JP2011512804A - 細胞酵素ベースのバイオセンサ - Google Patents
細胞酵素ベースのバイオセンサ Download PDFInfo
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- JP2011512804A JP2011512804A JP2010547960A JP2010547960A JP2011512804A JP 2011512804 A JP2011512804 A JP 2011512804A JP 2010547960 A JP2010547960 A JP 2010547960A JP 2010547960 A JP2010547960 A JP 2010547960A JP 2011512804 A JP2011512804 A JP 2011512804A
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Abstract
Description
本発明の更なる態様は、センサを生産する方法、詳細には、センサの上に表面層を作成する方法に関する。詳細には、表面層は、第1領域および第2領域を有し、これらは物理的に分離し、互いに近接するようにセンサの上に設けられる。
用語「領域」は、ある層を参照するものとしてここでは使用し、その層の一部を参照している。領域は、連続的でもよく、複数の小部品(例えば、ライン)で構築してもよい。こうした小部品は「エレメント」と称している。
本実施形態では、グルタミン酸オキシダーゼ(GLOD)およびグルタミン酸ピルビン酸トランスアミナーゼ(GPT)を、イオン感応電界効果トランジスタ(ISFET)の浮遊ゲートの金属表面に固定化する。
グルタミン酸オキシダーゼ(GLOD)とグルタミン酸ピルビン酸トランスアミナーゼ(GPT)の二酵素ペアは、イオン感応電界効果トランジスタのプロトン感応ゲート層の五酸化タンタル(Ta2O5)表面に固定化する。シラン・トリエトキシシリルウンデカナール(TESU)自己組織化単分子膜を酸化物表面に堆積する。酵素を固定化するためのこのシラン層の官能基はアルデヒドであり、これは酵素のアミノ基と直接に結合可能である。結合後、シアノホウ水素化物を用いてアミノ基とアルデヒド基の結合を減少させ、それを安定化させる。このアルデヒド単分子膜との酵素の結合を改善するために、シラン処理後、ポリ−L−リシン(PLL)を中間層として堆積する。その後、酵素は、グルタルアルデヒドによってPLLと結合する。
ポリ(ジメチルスルホキシド)(PDMS)と硬化剤の混合物で構成され、パターン原盤に対して金型セットの中に注入し、高温、典型的には110℃で硬化させたエラストマースタンプを用いて、マイクロコンタクト印刷を行う。PDMSと硬化剤の比率は、硬化したスタンプの所望の柔らかさまたは硬さに応じて変化する。典型的な比率は、10/1=PDMS/硬化剤である。硬化したスタンプは疎水性であるため、これらは親水性の分子およびポリマーを好都合な表面に堆積させるのに有力な候補である。これらはスタンプ表面に不可逆的に結合せず、より好都合な基板表面に転写するのが容易になるからである。しかしながら、こうした分子のインク付けを改善するためには、分子は、スタンプ内に拡散可能であることが必要になる。この拡散を改善する方法は、硬化したスタンプを、使用前に1週間水中に浸漬することを含む。
ポリ−L−リシン(PLL)層が細胞付着のための第1領域として機能する。それをトリエトキシシリルウンデカナール(TESU)表面層の上に、2mg/mL溶液でホウ酸緩衝液中で30分間堆積する。PLLのアミノ基はTESU層のアルデヒド基と反応して、イミド結合を生成する。この反応性イミド結合は、シアノホウ水素化物との第2反応を通じてより安定な単一結合に還元される。正に帯電したPLL表面は細胞の付着を促進し、細胞接着領域を作成する。
センサの表面層は、グルタミン酸オキシダーゼ(GLOD)およびグルタミン酸ピルビン酸トランスアミナーゼ(GPT)の二酵素系を持つポリ−L−リシン(PLL)層を含む。2つの酵素は、表面層に所定のパターンで整列する。さらに、2つの酵素は、充分なPLLの細胞接着領域が神経細胞にとって接近可能なように整列する。その結果、PLL領域にある細胞が所定パターンで発生する。PLL領域は、細胞接着領域として機能し、ニューロンの付着および有向(directed)成長を可能にする。このデバイスでは、GLODがニューロンに接近しており、分泌グルタミン酸を変換し、アンモニアと過酸化水素の発生とともに、局所的なpH変化を作成する。この局所的pH変化は、センサによって検出される。GPTの存在下で、グルタミン酸は再生され、GLODと再び反応して、増強された局所的pH変化をもたらす。こうして検出可能な部分が増幅され、信号は増大する。
Claims (21)
- センシング層(2)と、表面層(3)とを備え、
表面層は、細胞の接着成長に適した第1領域(4)と、前記第1領域に近接し、タンパク質の付着に適した第2領域(5)とを備え、
第1領域および第2領域はセンシング層(2)と接触しているセンサ(1)。 - 前記第1領域の上に、細胞(6)をさらに備える請求項1記載のセンサ。
- 前記第2領域(5)は、前記細胞(6)によって生産された化合物を、検出可能な信号を発生する生成物に変換するための少なくとも1つの酵素(7)を有する請求項1または2記載のセンサ。
- 前記第1領域及び/又は前記第2領域は、複数の線状エレメントからなる請求項1〜3のいずれかに記載のセンサ。
- 前記第1領域及び/又は前記第2領域は、複数の線状エレメントの格子状パターンからなる請求項1〜4のいずれかに記載のセンサ。
- 第1領域の前記線状エレメントは、約5〜20マイクロメータの幅を有する請求項4または5記載のセンサ。
- 第2領域の前記線状エレメントは、約2〜20マイクロメータの幅を有する請求項4または5記載のセンサ。
- 前記細胞は、神経細胞である請求項1〜7のいずれかに記載のセンサ。
- 前記少なくとも1つの酵素は、二酵素系である請求項1〜8のいずれかに記載のセンサ。
- 前記細胞によって生産される化合物は、神経伝達物質である請求項1〜9のいずれかに記載のセンサ。
- 神経伝達物質は、グルタミン酸である請求項10記載のセンサ。
- 二酵素系は、グルタミン酸オキシダーゼまたはグルタミン酸脱水素酵素と、L−グルタミン酸ピルビン酸トランスアミナーゼである請求項8記載のセンサ。
- 細胞による化合物生産の検出方法であって、
・第1領域における前記細胞と、第2領域において、前記細胞(6)によって生産された化合物を、検出可能な信号を発生する生成物に変換するための少なくとも1つの酵素(7)とを備える、請求項1〜12のいずれかに記載のセンサを用意するステップと、
・前記加工物を、検出可能な信号を発生する生成物に変換するための条件を提供するステップと、
・前記検出可能な信号を検出するステップとを含む方法。 - 検出可能な信号はpHの変化である請求項13記載の方法。
- センサを生産する方法であって、
・センサ表面にセンシング層(2)を設けるステップと、
・センシング層の上/内に、接着細胞成長に適した、表面層の第1領域(4)を設けるステップと、
・センシング層の上/内に、タンパク質の付着に適した、表面層の第2領域(5)を設けるステップとを含み、
第1領域および第2領域は、センシング層と接触するようにした方法。 - 第1領域および第2領域の少なくとも1つが、コンタクト印刷によって設けられる請求項15記載の方法。
- 酵素を前記第2領域に設けるステップをさらに含む請求項15または16記載の方法。
- 前記酵素は、コンタクト印刷によって設けられる請求項15または16記載の方法。
- 細胞を、前記第1領域の前記第1表面層に設けるステップをさらに含む請求項15〜18のいずれかに記載の方法。
- センシング層と、
表面層の第1領域において固定化した細胞と、表面層の第2領域において、前記細胞によって生産される化合物の変換が可能である、少なくとも1つの固定化した酵素とを含む表面層とを備え、
表面層の第1領域および第2領域は、前記細胞による前記化合物の生産の検出のために、センシング層と接触しているセンサの使用。 - 細胞は神経細胞であり、化合物は神経伝達物質である請求項20記載の使用。
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