comprising first exposing the agent and the binding partner to the enzyme, and then exposing the particles and beads to the agent and the binding partner. 64. The method of claim 63, comprising exposing the particles to the beads in the presence of an agent, a binding partner, and an enzyme, as well as in the presence of a candidate drug to modulate enzyme activity. 66. The method of claim 65, wherein the binding partner comprises an enzymatically cleavable protein or peptide. 67. The method of claim 66, wherein the protein is adapted for binding to colloid particles and to beads. 68. The method of claim 67, wherein the protein comprises a metal binding tag and biotin, one of the colloids or beads comprises a chelate that coordinates the metal, and the other colloid or beads comprises streptavidin. 68. The method of claim 67, wherein the protein comprises a metal binding tag and biotin, the colloid comprises a chelate for coordinating the metal, and the beads comprise streptavidin. 61. The method of claim 60, wherein the signaling entity is a metallocene and the binding partner comprises a protein that is cleavable by an enzyme, wherein the signaling partner is in the presence of an agent, a binding partner, and an enzyme to further modulate enzyme activity. Exposing the particles to the beads in the presence of the candidate drug, magnetically attracting the beads near the electrode, and activating the electrode to measure the proximity of the metallocene to the electrode, thereby determining the enzymatic activity of the enzyme. Such a method, comprising determining the effectiveness of a drug candidate that inhibits cleavage activity. Exposure of colloidal particles and non-colloidal structures to entities that are adapted to bind to both colloidal particles and non-colloidal structures in the presence of both enzymes capable of cleaving the entity and candidate drugs to modulate enzyme activity The method of claim 1, comprising: 73. The method of claim 71, wherein the non-colloidal structure is a magnetic bead and the colloidal particles have immobilized electroactive species, wherein the bead is magnetically attracted near the electrode and the electrode is activated. Measuring the proximity of the electroactive species to the electrode thereby determining the effectiveness of a drug candidate that inhibits the cleavage activity of the enzyme. 72. The method of claim 71, wherein the non-colloidal structure is a surface of the electrode and the colloidal particles have immobilized electroactive species, wherein the electrode is adapted for binding to colloidal particles, both colloidal particles and electrodes. Exposure to the identified entities, enzymes, and candidate drugs, and by measuring the proximity of the electroactive species to the electrodes by activating the electrodes, thereby determining the effectiveness of drug candidates that inhibit the cleavage activity of the enzyme The above method comprising: Includes exposing the colloidal particles and non-colloidal structures to a substrate of an enzyme adapted for binding to the non-colloidal structure, a species adapted for binding to the particles, capable of binding to the substrate by enzymatic activity, and an enzyme for the substrate. The method of claim 1, wherein 75. The method of claim 74, further comprising exposing the colloidal particles and non-colloidal structures to a candidate drug for modulating enzyme activity. 75. The method of claim 74, wherein the non-colloidal structure is a magnetic bead and the colloidal particle has an immobilized electroactive entity, wherein the bead is magnetically attracted near the electrode and the electrode is activated. Measuring the proximity of the electroactive entity to the electrode thereby determining the effectiveness of the drug candidate in modulating the activity of the enzyme. 75. The method of claim 74, wherein the non-colloidal structure is a surface of the electrode and the colloidal particles have immobilized electroactive entities, exposing the electrode surface to colloidal particles, substrates, enzymes, and candidate drugs. And measuring the proximity of the electroactive entity to the electrode by activating the electrode, thereby determining the effectiveness of the drug candidate in modulating the activity of the enzyme. 75. The method of claim 74, wherein the non-colloidal structure is a magnetic bead. 79. The method of claim 78, wherein the colloidal particles include an auxiliary signaling entity. 79. The method of claim 78, wherein the signaling entity is a metallocene. 79. The method of claim 78, wherein the signaling entity is ferrocene or a ferrocene derivative. 79. The method of claim 78, comprising exposing the particles to the beads in the presence of a substrate and a binding partner, as well as in the presence of a candidate drug to modulate enzyme activity. 83. The method of claim 82, wherein the binding partner is adapted to bind to the particle via a metal binding tag / metal / chelate bond and the substrate is adapted to bind to the beads via a biotin / streptavadin bond. 83. The method of claim 82, wherein the signaling entity is a metallocene, wherein magnetically attracting the beads near the electrode and activating the electrode determine the proximity of the metallocene to the electrode. Measuring the effectiveness of a drug candidate that modulates enzymatic activity. A method comprising signaling a single bond of a first biological or chemical agent to a second biological or chemical agent by a plurality of signaling entities. 86. The method of claim 85, comprising signaling a single binding by multiple signaling entities simultaneously. 86. Providing a first agent having a plurality of signaling entities, binding the first agent to a second agent, and measuring binding with the signaling entity. The method described in. 91. The method of claim 87, wherein the first agent binds to the particle to which the signaling entity has been immobilized. 91. The method of claim 87, wherein the first agent is attached to the signaling entity by the polymer. 91. The method of claim 87, wherein the first agent binds to the signaling entity via a dendrimer. 91. The method of claim 87, wherein the first agent is a biological or chemical ligand and the second agent is a cell that presents a receptor or protein on its surface. Such a method, comprising binding to a receptor or protein. 91. The method of claim 87, wherein the plurality of signaling entities comprises a plurality of signaling entities attached to a polymer attached to the first agent. 90. The method of claim 87, wherein the plurality of signaling entities comprises a plurality of signaling entities attached to the dendrimer attached to the first agent. 90. The method of claim 87, wherein the first agent is immobilized on a colloid particle that includes a plurality of immobilized signaling entities. 86. The method of claim 85, wherein the plurality of signaling entities comprises a plurality of electroactive molecules. 97. The method of claim 95, wherein the plurality of electroactive molecules comprises a plurality of metallocenes. 97. The method of claim 95, wherein the plurality of electroactive molecules comprises a plurality of ferrocenes or ferrocene derivatives. 93. The method of claim 92, wherein the polymer is attached to the first agent via an affinity tag / binding partner binding. 93. The method of claim 92, wherein the polymer is attached to the first agent via a binding tag / metal / chelate linkage. 100. The method of claim 99, wherein the first agent has a polyamino acid tag and the polymer has a chelate that coordinates the metal. 93. The method of claim 92, wherein the dendrimer binds to the first agent via a binding tag / metal / chelate bond. 102. The method of claim 101, wherein the first agent has a polyamino acid tag and the dendrimer has a chelate that coordinates the metal. Claims wherein at least one of the auxiliary signaling entities comprises a dye, a dye, an electroactive molecule, a fluorescent moiety, an up-regulating fluorophore, or an enzyme-linked signaling moiety comprising horseradish peroxidase and alkaline phosphatase. 85. The method according to 85. A method comprising measuring a protein / ligand interaction in the absence of SPR without labeling either the protein or the ligand. 104. Exposure to a protein suspected of interacting with the ligand, comprising exposing the ligand, i.e., a ligand in a certain close relationship to an electroactive entity, having an electroactive signal dependent on the proximity to the protein. The method described in. 105. The method of claim 104, wherein the electroactive entity has an electroactive signal that can change depending on a change in proximity between the electroactive entity and the protein. 105. The method of claim 104, comprising exposing the protein to a surface on which the ligand and the electroactive entity are both immobilized. 108. The method of claim 107, wherein the surface is a surface of an electrode. 108. The method of claim 107, wherein the ligand and the electroactive entity each form part of a surface self-assembled monolayer. 110. The method of claim 109, wherein the self-assembled monolayer comprises a species that enhances the permeability of the self-assembled monolayer for electrons. 112. The method of claim 110, wherein the species that promotes electron transmission comprises a conductive self-assembled monolayer-forming species. 112. The method of claim 110, wherein the species that promote electron permeability include species that create defect sites in the self-assembled monolayer. 105. The method of claim 104, wherein the electroactive entity comprises a metallocene. 105. The method of claim 104, wherein the electroactive entity comprises ferrocene or a ferrocene derivative. 105. The method of claim 104, wherein the electroactive entity comprises ferrocene dicarboxylic acid. 110. The method of claim 109, wherein the ligand binds to the self-assembled monolayer-forming species by affinity tag / binding partner binding. 110. The method of claim 109, wherein the ligand binds to the self-assembled monolayer forming species via a metal binding tag / metal / chelate bond. A method that includes:
a) a solution comprising i) a colloid, wherein the colloid comprises a ligand capable of interacting with a cell surface molecule; and ii) a composition comprising an electrode comprising growing cells, said cells comprising said ligand and said ligand. Including at least one cell surface molecule capable of interacting).
b) adding at least a portion of the colloid to the composition. 118. The method of claim 118, further comprising:
c) detecting the aggregation of the colloid as a measure of the interaction between the ligand and the cell surface molecule. 119. The method of claim 118, wherein said colloid is gold colloid. 121. The method of claim 120, wherein prior to step (a), the colloidal gold is treated to incorporate thiol groups. 119. The method of claim 118, wherein prior to step (a), the ligand is derivatized with a moiety having a binding partner. 126. The method of claim 122, wherein prior to step (a), the ligand is derivatized with a moiety capable of binding a metal chelate. 124. The method of claim 123, wherein said moiety comprises a histidine tag. A method that includes:
a) a solution comprising i) a colloid, wherein the colloid comprises a ligand capable of interacting with a cell surface molecule; ii) a candidate drug; and iii) a composition comprising an electrode comprising growing cells (the cells comprising Comprises at least one cell surface molecule capable of interacting with said ligand).
b) mixing at least a portion of the colloid with the drug and the composition. A method comprising recruiting an electronic signaling entity to an electrode using a magnetic material. 129. The method of claim 126, comprising partially recruiting the signaling entity to the electrode by a protein / protein bond involved in immobilization between the signaling entity and the magnetic beads. 130. The method of claim 127, wherein the protein / protein binding involves a protein that is not an antibody. Articles having a surface and a ligand suspected of interacting with a protein and an electroactive entity immobilized on the surface, respectively. 130. The article of claim 129, further comprising a species that promotes surface permeability of electrons immobilized with respect to the surface. An article comprising a first biological or chemical agent immobilized with respect to a plurality of signaling entities that is capable of biological or chemical binding to a second agent. 142. The article of claim 131, wherein the first agent binds to the particle to which the signaling entity is immobilized. 134. The article of claim 131, wherein the first agent is immobilized on the signaling entity by a polymer. 134. The article of claim 131, wherein the first agent is immobilized on the signaling entity by the dendrimer. An article having a surface and a self-assembled monolayer formed on the surface of the article, the monolayer comprising a first molecular species (such that the surface prevents electrical communication between the fluid to which it is exposed and the surface; A second molecular species (having a molecular structure that promotes self-assembly at the surface with other first molecular species in a tightly packed manner) and a second molecular species (having a different molecular structure from the first species and being tightly packed self-assembly) The above-described article causes a disruption of the structure, causing defects in the tightly packed structure, and allowing the surface to be in electrical communication with the fluid to which the surface is exposed. 36. The article of claim 35, wherein the first species is essentially linear and the second species includes at least one non-linear portion. A composition comprising said first molecule and one or more signaling entities bound to a solid support, wherein said first molecule is a ligand capable of interacting with a cell surface receptor or protein. 138. The composition of claim 137, wherein said solid support is a colloid. 139. The composition of claim 138, wherein said colloid is gold colloid. 139. The composition of claim 138, wherein said ligand is directly covalently attached to said colloid. 139. The composition of claim 138, wherein said signaling entity is an electroactive molecule. 142. The composition of claim 141, wherein said electroactive molecule comprises ferrocene or a ferrocene derivative. 138. The composition of claim 137, wherein said ligand is a peptide. 145. The composition of claim 143, wherein the peptide is derivatized with a moiety capable of binding a metal chelate. 153. The composition of claim 144, wherein said moiety comprises a histidine tag. 153. The composition of claim 144, wherein said solid support comprises a metal chelate, and wherein said peptide is attached to said solid support by attachment of said moiety to said metal chelate. 138. The composition of claim 137, wherein the solid support comprises a monolayer of the second molecule. 148. The composition of claim 147, wherein said monolayer is a self-assembled monolayer. 148. The composition of claim 147, wherein said second molecule is a thiol. A composition comprising a first molecule, a second molecule and a third molecule bound to a solid support, wherein said first molecule comprises a ligand capable of interacting with a cell surface receptor or protein, wherein said second molecule is Forms a monolayer on the solid support, wherein the third molecule is electroactive. 150. The composition of claim 150, wherein said solid support is a colloid. 151. The composition of claim 151, wherein the colloid is a gold colloid. 152. The composition of claim 151, wherein said ligand is directly covalently attached to said colloid. 153. The composition of claim 153, wherein said electroactive molecule comprises ferrocene or a ferrocene derivative. 153. The composition of claim 153, wherein said ligand is a peptide. 160. The composition of claim 155, wherein said peptide is derivatized with a moiety capable of binding to a metal chelate. 157. The composition of claim 156, wherein said moiety comprises a histidine tag. 157. The composition of claim 156, wherein said solid support comprises a metal chelate, and wherein said peptide is attached to said solid support by attachment of said moiety to said metal chelate. 138. The composition of claim 137, wherein said solid support is a liposome. 148. The composition of claim 147, wherein said liposome comprises at least one lipid containing a reactive group. Article comprising a metal support constructed and arranged to support cell growth on a surface: the metal support comprises a monolayer of at least one type of molecule, wherein the monolayer comprises a metal support. It is designed so that the body can be used as an electrode. 163. The article of claim 161, further comprising cells growing on a metal support. A composition comprising:
Colloid particles: signaling entities immobilized with respect to colloid particles; and proteins immobilized with respect to colloid particles. A composition comprising:
A polymer or dendrimer having a plurality of signaling entities adapted to bind to a biological or chemical agent. 166. The species of claim 164, wherein the polymer or dendrimer is adapted to bind to a chemical or biological agent via a metal binding tag / metal / chelate bond. 167. The species of claim 164, wherein the polymer or dendrimer has a chelate that can coordinate a metal. 165. The species of claim 164, wherein the polymer or dendrimer has more than one electroactive species. 167. The species of claim 164, wherein the polymer or dendrimer has multiple metallocenes. 165. The species of claim 164, wherein the polymer or dendrimer has more than one ferrocene or ferrocene derivative. Articles comprising colloid particles immobilized with respect to a glutathione derivative and at least one signaling entity. Articles comprising colloidal particles having on their surface a self-assembled monolayer comprising a glutathione derivative. 2. The method of claim 1, wherein the non-colloidal structure is a biological sample. 172. The method of claim 172, wherein the biological sample is taken from a human or animal. 173. The method of claim 173, wherein the biological sample comprises a cell or tissue section. Providing at least a first and a second biological sample;
Exposing the first and second samples to colloidal particles with immobilized species that are presumed to have or have the binding partner presented by the sample;
172. The method of claim 172 comprising: 177. The method of claim 175, wherein the first and second biological samples are samples with different states of infection or disease. 177. The method of claim 175, comprising measuring a difference between binding to a different sample and binding of the colloid particles to the first sample. 177. The method of claim 177, wherein the differences include differences in binding levels. 177. The method of claim 177, wherein the difference is in a colloid immobilization pattern. 177. The method of claim 175, further comprising measuring the immobilization of the colloid particles on either or both of the samples. 177. The method of claim 175, wherein the biological sample or source thereof is pre-treated with a candidate agent for modifying a disease state, which can be measured by the expression level and / or pattern of the binding partner, expressed by the sample. . The non-colloidal structure includes an article having a surface, and a self-assembled monolayer formed at the surface of the article, the monolayer comprising a first molecular species (electrical communication between the surface and the fluid to which the surface is exposed). And a mixture of a second molecular species (including a molecular wire) and a second molecular species (including a molecular wire) which prevents self-organization at the surface with other first molecular species in a tightly packed manner. 2. The method according to 1. The method according to claim 1, wherein the non-colloidal structure is a solid support. 183. The method of claim 183, wherein the surface is a surface of an electrode. 183. The method of claim 183, further comprising a ligand and an electroactive entity that respectively form a portion of the self-assembled monolayer at the surface of the colloidal particles. 185. The method of claim 185, wherein the self-assembled monolayer comprises a species that promotes the permeability of the self-assembled monolayer for electrons. 189. The method of claim 186, wherein the species that facilitate electron transmission comprises a conductive self-assembled monolayer-forming species. 189. The method of claim 186, wherein the species that promote electron permeability include species that create defect sites in the self-assembled monolayer. 105. The method of claim 104, wherein the electroactive entity comprises ferrocene dicarboxylic acid. 187. The method of claim 185, wherein the ligand binds to the self-assembled monolayer forming species via a metal binding tag / metal / chelate bond. 183. The method of claim 183, wherein the solid support comprises a substantially planar substrate. 183. The method of claim 183, wherein the surface of the article has a self-assembled monolayer. 192. The method of claim 192, wherein the self-assembled monolayer is a conductive self-assembled monolayer. 192. The method of claim 192, wherein the self-assembled monolayer includes a binding partner for an affinity tag. 199. The method of claim 194, wherein the binding partner is a chelate that can coordinate a metal. 195. The method of claim 194, wherein the binding partner of the affinity tag comprises glutathione or biotin. 194. The method of claim 193, further comprising a chemical or biological agent immobilized on the self-assembled monolayer by EDC / NHS coupling chemistry. 194. The method of claim 193, comprising a plurality of chemical or biological binding partners immobilized on the self-assembled monolayer and optionally distributed across the self-assembled monolayer. 194. The method of claim 193, wherein the self-assembled monolayer comprises an isolated region that further includes a unique chemical or biological binding partner immobilized thereon, respectively. Includes a chemical or biological agent immobilized on a colloidal structure that is suspected of being a binding partner or a chemical or biological agent immobilized on the surface of the article 182. The method of claim 182. 200. The method of claim 200, wherein the colloid particles include an auxiliary signaling entity. The method of claim 200, further comprising a self-assembled monolayer on the surface of the colloid particles. 220. The method of claim 201, wherein the auxiliary signaling entity is an electroactive signaling entity. 220. The method of claim 201, wherein the auxiliary signaling entity is a visible signaling entity.

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