JP3657790B2 - Metal thin film for SPR sensor, its production method, and measurement method using the same - Google Patents

Description

【００２４】
多糖類としては、アミノ糖を含有する多糖類が好ましく、アミノ糖が重合したものでも、アミノ糖が部分的に導入されているものでもよいが、キトサンのようにアミノ糖が重合した多糖類が好ましい。たま、多糖類にアミノ基などの反応性の官能基を導入したものであってもよい。
これらのポリマーの分子量は、ポリマーの種類によっても異なるが、一般的には、１，０００〜１，０００，０００、好ましくは５，０００〜１，０００，０００、より好ましくは５，０００〜５００，０００程度である。
【００２５】
有機リンカーとポリマーとを結合させる方法としては、これらを溶媒の存在下に直接反応させることもできるが、反応性が充分でない場合にはこれらの官能基を活性化、例えば、活性エステル化などにより活性化させて、反応させることもできる。
原料物質として、システインメチルエステル、グルタルアルデヒド及びポリエチレンイミンを用いた場合の本発明の金属薄膜の処理法を、図１に示す。金属薄膜を温めた水酸化カリウム水溶液に数回つけることで洗浄した後、Ｌ−システインメチルエステルのＤＭＦ溶液に浸すことにより図１の（１）の状態とし、これを、グルタルアルデヒド水溶液で処理して図１の（２）の状態にし、これにポリエチレンイミン水溶液を浸して図１の（３）のポリエチレンイミン膜を有するセンサープローブとすることができた。
【００２６】
また、原料として、システインメチルエステル、グルタルアルデヒド及びキトサンを用いた場合の本発明の金属薄膜の処理法を、図２に示す。図２では、さらに、スペーサーとしてα−ブロム酢酸が使用されている。
図１に示した方法と同様な方法により、金属薄膜を洗浄した後、Ｌ−システインメチルエステルＤＭＦ溶液及びグルタルアルデヒド水溶液で処理して有機リンカー部分を調製し、これに１％キトサン水溶液を接触させることによりキトサンを固定化してセンサープローブを調製した。次いで、キトサンをα−ブロム酢酸の水酸化ナトリウム溶液でカルボキシル化して、カルボキシメチル（スペーサー部分）化されたＣＭ−キトサンセンサープローブを調製した。
【００２７】
本発明の検出用試薬としては、試料中の特定の物質を検出、同定又は定量することができる試薬であって、前記ポリマーに吸着又は結合などにより固定できるものでれば特に制限はないが、カルボキシル基やアミノ基などの官能基を有するものが好ましい。好ましい検出用試薬として、蛋白質が挙げられる。より詳細には、抗原又は抗体が挙げられる。
本発明の検出用試薬を直接前記したポリマー層に固定することもできるが、固定するに当たって必要に応じてスペーサーとなる物質を用いて両者を固定することもできる。スペーサーとしては、多官能性の種々の物質を使用することができる。比較的長鎖の物質を使用することもできるが、炭素数が２〜１５、好ましくは２〜１０程度の短いものが好ましい。スペーサーとして使用される化合物としては、例えば、α−ブロム酢酸、α−クロル酢酸などのハロカルボン酸、グルタル酸、アジピン酸、スベリン酸などのジカルボン酸などが挙げられる。
【００２８】
図３に、図１に示したポリエチレンイミン膜を有する金属薄膜にスペーサーとしてスベリン酸を用い、活性エステル化法により検出用試薬（図３中ではリガンドとして示されている。）を固定する方法を示す。
図４に、図２に示したＣＭ−キトサンセンサープローブに検出用試薬（図４中ではリガンドとして示されている。）を固定する方法を示す。図４中のＥＤＣは１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミドを示し、ＮＨＳはＮ−ヒドロキシサクシンイミドを示す。
【００２９】
本発明の金属薄膜に使用される金属としては、自由電子を有する金属であればよく、例えば、金、銀、銅、アルミニウム、クロムなどが挙げられる。好ましい金属は、金であるが、銀、銅、アルミニウなども好ましい金属である。金属薄膜は金属を延ばして製造されるものであってもよく、また、基板に蒸着などの方法で形成されるものであってもよい。金属薄膜の厚さは、測定可能な厚さであれば特に制限はないが、５０〜１，０００ｎｍ、好ましくは５０〜５００ｎｍ、より好ましくは１００〜５００ｎｍ程度である。
【００３０】
表面プラズモン共鳴(Surface Plasmon Resonance :ＳＰＲ）装置に本発明の金属薄膜を使用する場合には、プリズムなどの基板に本発明の金属薄膜が密着されて使用される。
図５に本発明の金属薄膜の調製法と、表面プラズモン共鳴装置の概要を示す。図５に示されるように本発明の金属薄膜を調製法としては、バッチ法とオールフローインジェクト法（ＡＦＩ法）がある。バッチ法は、有機リンカー層、ポリマー層、検出用試薬などを形成させるための必要な物質（図５中ではＡ液、Ｂ液、Ｃ液として示されている。）で金属薄膜を処理して、処理された金属薄膜（センサープローブ）を調製した後、これをＳＰＲ装置にセットして試料を添加して測定する方法である。
【００３１】
また、ＡＦＩ法は、ＳＰＲ装置にセットされた金属薄膜に順次、処理溶液を金属薄膜に流すことにより、セットされた金属薄膜を所望の状態に処理するものである。より詳細には、図５に示されるように、ＨＰＬＣ用のポンプなどのポンプにより常時緩衝液など（例えば、ランニングバァファー（ｐＨ７．４ １０ｍＭＨＢＳ（ＨＥＰＥＳ Ｂｕｆｆｅｒ Ｓａｌｉｎｅ）／Ｔｕｅｅｎ））を、流速が０．１〜１．０、好ましくは０．２〜０．４ｍｌ／ｍｉｎで流しておき、必要な時に各反応試薬を含有する溶液をインジェクターから注入することで、金属薄膜（センサープローブ）に供給させることにより必要な処理を行うことができる。そして、この間測定を継続することも可能であり、金属薄膜の測定面の反対側で生起している反応を、反応系に直接光やマイクロ波などの測定線を照射することなく反応系を測定することができる。
【００３２】
ＡＦＩ法は、測定を継続しつつ金属薄膜の表面を処理することができるので、表面の状態をリアルタイムで把握することができる。また、金属薄膜の表面に固定された検出用試薬と試料中の物質との相互の動向（化学反応などの）を逐次測定し観察することができる。
本発明はバッチ法及びＡＦＩ法のいずれの方法をも包含するものである。
【００３３】
材料に目的とする性能、機能を付与するためには材料表面を分子レベルで設計し機能特性をもたせることが重要となってくる。そのため、今日の臨床化学分野における抗原-抗体反応や、セルレセプター認識を用いた生体物質のセンシングにおいてそのセンサー材料表面及び、生体物質間の相互作用を含めた反応プロセスの理解は大切な課題となっている。
【００３４】
本発明では、様々な官能基を有する高分子を用いてセンサー表面を構築することで、幅広い物質の固定化、検出に利用できるセンサープローブを構築することを目的とした。具体的には、金薄膜をセンサー基盤として用い、デバイスとして表面プラズモン共鳴(Surface Plasmon Resonance :ＳＰＲ)装置を使用して、センサー表面の設計を行った。
【００３５】
より具体的には、抗原-抗体反応を利用した高感度免疫センサーを開発するために、タンパク質を固定化するに当たって、金表面を修飾する膜として、異なる官能基を有する高分子であるポリエチレンイミン（polyethyleneimine(PEI)）、及びキトサン（chitosan）に着目した。ＰＥＩ膜は膜中に存在する一級アミンを、キトサン膜はカルボキシル基を活性化させて、タンパク質中のアミノ基とバインディングサイトを持たせ、そこに抗原を化学結合させてセンサ−膜を作製した。 本発明では、この作製方法として、従来から行われているバッチ法の他に、すべての試薬をフロ−系に導入して固定化するオ−ルフロ−型固定化法も開発した。作製したセンサ−膜に、抗体が特異的に結合した際のＳＰＲアングルシフトによりその定量を行った。
【００３６】
また、糖質結合タンパク（Concanavalin A :ConA)を利用した新規糖質センサーの開発においては、まず、糖質としてグルコースを測定対象とした。ＣｏｎＡの特性として、ｐＨ７付近でグルコースとの４つのバインディングサイトを持っているためグルコースに対する吸着性は高い。しかし、あらかじめＣｏｎＡとグルコースを混合し、バインディングサイトがいくつかグルコースと結合したものを作用させると、その選択的吸着性は低下し、この変化は、ＣｏｎＡの濃度を一定とすれば、混合するグルコース濃度に対応してくると考えた。この特性を利用し、Ａｕ表面をグルコースユニットで修飾してやることで、そこに吸着するＣｏｎＡの濃度から逆にグルコース濃度の測定を行った。
【００３７】
本発明の方法は、特にポリマー層に直接又はスペーサーを介して固定された抗原又は抗体と、試料中の抗体又は抗原との反応を測定するのに有利な方法でである。
例えば、抗体タンパク質を多量に固定化し、高感度に検出するため、金属表面を修飾する膜材としては、異なる官能基を有する高分子であるポリエチレンイミン（ＰＥＩ）、及びカルボキシメチル化キトサン（ＣＭ−ｃｈｉｔｏｓａｎ）が特に好ましい。ＰＥＩは膜中に存在する一級アミンを、ＣＭ−ｃｈｉｔｏｓａｎはカルボキシル基を活性化し、抗体と特異的に反応するリガンドを化学結合させてセンサ−プローブ表面を構築した。
【００３８】
本発明ではこれらの表面を作製する際、従来から行われているバッチ法に加え、すべての試薬をフロ−系に導入して固定化するオ−ルフロ−インジェクト（ＡＦＩ）法も検討した。作製したセンサ−プローブを利用して、具体的には、プロテインＡ−ヒト免疫グロブリンＧ（ｈＩｇＧ）、抗ヒト免疫グロブリンＧ（ａｎｔｉ−ｈＩｇＧ）−ヒト免疫グロブリンＧ（ｈＩｇＧ）系におけるＳＰＲ応答を検討した。
【００３９】
今回の実験に使用したＳＰＲセンサー（ＤＫＫ製）は、図５に示すフロースルーのシステムが実行できるものである。
ＡＦＩ法により実験を行う場合には、ＨＰＬＣ用のポンプによって常時ランニングバァファー（ｐＨ７．４ １０ｍＭＨＢＳ（ＨＥＰＥＳ Ｂｕｆｆｅｒ Ｓａｌｉｎｅ）／Ｔｕｅｅｎ）を流速０．２〜０．４ｍｌ／ｍｉｎで流しておき、各試料溶液をインジェクターから注入することで、ＳＰＲ装置中のセンサープローブと接触させ、そのデータをコンピューターによって出力している。
【００４０】
次に本発明をより具体的に説明するが、本発明はこれらの具体例に限定されるものではない。本発明のより具体的な説明において使用する略号を、以下にまとめて示しておく。
【００４１】
化合物
ＢＳＡ；ウシ血清アルブミン（Bovine serum albumin）。
ＢＳ３；スベリン酸ビス（スルホサクシンイミドイル）（Bis(sulfosuccinimi dyl) suberate。
ＣＡＰ；セルロースアセテートフタレート（Cellulose acetate phtalate）。
ＣＭ−ｃｈｉｔｏｓａｎ；カルボキシメチルキトサン（Carboxymeyhylated chitosan）。
ＥＤＣ；１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド （1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide）。
ＨＢＳ；ＨＥＰＥＳ緩衝液（HEPES Buffer Saline）。
ｈＩｇＧ；ヒト免疫グロブリンＧ（human immunoglobulin G）。
ＮＨＳ；Ｎ−ヒドロキシサクシンイミド（N-Hydroxysuccinimide）。
ＰＥＩ；ポリエチレンイミン（Polyethylene imine）。
【００４２】
溶媒
ＡｃＯＨ ；酢酸（Acetic Acid ）。
ＤＭＦ；Ｎ，Ｎ−ジメチルホルムアミド（N,N-Dimethylformamide）。
ＥｔＯＨ；エタノール（Ethanol）。
ｉ−ＰｒＯＨ；イソプロパノール（Isopropanol）。
【００４３】
まず、金薄膜を７０℃ほどに温めた１ＭＫＯＨ水溶液に数回つけることで洗浄した後、５ｍＭ Ｌ−システインメチルエステルのＤＭＦ溶液に、２４時間浸すことにより、図１の（１）の状態とした。これを、５％グルタルアルデヒド水溶液で２時間処理して図１の（２）の状態とし、次いで２％ＰＥＩ水溶液に２時間浸し図１の（３）で示されるのＰＥＩ膜センサープローブを得た。
【００４４】
このようにして調製されたセンサープローブの有機リンカー部分の表面構造は、一部のグタルアルデヒドが２分子のシステインのアミノ基と結合して２個のアルデヒド基を消費した状態のものなどがあり、分子が金属表面に規則正しく整列している自己吸着性単層膜（Self-Assembled Monolayer）の状態とは異なる状態を形成しているものと考えられる。詳細な金属表面の状態はＸＰＳなどの表面分析を利用しその厚さを測定することにより把握することができるであろう。
【００４５】
次に、作製したプローブをＳＰＲ装置にセットし、ｐＨ７．４ ＨＢＳ Ｔ／バッファーによるベースラインが安定した後、膜中に存在する未反応のグルタルアルデヒドを０．１Ｍ グリシン溶液をインジェクトすることでブロックした。この後、ＰＥＩ膜中の一級アミンをスペーサーであるＢＳ３で活性化させ、０．５ｍｇ／ｍｌ ＢＳＡを固定化した時の結果を図６に示し、次いで、０．１ｍｇ／ｍｌ ｈＩｇＧを添加した時の結果を図７に示す。
【００４６】
図６及び図７は、ＳＰＲの測定結果であり、横軸に時間（分）、縦軸にレスポンス（アーク秒（１度は３６００アーク秒））を示す。
図６中の、（１）はｐＨ７．４ ＨＢＳ Ｔ／バッファーを添加した時点を、（２）はＢＳ３を添加した時点を、（３）は０．５ｍｇ／ｍｌのＢＳＡを添加した時点を、（４）はグリシンｐＨ３を添加した時点をそれぞれ示している。ＢＳＡを添加した時点でのレスポンスΔθは６０４アーク秒であった。
図７中の、（１）はｐＨ７．４ ＨＢＳ Ｔ／バッファーを添加した時点を、（２）はＢＳ３を添加した時点を、（３）は０．１ｍｇ／ｍｌのｈＩｇＧを添加した時点を、（４）はグリシンｐＨ３を添加した時点をそれぞれ示している。ｈＩｇＧを添加した時点でのレスポンスΔθは１５０アーク秒であった。
【００４７】
次に、オールフロ−インジェクト法（ＡＦＩ法）による金属薄膜の処理を検討した。
金薄膜を洗浄した後、直接ＳＰＲ装置にセットし、そこからすべての試薬をフロ−系に導入して固定化した。即ち、１０ｍＭＬ−システインの水：エタノール１：４溶液、１０％グルタルアルデヒド水溶液、１％ＰＥＩ水溶液を順にインジェクトしてＰＥＩ膜プローブを作製し、ＢＳＡを固定化した。結果を図８及び図９に示す。
図８中の、（１）はｐＨ７．４ ＨＢＳ Ｔ／バッファーを添加した時点を、（２）は１０ｍＭＬ−システインを添加した時点を、（３）は１０％グルタルアルデヒドを添加した時点を、（４）は１％ＰＥＩを添加した時点をそれぞれ示している。
【００４８】
図９中の、（ａ）はｐＨ７．４ ＨＢＳ Ｔ／バッファーを添加した時点を、（ｂ）は１ｍＭのＢＳ３を添加した時点を、（ｃ）は５００μｇ／ｍｌのＢＳＡを添加した時点を、（ｄ）はグリシンｐＨ３を添加した時点を、（ｅ）はｐＨ８．５で０．１Ｍのエタノールアミンを添加した時点をそれぞれ示している。（ｄ）のグリシンを添加した時点でのレスポンスΔθは静電的相互作用に起因するもので３３４アーク秒であり、共有結合による相互作用に起因するものが１１３０アーク秒であった。
【００４９】
次に、ＰＥＩ膜センサープローブ表面への非特異的吸着の抑制について検討した。センサーを作製する際、体液中に存在する多種のタンパク質のセンサー膜表面への非特異的吸着は、検出感度やその選択性を低下させる主な原因として指摘されている。前記した実験においても、ＰＥＩ膜表面にＢＳＡを接触させた時、全吸着量の内の１／４から１／３程度の非特異的吸着（グリシン／塩酸ｐＨ３．０を接触させた時に脱離した分）が見られた。これは、膜中に多数残存するアミンがプロトン化することで持つプラスチャージによるＢＳＡとの静電的相互作用が主な原因であると考えられる。
【００５０】
そこで、タンパク質をＰＥＩ膜プローブに固定化する際、ｐＨ７．４ ＨＢＳＴ／バッファーの代わりにｐＨ５．３酢酸バッファーを使用し反応時のｐＨを変化させた時の結果を図１０に示す。
図１０中の、（１）はｐＨ５．３酢酸バッファーを添加した時点を、（２）はスペーサーのＢＳ３を１ｍＭ添加した時点を、（３）は０．５ｍｇ／ｍｌのＢＳＡ溶液を添加した時点を、（４）は０．１Ｍのグリシン（ｐＨ３．０）を添加した時点をそれぞれ示している。グリシンを添加した時点でのレスポンスΔθはそれぞれ８６０及び６２０アーク秒であった。
【００５１】
また、ｐＨ７．４バッファーで、ＢＳ３をスペーサーとして用いずに、膜中のアミノ基を１００ｍＭグリオキル酸（Ｇｌｙｏｘｙｌｉｃ Ａｃｉｄ）と反応させカルボキシル化し、膜全体のチャージバランスをとり、これを０．４Ｍ ＥＤＣ／０．１Ｍ ＮＨＳ １：１混合溶液で活性化した後、ＢＳＡを固定化した際の結果を図１１に示す。
図１１中の、（１）はｐＨ７．４ＨＢＳバッファーを添加した時点を、（２）は０．４ＭＥＤＣ／０．１ＭＮＨＳを添加した時点を、（３）は０．５ｍｇ／ｍｌのＢＳＡ溶液を添加した時点を、（４）は０．１Ｍのグリシン（ｐＨ３．０）を添加した時点をそれぞれ示している。ＢＳＡを添加した時点のレスポンスΔθは１２１１アーク秒であり、グリシンを添加した時点のレスポンスΔθは６７３アーク秒であった。
【００５２】
この結果、反応時のｐＨやセンサープローブ表面のチャージバランスを変えることにより、ＢＳＡの非特異的吸着を減少させることはできなかった。
ＢＳＡなどのタンパク質は、全体としてプラスかマイナスのどちらかの電荷をもっていることが多いが、各分子内にはプラスチャージとマイナスチャージの両方の部位をもっているため、ｐＨの変化や全体としてプローブ表面のチャージのバランスをとってみても、タンパク質分子中の各部位すべてを膜表面に吸着しにくい状態にするのは困難である。タンパク質の種類により、非特異的な吸着を防ぐ最適なｐＨ、及びチャージ状態は決まってくるのであろうが、血清中など多種のタンパク質が存在する中でＰＥＩ膜免疫センサープローブとして測定する際、ｐＨやチャージバランスを変えての測定は全く意味を持たない。
【００５３】
そこで、チャージを持つ膜中の未反応のアミノ基どうしを、グルタルアルデヒドで架橋することで不活性にしてチャージを抑制すると共に、残ったアルデヒド基による活性部位の増加を考えた。このプローブの活性化部位に検出する抗体と特異的に反応するリガンドを固定化し、これをＰＥＩ膜センサープローブとした。この反応を模式化して図１２に示す。
【００５４】
以上の実験から、ＰＥＩ膜センサープローブの基礎特性として次のように考えられる。アミノ基を持つリガンドの固定化が行えたことから、ＰＥＩ膜センサ−プローブの作製が従来から行われているバッチ法に加えて、すべての試薬をフロ−系に導入して作製するオ−ルフロ−インジェクト（ＡＦＩ）法でも行えることが確認できた。
フロー系による試料と表面との接触時間は１０分程度であるので、図１に示す反応はすべてがこの時間内で進行することがわかった。ＡＦＩ法は、これまでに確立されている作製に数十時間かかる他のセンサープローブと比較し、簡便、迅速なプローブの作製方法として有用な方法と考えられる。
バッチにより作製したＰＥＩ膜プローブとフローによるプローブのＢＳＡの固定化量を比較すると(図６及び図８、図９）、フロー系の方が２．８倍大きなレスポンスとして検出されている。しかし、ＢＳＡ固定化のための活性剤であるＢＳ３の応答は、フロー系の方が小さくなっており、ＢＳＡの固定化量と逆になっている。作成した２つのプローブの表面構造に何らかの違いがあることに起因していると考えられる。
【００５５】
また、問題となるＰＥＩ膜表面へのタンパク質の非特異的吸着量を小さくするため、可能なかぎり高い濃度のＢＳ３およびＥＤＣ／ＮＨＳを接触させて、膜中の官能基を活性化させることや、余ったアミノ基を十分にブロックしていく必要がある。タンパク質の吸着の少ない物質として知られている、エーテル結合を多く含んだエチレングリコールユニットを持つ試薬を膜表面の設計の際に導入することも１つの方法として考えられる。
【００５６】
次に、センサープローブのアプリケーションとして、ＰＥＩ膜中のバインディングサイトに抗ｈＩｇＧおよびｐｒｏｔｅｉｎＡを結合させ、これらに高い特異性と強い結合力をもつ人間の免疫グロブリン抗体、ｈＩｇＧを濃度を変化させて接触させたときのレスポンスをそれぞれ測定し、ｈＩｇＧの定量を行った。
また、ｐｒｏｔｅｉｎＡとの弱い結合性をもつマウス−ＩｇＧ、全く結合性を示さないチキンＩｇＧ（ｃｈｉｃｋｅｎ−ＩｇＧ）を接触させることでプローブ表面へのタンパク質の非特異吸着量について検討した。測定に用いた８種類のプローブを図１３にまとめる。
【００５７】
まず図１３のＮｏ．８のプローブを用いて測定を行った時のｈＩｇＧ検出のリアルタイムプロットを図１４に示した。
図１４中の、（１）はＨＢＳ／ＮＨＳバッファーを添加した時点を、（２）はＥＤＣ／ＮＨＳを添加した時点を、（３）は２００μｇ／ｍｌの抗ｈＩｇＧを添加した時点を、（４）はエタノールアミンを添加した時点を、（５）はグリシンを添加した時点を、（６）は０．１ｕｇ／ｍｌのｈＩｇＧを添加した時点を、（７）は１．０ｕｇ／ｍｌのｈＩｇＧを添加した時点をそれぞれ示している。
【００５８】
バッファーが定流している状態でＥＤＣ／ＮＨＳをインジェクトして（図１４の（２）の時点）、膜中のカルボキシル基を活性化し、抗ｈＩｇＧを接触させたところ、ＳＰＲアングルシフトで、２１００ａｒｃｓｅｃｏｎｄほどの固定化量がみられた。図１４の（４）、（５）の時点で、ｐＨ８．５エタノールアミン、ｐＨ３．０グリシン溶液により、チャージで吸着している抗ｈＩｇＧを脱離させると共に、残った膜中の活性化部位をブロックした。ここまでで、ｈＩｇＧ測定のためのセンサープローブの作製が完了し、ｈＩｇＧを濃度を変化させて膜に接触させ、得られるレスポンスを検出した。まず、０．１μｇ／ｍｌ ｈＩｇＧをインジェクトした結果、ほとんど検出されなかったので、塩酸を流して膜の状態を戻し、１０倍の濃度の１μｇ／ｍｌを接触させたところ、２８８ａｒｃ ｓｅｃｏｎｄのレスポンスが検出された。その後、６０、１６０、再び６０μｇ／ｍｌ ｈＩｇＧを順にインジェクトした結果、それぞれの濃度に対応したレスポンスを得ることができた（図１５参照）。
【００５９】
それぞれのプローブごとに得られたデータに基づき検量線を作成し、活性化方法による違い（図１３のＮｏ．２−Ｎｏ．６、Ｎｏ．４−Ｎｏ．８）、および作製方法による違い（図１３のＮｏ．５−Ｎｏ．６、Ｎｏ．７−Ｎｏ．８）について検討を行った（図１６及び図１７参照）。図１６中の左側はＢＳ３で活性化したバッチ法によるものであり、右側はＥＤＣ／ＮＨＳで活性化したバッチ法によるものであり、黒三角印及び白四角印はプロテインＡ−ｈＩｇＧ系を示し、黒丸印及びバツ印は抗ｈＩｇＧ−ｈＩｇＧ系を示す。図１７中の左側はＥＤＣ／ＮＨＳで活性化したバッチ法によるものであり、右側はＥＤＣ／ＮＨＳで活性化したＡＦＩ法のよるものであり、白四角印及び白丸印はプロテインＡ−ｈＩｇＧ系を示し、バツ印及び白菱形印は抗ｈＩｇＧ−ｈＩｇＧ系を示す。
また、非特異抗体の吸着量についてもＮｏ．１、５、６チップを比較することで検討した（図１８参照）。図１８中のグレーの棒はｈＩｇＧを示し、斜線の棒はマウスＩｇＧを示し、横線の棒はチキンＩｇＧを示す。いずれの濃度も９０μｇ／ｍｌである。
ＰＥＩ膜中のアミノ基をＢＳ３を用いて活性化した結果と、グリオキシル酸でカルボキシル基にし、膜表面全体のチャ−ジバランスをとり、これをＥＤＣ／ＮＨＳで活性化した場合について図１６で比較検討した。
【００６０】
その結果、プロテインＡ−ｈＩｇＧ系において、カルボキシル基を導入した表面の方が得られる検量線の直線性が高いうえに、検出感度も増加することがわかった。これは、図１８の非特異抗体の吸着量をみても、プロ−ブＮｏ．５のＥＤＣ／ＮＨＳ活性の方が、Ｎｏ．１のＢＳ３活性と比較してマウス（Ｍｏｕｓｅ）、チキン（Ｃｈｉｃｋｅｎ）−ＩｇＧともに、ｈＩｇＧの量と相対的にみて吸着量がおさえられ、検出精度が高くなった結果とも一致している。
前記したＢＳＡの固定化の実験においては、ＰＥＩにカルボン酸を導入することでＢＳＡの非特異的吸着量は増加したが、今回の抗原−抗体反応においては、等電点などタンパク質の複雑な構造の違いにより、マウス（Ｍｏｕｓｅ）、チキン（Ｃｈｉｃｋｅｎ）−ＩｇＧの膜への非特異吸着は膜にマイナスのチャージを導入することで防げることがわかった。
【００６１】
図１７で、このカルボン酸を導入した系についてＡＦＩ法、バッチ法と分けて検量線を作製した。その結果、フロー系で作製したプローブについても、バッチ法と同等の検出感度、直線性を得られた。また、図１８に示す様に、ＡＦＩ法で作製したＰＥＩ膜はバッチ法と比較して非特異的吸着が減少した。これはチップ表面の膜構造（アミノ基の配向性など）に何らかの違いがあるためだとおもわれる（図１９参照）。例えば、１９図に示されるように吸着ホモポリマー型や、投錨型ポリマー（cerminally-anchored）型などが考えられる。ＡＦＩ法による金属、特に金基盤表面へのタンパク質の固定化はこれまでに報告例がなく、簡便、迅速かつ再現性のよいセンサープローブの作製法として有用な方法であると考えられる。
これらの結果より、タンパク質の非特異的吸着の少ない膜表面の設計には膜表面の電荷の制御が1つの重要なファクターであることがわかり、膜材や作製方法をさらに検討することで、検出特性や検出能はさらに改善される。
【００６２】
また、タンパク質は一般に高分子表面に接触すると徐々に接点を増やし、そのコンフォメーションを変化させることが有り、自然な状態の性質が損なわれる。さらに、その活性サイトが表面との接点になっていたり、立体的に障害されるように配置されても活性を落としてしまう。リガンド固定化の際にその配向性を制御していくことも、検出精度の増加につながる。
【００６３】
次に、アミノ糖を含有するポリマーを膜に使用することについて検討した。
キチン（chitin）はポリアミノ糖であり、キトサン（chitosan）はキチンのアセチル基が部分的に加水分解されたもので、Ｄ−グルコースアミンのβ−（１，４）−重合体である。
chitin、chitosanは甲殻類、昆虫類の組織支持体であるため、生体との親和性がよく、酵素によって生体内で分解される。このことを利用して、医療材料として吸収性縫合糸や人工皮膚などに用いられている。また、免疫活性を有すること、タンパク質などの体液中成分の吸着が少ないことからドラッグデリバリーシステムの担体としても利用されている。さらにchitosanの誘導体は、アシル基の長さにより光学異性体の選択的吸着性があるので、ＨＰＬＣでラセミ混合物の分離に用いられている。
【００６４】
本発明では、キトサンの高い生体適合性と、ＰＥＩ膜センサープローブで問題となったタンパク質の非特異的吸着のない性質を利用して、キトサン膜免疫センサープローブの作製を試みた。
まず、ＰＥＩの時と同様に、金薄膜を７０℃ほどに温めた１Ｍ、ＫＯＨ水溶液に数回つけることで洗浄した後、５ｍＭ Ｌ−システインメチルエステルのＤＭＦ溶液に２４時間浸した後、これを５％グルタルアルデヒド水溶液に２時間浸けた。これに１％キトサン水溶液を２４時間接触させることでキトサンを固定化した。次に、キトサン中のＯＨ基を２ＭのＢｒＣＨ_２ＣＯＯＨ／１Ｍ ＮａＯＨ溶液に１２時間浸すことでカルボキシル化し、カルボキシメチル化キトサン（ＣＭ−ｃｈｉｔｏｓａｎ）センサープローブを作製した（図２参照）。
【００６５】
ＰＥＩ膜プローブによる測定と同様に、作製したプローブをＳＰＲ装置にセットし、ｐＨ７．４ ＨＢＳ Ｔ／バッファーによるベースラインが安定した後、膜中の−ＣＯＯＨ基を、０．４Ｍ ＥＤＣ／０．１Ｍ ＮＨＳ １：１混合溶液をインジェクトして活性化した後、０．５ｍｇ／ｍｌ ＢＳＡを固定化した時の結果を図２０に示す。
図２０中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）は０．４ＭＥＤＣ／０．１ＭＮＨＳを添加した時点を、（３）は０．５ｍｇ／ｍｌのＢＳＡ溶液を添加した時点を、（４）はエタノールアミンを添加した時点をそれぞれ示している。ＢＳＡを添加した時点のレスポンスΔθは１５０アーク秒であった。
【００６６】
キトサン膜にＢｒＣＨ_２ＣＯＯＨを浸すことで膜中の−ＯＨ基を−ＣＯＯＨ基にし、これを活性化した後、ＢＳＡを固定化したところ、図２０に示すように１５０アーク秒のレスポンスが得られた。その際、ＢＳＡの固定化量はＣＭ−ｃｈｉｔｏｓａｎ膜プローブ中に存在するＣＯＯＨ基の量に依存すると考えられる。そこで、キトサンをカルボキシル化する際に、バッチ法によりプローブ表面で行うものから、あらかじめカルボキシル化率の高いＣＭ−ｃｈｉｔｏｓａｎを合成し、これを表面に固定化することでリガンドの固定化量増加を試みた。
【００６７】
キトサン１．０ｇ、ラウリルスルホン酸ナトリウム０．１ｇを４５％ＮａＯＨ水溶液７０ｍｌ中にゆっくり加え、４℃に保ちながら溶解するまで１時間ほど攪拌した。これを−２０℃で、１２時間ほど放冷した後、ｉ−ＰｒＯＨ１２５ｍｌ、ＣｌＣＨ_２ＣＯＯＨ２８．４ｇを加えて７２時間反応させた。続いて、２ＭＨＣｌを加えていき、白色の結晶を９．８７２７ｇ得た。
次に、この結晶０．５ｇを１０％ＮａＯＨ水溶液２０ｍｌに溶解させた後、４ＭＨＣｌをｐＨ１になるまで加えた。これに、大量のアセトンを加えることで結晶を析出させ、吸引ろ過により０．７ｇのＣＭ−ｃｈｉｔｏｓａｎを得た。
【００６８】
前記の方法で合成したＣＭ−ｃｈｉｔｏｓａｎを前と前記したスキームと同様な方法でセンサープローブに固定化しカルボキシル基を活性化したが、この系ではＢＳＡを固定化することはできなかった。スキーム中に反応のいかないステップがあると考えられる。グルタルアルデヒドの固定化までは反応が進むことがわかっているので、ＣＭ−ｃｈｉｔｏｓａｎをモノレイヤ−に固定化する反応がネックになっていると予想された。ＣＭ−ｃｈｉｔｏｓａｎを合成する際、糖のアミノ基もカルボキシル化されるため、ポリマー中の−ＮＨ_２基の量が少なくなっていること、−ＮＨ_２基がポリマーの主鎖に直接ついているため自由度がなく、反応性が低いことが原因になっていると思われる。そこで、別の反応径路に変えてキトサン膜センサ−プローブの作製を行った。
【００６９】
金薄膜を洗浄した後、１０^−３ＭのＨＳ−Ｃ_１０Ｈ_２０ＣＯＯＨ／ＥｔＯＨ溶液に２時間、０．４Ｍ ＥＤＣ／０．１Ｍ ＮＨＳの１：１混合溶液に４０分浸した後、先程の２％ＣＭ−ｃｈｉｔｏｓａｎ水溶液に２時間つけることで、ｃｈｉｔｏｓａｎ膜センサープローブＩＩの作製を行った。このプローブを用いて、膜中の−ＣＯＯＨ基を、０．４Ｍ ＥＤＣ／０．１Ｍ ＮＨＳ １：１混合溶液で活性化し、２００μｇ／ｍｌ ｈＩｇＧを固定化した後、５００μｇ／ｍｌのＢＳＡの膜への吸着を測定した結果を図２１に示す。
図２１中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）はＥＤＣ／ＮＨＳを添加した時点を、（３）は０．２ｍｇ／ｍｌのＩｇＧ溶液を添加した時点を、（４）はエタノールアミンを添加した時点を、（５）は５００μｇ／ｍｌのＢＳＡを添加した時点をそれぞれ示している。ＢＳＡを添加した時点のレスポンスΔθは７２０アーク秒であった。
【００７０】
反応系を変えることで、ＣＭ−ｃｈｉｔｏｓａｎを基盤に固定化することができた。キトサンの性質から、ＣＭ−ｃｈｉｔｏｓａｎ膜プローブではＰＥＩ膜にみられたタンパク質（ＢＳＡ）の膜への非特異的吸着は、ほとんどみられなかった。それを確かめるため、プローブにキトサンおよびＣＭ−ｃｈｉｔｏｓａｎを固定化し、それらを活性化しないでＢＳＡを接触させた時の様子を図２２及び図２３に示す。
図２２中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）はグリシン／ＨＣｌ（ｐＨ３）を添加した時点を、（３）は５００μｇ／ｍｌのＢＳＡを添加下時点をそれぞれ示している。ＢＳＡを添加した時点のレスポンスΔθは５０アーク秒であった。
図２３中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）は５００μｇ／ｍｌのＢＳＡを添加した時点をそれぞれ示している。ＢＳＡを添加した時点のレスポンスΔθは１００アーク秒であった。
【００７１】
キトサンはその性質のとおり非特異吸着はほとんどみられなかったが、その誘導体であるＣＭ−ｃｈｉｔｏｓａｎも、膜中に多量のカルボキシル基が存在しているにも関わらず、５００ｕｇ／ｍｌというかなり高濃度のＢＳＡを接触させた時のレスポンスは１００アーク秒と非常に小さな値となった。これによりＰＥＩ膜プローブ作製課程で行ったプローブ表面の電荷のブッロクはＣＭ−ｃｈｉｔｏｓａｎ膜では必要ないことが確認できた。
【００７２】
次に、ＰＥＩと同様にＡＦＩ法によるキトサン膜センサープローブの調製を行った。ＰＥＩ膜センサープローブの時と同様に金薄膜をＳＰＲ装置にセットし、そこからすべての試薬をフロ−系に導入して固定化するオ−ルフロ−系固定化法を試みた。結果を図２４に示す。
図２４中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）はモノレイヤーのエタノール溶液を添加した時点を、（３）はＥＤＣ／ＮＨＳの０．２Ｍ／０．０５Ｍを添加した時点を、（４）は１％ＣＭ−キトサンを添加した時点を、（５）は０．５ｍｇ／ｍｌのＢＳＡを添加した時点をそれぞれ示している。ＢＳＡを添加した時点のレスポンスΔθが観測された。
【００７３】
ＣＭ−ｃｈｉｔｏｓａｎ膜センサ−プローブの作製をフロ−系で試みた結果、フローで行った系でもＢＳＡを固定化することができたが、そのレスポンスわ僅かであった。これは、フロ−系での各試薬と膜表面との接触時間が約１０分であることから、時間的に反応が充分ではないステップがあると考えられる。フロ−でのＰＥＩ膜の作製が行えることから、ＣＭ−ｃｈｉｔｏｓａｎをモノレイヤ−に固定化する反応がネックになっていると予想され、これはおそらく問題となった固定化部位であるＣＭ−ｃｈｉｔｏｓａｎ中の−ＮＨ_２基がポリマーの主鎖に直接ついているため自由度がなく、反応性が低いことが原因になっていると考えられる。ポリマーの主鎖ではなく、側鎖に自由度の大きなアミノ基などの官能基を有するようにポリマーを修飾することにより、より反応性のあるポリマーとすることができると考えられる。
【００７４】
これまでの実験結果より、合成したCM-chitosanを基盤に固定化したセンサープローブの作製がバッチ法で行え、アミノ基を持つリガンドの検出がＳＰＲシグナルとして確認できた。その際、ＰＥＩ膜プローブにみられたタンパク質（ＢＳＡ）の膜への非特異的吸着は、ほとんどみられなかった。
このプローブのＡＦＩ法へ適用することもできるが、−ＮＨ_２基がポリマーの主鎖に直接ついているため自由度がなく、反応性が低いことが原因となって、バッチほうに比べて充分な反応性がみられないことがある。ＣＭ−ｃｈｉｔｏｓａｎ合成にもう１ステップ加えて、アミノ基に親水性の高いスぺーサーを導入し、モノレイヤーとの結合部位の自由度を高めることによっても、この問題は解決できると考えられる。
【００７５】
また、CM-chitosanの水溶性が低いことも、プローブを作製するうえで問題点のひとつになっている。一般にキトサンは数十万の分子量をもっており、これが小さくなるにつれて水溶性が増していくことがわかっている。分子量の小さいキトサンを使用して、CM-chitosanを合成することで、基盤に固定化されやすくなると予想される。その時の反応条件として酸性度が高すぎると、アミノ基の求核性がプロトン化することで失われ反応が進まなくなるので、中性よりやや塩基性側で反応を行うのが適当だと考えられる。
【００７６】
さらに、本発明は抗原-抗体反応を利用した高感度免疫センサーを提供する。ＣＭ−ｃｈｉｔｏｓａｎセンサープローブのアプリケーションとして、ＰＥＩ膜と比較するためバインディングサイトにプロテインＡを結合させ、これらに高い特異性と強い結合力をもつ人間の免疫グロブリン抗体、ｈＩｇＧを濃度を変化させて接触させたときのレスポンスをそれぞれ測定し、ｈＩｇＧの定量を行った。Ｎｏ．５、６のＰＥＩ膜プローブの結果と合わせた検量線を図２５に示す。図２５中の丸印はＰＥＩ膜プローブを用いたＡＦＩ法によるものであり、四角印はＰＥＩ膜プローブを用いたバッチ法によるものであり、三角印はＣＭ−キトサン膜プローブを用いたバッチ法によるものである。
【００７７】
また同様に、プロテインＡとの弱い結合性をもつマウス−ＩｇＧ、全く結合性を示さないチキン（ｃｈｉｃｋｅｎ）−ＩｇＧを接触させることでプローブ表面へのタンパク質の非特異吸着量について検討し、ＰＥＩ及び市販品であるＣＭＤ膜プローブと比較したデータを図２６に示す。図２６中のグレーの棒はｈＩｇＧを示し、斜線の棒はマウスＩｇＧを示し、横線の棒はチキンＩｇＧを示す。いずれの濃度も９０μｇ／ｍｌである。
【００７８】
ＰＥＩ膜と比較し応答感度は１／４程度に低下したが、この膜は以前に考察したように非特異的抗体の吸着が全くみられず、市販品であるＣＭＤ膜プローブと比較しても、高い検出精度を得ることができた。
これまでに、オリゴエチレンモノレイヤーで表面を被服されたプローブは、タンパク質の非特異的な吸着を抑制することが報告されており、同様な傾向がＣＭ−ｃｈｉｔｏｓａｎ、ＣＭＤ膜プローブでもみられた。
【００７９】
これまでは、タンパク質の吸着性を電荷と親疎水性の点から論じてきたが、この特異的性質を表面付近の水と膜の相互作用という観点から考えてみたい。
水は生命に不可欠な溶媒としての重要性から、また４℃付近で最も密度が高くなり、他の液体に比べて著しく大きな比熱を持つといった特異的性質のため、古くから非常によく研究されてきた。
この水の性質は、分子同士が互いに水素結合することにより形づくられる「構造」に由来するが、その構造に関しては未解決の問題が多く残されている。また、タンパク質や高分子をはじめとする水溶性高分子が、水との相互作用によってその構造や性質、機能に大きく影響を受けることや、逆に高分子の水和水や高分子ゲル中の水が、運動性、凍結−融解挙動等の性質においてバルク水と異なることが知られている。
一般に、高分子表面の水は、自由水とポリマーに束縛された水に分類することができ、束縛の強さによって、ポリマー表面に形成される水和層の状態が異なってくる。この表面水和層の状態が、タンパク質の表面への吸着性を決定する要因の１つであるといわれている。
【００８０】
ポリエチレングリコール、ＣＭ−ｃｈｉｔｏｓａｎ、ＣＭＤの化学構造に共通する点として、いずれもエーテル結合を分子内部に持っており、この部位により膜表面に形成される水和層が、タンパク質の非特異的な吸着を抑制するのではないかと予想される。
【００８１】
本発明は、ＰＥＩ膜センサープローブの作製法として従来のバッチ法に加え、オールフローインジェクト（ＡＦＩ）法を提供したものであり、ＡＦＩ法で作製したＰＥＩ膜は、バッチ法と同等の高い応答感度が得られ、非特異抗体の吸着量も小さかった。また、タンパク質の非特異的吸着の少ない膜表面の設計には膜表面の電荷の制御が１つの重要なファクターであることがわかり、膜材や作製方法をさらに検討することで、検出特性や検出能はさらに改善されると考えられる。さらに、ＡＦＩ法で作製したＰＥＩ膜は、バッチ法と比較して非特異的吸着が少ないうえ、検出精度も高くなることがわかった。これはプローブ表面の膜構造（アミノ基の配向性など）に何らかの違いがあるためだとおもわれる。ＡＦＩ法による、金属表面へのタンパク質の固定化はこれまでに報告例がなく、簡便、迅速かつ再現性のよいセンサープローブの作製法として有用な方法と考えられる。
【００８２】
本発明のＣＭ−ｃｈｉｔｏｓａｎ膜に基づくセンサープローブの検討では、ＡＦＩ法及びバッチ法による作製を行い、プロテインＡを固定化してｈＩｇＧを検出した。その結果、ＰＥＩ膜の場合と比較して応答感度は１／４程度に低下したが、ＣＭ−ｃｈｉｔｏｓａｎ膜は非特異的吸着が全くみられなかった。
これらの結果より、タンパク質の非特異的吸着の少ない膜表面の設計には膜表面の電荷の制御が１つの重要なファクターであることがわかり、膜材や作製方法をさらに検討することで、検出特性や検出能はさらに改善されると考えられる。
【００８３】
本発明のセンサープローブの効果をより明確にするために、公知のデキストラン膜センサーチップとの比較試験を行った。デキストラン膜は既にカルボキシメチル化されているので、本発明のＰＥＩ膜と同様に０．４ＭＥＤＣ／０．１ＭＮＨＳの１：１混合液で活性化して両者にプロテインＡを固定した。
これに、濃度０．１、１．０、１０、４５、９０、２００μｇ／ｍｌのｈＩｇＧをそれぞれのセンサーに流した。本発明のＰＥＩ膜の経時変化を図２７に示す。また、これらの実験で得られた両者の検量線を図２８に示す。図２８の左側が本発明のＰＥＩ膜のものであり、右側はデキストラン膜のものである。
【００８４】
この結果、図２８からもわかるように、本発明のＰＥＩ膜ではｈＩｇＧ濃度が０．１〜９０μｇ／ｍｌ、デキストラン膜では１０〜２００μｇ／ｍｌの範囲でほぼ直線性を示すことがわかり、本発明のＰＥＩ膜のほうがその応答感度において約２０倍も大きく、そのために検出限界も低くなっている。
このように、本発明のポリマー膜、特にＰＥＩ膜は、比較的簡単な化学構造を有するポリマーを使用することにより、簡便で、安価な、しかも高感度のセンサープローブであることがわかる。
【実施例】
次に実施例により本発明をさらに具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
【００８５】
実施例１
金薄膜を７０℃ほどに温めた１ＭＫＯＨ水溶液に数回つけることで洗浄した後、５ｍＭ Ｌ−システインメチルエステルのＤＭＦ溶液に、２４時間浸すことにより、図１の（１）の状態とした。これを、５％グルタルアルデヒド水溶液で２時間処理して図１の（２）の状態とし、次いで２％ＰＥＩ水溶液に２時間浸し図１の（３）で示されるのＰＥＩ膜センサープローブを得た。
【００８６】
実施例２
実施例１で作製したプローブをＳＰＲ装置にセットし、ｐＨ７．４ ＨＢＳ Ｔ／バッファーによるベースラインが安定した後、膜中に存在する未反応のグルタルアルデヒドを０．１Ｍ グリシン溶液をインジェクトすることでブロックした。この後、ＰＥＩ膜中の一級アミンをスペーサーであるＢＳ３で活性化させ、０．５ｍｇ／ｍｌ ＢＳＡを固定化した時の結果を図６に示し、次いで、０．１ｍｇ／ｍｌ ｈＩｇＧを添加した時の結果を図７に示す。
【００８７】
実施例３
実施例２において、タンパク質をＰＥＩ膜プローブに固定化する際、ｐＨ７．４ ＨＢＳ Ｔ／バッファーの代わりにｐＨ５．３酢酸バッファーを使用し反応時のｐＨを変えて実施例２と同様に行った。結果を図１０に示した。
【００８８】
実施例４
また、実施例２においてｐＨ７．４ ｂｕｆｆｅｒで、スペーサーとしてＢＳ３の代わりに１００ｍＭ グリオキシ酸ル（ＯＨＣ−ＣＯＯＨ）と反応させカルボキシル基を導入し、膜全体のチャージバランスをとり、これを０．４Ｍ ＥＤＣ／０．１Ｍ ＮＨＳ １：１混合溶液で活性化した後、ＢＳＡを固定化した。この結果を図１１に示した。
【００８９】
実施例５
スペーサーによりカルボキシル基を導入したセンサープローブに緩衝液を流し、バッファーが定流している状態でＥＤＣ／ＮＨＳをインジェクトして、膜中のカルボキシル基を活性化し、抗ｈＩｇＧを接触させたところ、ＳＰＲアングルシフトで、２１００ａｒｃｓｅｃｏｎｄほどの固定化量がみられた。次いで、ｐＨ８．５エタノールアミン、ｐＨ３．０グリシン溶液により、チャージで吸着している抗ｈＩｇＧを脱離させると共に、残った膜中の活性化部位をブロックした。ここまでで、ｈＩｇＧ測定のためのセンサープローブの作製が完了した。
ｈＩｇＧを濃度を変化させて膜に接触させ、得られるレスポンスを検出した。１μｇ／ｍｌを接触させたところ、２８８ａｒｃ ｓｅｃｏｎｄのレスポンスが検出された。その後、６０、１６０、再び６０μｇ／ｍｌ ｈＩｇＧを順にインジェクトした結果、それぞれの濃度に対応したレスポンスを得ることができた。
結果を図１４及び図１５に示した。
【００９０】
実施例６
それぞれのプローブごとに実施例５と同様にして得られたデータに基づき検量線を作成し、活性化方法による違い（図１３のＮｏ．２−Ｎｏ．６、Ｎｏ．４−Ｎｏ．８）、および作製方法による違い（図１３のＮｏ．５−Ｎｏ．６、Ｎｏ．７−Ｎｏ．８）について同様にして行った。結果を図１６及び図１７に検量線として示した。
また、非特異抗体の吸着量についても図１３のＮｏ．１、５、６チップを比較することで検討した。結果を図１８に示した。
【００９１】
実施例７
実施例１のＰＥＩの時と同様に、金薄膜を７０℃ほどに温めた１Ｍ、ＫＯＨ水溶液に数回つけることで洗浄した後、５ｍＭ Ｌ−システインメチルエステルのＤＭＦ溶液に２４時間浸した後、これを５％グルタルアルデヒド水溶液に２時間浸けた。これに１％キトサン水溶液を２４時間接触させることでキトサンを固定化した。
次に、キトサン中のＯＨ基を２ＭのＢｒＣＨ_２ＣＯＯＨ／１Ｍ ＮａＯＨ溶液に１２時間浸すことでカルボキシル化し、カルボキシメチル化キトサン（ＣＭ−ｃｈｉｔｏｓａｎ）センサープローブを作製した（図２参照）。
【００９２】
実施例８
ＰＥＩ膜プローブによる測定と同様に、作製したプローブをＳＰＲ装置にセットし、ｐＨ７．４ ＨＢＳ Ｔ／バッファーによるベースラインが安定した後、膜中の−ＣＯＯＨ基を、０．４Ｍ ＥＤＣ／０．１Ｍ ＮＨＳ １：１混合溶液をインジェクトして活性化した後、０．５ｍｇ／ｍｌ ＢＳＡを固定化した時の結果を図２０に示す。
【００９３】
実施例９
キトサン１．０ｇ、ラウリルスルホン酸ナトリウム０．１ｇを４５％ＮａＯＨ水溶液７０ｍｌ中にゆっくり加え、４℃に保ちながら溶解するまで１時間ほど攪拌した。これを−２０℃で、１２時間ほど放冷した後、ｉ−ＰｒＯＨ１２５ｍｌ、ＣｌＣＨ_２ＣＯＯＨ２８．４ｇを加えて７２時間反応させた。続いて、２ＭＨＣｌを加えていき、白色の結晶を９．８７２７ｇ得た。
次に、この結晶０．５ｇを１０％ＮａＯＨ水溶液２０ｍｌに溶解させた後、４ＭＨＣｌをｐＨ１になるまで加えた。これに、大量のアセトンを加えることで結晶を析出させ、吸引ろ過により０．７ｇのＣＭ−ｃｈｉｔｏｓａｎを得た。
【００９４】
実施例１０
金薄膜を洗浄した後、１０^−３ＭのＨＳ−Ｃ_１０Ｈ_２０ＣＯＯＨ／ＥｔＯＨ溶液に２時間、０．４Ｍ ＥＤＣ／０．１Ｍ ＮＨＳの１：１混合溶液に４０分浸した後、実施例９で製造した２％ＣＭ−ｃｈｉｔｏｓａｎ水溶液に２時間つけることで、キトサン膜センサープローブＩＩを製造した。このプローブを用いて、膜中の−ＣＯＯＨ基を、０．４Ｍ ＥＤＣ／０．１Ｍ ＮＨＳ １：１混合溶液で活性化し、２００μｇ／ｍｌ ｈＩｇＧを固定化した後、５００μｇ／ｍｌのＢＳＡの膜への吸着を測定した結果を図２１に示す。
【００９５】
実施例１１
実施例２と同様にして、ＡＦＩ法によりキトサン膜センサープローブの調製を行った。ＰＥＩ膜センサープローブの時と同様に金薄膜をＳＰＲ装置にセットし、そこからすべての試薬をフロ−系に導入して固定化するオ−ルフロ−系で固定化法を行った。結果を図２４に示す。
図２４中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）はモノレイヤーのエタノール溶液を添加した時点を、（３）はＥＤＣ／ＮＨＳの０．２Ｍ／０．０５Ｍを添加した時点を、（４）は１％ＣＭ−キトサンを添加した時点を、（５）は０．５ｍｇ／ｍｌのＢＳＡを添加した時点をそれぞれ示している。ＢＳＡを添加した時点のレスポンスΔθが観測された。
【００９６】
実施例１２
ＰＥＩ膜を０．４ＭＥＤＣ／０．１ＭＮＨＳの１：１混合液で活性化して、これにプロテインＡを固定した。
これに、濃度０．１、１．０、１０、４５、９０、２００μｇ／ｍｌのｈＩｇＧをそれぞれのセンサーに流した。ＡＦＩ法によるＰＥＩ膜の経時変化の結果を図２７に示す。図２７中の１，０００Ｒ．Ｕは１ｎｇ／ｍｍ^２である。
また、この実験で得られた検量線を図２８の左側に示す。
【００９７】
比較例１
デキストラン膜を用いて実施例１２と同様に行った。その結果得られた検量線を、図２８の右側に示す。
【００９８】
【発明の効果】
本発明は、簡便、迅速かつ再現性のよいＳＰＲ測定用のセンサープローブとして有用な金属薄膜を提供するものであり、本発明のセンサープローブは高感度で、高精度のものであり、かつ、簡便で安価に製造することができる。
【図面の簡単な説明】
【図１】図１は、原料物質として、システインメチルエステル、グルタルアルデヒド及びポリエチレンイミンを用いた場合の本発明の金属薄膜の処理法を示す。
【図２】図２は、原料として、システインメチルエステル、グルタルアルデヒド及びキトサンを用いた場合の本発明の金属薄膜の処理法を示す。
【図３】図３は、本発明のポリエチレンイミン膜を有する金属薄膜にスペーサーとしてスベリン酸を用い、活性エステル化法により検出用試薬（図３中ではリガンドとして示されている。）を固定する方法を示す。
【図４】図４は、図２に示したＣＭ−キトサンセンサープローブに検出用試薬（図４中ではリガンドとして示されている。）を固定する方法を示す。
【図５】図５は、本発明の金属薄膜の調製法と、表面プラズモン共鳴装置の概要と、本発明の金属薄膜のバッチ法とオールフローインジェクト法（ＡＦＩ法）の概要を示す。
【図６】図６は、本発明のＰＥＩ膜を用いたＳＰＲの測定結果であり、横軸に時間（分）、縦軸にレスポンス（アーク秒（１度は３６００アーク秒））を示す。図６中の、（１）はｐＨ７．４ ＨＢＳ Ｔ／バッファーを添加した時点を、（２）はＢＳ３を添加した時点を、（３）は０．５ｍｇ／ｍｌのＢＳＡを添加した時点を、（４）はグリシンｐＨ３を添加した時点をそれぞれ示している。ＢＳＡを添加した時点でのレスポンスΔθは６０４アーク秒であった。
【図７】図７は、本発明のＰＥＩ膜を用いたＳＰＲの測定結果であり、横軸に時間（分）、縦軸にレスポンス（アーク秒（１度は３６００アーク秒））を示す。図７中の、（１）はｐＨ７．４ ＨＢＳ Ｔ／バッファーを添加した時点を、（２）はＢＳ３を添加した時点を、（３）は０．１ｍｇ／ｍｌのｈＩｇＧを添加した時点を、（４）はグリシンｐＨ３を添加した時点をそれぞれ示している。ｈＩｇＧを添加した時点でのレスポンスΔθは１５０アーク秒であった。
【図８】図８は、本発明のＰＥＩ膜を用いたＡＦＩ法によるＳＰＲの測定結果である。図８中の、（１）はｐＨ７．４ ＨＢＳ Ｔ／バッファーを添加した時点を、（２）は１０ｍＭＬ−システインを添加した時点を、（３）は１０％グルタルアルデヒドを添加した時点を、（４）は１％ＰＥＩを添加した時点をそれぞれ示している。
【図９】図９は、本発明のＰＥＩ膜を用いたＡＦＩ法によるＳＰＲの測定結果である。図９中の、（ａ）はｐＨ７．４ ＨＢＳ Ｔ／バッファーを添加した時点を、（ｂ）は１ｍＭのＢＳ３を添加した時点を、（ｃ）は５００μｇ／ｍｌのＢＳＡを添加した時点を、（ｄ）はグリシンｐＨ３を添加した時点を、（ｅ）はｐＨ８．５で０．１Ｍのエタノールアミンを添加した時点をそれぞれ示している。
【図１０】図１０は、本発明のＰＥＩ膜を用い、緩衝液としてｐＨ５．３酢酸バッファーを用いたときのＳＰＲの測定結果である。図１０中の、（１）はｐＨ５．３酢酸バッファーを添加した時点を、（２）はスペーサーのＢＳ３を１ｍＭ添加した時点を、（３）は０．５ｍｇ／ｍｌのＢＳＡ溶液を添加した時点を、（４）は０．１Ｍのグリシン（ｐＨ３．０）を添加した時点をそれぞれ示している。
【図１１】図１１は、本発明のＰＥＩ膜を用い、膜中のアミノ基を１００ｍＭグリオキル酸と反応させカルボキシル化したときのＳＰＲの測定結果である。図１１中の、（１）はｐＨ７．４ＨＢＳバッファーを添加した時点を、（２）は０．４ＭＥＤＣ／０．１ＭＮＨＳを添加した時点を、（３）は０．５ｍｇ／ｍｌのＢＳＡ溶液を添加した時点を、（４）は０．１Ｍのグリシン（ｐＨ３．０）を添加した時点をそれぞれ示している。
【図１２】図１２は、本発明のＰＥＩ膜の未反応のアミノ基どうしを、グルタルアルデヒドで架橋することで不活性化するようすを模式的に示したものである。
【図１３】図１３は、本発明の測定に用いた８種類のプローブをまとめて示したものである。
【図１４】図１４は、本発明の図１３に記載のＮｏ．８のプローブを用いて測定を行った時のｈＩｇＧ検出のリアルタイムプロットを示したものである。図１４中の、（１）はＨＢＳ／ＮＨＳバッファーを添加した時点を、（２）はＥＤＣ／ＮＨＳを添加した時点を、（３）は２００μｇ／ｍｌの抗ｈＩｇＧを添加した時点を、（４）はエタノールアミンを添加した時点を、（５）はグリシンを添加した時点を、（６）は０．１ｕｇ／ｍｌのｈＩｇＧを添加した時点を、（７）は１．０ｕｇ／ｍｌのｈＩｇＧを添加した時点をそれぞれ示している。
【図１５】図１５は、ｈＩｇＧを濃度を６０、１６０、再び６０μｇ／ｍｌｈＩｇＧに変化させて膜に接触させ、得られるレスポンスを示したものである。
【図１６】図１６は、本発明のセンサープローブを用いた検量線を示す。図１６中の左側はＢＳ３で活性化したバッチ法によるものであり、右側はＥＤＣ／ＮＨＳで活性化したバッチ法によるものであり、黒三角印及び白四角印はプロテインＡ−ｈＩｇＧ系を示し、黒丸印及びバツ印は抗ｈＩｇＧ−ｈＩｇＧ系を示す。
【図１７】図１７は、本発明のセンサープローブを用いた検量線を示す。図１７中の左側はＥＤＣ／ＮＨＳで活性化したバッチ法によるものであり、右側はＥＤＣ／ＮＨＳで活性化したＡＦＩ法のよるものであり、白四角印及び白丸印はプロテインＡ−ｈＩｇＧ系を示し、バツ印及び白菱形印は抗ｈＩｇＧ−ｈＩｇＧ系を示す。
【図１８】図１８は、本発明のセンサープローブを用いたときの非特異抗体の吸着量を示す。図１８中のグレーの棒はｈＩｇＧを示し、斜線の棒はマウスＩｇＧを示し、横線の棒はチキンＩｇＧを示す。いずれの濃度も９０μｇ／ｍｌである。
【図１９】図１９は、センサープローブの表面の膜構造をもし九滴に示したものである。
【図２０】図２０は、本発明のＣＭ−キトサン膜を用いたＳＰＲ測定の結果を示す。図２０中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）は０．４ＭＥＤＣ／０．１ＭＮＨＳを添加した時点を、（３）は０．５ｍｇ／ｍｌのＢＳＡ溶液を添加した時点を、（４）はエタノールアミンを添加した時点をそれぞれ示している。
【図２１】図２１は、本発明のＣＭ−キトサン膜を用いたＳＰＲ測定の結果を示す。図２１中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）はＥＤＣ／ＮＨＳを添加した時点を、（３）は０．２ｍｇ／ｍｌのＩｇＧ溶液を添加した時点を、（４）はエタノールアミンを添加した時点を、（５）は５００μｇ／ｍｌのＢＳＡを添加した時点をそれぞれ示している。
【図２２】図２２は、活性化をせずに本発明のＣＭ−キトサン膜を用いたＳＰＲ測定の結果を示す。図２２中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）はグリシン／ＨＣｌ（ｐＨ３）を添加した時点を、（３）は５００μｇ／ｍｌのＢＳＡを添加下時点をそれぞれ示している。
【図２３】図２３は、活性化をせずに本発明のＣＭ−キトサン膜を用いたＳＰＲ測定の結果を示す。図２３中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）は５００μｇ／ｍｌのＢＳＡを添加した時点をそれぞれ示している。
【図２４】図２４は、本発明のＣＭ−キトサン膜を用いたＡＦＩ法によるＳＰＲ測定の結果を示す。図２４中の、（１）はｐＨ７．４ＨＢＳ／Ｔバッファーを添加した時点を、（２）はモノレイヤーのエタノール溶液を添加した時点を、（３）はＥＤＣ／ＮＨＳの０．２Ｍ／０．０５Ｍを添加した時点を、（４）は１％ＣＭ−キトサンを添加した時点を、（５）は０．５ｍｇ／ｍｌのＢＳＡを添加した時点をそれぞれ示している。
【図２５】図２５は、本発明のプローブを用いたときの検量線を示す。図２５中の丸印はＰＥＩ膜プローブを用いたＡＦＩ法によるものであり、四角印はＰＥＩ膜プローブを用いたバッチ法によるものであり、三角印はＣＭ−キトサン膜プローブを用いたバッチ法によるものである。
【図２６】図２６は、本発明のセンサープローブを用いたときの非特異抗体の吸着量を示す。図２６中のグレーの棒はｈＩｇＧを示し、斜線の棒はマウスＩｇＧを示し、横線の棒はチキンＩｇＧを示す。いずれの濃度も９０μｇ／ｍｌである。
【図２７】図２７は、本発明のＰＥＩ膜にプロテインＡを固定したセンサーを用いた、各種濃度のｈＩｇＧにおける経時変化を示したものである。図２７中の１，０００Ｒ．Ｕは１ｎｇ／ｍｍ^２である。
【図２８】図２８は、実施例１２及び比較例１で得られた検量線を示したものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a metal thin film surface-treated with a polymer-bound organic linker, a surface plasmon resonance (SPR) apparatus using the same, and a method for detecting, identifying or quantifying a sample thereby.
[0002]
[Prior art]
Today, immunoassays are widely used in the field of infectious disease testing and diagnosis. In general, a large amount of antibody is produced in the body in a normal infection, and diagnosis is performed by identifying the presence or absence of this antibody. In addition, the values of components (blood glucose level, cholesterol level, etc.) in body fluids such as serum, urine, and saliva are important parameters for grasping the patient's pathological condition and examining the treatment policy.
At the time of diagnosis, the amount of sample collected is limited, and frequent collection is difficult, so the measurement method requires high accuracy (reproducibility) and sensitivity. Moreover, since the target component is usually contained in a very small amount in a complex matrix called a biological sample, it is extremely important to obtain a highly reliable analysis result accurately.
[0003]
On the other hand, a surface plasmon resonance (SPR) sensor is a device that detects a surface plasmon resonance phenomenon that occurs on the surface of a metal thin film such as gold or silver. Surface plasmon is a kind of electron density wave generated at the metal-dielectric interface, and the wave number varies depending on the thickness and optical characteristics (dielectric constant, refractive index) of the sample up to several hundred nm in contact with the metal thin film surface. Since it is impossible to measure this change directly, the SPR sensor generates an evanescent wave by applying a laser beam from the opposite side of the sample, and changes the incident angle of the laser (SPR angle shift) when it resonates with the surface plasmon. It is a common method to indirectly measure changes in the surface state by measuring.
[0004]
In 1971, Kretschmann established the surface plasmon excitation method by photoexcitation, and 11 years later, NPR was first applied to gas sensing as a sensor by Nylander et al. . This sensor was a simple one that captured the change in the refractive index of the bulk due to the presence of the sample. By around 1990, however, this type of sensor for the concentration of alcohol in the sample solution, etc., was developed extensively. It was broken.
[0005]
In the late 1980s, research on self-adsorption on gold surfaces such as thiols and sulfides was conducted, and self-adsorbing monolayers modified with gold thiols having various functional groups such as carboxylic acids and amino groups. A research example of a membrane (Self-Assembled Monolayer) has been reported.
In 1993, a specific reaction such as an antigen-antibody occurred in the active site of a two-layer structure in which another layer with charge was formed on a self-adsorbing monolayer on the gold surface. Biosensors with immobilized ligands have been developed and used for qualitative and quantitative analysis and elucidation of reaction processes (Stelzle, M., et al., J. Phys. Chem., 97, 2974- 2981 (1993)).
[0006]
In addition, research on optical fiber type SPR sensors aimed at miniaturization began to be reported from around this time, and the method of measuring the incident angle at which resonance with the sample occurs while keeping the wavelength of the conventional light source constant, A sensor device has been developed that keeps the wavelength constant. Since 1995, many examples of sensing using an SPR sensor have been reported, and in particular, in the field of biosensors, many examples of research aimed at increasing the sensitivity of a sensor and measuring a new ligand have been reported.
[0007]
In the development of the SPR chemical sensor, it is required to apply some modification to the metal thin film of the sensing part. In order to apply the SPR sensor to a biosensor, modification of a polymer having a functional group to the metal thin film can be considered.
In the case of using a gold thin film as a metal thin film, a modification method using the self-adsorption property of thiols and sulfides to gold (Self-Assembly) is conceivable. Actually, in the SPR biosensor, the ligand is immobilized on the sensor surface. There has been reported a method in which a carboxymethyldextran (CMD) layer is introduced and immobilized on the CMD layer using alkanethiol self-adsorbed on the Au thin film as a linker layer. The role of the CMD layer is to increase the amount of ligand to be immobilized and to prevent nonspecific binding of biomolecules to the gold thin film. Methods for modifying thiols and sulfides are described in Claire. In a paper by Jordan et al. (Jordan, CE, et al., Langmuir, 1994, 10,3642-3648), an adsorption rate of 90% is obtained by simply immersing a gold thin film in an ethanol solution of 1 mM thiol for 1 h. Adsorption can be evaluated by looking at XPS (X-ray photoelectron spectroscopy) and SPR angle change.
[0008]
Japanese Patent Application Laid-Open No. 4-501605 describes an invention relating to a detection surface in which a layer of a biocompatible porous matrix such as a hydrogel is provided on a layer bonded to a metal surface. Japanese Patent No. 4-501606 describes an invention about a sensor unit using a bifunctional or polyfunctional molecule, and Japanese Patent Publication No. 4-501607 discloses a macromolecule using a sensor in which a ligand is bound to the sensor surface. An invention relating to a method for determining the characteristics of the above is described. Japanese Patent Publication No. 7-507865 describes an invention for a method and apparatus for detecting an analyte of interest in a sample using a solid support containing an immobilized binding partner containing a reversible binding receptor. Has been. WO96 / 10178 describes an invention about a method for producing a temperament surface in which a film-forming protein or lipid is bound on a self-adsorbing monolayer.
[0009]
Since the SPR sensor is a device that indirectly measures the state change of the sample using an evanescent wave, the part where the laser beam used for measurement passes and the part where the sample exists are different, so compared to conventional optical sensors. There are the following advantages.
1. Since the change in the refractive index of the sample occurring on the sensor surface can be detected in real time, the progress of the experiment and the kinetic relationship can be known.
2. Since the laser beam used for measurement does not pass through the sample solution, it is not easily affected by coloring, turbidity or bubbles of the sample solution.
3. Since the optical characteristics in a very narrow region of about 1 μm from the sensor substrate surface are detected, the amount of the sample solution required is very small and is not easily affected by background noise.
However, the conventional sensor surface treatment does not have good SPR sensitivity and is not sufficiently reactive, and is not practical for real-time measurement.
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a sensor device that can detect more biological components by chemically modifying the sensor surface with polymers having various characteristics and functions. Specifically, it is intended to provide a highly sensitive immunosensor using an antigen-antibody reaction using a metal thin film as a sensor substrate and using a surface plasmon resonance (SPR) device as a device. . Furthermore, it aims at providing the novel carbohydrate sensor using carbohydrate binding protein (Concanavalin A: ConA).
The present invention also provides a metal thin film for these novel sensors, an SPR device using the same, and a method for detecting, identifying or quantifying a sample using the same.
[0011]
[Means for Solving the Problems]
In the present invention, the surface of a metal thin film having a free electron metal surface is directly applied to (1) an organic linker having a functional group that can be immobilized on the metal surface and a functional group that can bind to a polymer, and (2) a detection reagent. The present invention relates to a metal thin film treated with a functional group capable of binding or a functional group capable of binding to a detection reagent via a spacer and a polymer having a functional group capable of binding to the organic linker. More specifically, the present invention relates to the metal thin film, wherein the polymer is a polymer having an amino group such as chitosan or polyethyleneimine.
[0012]
The present invention further relates to a metal thin film in which the detection reagent is bonded to the polymer directly or via a spacer having a functional group capable of binding to the detection reagent.
The metal thin film of the present invention is useful as a highly sensitive sensor thin film for measuring surface plasmon resonance (SPR).
[0013]
The present invention also relates to a method for producing the metal thin film, a measurement method using surface plasmon resonance (SPR) using the metal thin film, a kit therefor, and a surface plasmon resonance (SPR) measurement apparatus.
[0014]
The surface of the metal thin film of the present invention has (1) one organic linker layer fixed to the metal thin film surface, (2) a polymer layer bonded to the organic linker layer, and (3) a polymer layer directly or via a spacer. It consists of a detection reagent layer that binds.
[0015]
The organic linker layer of the present invention is composed of atoms selected from the group consisting of a carbon atom, an oxygen atom, and a nitrogen atom between the functional group that can be fixed on the metal surface and the functional group that can be bonded to the polymer. The moiety may be a single molecular species as long as it has a linear, branched or cyclic chemical structure of 2 to 20 atoms, preferably 2 to 15 atoms, more preferably 2 to 10 atoms. However, it may be designed using two or more molecular species. When two or more molecular species are used, a functional group having a relatively high reactivity can be used, and an organic linker layer can be formed in a short time and at a low cost. It becomes.
[0016]
The “functional group that can be immobilized on the metal surface” of the organic linker having the functional group that can be immobilized on the metal surface and the functional group that can be bonded to the polymer forming the organic linker layer of the present invention is immobilized by chemical bonding to the metal surface The functional group may be a functional group that can be fixed by adsorption. As such a functional group, those containing a sulfur atom such as a mercapto group, sulfide group or disulfide group are preferable. In addition, as the “functional group capable of binding to the polymer”, various functional groups can be selected depending on the functional group contained in the polymer to be used. For example, when the polymer contains an amino group, a carboxyl group or a carbonyl group can be selected, and when the polymer contains a carboxyl group, an amino group or a hydroxyl group is selected. be able to.
The carbonyl group in the present invention includes an aldehyde group and a keto group, and is preferably an aldehyde group.
[0017]
When a single molecular species is used as the organic linker of the present invention, for example, it has 2 to 20 carbon atoms, preferably 2 to 15 carbon atoms having a functional group containing a sulfur atom such as a mercapto group, sulfide group or disulfide group. More preferably, 2 to 12 fatty acids can be used. More specifically, for example, 11-mercapto-undecanoic acid can be mentioned.
[0018]
More preferred organic linkers of the present invention include those formed using two or more molecular species. The first molecular species immobilized on the metal surface has a functional group containing a sulfur atom such as a mercapto group, sulfide group or disulfide group as the “functional group capable of being immobilized on the metal surface”, and Organic compounds having other reactive functional groups capable of forming a chemical bond with the two molecular species. Any reactive functional group may be used as long as it can form a chemical bond with the second molecular species, and examples thereof include an amino group, a hydroxyl group, and a carboxyl group. In view of reactivity and availability, an amino acid having a functional group containing a sulfur atom such as a mercapto group, sulfide group or disulfide group or a derivative thereof is preferred, and more specifically, a cysteine alkyl ester is preferred. As the alkyl ester, an alkyl ester having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms is preferable. More specifically, cysteine methyl ester, cysteine ethyl ester and the like can be mentioned.
[0019]
The second molecular species bonded to the first molecular species forming the organic linker of the present invention has a functional group capable of chemically bonding to the reactive functional group of the first molecular species, and the following polymer layer An organic compound having a functional group that can be bonded to the polymer that forms, and examples of these functional groups include an amino group, a carboxyl group, a hydroxyl group, and a carbonyl group. As the preferred second molecular species, for example, a compound having a highly reactive carbonyl group is preferred, and more specifically, glutaraldehyde and the like can be mentioned.
As the molecular species forming the organic linker of the present invention, there are various combinations of organic compounds. As a preferred combination, cysteine methyl ester is used as the first molecular species, and glutaraldehyde is used as the second molecular species. This is a preferred embodiment of the present invention because the reaction proceeds without special activation of the functional group (for example, converting the carboxyl group to an active ester). it can.
[0020]
The method for fixing the organic linker of the present invention to the metal surface can be carried out by bringing a compound to be the organic linker into contact with the metal. It is preferable to wash the metal surface with an alkali such as potassium hydroxide before contacting. The contact with the metal surface is preferably performed in the presence of an organic solvent such as dimethylformamide (DMF) or water.
Next, an organic linker layer can be formed by reacting the second molecular species as necessary. In this case, the functional group involved in the reaction can be activated as necessary.
[0021]
The polymer that forms the polymer layer of the present invention has a reactive functional group that can be bonded to a “functional group that can be bonded to a polymer” in an organic linker such as an amino group, a carboxyl group, a hydroxyl group, and a carbonyl group. And it is a polymer which has a functional group which can couple | bond the detection reagent or the spacer for couple | bonding a detection reagent. The “reactive functional group” and “functional group capable of binding to a detection reagent” in these polymers may be different functional groups, or the same functional group. Preferred functional groups include amino groups, carboxyl groups, hydroxyl groups, carbonyl groups and the like.
When the “functional group capable of binding to the polymer” in the organic linker is a carbonyl group such as an aldehyde group, a polymer having a primary amino group is preferred. The primary amino group in the polymer may be bonded to the main chain of the polymer, but is preferably bonded to the side chain of the polymer.
[0022]
Examples of the polymer of the present invention include polyalkyleneimines, polysaccharides, polylysine and the like, but polylower alkyleneimines and polysaccharides containing amino sugars are preferred.
Examples of the polyalkyleneimine include a polymer of alkylenediamine, and a polymer in which a primary amino group is present in the side chain portion is preferable, but a primary amino group is introduced into the side chain portion by chemical modification after polymerization. It may be a thing. The alkylene diamine is a diamine having an alkylene group having 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, preferably ethylene diamine. Preferable examples of the alkylenediamine polymer include polyethyleneimine having a repeating unit represented by the following formula.
[0023]
[Chemical 1]

[0024]
Polysaccharides containing amino sugars are preferred as polysaccharides, and amino sugars may be polymerized or amino sugars may be partially introduced. However, polysaccharides obtained by polymerizing amino sugars such as chitosan may be used. preferable. Occasionally, a reactive functional group such as an amino group may be introduced into a polysaccharide.
The molecular weight of these polymers varies depending on the type of polymer, but is generally 1,000 to 1,000,000, preferably 5,000 to 1,000,000, more preferably 5,000 to 500. About 1,000.
[0025]
As a method for bonding the organic linker and the polymer, they can be reacted directly in the presence of a solvent. However, when the reactivity is not sufficient, these functional groups are activated, for example, by active esterification or the like. It can also be activated and reacted.
FIG. 1 shows a method for treating a metal thin film of the present invention when cysteine methyl ester, glutaraldehyde, and polyethyleneimine are used as raw materials. After washing the metal thin film several times in a warmed aqueous potassium hydroxide solution, it is immersed in a DMF solution of L-cysteine methyl ester to obtain the state shown in (1) of FIG. 1, which is treated with an aqueous glutaraldehyde solution. The sensor probe having the polyethyleneimine film of (3) in FIG. 1 was obtained by immersing a polyethyleneimine aqueous solution in the state of (2) in FIG.
[0026]
Moreover, the processing method of the metal thin film of this invention at the time of using cysteine methyl ester, glutaraldehyde, and chitosan as a raw material is shown in FIG. In FIG. 2, α-bromoacetic acid is further used as a spacer.
After washing the metal thin film by the same method as shown in FIG. 1, an organic linker part is prepared by treating with an L-cysteine methyl ester DMF solution and a glutaraldehyde aqueous solution, and contacting this with a 1% chitosan aqueous solution As a result, a sensor probe was prepared by immobilizing chitosan. Next, chitosan was carboxylated with sodium hydroxide solution of α-bromoacetic acid to prepare a carboxymethyl (spacer moiety) CM-chitosan sensor probe.
[0027]
The detection reagent of the present invention is a reagent that can detect, identify or quantify a specific substance in a sample, and is not particularly limited as long as it can be fixed to the polymer by adsorption or binding, Those having a functional group such as a carboxyl group and an amino group are preferred. A preferred detection reagent is a protein. More specifically, an antigen or an antibody can be mentioned.
Although the detection reagent of the present invention can be directly fixed to the polymer layer, both can be fixed using a substance serving as a spacer if necessary. As the spacer, various multifunctional substances can be used. Although relatively long-chain substances can be used, those having 2 to 15 carbon atoms, preferably about 2 to 10 carbon atoms are preferable. Examples of the compound used as the spacer include halocarboxylic acids such as α-bromoacetic acid and α-chloroacetic acid, and dicarboxylic acids such as glutaric acid, adipic acid, and suberic acid.
[0028]
FIG. 3 shows a method of immobilizing a detection reagent (shown as a ligand in FIG. 3) by active esterification using suberic acid as a spacer on the metal thin film having the polyethyleneimine film shown in FIG. Show.
FIG. 4 shows a method of immobilizing a detection reagent (shown as a ligand in FIG. 4) on the CM-chitosan sensor probe shown in FIG. In FIG. 4, EDC represents 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and NHS represents N-hydroxysuccinimide.
[0029]
As a metal used for the metal thin film of this invention, what is necessary is just a metal which has a free electron, For example, gold | metal | money, silver, copper, aluminum, chromium etc. are mentioned. The preferred metal is gold, but silver, copper, aluminum and the like are also preferred metals. The metal thin film may be produced by extending a metal, or may be formed on a substrate by a method such as vapor deposition. The thickness of the metal thin film is not particularly limited as long as it is a measurable thickness, but is about 50 to 1,000 nm, preferably 50 to 500 nm, and more preferably about 100 to 500 nm.
[0030]
When the metal thin film of the present invention is used in a surface plasmon resonance (SPR) apparatus, the metal thin film of the present invention is used in close contact with a substrate such as a prism.
FIG. 5 shows an outline of a method for preparing a metal thin film of the present invention and a surface plasmon resonance apparatus. As shown in FIG. 5, there are a batch method and an all-flow injection method (AFI method) as methods for preparing the metal thin film of the present invention. In the batch method, a metal thin film is treated with substances necessary for forming an organic linker layer, a polymer layer, a detection reagent, and the like (shown as A liquid, B liquid, and C liquid in FIG. 5). In this method, after a treated metal thin film (sensor probe) is prepared, it is set in an SPR device, and a sample is added to perform measurement.
[0031]
In the AFI method, the set metal thin film is processed into a desired state by sequentially flowing a treatment solution to the metal thin film set in the SPR apparatus. More specifically, as shown in FIG. 5, a buffer such as a pump for HPLC is constantly used (for example, a running buffer (pH 7.4 10 mM HBS (HEPES Buffer Saline) / Tween)) with a flow rate of 0. .1 to 1.0, preferably 0.2 to 0.4 ml / min, and when necessary, a solution containing each reaction reagent is injected from the injector to be supplied to the metal thin film (sensor probe). Therefore, necessary processing can be performed. During this time, measurement can be continued, and the reaction occurring on the opposite side of the measurement surface of the metal thin film can be measured without irradiating the measurement line such as light or microwave directly on the reaction system. can do.
[0032]
Since the AFI method can treat the surface of the metal thin film while continuing the measurement, the state of the surface can be grasped in real time. In addition, the mutual trend (such as chemical reaction) between the detection reagent fixed on the surface of the metal thin film and the substance in the sample can be sequentially measured and observed.
The present invention includes both the batch method and the AFI method.
[0033]
In order to impart the desired performance and function to the material, it is important to design the material surface at the molecular level to provide functional properties. Therefore, understanding the reaction process including the surface of the sensor material and the interaction between biological substances is an important issue in the sensing of biological substances using antigen-antibody reactions and cell receptor recognition in today's clinical chemistry field. ing.
[0034]
An object of the present invention is to construct a sensor probe that can be used for immobilization and detection of a wide range of substances by constructing a sensor surface using polymers having various functional groups. Specifically, the surface of the sensor was designed using a gold thin film as a sensor substrate and using a surface plasmon resonance (SPR) device as a device.
[0035]
More specifically, in order to develop a highly sensitive immunosensor using an antigen-antibody reaction, in immobilizing a protein, as a film for modifying a gold surface, polyethyleneimine (polymer having different functional groups) We focused on polyethyleneimine (PEI)) and chitosan. A PEI membrane activated a primary amine present in the membrane, and a chitosan membrane activated a carboxyl group to have an amino group and a binding site in a protein, and an antigen was chemically bonded thereto to produce a sensor membrane. In the present invention, in addition to the conventional batch method, an all-flow type immobilization method has been developed in which all reagents are introduced into the flow system and immobilized. The quantification was performed by SPR angle shift when the antibody was specifically bound to the produced sensor membrane.
[0036]
In the development of a new carbohydrate sensor using a carbohydrate-binding protein (Concanavalin A: ConA), glucose was first measured as a carbohydrate. ConA has a high adsorptivity to glucose because it has four binding sites with glucose around pH 7. However, when ConA and glucose are mixed in advance and some of the binding sites bind to glucose, the selective adsorptivity decreases, and this change is observed when the concentration of ConA is constant. I thought it would correspond to the concentration. Utilizing this property, the surface of Au was modified with a glucose unit, so that the glucose concentration was measured in reverse from the concentration of ConA adsorbed thereto.
[0037]
The method of the present invention is an advantageous method for measuring the reaction between an antigen or an antibody immobilized on a polymer layer directly or via a spacer and an antibody or an antigen in a sample.
For example, in order to immobilize a large amount of antibody protein and detect it with high sensitivity, as a membrane material for modifying a metal surface, polyethyleneimine (PEI), which is a polymer having different functional groups, and carboxymethylated chitosan (CM- chitosan) is particularly preferred. PEI activated the primary amine present in the membrane, CM-chitosan activated the carboxyl group, and a ligand specifically reacting with the antibody was chemically bonded to construct a sensor-probe surface.
[0038]
In the present invention, when producing these surfaces, in addition to the conventional batch method, an all-flow injection (AFI) method in which all reagents are introduced into the flow system and immobilized is examined. Specifically, SPR responses in protein A-human immunoglobulin G (hIgG) and anti-human immunoglobulin G (anti-hIgG) -human immunoglobulin G (hIgG) systems were examined using the prepared sensor-probes. did.
[0039]
The SPR sensor (manufactured by DKK) used in this experiment can execute the flow-through system shown in FIG.
When the experiment is performed by the AFI method, a running buffer (pH 7.4 10 mM HBS (HEPES Buffer Saline) / Tween) is always allowed to flow at a flow rate of 0.2 to 0.4 ml / min with an HPLC pump. By injecting the solution from the injector, it is brought into contact with the sensor probe in the SPR device, and the data is output by a computer.
[0040]
Next, the present invention will be described more specifically, but the present invention is not limited to these specific examples. Abbreviations used in a more specific description of the present invention are summarized below.
[0041]
Compound
BSA; Bovine serum albumin.
BS3; Bis (sulfosuccinimidyl) suberate.
CAP: Cellulose acetate phtalate.
CM-chitosan; Carboxymeyhylated chitosan.
EDC; 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide.
HBS; HEPES Buffer Saline.
hIgG; human immunoglobulin G.
NHS; N-Hydroxysuccinimide.
PEI; Polyethyleneimine.
[0042]
solvent
AcOH; acetic acid.
DMF; N, N-dimethylformamide.
EtOH; Ethanol.
i-PrOH; Isopropanol.
[0043]
First, the gold thin film was washed by applying it several times to a 1 M KOH aqueous solution heated to about 70 ° C., and then immersed in a DMF solution of 5 mM L-cysteine methyl ester for 24 hours to obtain the state of (1) in FIG. . This was treated with a 5% glutaraldehyde aqueous solution for 2 hours to obtain the state of (2) in FIG. 1, and then immersed in a 2% PEI aqueous solution for 2 hours to obtain the PEI membrane sensor probe shown in (3) of FIG. .
[0044]
The surface structure of the organic linker portion of the sensor probe prepared in this way includes a state in which some aldehydes are bound to the amino groups of two cysteines and consume two aldehyde groups. It is considered that a state different from the state of a self-adsorptive monolayer in which molecules are regularly arranged on the metal surface is formed. The detailed state of the metal surface can be grasped by measuring its thickness using a surface analysis such as XPS.
[0045]
Next, the prepared probe was set in an SPR device, and after the baseline of pH 7.4 HBST / buffer was stabilized, unreacted glutaraldehyde present in the membrane was injected with a 0.1 M glycine solution. Blocked. Thereafter, the primary amine in the PEI membrane was activated with BS3 as a spacer, and the result when 0.5 mg / ml BSA was immobilized is shown in FIG. 6, and then when 0.1 mg / ml hIgG was added The results are shown in FIG.
[0046]
6 and 7 show SPR measurement results, with the horizontal axis representing time (minutes) and the vertical axis representing responses (arc seconds (1 degree is 3600 arc seconds)).
In FIG. 6, (1) shows the time when pH 7.4 HBS T / buffer was added, (2) shows the time when BS3 was added, and (3) shows the time when 0.5 mg / ml BSA was added. (4) shows the time when glycine pH 3 was added. The response Δθ at the time when BSA was added was 604 arc seconds.
In FIG. 7, (1) shows the time when pH 7.4 HBST / buffer was added, (2) shows the time when BS3 was added, (3) shows the time when 0.1 mg / ml hIgG was added, (4) shows the time when glycine pH 3 was added. The response Δθ at the time when hIgG was added was 150 arc seconds.
[0047]
Next, processing of metal thin films by the all-flow injection method (AFI method) was examined.
After washing the gold thin film, it was set directly on the SPR device, from which all reagents were introduced into the flow system and immobilized. That is, 10 mM L-cysteine water: ethanol 1: 4 solution, 10% glutaraldehyde aqueous solution, 1% PEI aqueous solution were sequentially injected to prepare a PEI membrane probe, and BSA was immobilized. The results are shown in FIGS.
In FIG. 8, (1) is the time when pH 7.4 HBST / buffer was added, (2) was the time when 10 mM L-cysteine was added, (3) was the time when 10% glutaraldehyde was added ( 4) shows the time points when 1% PEI was added.
[0048]
In FIG. 9, (a) shows the time when pH 7.4 HBST / buffer was added, (b) shows the time when 1 mM BS3 was added, and (c) shows the time when 500 μg / ml BSA was added. (D) shows the time when glycine pH 3 was added, and (e) shows the time when 0.1 M ethanolamine was added at pH 8.5. The response Δθ at the time of addition of glycine in (d) was attributed to electrostatic interaction and was 334 arc seconds, and attributed to covalent bond interaction was 1130 arc seconds.
[0049]
Next, suppression of nonspecific adsorption to the PEI membrane sensor probe surface was examined. In producing a sensor, non-specific adsorption of various proteins present in body fluids on the surface of the sensor membrane has been pointed out as a main cause of lowering detection sensitivity and selectivity. Also in the experiment described above, when BSA is brought into contact with the PEI membrane surface, non-specific adsorption of about 1/4 to 1/3 of the total adsorption amount (desorption when glycine / hydrochloric acid pH 3.0 is brought into contact). Was seen). It is considered that this is mainly due to electrostatic interaction with BSA due to the positive charge that many amines remaining in the membrane protonate.
[0050]
Therefore, FIG. 10 shows the results when the pH during the reaction was changed using pH 5.3 acetate buffer instead of pH 7.4 HBST / buffer when the protein was immobilized on the PEI membrane probe.
In FIG. 10, (1) shows the time when pH 5.3 acetate buffer was added, (2) shows the time when 1 mM of spacer BS3 was added, and (3) shows the time when 0.5 mg / ml BSA solution was added. (4) shows the time when 0.1 M glycine (pH 3.0) was added. The responses Δθ at the time when glycine was added were 860 and 620 arc seconds, respectively.
[0051]
In addition, with the pH 7.4 buffer, without using BS3 as a spacer, the amino group in the membrane is reacted with 100 mM glyoxylic acid to carboxylate to obtain a charge balance of the entire membrane, which is 0.4 M EDC / FIG. 11 shows the results when BSA was immobilized after activation with a 0.1 M NHS 1: 1 mixed solution.
In FIG. 11, (1) shows the time when pH 7.4 HBS buffer was added, (2) shows the time when 0.4 MEDC / 0.1 MNHS was added, and (3) shows the addition of 0.5 mg / ml BSA solution. (4) shows the time when 0.1 M glycine (pH 3.0) was added. The response Δθ when BSA was added was 1211 arc seconds, and the response Δθ when glycine was added was 673 arc seconds.
[0052]
As a result, nonspecific adsorption of BSA could not be reduced by changing the pH during the reaction or the charge balance of the sensor probe surface.
Proteins such as BSA often have either a positive or negative charge as a whole, but each molecule has both a positive charge and a negative charge. Even when balancing the charge, it is difficult to make all the sites in the protein molecule difficult to adsorb on the membrane surface. The optimum pH to prevent non-specific adsorption and the charge state will be determined depending on the type of protein, but when measuring as a PEI membrane immunosensor probe in the presence of various proteins such as serum, pH And changing the charge balance makes no sense at all.
[0053]
Therefore, the unreacted amino groups in the charged film were inactivated by cross-linking with glutaraldehyde to suppress the charge, and the increase of active sites due to the remaining aldehyde groups was considered. A ligand that specifically reacts with the antibody to be detected was immobilized at the activation site of this probe, and this was used as a PEI membrane sensor probe. This reaction is schematically shown in FIG.
[0054]
From the above experiments, the basic characteristics of the PEI membrane sensor probe are considered as follows. Since the ligand having an amino group has been immobilized, the PEI membrane sensor probe can be prepared by introducing all reagents into the flow system in addition to the conventional batch method. -It was confirmed that the injection (AFI) method can be used.
Since the contact time between the sample and the surface by the flow system is about 10 minutes, it was found that all the reactions shown in FIG. 1 proceed within this time. The AFI method is considered to be a useful method as a simple and rapid method for producing a probe as compared with other sensor probes that take several tens of hours to produce.
Comparing the amount of BSA immobilized on the PEI membrane probe produced by the batch and the probe by the flow (FIGS. 6, 8, and 9), the flow system was detected as a response 2.8 times larger. However, the response of BS3, which is an activator for immobilizing BSA, is smaller in the flow system and is opposite to the amount of immobilization of BSA. This is considered to be caused by some difference in the surface structure of the two probes prepared.
[0055]
In addition, in order to reduce the nonspecific adsorption amount of the protein on the surface of the PEI membrane in question, the highest possible concentration of BS3 and EDC / NHS is contacted to activate the functional group in the membrane, It is necessary to block the remaining amino group sufficiently. One possible method is to introduce a reagent having an ethylene glycol unit containing a large amount of ether bonds, which is known as a substance with low protein adsorption, when designing the membrane surface.
[0056]
Next, as an application of the sensor probe, anti-hIgG and proteinA are bound to the binding site in the PEI membrane, and human immunoglobulin antibody hIgG having high specificity and strong binding force is contacted with varying concentrations. The response was measured and hIgG was quantified.
Further, non-specific adsorption amount of the protein on the probe surface was examined by contacting mouse-IgG having weak binding property with protein A and chicken IgG (chicken-IgG) showing no binding property at all. The eight types of probes used for measurement are summarized in FIG.
[0057]
First, in FIG. FIG. 14 shows a real-time plot of hIgG detection when measurement was performed using 8 probes.
In FIG. 14, (1) shows the time when HBS / NHS buffer was added, (2) shows the time when EDC / NHS was added, (3) shows the time when 200 μg / ml anti-hIgG was added (4 ) Shows the time when ethanolamine was added, (5) shows the time when glycine was added, (6) shows the time when 0.1 ug / ml hIgG was added, and (7) shows 1.0 ug / ml hIgG. Each time point of addition is shown.
[0058]
EDC / NHS was injected while the buffer was in constant flow (at time (2) in FIG. 14), the carboxyl group in the membrane was activated, and contacted with anti-hIgG, the SPR angle shift caused 2100 arcseconds. A moderate amount of immobilization was observed. At the time of (4) and (5) in FIG. 14, the anti-hIgG adsorbed by the charge is desorbed with pH 8.5 ethanolamine and pH 3.0 glycine solution, and the remaining activated sites in the membrane are removed. Blocked. Up to this point, the preparation of a sensor probe for measuring hIgG was completed, and hIgG was brought into contact with the membrane while changing its concentration, and the resulting response was detected. First, as a result of injecting 0.1 μg / ml hIgG, almost no detection was made. Therefore, when the membrane state was returned by flowing hydrochloric acid and contacted with 1 μg / ml of 10-fold concentration, a response of 288 arc seconds was obtained. was detected. Thereafter, 60, 160, and 60 μg / ml hIgG were sequentially injected. As a result, responses corresponding to the respective concentrations were obtained (see FIG. 15).
[0059]
A calibration curve is created based on the data obtained for each probe, and the difference depending on the activation method (No. 2-No. 6, No. 4-No. 8 in FIG. 13) and the difference depending on the preparation method (Fig. No. 13 No. 5-No. 6, No. 7-No. 8) were examined (see FIGS. 16 and 17). The left side in FIG. 16 is based on the batch method activated with BS3, the right side is based on the batch method activated with EDC / NHS, the black triangle mark and the white square mark indicate the protein A-hIgG system, Black circles and crosses indicate anti-hIgG-hIgG system. The left side in FIG. 17 is based on the batch method activated with EDC / NHS, the right side is based on the AFI method activated with EDC / NHS, and the white square mark and the white circle mark indicate the protein A-hIgG system. The cross and white diamond marks indicate the anti-hIgG-hIgG system.
The amount of nonspecific antibody adsorbed is also No. The comparison was made by comparing 1, 5, and 6 chips (see FIG. 18). The gray bar in FIG. 18 indicates hIgG, the hatched bar indicates mouse IgG, and the horizontal bar indicates chicken IgG. Both concentrations are 90 μg / ml.
FIG. 16 compares the results of activation of the amino group in the PEI film with BS3, and the case where the carboxyl group is made with glyoxylic acid, the charge balance of the entire film surface is taken, and this is activated with EDC / NHS. investigated.
[0060]
As a result, in the protein A-hIgG system, it was found that the surface of the carboxyl group-introduced surface has a higher linearity of the calibration curve and the detection sensitivity also increases. Even when the amount of nonspecific antibody adsorbed in FIG. No. 5 EDC / NHS activity is No.5. Compared with the BS3 activity of 1, both mouse and chicken-IgG were able to suppress the amount of adsorption relative to the amount of hIgG, which is consistent with the results of higher detection accuracy.
In the BSA immobilization experiment described above, the amount of non-specific adsorption of BSA increased by introducing carboxylic acid into PEI. However, in this antigen-antibody reaction, the complex structure of protein such as isoelectric point is used. Thus, it was found that nonspecific adsorption of mouse and chicken-IgG to the membrane can be prevented by introducing a negative charge into the membrane.
[0061]
In FIG. 17, a calibration curve was prepared for the system into which this carboxylic acid was introduced separately from the AFI method and the batch method. As a result, the detection sensitivity and linearity equivalent to those of the batch method were also obtained for the probe produced in the flow system. Further, as shown in FIG. 18, the nonspecific adsorption of the PEI film produced by the AFI method was reduced as compared with the batch method. This is thought to be because there is some difference in the film structure (amino group orientation, etc.) on the chip surface (see FIG. 19). For example, as shown in FIG. 19, an adsorption homopolymer type, a throwing type polymer (cerminally-anchored) type, or the like can be considered. There has been no report on the immobilization of proteins on the surface of metals, particularly gold bases, by the AFI method, and it is considered to be a useful method for producing a sensor probe that is simple, rapid, and reproducible.
From these results, it can be seen that controlling the charge on the membrane surface is one important factor in designing membrane surfaces with less nonspecific adsorption of proteins. Properties and detectability are further improved.
[0062]
In general, when a protein comes into contact with the surface of a polymer, the number of contacts gradually increases, and its conformation may be changed. Furthermore, even if the active site is in contact with the surface or arranged so as to be sterically hindered, the activity is reduced. Controlling the orientation during ligand immobilization also leads to an increase in detection accuracy.
[0063]
Next, the use of polymers containing amino sugars in the membrane was studied.
Chitin is a polyaminosaccharide, and chitosan is a β- (1,4) -polymer of D-glucosamine, which is a partially hydrolyzed acetyl group of chitin.
Since chitin and chitosan are crustacean and insect tissue supports, they have good affinity with living organisms and are degraded in vivo by enzymes. Utilizing this fact, it is used as a medical material for absorbent sutures, artificial skin, and the like. In addition, it is also used as a carrier for drug delivery systems because of its immunological activity and low adsorption of components in body fluids such as proteins. Furthermore, chitosan derivatives have selective adsorption of optical isomers depending on the length of the acyl group, and are therefore used for separation of racemic mixtures by HPLC.
[0064]
In the present invention, an attempt was made to produce a chitosan membrane immunosensor probe by taking advantage of the high biocompatibility of chitosan and the property of non-specific adsorption of proteins that has been a problem with PEI membrane sensor probes.
First, as in the case of PEI, the gold thin film was washed by applying it several times to a 1M KOH aqueous solution heated to about 70 ° C., and then immersed in a DMF solution of 5 mM L-cysteine methyl ester for 24 hours. It was immersed in a 5% glutaraldehyde aqueous solution for 2 hours. The chitosan was fixed by contacting this with a 1% chitosan aqueous solution for 24 hours. Next, the OH group in chitosan was changed to 2M BrCH. ₂ Carboxylation was performed by soaking in a COOH / 1M NaOH solution for 12 hours to prepare a carboxymethylated chitosan sensor probe (see FIG. 2).
[0065]
Similar to the measurement with the PEI membrane probe, the prepared probe was set in the SPR device, and after the baseline with pH 7.4 HBS T / buffer was stabilized, the —COOH group in the membrane was changed to 0.4 M EDC / 0.1 M. FIG. 20 shows the result when 0.5 mg / ml BSA was immobilized after injecting and activating NHS 1: 1 mixed solution.
In FIG. 20, (1) is the time when pH 7.4 HBS / T buffer is added, (2) is the time when 0.4 MEDC / 0.1MNHS is added, and (3) is the 0.5 mg / ml BSA solution. (4) shows the time when ethanolamine was added. The response Δθ at the time when BSA was added was 150 arc seconds.
[0066]
BrCH on chitosan membrane ₂ After immersing COOH to convert —OH groups in the film to —COOH groups and activating them, BSA was immobilized. As shown in FIG. 20, a response of 150 arc seconds was obtained. At that time, the amount of BSA immobilized is considered to depend on the amount of COOH groups present in the CM-chitosan membrane probe. Therefore, when carboxylating chitosan, a CM-chitosan with a high carboxylation rate was synthesized in advance from what was performed on the probe surface by a batch method, and this was immobilized on the surface to try to increase the amount of ligand immobilized. It was.
[0067]
1.0 g of chitosan and 0.1 g of sodium lauryl sulfonate were slowly added to 70 ml of 45% NaOH aqueous solution and stirred for about 1 hour until dissolved while keeping at 4 ° C. This was allowed to cool at −20 ° C. for about 12 hours, then 125 ml of i-PrOH, ClCH ₂ 28.4 g of COOH was added and reacted for 72 hours. Subsequently, 2M HCl was added to obtain 9.8727 g of white crystals.
Next, 0.5 g of this crystal was dissolved in 20 ml of 10% NaOH aqueous solution, and then 4M HCl was added until the pH reached 1. Crystals were precipitated by adding a large amount of acetone to this, and 0.7 g of CM-chitosan was obtained by suction filtration.
[0068]
CM-chitosan synthesized by the above method was immobilized on the sensor probe and activated the carboxyl group by the same method as in the previous and previous schemes, but BSA could not be immobilized in this system. There may be unresponsive steps in the scheme. Since it is known that the reaction proceeds until immobilization of glutaraldehyde, it was expected that the reaction of immobilizing CM-chitosan on the monolayer was a bottleneck. When synthesizing CM-chitosan, the amino group of the sugar is also carboxylated. ₂ The amount of groups is reduced, -NH ₂ The group is directly attached to the main chain of the polymer, so there is no degree of freedom and it seems to be caused by low reactivity. Therefore, a chitosan film sensor-probe was prepared by changing to another reaction path.
[0069]
After cleaning the gold thin film, ^-3 M HS-C ₁₀ H ₂₀ After immersing in a 1: 1 mixed solution of 0.4M EDC / 0.1M NHS in COOH / EtOH solution for 40 minutes and then immersing in the previous 2% CM-chitosan aqueous solution for 2 hours, chitosan membrane sensor probe II Was made. Using this probe, the —COOH group in the membrane was activated with a 0.4 M EDC / 0.1 M NHS 1: 1 mixed solution, 200 μg / ml hIgG was immobilized, and then to the membrane of 500 μg / ml BSA. The result of measuring the adsorption of is shown in FIG.
In FIG. 21, (1) is the time when pH 7.4 HBS / T buffer is added, (2) is the time when EDC / NHS is added, and (3) is the time when 0.2 mg / ml IgG solution is added. (4) shows the time when ethanolamine was added, and (5) shows the time when 500 μg / ml BSA was added. The response Δθ when BSA was added was 720 arc seconds.
[0070]
By changing the reaction system, CM-chitosan could be immobilized on the base. Due to the nature of chitosan, non-specific adsorption of protein (BSA) observed in the PEI membrane to the membrane was hardly observed with the CM-chitosan membrane probe. In order to confirm this, FIGS. 22 and 23 show the state when chitosan and CM-chitosan were immobilized on the probe and BSA was contacted without activating them.
In FIG. 22, (1) shows the time when pH 7.4 HBS / T buffer was added, (2) shows the time when glycine / HCl (pH 3) was added, and (3) shows the time when 500 μg / ml BSA was added. Respectively. The response Δθ at the time when BSA was added was 50 arc seconds.
In FIG. 23, (1) shows the time when pH 7.4 HBS / T buffer was added, and (2) shows the time when 500 μg / ml BSA was added. The response Δθ at the time when BSA was added was 100 arc seconds.
[0071]
Chitosan showed almost no non-specific adsorption according to its properties, but its derivative, CM-chitosan, also has a fairly high concentration of 500 ug / ml despite the presence of a large amount of carboxyl groups in the membrane. When BSA was contacted, the response was as small as 100 arc seconds. Accordingly, it was confirmed that the charge block on the probe surface performed in the PEI film probe preparation process is not necessary for the CM-chitosan film.
[0072]
Next, the chitosan film | membrane sensor probe by AFI method was prepared similarly to PEI. As in the case of the PEI membrane sensor probe, an all flow system immobilization method was attempted in which a gold thin film was set in an SPR device, and then all reagents were introduced into the flow system and immobilized. The results are shown in FIG.
In FIG. 24, (1) is the time when the pH 7.4 HBS / T buffer was added, (2) was the time when the monolayer ethanol solution was added, and (3) was EDC / NHS 0.2M / 0.00. The time when 05M was added, (4) shows the time when 1% CM-chitosan was added, and (5) shows the time when 0.5 mg / ml BSA was added. A response Δθ at the time when BSA was added was observed.
[0073]
As a result of the production of the CM-chitosan membrane sensor probe in the flow system, BSA could be immobilized even in the flow system, but the response was small. This is considered that there is a step in which the reaction is not sufficient in time because the contact time between each reagent and the membrane surface in the flow system is about 10 minutes. Since it is possible to produce a PEI membrane in the flow, it is expected that the reaction of immobilizing CM-chitosan to the monolayer is a bottleneck, which is probably the problem in the immobilization site CM-chitosan -NH ₂ The group is directly attached to the main chain of the polymer, so there is no degree of freedom and it is thought that this is due to low reactivity. It is considered that a more reactive polymer can be obtained by modifying the polymer to have a functional group such as an amino group having a high degree of freedom in the side chain instead of the main chain of the polymer.
[0074]
From the experimental results so far, a sensor probe immobilized on the basis of the synthesized CM-chitosan could be prepared by a batch method, and detection of a ligand having an amino group could be confirmed as an SPR signal. At that time, the nonspecific adsorption of the protein (BSA) observed in the PEI membrane probe to the membrane was hardly observed.
Although it can be applied to the AFI method of this probe, -NH ₂ Since the group is directly attached to the main chain of the polymer, there is no degree of freedom, and due to low reactivity, sufficient reactivity may not be seen compared to the batch method. It is thought that this problem can also be solved by introducing another step to the CM-chitosan synthesis and introducing a highly hydrophilic spacer to the amino group to increase the degree of freedom of the binding site with the monolayer.
[0075]
In addition, the low water solubility of CM-chitosan is one of the problems in preparing probes. In general, chitosan has a molecular weight of several hundred thousand, and it is known that water solubility increases as the molecular weight decreases. By synthesizing CM-chitosan using chitosan with low molecular weight, it is expected to be immobilized on the substrate. If the acidity is too high as the reaction conditions at that time, the nucleophilicity of the amino group is lost due to protonation and the reaction does not proceed, so it is considered appropriate to carry out the reaction slightly more basic than neutral. .
[0076]
Furthermore, the present invention provides a highly sensitive immunosensor utilizing an antigen-antibody reaction. As an application of CM-chitosan sensor probe, protein A is bound to the binding site for comparison with the PEI membrane, and human immunoglobulin antibody hIgG having high specificity and strong binding force is contacted with varying concentration. The response was measured and hIgG was quantified. No. A calibration curve combined with the results of the PEI membrane probes 5 and 6 is shown in FIG. The circles in FIG. 25 are based on the AFI method using the PEI membrane probe, the squares are based on the batch method using the PEI membrane probe, and the triangles are based on the batch method using the CM-chitosan membrane probe. Is.
[0077]
Similarly, non-specific adsorption amount of the protein on the probe surface was examined by contacting mouse-IgG having weak binding property with protein A and chicken-IgG not showing binding property at all. The data compared with the commercially available CMD membrane probe is shown in FIG. In FIG. 26, gray bars indicate hIgG, hatched bars indicate mouse IgG, and horizontal bars indicate chicken IgG. Both concentrations are 90 μg / ml.
[0078]
Compared with the PEI membrane, the response sensitivity decreased to about 1/4, but this membrane did not show any nonspecific antibody adsorption as previously discussed, and even compared with the commercially available CMD membrane probe. High detection accuracy could be obtained.
So far, it has been reported that the probe coated on the surface with an oligoethylene monolayer suppresses non-specific adsorption of proteins, and the same tendency was observed with CM-chitosan and CMD membrane probes.
[0079]
So far, we have discussed protein adsorption from the viewpoint of charge and hydrophilicity / hydrophobicity, but I would like to consider this specific property from the viewpoint of the interaction between water and membranes near the surface.
Water has been studied very well for a long time because of its importance as a vital solvent, and because of its unique property that it has the highest density around 4 ° C and has a significantly higher specific heat than other liquids. It was.
The nature of this water originates from the “structure” formed by hydrogen bonding between molecules, but many unresolved problems remain regarding the structure. In addition, water-soluble polymers such as proteins and polymers are greatly affected by their structure, properties, and functions due to their interaction with water. It is known that water differs from bulk water in properties such as motility and freeze-thaw behavior.
In general, the water on the polymer surface can be classified into free water and water bound to the polymer, and the state of the hydrated layer formed on the polymer surface varies depending on the strength of the binding. It is said that the state of the surface hydrated layer is one of the factors that determine the adsorptivity of the protein to the surface.
[0080]
As a common point to the chemical structures of polyethylene glycol, CM-chitosan, and CMD, all have an ether bond inside the molecule, and the hydrated layer formed on the membrane surface by this site is the nonspecific adsorption of proteins. It is expected that this will be suppressed.
[0081]
The present invention provides an all-flow injection (AFI) method as a method for producing a PEI membrane sensor probe in addition to the conventional batch method, and the PEI membrane produced by the AFI method has a high response equivalent to the batch method. Sensitivity was obtained and the amount of nonspecific antibody adsorbed was small. In addition, it is clear that the control of the charge on the membrane surface is one important factor in designing a membrane surface with less nonspecific adsorption of proteins. By further studying the membrane material and production method, detection characteristics and detection Performance is expected to improve further. Further, it was found that the PEI film produced by the AFI method has less non-specific adsorption and higher detection accuracy than the batch method. This is thought to be due to some difference in the film structure (such as amino group orientation) on the probe surface. The immobilization of proteins on metal surfaces by the AFI method has not been reported so far, and is considered to be a useful method for producing a sensor probe that is simple, rapid, and reproducible.
[0082]
In the examination of the sensor probe based on the CM-chitosan membrane of the present invention, production was performed by AFI method and batch method, protein A was immobilized and hIgG was detected. As a result, the response sensitivity was reduced to about ¼ compared with the case of the PEI film, but the non-specific adsorption was not observed at all in the CM-chitosan film.
From these results, it is clear that the control of the charge on the membrane surface is one important factor for designing the membrane surface with less non-specific protein adsorption. Properties and detectability are expected to be further improved.
[0083]
In order to clarify the effect of the sensor probe of the present invention, a comparative test with a known dextran membrane sensor chip was performed. Since the dextran membrane was already carboxymethylated, it was activated with a 1: 1 mixture of 0.4MEDC / 0.1MNHS to immobilize protein A on both as in the PEI membrane of the present invention.
To this, hIgG at a concentration of 0.1, 1.0, 10, 45, 90, and 200 μg / ml was passed through each sensor. FIG. 27 shows the change over time of the PEI film of the present invention. Moreover, both calibration curves obtained in these experiments are shown in FIG. The left side of FIG. 28 is the PEI film of the present invention, and the right side is the dextran film.
[0084]
As a result, as can be seen from FIG. 28, it can be seen that the PEI membrane of the present invention exhibits almost linearity in the range of hIgG concentration of 0.1 to 90 μg / ml, and the dextran membrane of 10 to 200 μg / ml. The PEI film is about 20 times larger in response sensitivity, and therefore the detection limit is lower.
Thus, it can be seen that the polymer film of the present invention, particularly the PEI film, is a simple, inexpensive and highly sensitive sensor probe by using a polymer having a relatively simple chemical structure.
【Example】
EXAMPLES Next, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples.
[0085]
Example 1
The gold thin film was washed by applying it several times to a 1M KOH aqueous solution heated to about 70 ° C., and then immersed in a DMF solution of 5 mM L-cysteine methyl ester for 24 hours to obtain the state of (1) in FIG. This was treated with a 5% glutaraldehyde aqueous solution for 2 hours to obtain the state of (2) in FIG. 1, and then immersed in a 2% PEI aqueous solution for 2 hours to obtain the PEI membrane sensor probe shown in (3) of FIG. .
[0086]
Example 2
After setting the probe prepared in Example 1 to the SPR device and stabilizing the baseline with pH 7.4 HBS T / buffer, inject 0.1 M glycine solution into unreacted glutaraldehyde present in the membrane. Blocked. Thereafter, the primary amine in the PEI membrane was activated with BS3 as a spacer, and the result when 0.5 mg / ml BSA was immobilized is shown in FIG. 6, and then when 0.1 mg / ml hIgG was added The results are shown in FIG.
[0087]
Example 3
In Example 2, the protein was immobilized on the PEI membrane probe in the same manner as in Example 2 except that pH 5.3 acetate buffer was used instead of pH 7.4 HBST / buffer and the pH during the reaction was changed. The results are shown in FIG.
[0088]
Example 4
Further, in Example 2, the pH is 7.4 buffer, and a carboxyl group is introduced by reacting with 100 mM glyoxylate (OHC-COOH) instead of BS3 as a spacer to obtain a charge balance of the entire membrane, which is 0.4 M EDC. After activation with a 0.1 M NHS 1: 1 mixed solution, BSA was immobilized. The results are shown in FIG.
[0089]
Example 5
When the buffer solution was passed through the sensor probe into which the carboxyl group was introduced by the spacer, EDC / NHS was injected while the buffer was in constant flow, the carboxyl group in the membrane was activated, and anti-hIgG was contacted. As a result of the angle shift, an immobilization amount of about 2100 arcseconds was observed. Next, the anti-hIgG adsorbed by charge was desorbed with pH 8.5 ethanolamine and pH 3.0 glycine solution, and the activated sites in the remaining membrane were blocked. Thus far, the production of a sensor probe for measuring hIgG has been completed.
hIgG was brought into contact with the membrane at different concentrations, and the resulting response was detected. When contacted at 1 μg / ml, a response of 288 arc seconds was detected. Thereafter, as a result of sequentially injecting 60, 160 and 60 μg / ml hIgG sequentially, responses corresponding to the respective concentrations could be obtained.
The results are shown in FIGS.
[0090]
Example 6
A calibration curve is created for each probe based on the data obtained in the same manner as in Example 5, and the difference depending on the activation method (No. 2-No. 6, No. 4-No. 8 in FIG. 13), Further, the difference in the manufacturing method (No. 5-No. 6, No. 7-No. 8 in FIG. 13) was performed in the same manner. The results are shown as calibration curves in FIGS.
In addition, the amount of nonspecific antibody adsorbed is also No. 1 in FIG. The comparison was made by comparing 1, 5, and 6 chips. The results are shown in FIG.
[0091]
Example 7
As in the case of PEI in Example 1, the gold thin film was washed by applying it several times to a 1M KOH aqueous solution heated to about 70 ° C., and then immersed in a DMF solution of 5 mM L-cysteine methyl ester for 24 hours. This was immersed in a 5% aqueous solution of glutaraldehyde for 2 hours. The chitosan was fixed by contacting this with a 1% chitosan aqueous solution for 24 hours.
Next, the OH group in chitosan was changed to 2M BrCH. ₂ Carboxylation was performed by soaking in a COOH / 1M NaOH solution for 12 hours to prepare a carboxymethylated chitosan sensor probe (see FIG. 2).
[0092]
Example 8
Similar to the measurement with the PEI membrane probe, the prepared probe was set in the SPR device, and after the baseline with pH 7.4 HBS T / buffer was stabilized, the —COOH group in the membrane was changed to 0.4 M EDC / 0.1 M. FIG. 20 shows the result when 0.5 mg / ml BSA was immobilized after injecting and activating NHS 1: 1 mixed solution.
[0093]
Example 9
1.0 g of chitosan and 0.1 g of sodium lauryl sulfonate were slowly added to 70 ml of 45% NaOH aqueous solution and stirred for about 1 hour until dissolved while keeping at 4 ° C. This was allowed to cool at −20 ° C. for about 12 hours, then 125 ml of i-PrOH, ClCH ₂ 28.4 g of COOH was added and reacted for 72 hours. Subsequently, 2M HCl was added to obtain 9.8727 g of white crystals.
Next, 0.5 g of this crystal was dissolved in 20 ml of 10% NaOH aqueous solution, and then 4M HCl was added until the pH reached 1. Crystals were precipitated by adding a large amount of acetone to this, and 0.7 g of CM-chitosan was obtained by suction filtration.
[0094]
Example 10
After cleaning the gold thin film, ^-3 M HS-C ₁₀ H ₂₀ After being immersed in a 1: 1 mixed solution of 0.4 M EDC / 0.1 M NHS for 40 minutes in a COOH / EtOH solution and then immersed in the 2% CM-chitosan aqueous solution prepared in Example 9 for 2 hours, chitosan Membrane sensor probe II was manufactured. Using this probe, the —COOH group in the membrane was activated with a 0.4 M EDC / 0.1 M NHS 1: 1 mixed solution, 200 μg / ml hIgG was immobilized, and then to the membrane of 500 μg / ml BSA. The result of measuring the adsorption of is shown in FIG.
[0095]
Example 11
In the same manner as in Example 2, a chitosan membrane sensor probe was prepared by the AFI method. As in the case of the PEI membrane sensor probe, the gold thin film was set in the SPR device, and the immobilization method was carried out in an all flow system in which all reagents were introduced into the flow system and immobilized. The results are shown in FIG.
In FIG. 24, (1) is the time when the pH 7.4 HBS / T buffer was added, (2) was the time when the monolayer ethanol solution was added, and (3) was EDC / NHS 0.2M / 0.00. The time when 05M was added, (4) shows the time when 1% CM-chitosan was added, and (5) shows the time when 0.5 mg / ml BSA was added. A response Δθ at the time when BSA was added was observed.
[0096]
Example 12
The PEI membrane was activated with a 1: 1 mixture of 0.4 MEDC / 0.1 MNHS, and protein A was immobilized thereon.
To this, hIgG at a concentration of 0.1, 1.0, 10, 45, 90, and 200 μg / ml was passed through each sensor. FIG. 27 shows the results of changes over time of the PEI film by the AFI method. In FIG. U is 1 ng / mm ² It is.
The calibration curve obtained in this experiment is shown on the left side of FIG.
[0097]
Comparative Example 1
The same operation as in Example 12 was performed using a dextran membrane. The calibration curve obtained as a result is shown on the right side of FIG.
[0098]
【The invention's effect】
The present invention provides a metal thin film that is useful as a sensor probe for SPR measurement that is simple, rapid, and reproducible. The sensor probe of the present invention is highly sensitive, highly accurate, and simple. And can be manufactured inexpensively.
[Brief description of the drawings]
FIG. 1 shows a method for treating a metal thin film of the present invention when cysteine methyl ester, glutaraldehyde and polyethyleneimine are used as raw materials.
FIG. 2 shows a method for treating a metal thin film of the present invention when cysteine methyl ester, glutaraldehyde and chitosan are used as raw materials.
FIG. 3 is an illustration of immobilizing a detection reagent (shown as a ligand in FIG. 3) by active esterification using suberic acid as a spacer on a metal thin film having a polyethyleneimine film of the present invention. The method is shown.
FIG. 4 shows a method of immobilizing a detection reagent (shown as a ligand in FIG. 4) on the CM-chitosan sensor probe shown in FIG. 2;
FIG. 5 shows a method for preparing a metal thin film of the present invention, an outline of a surface plasmon resonance apparatus, and an outline of a batch method and an all-flow injection method (AFI method) of the metal thin film of the present invention.
FIG. 6 shows SPR measurement results using the PEI film of the present invention, with the horizontal axis representing time (minutes) and the vertical axis representing response (arc seconds (1 degree is 3600 arc seconds)). In FIG. 6, (1) shows the time when pH 7.4 HBS T / buffer was added, (2) shows the time when BS3 was added, and (3) shows the time when 0.5 mg / ml BSA was added. (4) shows the time when glycine pH 3 was added. The response Δθ at the time when BSA was added was 604 arc seconds.
FIG. 7 shows SPR measurement results using the PEI film of the present invention, with the horizontal axis representing time (minutes) and the vertical axis representing response (arc seconds (1 degree is 3600 arc seconds)). In FIG. 7, (1) shows the time when pH 7.4 HBST / buffer was added, (2) shows the time when BS3 was added, (3) shows the time when 0.1 mg / ml hIgG was added, (4) shows the time when glycine pH 3 was added. The response Δθ at the time when hIgG was added was 150 arc seconds.
FIG. 8 is a measurement result of SPR by the AFI method using the PEI film of the present invention. In FIG. 8, (1) is the time when pH 7.4 HBST / buffer was added, (2) was the time when 10 mM L-cysteine was added, (3) was the time when 10% glutaraldehyde was added ( 4) shows the time points when 1% PEI was added.
FIG. 9 is a measurement result of SPR by the AFI method using the PEI film of the present invention. In FIG. 9, (a) shows the time when pH 7.4 HBST / buffer was added, (b) shows the time when 1 mM BS3 was added, and (c) shows the time when 500 μg / ml BSA was added. (D) shows the time when glycine pH 3 was added, and (e) shows the time when 0.1 M ethanolamine was added at pH 8.5.
FIG. 10 is a measurement result of SPR when the PEI membrane of the present invention is used and a pH 5.3 acetate buffer is used as a buffer solution. In FIG. 10, (1) shows the time when pH 5.3 acetate buffer was added, (2) shows the time when 1 mM of spacer BS3 was added, and (3) shows the time when 0.5 mg / ml BSA solution was added. (4) shows the time when 0.1 M glycine (pH 3.0) was added.
FIG. 11 is a measurement result of SPR when using the PEI membrane of the present invention and reacting the amino group in the membrane with carboxylate by reacting with 100 mM glyoxylic acid. In FIG. 11, (1) shows the time when pH 7.4 HBS buffer was added, (2) shows the time when 0.4 MEDC / 0.1 MNHS was added, and (3) shows the addition of 0.5 mg / ml BSA solution. (4) shows the time when 0.1 M glycine (pH 3.0) was added.
FIG. 12 schematically shows that unreacted amino groups of the PEI film of the present invention are inactivated by crosslinking with glutaraldehyde.
FIG. 13 collectively shows eight types of probes used in the measurement of the present invention.
FIG. 14 is a diagram of No. 1 described in FIG. 8 shows a real-time plot of hIgG detection when measurement was performed using 8 probes. In FIG. 14, (1) shows the time when HBS / NHS buffer was added, (2) shows the time when EDC / NHS was added, (3) shows the time when 200 μg / ml anti-hIgG was added (4 ) Shows the time when ethanolamine was added, (5) shows the time when glycine was added, (6) shows the time when 0.1 ug / ml hIgG was added, and (7) shows 1.0 ug / ml hIgG. Each time point of addition is shown.
FIG. 15 shows the response obtained when the concentration of hIgG is changed to 60, 160 and again to 60 μg / ml hIgG, and contacted with the membrane.
FIG. 16 shows a calibration curve using the sensor probe of the present invention. The left side in FIG. 16 is based on the batch method activated with BS3, the right side is based on the batch method activated with EDC / NHS, the black triangle mark and the white square mark indicate the protein A-hIgG system, Black circles and crosses indicate anti-hIgG-hIgG system.
FIG. 17 shows a calibration curve using the sensor probe of the present invention. The left side in FIG. 17 is based on the batch method activated with EDC / NHS, the right side is based on the AFI method activated with EDC / NHS, and the white square mark and the white circle mark indicate the protein A-hIgG system. The cross and white diamond marks indicate the anti-hIgG-hIgG system.
FIG. 18 shows the amount of nonspecific antibody adsorbed when the sensor probe of the present invention is used. The gray bar in FIG. 18 indicates hIgG, the hatched bar indicates mouse IgG, and the horizontal bar indicates chicken IgG. Both concentrations are 90 μg / ml.
FIG. 19 shows nine drops of the film structure on the surface of the sensor probe.
FIG. 20 shows the results of SPR measurement using the CM-chitosan membrane of the present invention. In FIG. 20, (1) is the time when pH 7.4 HBS / T buffer is added, (2) is the time when 0.4 MEDC / 0.1MNHS is added, and (3) is the 0.5 mg / ml BSA solution. (4) shows the time when ethanolamine was added.
FIG. 21 shows the results of SPR measurement using the CM-chitosan membrane of the present invention. In FIG. 21, (1) is the time when pH 7.4 HBS / T buffer is added, (2) is the time when EDC / NHS is added, and (3) is the time when 0.2 mg / ml IgG solution is added. (4) shows the time when ethanolamine was added, and (5) shows the time when 500 μg / ml BSA was added.
FIG. 22 shows the results of SPR measurement using the CM-chitosan membrane of the present invention without activation. In FIG. 22, (1) shows the time when pH 7.4 HBS / T buffer was added, (2) shows the time when glycine / HCl (pH 3) was added, and (3) shows the time when 500 μg / ml BSA was added. Respectively.
FIG. 23 shows the results of SPR measurement using the CM-chitosan membrane of the present invention without activation. In FIG. 23, (1) shows the time when pH 7.4 HBS / T buffer was added, and (2) shows the time when 500 μg / ml BSA was added.
FIG. 24 shows the result of SPR measurement by AFI method using the CM-chitosan film of the present invention. In FIG. 24, (1) is the time when the pH 7.4 HBS / T buffer was added, (2) was the time when the monolayer ethanol solution was added, and (3) was EDC / NHS 0.2M / 0.00. The time when 05M was added, (4) shows the time when 1% CM-chitosan was added, and (5) shows the time when 0.5 mg / ml BSA was added.
FIG. 25 shows a calibration curve when using the probe of the present invention. The circles in FIG. 25 are based on the AFI method using the PEI membrane probe, the squares are based on the batch method using the PEI membrane probe, and the triangles are based on the batch method using the CM-chitosan membrane probe. Is.
FIG. 26 shows the amount of nonspecific antibody adsorbed when the sensor probe of the present invention is used. In FIG. 26, gray bars indicate hIgG, hatched bars indicate mouse IgG, and horizontal bars indicate chicken IgG. Both concentrations are 90 μg / ml.
FIG. 27 shows changes over time in various concentrations of hIgG using a sensor in which protein A is immobilized on a PEI membrane of the present invention. In FIG. U is 1 ng / mm ² It is.
FIG. 28 shows the calibration curves obtained in Example 12 and Comparative Example 1.

Claims (29)

  The surface of the metal thin film having a free electron metal surface is (1) an organic linker having a functional group capable of being immobilized on the metal surface and a functional group capable of binding to a polymer, and (2) a function capable of directly binding to a detection reagent. A metal thin film treated with a polymer having a functional group capable of binding to a group or a detection reagent via a spacer and a functional group capable of binding to the organic linker, wherein the organic linker of (1) is 2 The first molecular species that is formed using more than one species is an amino acid having a functional group containing a sulfur atom or a derivative thereof, and the second molecule A metal thin film wherein the seed is glutaraldehyde, and the polymer of (2) is a polymer having an amino group in the main chain or side chain of the polymer.   The metal thin film according to claim 1, wherein the first molecular species is cysteine or an ester derivative thereof.   The metal thin film according to claim 1 or 2, wherein the polymer is a polysaccharide containing an amino sugar.   The metal thin film according to claim 3, wherein the polymer is chitosan.   The metal thin film according to claim 1 or 2, wherein the polymer is a poly-lower alkyleneimine.   The metal thin film according to any one of claims 1 to 5, wherein only one surface of the metal thin film is treated.   The metal thin film according to any one of claims 1 to 6, wherein the metal thin film has a thickness of 50 to 1000 nm.   The metal thin film according to claim 1, wherein the metal of the metal thin film is any one of gold, silver, copper, aluminum, or chromium.   The metal thin film according to claim 8, wherein the metal of the metal thin film is gold.   The metal thin film according to any one of claims 1 to 9, wherein the detection reagent is further bonded to the polymer directly or via a spacer having a functional group capable of binding to the detection reagent.   The metal thin film according to claim 10, wherein the spacer is an organic compound having a functional group capable of binding to a polymer and a functional group capable of binding to a detection reagent.   The metal thin film according to claim 11, wherein the functional group capable of binding to the detection reagent is a carboxyl group or a reactive derivative thereof.   The metal thin film according to any one of claims 10 to 12, wherein the detection reagent is a protein.   The metal thin film according to claim 13, wherein the protein is an antigen or an antibody.   The metal thin film according to claim 1, wherein the metal thin film is in close contact with the substrate.   The metal thin film according to claim 15, wherein the substrate is a prism.   The metal thin film according to claim 1, wherein the metal thin film is a metal thin film for surface plasmon resonance (SPR).   Treating the surface of the metal thin film having a free electron metal surface with a first organic compound having a functional group that can be immobilized on the metal surface and other functional groups, and immobilizing the first organic compound on the metal surface; Next, a second organic compound that can chemically react with another functional group that is not fixed to the metal surface of the first organic compound is reacted, and the first organic compound and the second organic compound are reacted with each other. The method for producing a metal thin film according to any one of claims 1 to 17, wherein an organic linker comprising bonding is introduced.   (1) An organic linker having a functional group that can be immobilized on the metal surface and a functional group that can bind to the polymer is introduced on the surface of the metal thin film having a free electron metal surface, and then (2) a detection reagent. A polymer having a functional group that can be directly bonded or a functional group that can be bonded to a detection reagent via a spacer and a functional group that can be bonded to the organic linker is reacted with the organic linker to bind it, and the polymer. The method for producing a metal thin film according to claim 18, wherein the detection reagent is bound or adsorbed directly or via a spacer having a functional group capable of binding to the detection reagent.   The method according to claim 19, wherein the detection reagent is a protein.   The method according to claim 20, wherein the protein is an antigen or an antibody.   When a sample is detected, identified or quantified by surface plasmon resonance (SPR), the metal thin film for measuring surface plasmon resonance (SPR) uses the metal thin film according to any one of claims 1 to 17. A method for detecting, identifying or quantifying a sample by surface plasmon resonance (SPR).   In detecting, identifying or quantifying a sample by surface plasmon resonance (SPR), a metal thin film for measuring surface plasmon resonance (SPR) is treated during measurement by the method according to any one of claims 18 to 21; The method for detecting, identifying or quantifying a sample by surface plasmon resonance (SPR) according to claim 22, wherein the measurement is performed by adding the sample.   A method for detecting, identifying or quantifying a sample by surface plasmon resonance (SPR), comprising using the metal thin film according to claim 1.   A detection reagent and a sample are added sequentially or simultaneously to a metal thin film in which a spacer having a functional group capable of binding a detection reagent is bonded to the metal thin film or polymer portion thereof according to claim 1 to 17 for detection. A method for detecting, identifying, or quantifying a trend between a reagent and a substance in a sample by surface plasmon resonance (SPR).   26. The method according to claim 25, wherein the trend between the detection reagent and the substance in the sample is an antigen-antibody reaction. (1) An organic linker having a functional group capable of fixing to a metal surface and a functional group capable of binding to a polymer, wherein the organic linker is formed using two or more molecular species, An organic linker in which the first molecular species immobilized on the surface is an amino acid having a functional group containing a sulfur atom or a derivative thereof, and the second molecular species is glutaraldehyde;
(2) A polymer having a functional group that can be directly bonded to the detection reagent or a functional group that can be bonded to the detection reagent via a spacer and a functional group that can be bonded to the organic linker, the main chain of the polymer or A polymer having an amino group in the side chain; and
(3) A kit for detecting, identifying or quantifying a sample by surface plasmon resonance (SPR) comprising a metal thin film.   28. The kit according to claim 27, further comprising (4) a detection reagent and, if necessary, a spacer for binding it to the polymer.   A surface plasmon resonance (SPR) apparatus using the metal thin film according to claim 1.

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