JP4929337B2 - 再構成エネルギーを使用した検体の検出 - Google Patents
再構成エネルギーを使用した検体の検出 Download PDFInfo
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- JP4929337B2 JP4929337B2 JP2009256449A JP2009256449A JP4929337B2 JP 4929337 B2 JP4929337 B2 JP 4929337B2 JP 2009256449 A JP2009256449 A JP 2009256449A JP 2009256449 A JP2009256449 A JP 2009256449A JP 4929337 B2 JP4929337 B2 JP 4929337B2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
Description
本発明は、電子移送過程の核再構成エネルギー"λ"の変化に基づく、検体検出用の新規な方法および組成物に関する。
電子移送反応は、光合成から好気性呼吸までの範囲の様々な生物学的変換において、重要なステップ群である。化学的および生物学的システム両方における電子移送反応の研究は、多大な知識の蓄積発展と、定義できるパラメーターのセットによる電子移送の速度を叙述する強力な理論的基盤を築いた。
に従うものと予測する。上記等式の核再構成エネルギー"λ"は、生成物の平衡核配置における反応物のエネルギーとして定義する。λには2つの成分があり;"領域外"効果(λ0)および"領域内"効果(λi)である。極性溶媒中での電子移送反応では、λへの最も有力な寄与は、反応物の電荷における変化に応じた溶媒分子の再配向から生じる。第2のλ成分は、ドナーとアクセプターの酸化状態の変化による、結合長さおよび角度の変化から生じる。
上記目的に従い、本発明は、試験サンプル中の標的検体の検出方法を提供する。この方法は、検体のレドックス活性錯体への結合を含む。レドックス活性錯体は、極性配位基によって占有されている、少なくとも一配位の部位、および、標的検体と結合する結合配位子とを有する、溶媒が近づき易い遷移金属錯体を含む。この錯体は電極と結合している。結合により、溶媒阻害された遷移金属錯体が形成され、溶媒阻害された金属錯体と電極の間で電子移送が検出される。本方法は、少なくとも第一入力シグナルの溶媒阻害された遷移金属錯体への利用も含む。
本発明は、検体との結合により生ずる、遷移金属錯体と電極の間の電子移送を促進する遷移金属錯体の溶媒再構成エネルギーの変化を使用した、標的検体の検出方法およびその組成物を提供するものである。本発明は、遷移金属イオンなどのレドックス活性分子の酸化状態の変化、すなわち、電子の受け入れまたは提供により、金属イオンの電荷およびサイズの変化を生ずるという事実に基づく。この電荷およびサイズの変化は、酸化状態におけるこの変化により、周囲の溶媒の温度を変化させて再構成させる。
一般的に、結合配位子の金属イオンまたはスペーサーへの結合は、結合リガンド上に天然に見出されるかまたはよく知られた技術で付加される官能基を使用して行なうことができる。これらの基は、例えばタンパク質のN-またはC-末端結合配位子の末端、またはいずれかの内部位置にある。例えば、アミノ、チオ、カルボキシル、またはアミド基を結合に使用することができる。当業者には理解されるように、同じく、通常の配位子の結合に使われるピリジンまたはフェナントロリンも使用することができる。例えば、タンパク質結合配位子の結合は、一般的に、アミノ酸側鎖またはN-またはC-末端にある官能基を使用して行なわれる;例えばN-末端またはヒスチジンなどの側鎖の基を金属イオンの配位子として供給し得る。同様に、炭水化物結合配位子の結合を、金属イオン配位子として作用し得るように糖を誘導体化する方法で行なう。さらに、これらの基をよく知られた技術を用いてスペーサーへの結合に使用し得る。これらの実施態様のいずれにおいても、付加的なコネクターまたはリンカーが存在し得る。例えば、結合配位子がタンパク質酵素基質または阻害剤であるとき、金属イオン配位子と機能的基質または阻害剤との間に、付加的なアミノ酸またはアルキル基などがあり得る。
“絶縁体”は、実質的に非導電性オリゴマーであり、好ましくは直鎖状である。本明細書中の“実質的に非導電性”とは、絶縁体を介した電子移送の速度が、導電性オリゴマーを介した電子移送の速度よりも遅いことを意味する。言いかえると、絶縁体の電気抵抗は、導電性オリゴマーの電気抵抗より高い。しかしながら、実施例に概説しているように、一般にオリゴマーが、−(CH2)16分子のような、絶縁体であるとみなされても、例え遅い速度でもなお電子を移送し得ることを承知しておくべきである。
一般的に、電子移送は、好ましい電位で電子的に開始させる。ある電位を、修飾核酸プローブを含むサンプルに適用する。適用した電位で、正確な制御と変化が、定電位装置、および三電極システム(1つは対照、1つはサンプルおよび1つはカウンター電極)または二電極システム(1つはサンプルおよび1つはカウンター電極)のどちらかを介して行なわれる。これは適要した電位と、一部は遷移金属錯体の選択に、そして一部は使用する伝導オリゴマーに依存するシステムの電子移送電位を最高点との調和を可能とする。
装置は、試験室すなわち測定電極に電気的に連結した電圧源を含む。好ましくは、電圧源は、必要であれば、交流および直流電圧も同様に放出し得る。
本実施例で、ルテニウム錯体およびビオチン結合配位子を含むレドックス活性錯体を使用して、センサーの基礎である溶液を調製した。小極性配位配位子(NH3)を持つルテニウムの遷移金属錯体を調製した。配位原子の1つは、結合配位子ノルビオチン(4炭素リンカーを介して第一級アミンと結合したビオチン)から得、それはアビジンと結合すると、遷移金属錯体が溶媒が近づきやすいものから溶媒阻害へ向かうものである。あるいは、ルテニウムのレドックス活性錯体とビオチンを、活性化させ、表面に加えて、電極-ベースセンサーを形成させる。
温度計、還流冷却器、ガス注入口を備えた3首丸底フラスコで予加熱した水(〜70℃)65ml中で、[(NH3)5RuIIICl]Cl22.5gr(8.5mmoles)をスラリーにした。このフラスコにNaHSO3 3.55gr(2.4mmoles)を加え、すぐに連続的なSOガス流を、溶液中に吹き込み、混合物は83℃に温まるまでおいた。反応を90分間進行させた。溶液を0℃に冷却し、生成物を集めアセトンで数回洗浄した。
Claims (29)
- a)電極に標的検体を添加すること、
ここで前記電極は:
i)第一のレドックス電位(E0 )を有するレドックス活性分子であって、マンガン、テクネチウム、レニウム、鉄、ルテニウム、オスミウム、コバルト、ロジウム、イリジウム、ニッケル、パラジウム、白金、銅、銀および金からなる群から選択される金属を含むレドックス活性分子;および
ii)前記標的検体に結合する結合配位子
を含み、前記標的検体が前記結合配位子に結合すると、異なる第2のレドックス電位を有するレドックス活性分子が形成される;および、
b)前記標的検体の存在の指標として前記第2のレドックス電位を測定すること、
を含む、試験サンプル中の標的検体を検出する方法。 - 前記測定は、少なくとも第一入力シグナルを前記レドックス活性分子に適用し、出力シグナルを受け取ることを含む、請求項1に記載の方法。
- 標的検体が存在しないと、前記出力シグナルが、前記第2のレドックス電位で有意な電子移送を示さない、請求項2に記載の方法。
- 前記第一入力シグナルが、少なくとも交流成分を含む、請求項2に記載の方法。
- 複数周波数で前記入力シグナルを適用することをさらに含む、請求項2に記載の方法。
- 前記入力シグナルが、少なくとも直流成分を含む、請求項2または4に記載の方法。
- 複数電圧で前記入力シグナルを適用することをさらに含む、請求項6に記載の方法。
- 前記出力シグナルが電流である、請求項2に記載の方法。
- 前記電流が交流電流である、請求項8に記載の方法。
- 前記電極が金を含む、請求項1に記載の方法。
- 前記電極が自己集合単層をさらに含む、請求項1に記載の方法。
- 前記自己集合単層が導電性オリゴマーを含む、請求項11に記載の方法。
- 前記自己集合単層が、絶縁体を含む、請求項11に記載の方法。
- 前記レドックス活性分子が、極性配位基により占有されている少なくとも1つの配位部位を有する、請求項1に記載の方法。
- 前記レドックス活性分子が、水分子により占有されている少なくとも1つの配位部位を有する、請求項1に記載の方法。
- 前記レドックス活性分子が、極性配位基により占有されている少なくとも2つの配位部位を有する、請求項1に記載の方法。
- 前記結合リガンドが、前記レドックス活性分子に共有結合されている、請求項1に記載の方法。
- 前記結合リガンドが、前記電極に共有結合されている、請求項1に記載の方法。
- 前記レドックス活性分子が、前記電極に共有結合されている、請求項1に記載の方法。
- 前記共有結合が、スペーサーを介している、請求項18に記載の方法。
- 前記共有結合が、スペーサーを介している、請求項19に記載の方法。
- 前記スペーサーが、導電性オリゴマーである、請求項20または21に記載の方法。
- 前記スペーサーが、絶縁体である、請求項20または21に記載の方法。
- 前記金属が、鉄である、請求項1に記載の方法。
- 前記金属が、ルテニウムである、請求項1に記載の方法。
- 前記金属が、オスミウムである、請求項1に記載の方法。
- 前記標的検体が、タンパク質である、請求項1に記載の方法。
- 前記タンパク質が、ペプチドである、請求項27に記載の方法。
- 前記標的検体が、酵素である、請求項27に記載の方法。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US08/873,977 US6013459A (en) | 1997-06-12 | 1997-06-12 | Detection of analytes using reorganization energy |
US08/873,977 | 1997-06-12 |
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JP50318499A Division JP2002504230A (ja) | 1997-06-12 | 1998-06-12 | 再構成エネルギーを使用した検体の検出 |
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JP2010091573A JP2010091573A (ja) | 2010-04-22 |
JP4929337B2 true JP4929337B2 (ja) | 2012-05-09 |
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JP50318499A Pending JP2002504230A (ja) | 1997-06-12 | 1998-06-12 | 再構成エネルギーを使用した検体の検出 |
JP2009256449A Expired - Lifetime JP4929337B2 (ja) | 1997-06-12 | 2009-11-09 | 再構成エネルギーを使用した検体の検出 |
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JP50318499A Pending JP2002504230A (ja) | 1997-06-12 | 1998-06-12 | 再構成エネルギーを使用した検体の検出 |
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US (11) | US6013459A (ja) |
EP (2) | EP2312304A1 (ja) |
JP (2) | JP2002504230A (ja) |
AU (1) | AU747345B2 (ja) |
CA (1) | CA2292696A1 (ja) |
WO (1) | WO1998057158A1 (ja) |
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US7267939B2 (en) | 2007-09-11 |
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US7018523B2 (en) | 2006-03-28 |
US20080135420A1 (en) | 2008-06-12 |
JP2010091573A (ja) | 2010-04-22 |
US20060073532A1 (en) | 2006-04-06 |
AU747345B2 (en) | 2002-05-16 |
US6248229B1 (en) | 2001-06-19 |
WO1998057158A1 (en) | 1998-12-17 |
US7582419B2 (en) | 2009-09-01 |
JP2002504230A (ja) | 2002-02-05 |
US20080179196A1 (en) | 2008-07-31 |
EP2312304A1 (en) | 2011-04-20 |
US20020033345A1 (en) | 2002-03-21 |
US6013170A (en) | 2000-01-11 |
US7514228B2 (en) | 2009-04-07 |
US7566534B2 (en) | 2009-07-28 |
US20100038262A1 (en) | 2010-02-18 |
US7579145B2 (en) | 2009-08-25 |
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