JP2004226381A - Sensor chip having brush-like structure surface carrying block polymer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sensor chip for improving a detection sensitivity of a biosensor. <P>SOLUTION: One, two, or more types of block polymer derivatives expressed by the general formula (1) A-L-P<SP>1</SP>/P<SP>2</SP>(1) [wherein, A indicates a hydrogen atom, a functional group, a protected functional group, or a ligand; L indicates a linking group or valence bond; P<SP>1</SP>/P<SP>2</SP>is a block polymer constituted of a block P<SP>1</SP>and a block P<SP>2</SP>; the block P<SP>1</SP>is a polymer block containing a hydrophilic straight chain monomer unit; and the block P<SP>2</SP>is a polymer block containing a monomer unit having a functional group bondable to a support body of the surface of the sensor chip] are bonded to the support body of the surface of the sensor chip via the functional group of the block P<SP>2</SP>of the block polymer derivative bondable to the support body of the surface of the sensor chip. By providing such a surface etc. for the sensor chip, it is possible to achieve the sensor chip for improving the detection sensitivity of the biosensor. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００２７】
［式中、Ｒ^１は、互いに独立して、水素原子または炭素原子数が１〜６のアルキル基であり、
Ｒ^２は、互いに独立して、炭素原子数が１〜６のアルキルオキシ基（ケタール基）、若しくは、２つのＲ^２が互いに一緒になって、オキシ基、炭素原子数が１〜６のアルキルで置換されてもよいエチレン基である］
、カルボキシル基、アセタール基、アルデヒド基、トシル基、メルカプト基、または、マレイミド基である。
【００２８】
式（２）において、Ｒ^１が、互いに独立してとり得る、炭素原子数が１〜６のアルキル基としては、例えば、メチル基、エチル基、ｎ−プロピル基、ｉｓｏ−プロピル基、ｎ−ブチル基、ｓｅｃ−ブチル基、ｎ−ヘキシル基等が挙げられるが、メチル基であることが好適である。また、Ｒ^２は、上述の通り、互いに独立して、炭素原子数が１〜６のアルキルオキシ基（ケタール基）をとることが可能であり、２つのＲ^２が互いに一緒になって、オキシ基（全体としてアルデヒド基となる）であることも可能であり、炭素原子数が１〜６のアルキルで置換されてもよいエチレン基（環状ケタールが形成される）であってもよいが、アルデヒド基、または、保護されたアルデヒド基（ケタール基）が好適である。
【００２９】
また、Ｌは、上に示したとおり、Ａと、ブロックＰ^１とを連結するものであり、上述の通り、原子価結合、または、炭素原子数が１〜６のアルキレン基であるが、メチレン基またはエチレン基であることが好適である。
【００３０】
また、ブロックポリマーＰ^１／Ｐ^２のうち、ブラシ状構造の主要部分を占める、ブロックＰ^１は、親水性直鎖状モノマー単位を含むポリマー（以下、親水性直鎖状ポリマーともいう）である。親水性直鎖状ポリマーは、一般的に可動性に富んでおり、かつ、良好な生体適合性が認められることが多く、ブラシ状構造の主要部分として好適な性質を有している。親水性直鎖状ポリマーとしては、例えば、ポリアルキレンオキシド、ポリアクリルアミド、ポリアクリル酸等を挙げることができるが、ポリアルキレンオキシドが好適である。また、親水性直鎖状ポリマーが、ポリアルキレンオキシドである場合、これを構成するアルキレンオキシドモノマー単位の炭素原子数は、２〜３、すなわち、ポリエチレンオキシド、または、ポリプロピレンオキシドであることが好適である。また、当該炭素原子数が、２、すなわち、ポリエチレンオキシドであることが最適である。また、ポリアルキレンオキシドにおける、アルキレンオキシド単位の重合数は、２〜１００００であることが好適であり、さらに好適には、１０〜１０００である。この重合数が１００００を超えると、これを用いた本チップ表面の感度が低下する傾向が認められる。
【００３１】
また、ブロックポリマーＰ^１／Ｐ^２のうち、本チップ表面に結合しているブロックＰ^２は、本チップ表面の支持体と配位可能な官能基を有するモノマー単位を含むポリマーのブロックである。このチップ表面の支持体と配位可能な官能基としては、例えば、アミノ基、カルボキシル基、メルカプト基、または、スルフィド基が挙げられる。これらの官能基を有するモノマー単位としては、例えば、メタクリル酸（２−ジエチルアミノエチル）、メタクリル酸（２−ジメチルアミノエチル）、ポリシステイン、ポリリジン、ポリグルタミン、メタクリル酸（３−トリメトキシシリルプロピル）等を挙げることができる。これらのモノマー単位を、常法に従い、重合反応を、ブロックポリマー誘導体（１）の製造工程において行うことにより、所望する官能基を有するブロックＰ^２を形成することができる。
【００３２】
ブロックＰ^２の好適な態様の一つとして、式（３）
【００３３】
【化５】

【００３４】
［式中、ｍは１〜１０の整数、ｎは２〜２００の整数を表し、Ｒ^３およびＲ^４は、互いに独立して、炭素原子数が１〜５のアルキル基、アミノ基、メルカプト基、または、スルフィド基を表す］で表される、メタクリル酸ポリマーを挙げることができる。
【００３５】
このメタクリル酸ポリマー（３）をブロックＰ^２とする、ブロックポリマー誘導体（１）の製造工程については、特開２００２−８０９０３号公報（特許文献４）において詳細に開示されており、これに従い、所望するメタクリル酸ポリマー（３）をブロックＰ^２とする、ブロックポリマー誘導体（１）を製造することができる。
【００３６】
すなわち、例えば、式
Ａ−Ｌ−Ｐ^１−ＯＨ （ａ）
［式中のＡ、Ｌ、Ｐ^１の定義は、それぞれ、ブロックポリマー誘導体（１）における定義と同一である］
の化合物（ａ）［この化合物（ａ）の製造方法自体も、公知である（例えば、特開平７−３１６２８５号公報：特許文献１、特開平１１−３２２９１６号公報：特許文献２、特開２００１−２０００５０号公報：特許文献３等を参照のこと）］に対して、ポリメタクリル酸ジメチルアミノエチルを、所望する重合度になるように、重合反応することにより、ブロックポリマー誘導体（１）を得ることができる。
【００３７】
ここに、このブロックポリマー誘導体（１）として、アセタール−ＰＥＧ／ＰＡＭＡの製造工程の一例を開示する。なお、この製造工程において、ｓおよびｔは、それぞれ正の整数を意味する。
【００３８】
【化６】

【００３９】
本チップ表面等
本チップ表面の素材、すなわち、本チップ表面の支持体の素材は、本チップの基板の素材と同一（すなわち、基板自体が、本チップ表面を形成する）とすることも可能であり、基板の表面に、金属等の薄膜を形成して、この金属等を支持体とすることも可能である。支持体として用い得る素材として、例えば、金、銀、銅、アルミニウム等の金属とすることができる。また、ガラス；ＣｄＳ、ＺｎＳ等の半導体；ＺｒＯ_２等のセラミック；グラファイト等のカーボン；酸化チタン、酸化アルミニウム等の金属酸化物；酸化イリジウムスズ等の合金酸化物；とすることができる。
【００４０】
これらの本チップ表面の支持体の素材に応じて、適切な官能基を有するブロックＰ^２をセグメントとするブロックポリマー誘導体（１）を選択して用いることが好ましい。すなわち、各本チップ表面の支持体の素材に配位可能な官能基を有するブロックＰ^２をセグメントとするブロックポリマー誘導体（１）を用いることで、容易に、ブロックポリマー誘導体（１）をチップ表面上に固定することができる。
【００４１】
例えば、上記の官能基が、アミノ基、または、メルカプト基である場合には、本チップ表面の支持体の素材は、金、銀、銅、アルミニウム等の金属；ＣｄＳ、ＺｎＳ等の半導体；酸化チタン、酸化アルミニウム等の金属酸化物；であることが好適である。また、同官能基が、シラノール基である場合には、チップの支持体の素材は、ガラス、シリコーン、酸化チタン、セラミックであることが好適である。また、同官能基が、カルボキシル基である場合には、チップの支持体の素材は、セラミック、カーボンが好適である。
【００４２】
本チップ表面を製造する際には、チップ表面の支持体に配位して、結合可能な官能基を有するポリマーをブロックＰ^２とするブロックポリマー誘導体（１）とチップ表面を、例えば、このブロックポリマー誘導体（１）を含有する溶液の中に、チップを浸漬させて、接触させることにより、ブロックＰ^２の前記官能基がチップ表面に結合して、ブロックポリマー誘導体（１）を、チップ表面に固定することができる。その結果、ブロックポリマー誘導体（１）のブロックＰ^１側が、表面上にブラシ状に起立した、本チップ表面を製造することができる。
【００４３】
すなわち、適切な緩衝化した水溶液に、ブロックポリマー誘導体（１）を溶解し、このポリマー溶液を、適温、例えば、２０〜３７℃程度で、浸漬等により、チップの支持体表面に接触させた状態で、数１０分〜数時間のインキュベートを行うことにより、本チップ表面を製造することができる。このポリマー溶液における、ブロックポリマー誘導体（１）の濃度は、用いるブロックポリマーの分子量により異なるが、通常、溶液の０．１〜５ｍｇ／ｍｌ、好適には１ｍｇ／ｍｌ程度である。
【００４４】
なお、上記のポリマー溶液におけるブロックポリマー誘導体（１）の濃度を調整することにより、本チップ表面上のブロックポリマー誘導体（１）の担持密度を設定することができる。すなわち、ポリマー溶液におけるブロックポリマー誘導体（１）の濃度が高ければ、本チップ表面上のブロックポリマー誘導体（１）の担持密度が高くなり、逆に前記濃度が低ければ、同担持密度は低くなる。本チップ表面においては、ブロックポリマー誘導体（１）分子が、０．０１〜１０分子／ｎｍ^２の密度で担持されていることが好適である。この担持密度が、０．０１分子／ｎｍ^２未満であると、本発明の目的である、非特異的な反応を十分に抑制することが困難な傾向があり、１０分子／ｎｍ^２を超えると、分子同士が一層のみに並ばずに、多層化する傾向が認められ、チップによる検出感度が低下する傾向が認められるようになる。なお、このブラシ状構造物の密度は、原子間力顕微鏡（ＡＦＭ）により確認することが可能であり、さらに、ＳＰＲ等により確認することも可能である。
【００４５】
また、本チップ表面におけるブラシ状構造物の嵩高は、すべて、実質的に同一であってもよいし、異なってもよい。
特に、異なる嵩高のブラシ状構造物を、本チップ表面に設ける場合には、例えば、異なる鎖長のブロックＰ^１を有するブロックポリマー誘導体（１）を含有するポリマー溶液で、上記の固定化工程を行うことも可能であり、各々に、異なる鎖長のブロックＰ^１を有するブロックポリマー誘導体（１）を含有するポリマー溶液を調製して、異なるポリマー溶液について、上記の固定化工程を、再度、行うことも可能である。後者の再度の固定化工程を行う場合には、最初の固定化工程に用いるポリマー溶液は、ブロックポリマー誘導体（１）のブロックＰ^１の鎖長が長いものを用い、後から行う固定化処理では、処理の順番が後になるほど、同ブロックＰ^１の鎖長を短く設定することが好適である。
【００４６】
なお、１回の固定化工程を行う場合には、その後に、２回以上の固定化工程を行う場合には、各々の固定化工程の後に、チップ表面の洗浄を、１回以上行い、未反応物等を除去することが必要である。洗浄は、未結合のブロックポリマー誘導体（１）等の未反応物を効率よく除去できるものであれば、特に限定されないが、希薄水酸化ナトリウム溶液を用いるのが好適である。
【００４７】
マスキング処理
上記 のようにして提供される、本チップ表面に、さらにマスキング処理を行うことにより、さらに非特異的な反応を抑制することができる。
【００４８】
このマスキング処理は、上述のようにして得られた、ブロックポリマー（１）が担持されている、本チップ表面に、マスキング剤を接触させて、当該マスキング剤を本チップ表面上に固定化することにより行われる。
【００４９】
マスキング剤としては、式（４）
【００５０】
【化７】

【００５１】
［式中、ｐは、１〜２００の整数であり、Ｒ^５は、水酸基、炭素原子数が１〜６のアルキル基であり、Ｒ^６は、水素原子、炭素原子数が１〜６のアルキル基、または、ピリジルチオ基である］
で表されるチオ化合物を挙げることができる。また、マスキング処理の好適な態様の一つとして、マスキング剤を２−メルカプトエタノールとする態様を挙げることができる。
【００５２】
マスキング処理は、上述のようにして製造され得る本チップ表面を、マスキング剤の溶液と接触させることにより行うことができる。この場合のマスキング剤の溶液におけるマスキング剤の濃度は、マスキング剤の種類等によっても異なるが、概ね、０．００１〜１０００ｍＭ，好適には、０．１〜１０ｍＭ程度である。また、マスキング処理の時間は、好適には、３分〜１日程度である。さらに、マスキング処理は、２０〜３７℃程度の温度下で行うことが好適である。
【００５３】
このようにして、ブロックＰ^２が平面固定され、かつ、ブロックＰ^１が嵩高に起立し、ブラシ状構造物の主要部を構成している、ブロックポリマー誘導体（１）が担持された本チップ表面、および、本チップ表面を有する本チップが提供される。本チップ表面において、生物学的な結合反応を行うと、非特異的な反応を著しく抑制することが可能となり、本チップを用いたセンサによる検出感度を向上させることができる。
【００５４】
【実施例】以下に、本発明の実施例を記載するが、この実施例により、本発明の技術的範囲が限定されるものではない。
〔参考例〕 ブロックポリマー誘導体（１）等の製造
（１）アセタール−ＰＥＧ−ＳＨ（Ｍｎ＝５，０００）の合成
アルゴン（Ａｒ）置換下、室温のフラスコ中、に溶媒としてＴＨＦ６０ｍｌを入れ、これに開始剤３，３−ｄｉｅｔｈｏｘｙ−１−ｐｒｏｐａｎｏｌ １ｍｍｏｌとＫ−Ｎａｐｈ（０．３１６８ｍｏｌ／ｌ） １ｍｍｏｌを攪拌しながら加え、メタル化を行った。充分攪拌後、ＥＯを１１２．９９ｍｍｏｌ加え水冷しながら二日間攪拌し、重合を行った。
【００５５】
二日間攪拌後、この溶液に再メタル化を目的としてＫ−Ｎａｐｈ ０．５ｍｍｏｌとｔｒｉｅｔｈｙｌａｍｉｎｅ ４．５ｍｍｏｌを加えた。Ａｒ置換下のナスフラスコ中にＴＨＦ溶媒１０ｍｌと停止剤として、ｍｅｔｈｙｌｓｕｌｆｏｎｙｌ ｃｈｌｏｒｉｄｅ ３．５ｍｍｏｌを溶解させ、これに等圧滴下漏斗を用いてＰＥＧ重合溶液を滴下した。滴下後、ｅｔｈｅｒ再沈により回収し、その後、ｃｈｌｏｒｏｆｏｒｍと飽和食塩水で抽出を行い、無水Ｎａ_２ＳＯ_４により脱水、ベンゼン凍結乾燥にて回収した。
【００５６】
減圧乾燥されたｐｏｔａｓｓｉｕｍ ｏ −ｅｔｈｙｌｄｉｔｈｉｏｃａｒｂｏｎａｔｅ ０．４４ｍｍｏｌにＡｒ下で溶媒としてＴＨＦ ５０ｍｌとＤＭＦ３．６ｍｌを加え攪拌した。この溶液を減圧乾燥させたａｃｅｔａｌ−ＰＥＧ−ＭＳ ０．２ｇに加え、室温で４時間反応させた。反応後、ｃｈｌｏｒｏｆｏｒｍと飽和食塩水で抽出を行い、無水Ｎａ_２ＳＯ_４により脱水、ｅｔｈｅｒ再沈により精製、ベンゼン凍結乾燥にて回収した。
【００５７】
更に、減圧乾燥させたａｃｅｔａｌ−ＰＥＧ−ｄｉｔｈｉｏｃａｒｂｏｎａｔｅ ０．１ｇにＡｒ下で溶媒としてＴＨＦ １０ｍｌを加えた、ここにｎ−ｐｒｏｐｙｌａｍｉｎｅを１．４Ｍ ＴＨＦ溶液になるように加え、室温で３時間攪拌し反応させた。反応後、ｃｈｌｏｒｏｆｏｒｍと飽和食塩水で抽出を行い、無水Ｎａ_２ＳＯ_４により脱水、ｅｔｈｅｒ再沈により精製、ベンゼン凍結乾燥にて回収した。
回収後、^１Ｈ−ＮＭＲにより構造解析、ＧＰＣ測定を行った。
【００５８】
（２）アセタール−ＰＥＧ／ＰＡＭＡ（ＰＥＧ＝６，０００， ＰＡＭＡ＝１０，０００）の合成
Ａｒ下の反応容器中に、反応溶媒としてテトラヒドロキシフラン（ＴＨＦ）を４５ｍｌ、反応開始物質として３，３−ジエトキシ‐１−プロパノールを１ｍｍｏｌ加え、等モル量のカリウムナフタレン（Ｋ−Ｎａｐｈ）ＴＨＦ溶液を加えて開始物質をメタル化し、開始剤を調製した。続けてエチレンオキシド（ＥＯ）１３７ｍｍｏｌを液体窒素で冷却したシリンジを用いて加え、室温下で２日間攪拌し、重合を行なった。ＥＯ重合後、シリンジでＧＰＣ測定用のサンプルを少量抜き取り、２‐メタクリル酸ジメチルアミノエチル（ＡＭＡ）３１ｍｍｏｌをシリンジで素早く加え、水冷下で２０分間反応させ、少量の酢酸を用いて反応を停止した。生成物は−２０℃に冷却した２−プロパノールに沈殿させ、遠心分離（−１０℃， ５０００ ｒｐｍ， ６０ｍｉｎ）により沈殿物を分離し、沈殿物はエバポレーターにより溶媒を除去した後、ベンゼン凍結乾燥を経て白色粉末として回収した。この回収したポリマー１ｇを蒸留水約１００ｍｌに溶解させ、塩酸を用いてｐＨ５に調整し、凍結乾燥した。さらにこのポリマーをＴＨＦ約１００ｍｌに溶解させ、ポリマーの沈殿物を吸引ろ過で濾別し、ＴＨＦで数回リンスした。濾別した沈殿物は蒸留水約１００ｍｌに溶解させ、水酸化ナトリウム水溶液を用いてｐＨ９に調整した。その後、ベンゼンに溶解させ凍結乾燥を経て白色粉末を回収した。ＧＰＣ，Ｈ−ＮＭＲ測定により生成物の構造を確認した。
【００５９】
（３）ＰＡＭＡ（Ｍｎ＝２２，５００）の合成
Ａｒ下の反応容器中に、溶媒としてテトラヒドロキシフラン（ＴＨＦ）を２０ｍｌ、開始剤としてジフェニルエチレン（ＤＰＥ） ０．５ｍｍｏｌ、等モル量のブチルリチウム（ＢｕＬｉ）を加えメタル化を行なった。そして、塩化リチウム（ＬｉＣｌ）のＴＨＦ溶液（０．７ｍｏｌ／ｌ）を開始剤の５倍モル量加えて５分間攪拌し、２−（ｄｉｍｅｔｈｙｌａｍｉｎｏ）ｅｔｈｙｌ ｍｅｔｈａｃｒｙｌａｔｅ （ＤＭＡＥＭＡ）７１ｍｍｏｌを加え１時間反応させ，少量のメタノールを用いて反応を停止した。これらの反応は全て−７８℃下において行った。合成したポリマーはヘキサン中に生成物を再沈させ、沈殿物をデシケーターで減圧乾燥した後、ベンゼン凍結乾燥を経て白色粉末を回収した。ＧＰＣ，Ｈ−ＮＭＲ測定により生成物の構造を確認した。
【００６０】
〔製造例〕 本チップ等の製造
（１）比較例
まず、金表面のＳＰＲセンサチップ（ＳＩＡキット：ＢＩＡＣＯＲＥ社、以下、同様である）の、いわゆるピラニア洗浄を行った。洗浄液として、特級濃硫酸：特級過酸化水素水＝３：１（容量比）とし、洗浄は、室温で１分間、洗浄液中にチップを浸漬することにより行った。次に、上記の参考例において製造した、Ａｃｅｔａｌ−ＰＥＧ−ＳＨを、１．０Ｍ ＮａＣｌ含有の５０ｍＭ ＰＢＳに溶解し、０．２ｍＭになるように調整した。次に、チップを、このＡｃｅｔａｌ−ＰＥＧ−ＳＨ溶液に、２０分間浸漬して、チップ表面におけるＡｃｅｔａｌ−ＰＥＧ−ＳＨの固定を行った。この固定化工程を、計３回行った。なお、各固定化の工程終了後に、チップの洗浄を行ったが、各固定化工程後の洗浄は、１．０Ｍ ＮａＣｌ含有５０ｍＭ ＰＢＳで１回行い、次いで、５０ｍＭ ＮａＣｌで１回行い、次いで、１．０Ｍ ＮａＯＨ含有５０ｍＭ ＰＢＳで２回行った。
【００６１】
このようにして得た、Ａｃｅｔａｌ−ＰＥＧ−ＳＨを担持させたチップを、比較例１のチップとした。比較例１のチップ表面を，ＡＦＭで解析したところ、チップ表面上に、０．３本／ｎｍ^２程度の密度で、ブラシ状構造物が認められた。
【００６２】
さらに、比較例１のチップ表面に、さらに、２−メルカプトエタノールをマスキング剤として用いて、表面のマスキングを行った。すなわち、比較例１のチップを、１．０ｍＭ ２−メルカプトエタノール（ＭＣＥ）水溶液中に、室温下で５分間浸漬し、Ａｃｅｔａｌ−ＰＥＧ−ＳＨがブラシ状構造物として担持された表面に、さらに、２−メルカプトエタノールを固定した。この２−メルカプトエタノールによるマスキング工程は１回行った。このマスキング工程終了後、未反応の２−メルカプトエタノールを除去するために、チップの洗浄を、超純水で、１８００振動／分で、２０秒間、２回行った。
【００６３】
（２）実施例
まず、比較例において用いたのと同じＳＰＲセンサチップの、いわゆるピラニア洗浄を行った。洗浄液として、特級濃硫酸：特級過酸化水素水＝３：１（容量比）とし、洗浄は、室温で１分間、洗浄液中にチップを浸漬することにより行った。次に、上記の参考例において製造した、Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡ（ＰＥＧ：ＭＷ６０００、ＰＡＭＡ：ＭＷ１００００）を、５０ｍＭ リン酸Ｎａ緩衝液に溶解し、０．２ｍＭになるように調整した。次に、チップを、このＡｃｅｔａｌ−ＰＥＧ／ＰＡＭＡ 溶液に、４時間浸漬して、チップ表面におけるＡｃｅｔａｌ−ＰＥＧ／ＰＡＭＡ の固定を行った。この固定化工程は、１回行った。なお、この固定化の工程終了後に、チップの洗浄を行ったが、固定化工程後の洗浄は、５０ｍＭ ＮａＯＨで１回行い、次いで、５０ｍＭ リン酸Ｎａ緩衝液で２回行った。
【００６４】
このようにして得た、Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡを担持させたチップを、実施例１のチップとした。実施例１のチップ表面を、ＡＭＦで解析したところ、チップ表面上に、０．２５本／ｎｍ^２程度の密度で、ブラシ状構造物が認められた。
【００６５】
さらに、実施例１のチップ表面に、さらに、２−メルカプトエタノールをマスキング剤として用いて、表面のマスキングを行った。すなわち、実施例１のチップを、１．０ｍＭ ２−メルカプトエタノール（ＭＣＥ）水溶液中に、室温下で５分間浸漬し、Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡ のＰＥＧ側が、いわゆるブラシ状構造物として担持された表面に、さらに、２−メルカプトエタノールを固定した。この２−メルカプトエタノールによるマスキング工程は１回行った。このマスキング工程終了後、未反応の２−メルカプトエタノールを除去するために、チップの洗浄を、超純水で、１８００振動／分で、２０秒間、２回行った。
【００６６】
［試験例］
（１）各表面のルテニウムビピリジン錯体の非特異的吸着抑制効果の検討−１
実施例１、２、および、比較例１、２のチップ表面に対して、ルテニウムビピリジン錯体（以下、Ｒｕ錯体ともいう）溶液（２μＭ）を添加し、室温で１０分間静置した。その後、各表面から、Ｒｕ錯体を除去し、ＨＥＰＥＳｂｕｆｆｅｒ（ｐＨ
７．４）で１回洗浄後、電子供給物質であるｎ−ｔｒｉｐｒｏｐｙｌａｍｉｎｅ（ＴＰＡ）１００ｍＭでリンスし、Ｒｕ錯体の発光強度の測定を行った。なお、比較のために、未修飾のチップ表面についても、同様の測定を行った。すなわち、上述のピラニア洗浄後、Ｒｕ錯体溶液を添加し、ルテニウムキレート型電気化学発光装置（松下電器産業に対する注文生産品）による発光強度の測定を行った。
【００６７】
その結果を、第１図（縦軸に、Ｒｕ錯体の発光強度を示す。この発光強度が強いほど、非特異的な吸着が強く、弱いほど、非特異的な吸着が抑制されていることを示す）に示す。第１図に示すように、比較例１と実施例１のチップを比較すると、ＰＥＧ部分のみが担持されている比較例１のチップよりも、ブロックポリマー誘導体を担持させた実施例１のチップの方が、非特異的吸着が抑制されていることがわかる。また、実施例においても、比較例においても、２−メルカプトエタノールによるマスキング処理を行った方が、非特異的な吸着が抑制され、殊に、実施例２のチップにおいては、非特異的な吸着が、ほぼ完全に抑制されていることがわかる。
【００６８】
（２）ＳＰＲによるチップ表面のマスキングの評価
上記の比較例等で用いたと同じ金表面のＳＰＲセンサチップに、上述したと同様の内容のピラニア洗浄を施し、Ａｃｅｔａｌ−ＰＥＧ−ＳＨ（ＭＷ．５０００）を、１．０ＭＮａＣｌ含有５０ｍＭＰＢＳ（ｐＨ７．４）に溶解し、０．２ｍＭとなるように調整した。このＡｃｅｔａｌ−ＰＥＧ−ＳＨ（ＭＷ．５０００）溶液を、ＳＰＲ装置（ＢｉａＣｏｒｅ３０００：ＢＩＡＣＯＲＥ社）にセットし、流速２０μｌ／分で、１回に付き２０分間流し、この流す操作を３回行い、Ａｃｅｔａｌ−ＰＥＧ−ＳＨ（ＭＷ．５０００）のチップ表面への固定化を行った。その後、１．０ｍＭの２−メルカプトエタノールを、流速１０μｌ／分で２０分間流し、ＳＰＲのレスポンスの変化を観察した。
【００６９】
Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡ（ＰＥＧ：ＭＷ６０００、ＰＡＭＡ：ＭＷ１００００）を、５０ｍＭ リン酸Ｎａ緩衝液に溶解し、０．２ｍＭになるように調整した。このＡｃｅｔａｌ−ＰＥＧ／ＰＡＭＡ溶液を、上記のＳＰＲ装置にセットして、流速５μｌ／分で、１回に付き１時間流し、この流す操作を４回行い、Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡのチップ表面への固定化を行った。その後、１．０ｍＭの２−メルカプトエタノールを、流速１０μｌ／分で２０分間流し、ＳＰＲのレスポンスの変化を観察した。その結果を、第２図［縦軸：アングルシフト（角度変化量）（Δθ×１０^−４［°］）、横軸：経過時間］に示す。
【００７０】
上記の２−メルカプトエタノール処理に伴う、ＳＰＲのレスポンスの変化を、Ａｃｅｔａｌ−ＰＥＧ−ＳＨを用いた例と、Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡを用いた例とを比較すると、Ａｃｅｔａｌ−ＰＥＧ−ＳＨは、レスポンス変化がマイナスになってしまったのに対し、Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡは、若干プラスに推移した。これは、Ａｃｅｔａｌ−ＰＥＧ−ＳＨの系では、２−メルカプトエタノールとＰＥＧ−ＳＨのＳＨ基とのＳＨ交換反応が起こってしまうが、Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡの系には、このような交換反応は起こらず、むしろ、２−メルカプトエタノール自体が、ＰＥＧのブラシ状構造物の間に入り込んでいると推測される。
【００７１】
このことから、ＰＥＧ／ＰＡＭＡと２−メルカプトエタノールを併用して、チップ表面の処理を行うことによって、より緻密なコーティング面を形成することが可能であることが明らかになった。
（３）各表面のルテニウムビピリジン錯体の非特異的吸着抑制効果の検討−２
ａ）金表面、ｂ）比較例１の表面、ｃ）参考例において製造したＰＡＭＡで被覆した表面、ｄ）実施例１の表面を用意し、それぞれのチップ表面に、１ｍｇ／ｍｌ ＢＳＡ溶液（溶媒：ＨＥＰＥＳバッファー）を流した際の、各表面におけるＢＳＡの吸着量（流速２０μｌ／分、時間１０分後、ランニングバッファーを３分間流した際の角度変化量）を、ＳＰＲにより計測した。Ｒｕ錯体溶液を流した場合の結果を、第３図に示す（縦軸は、角度変化量（Δθ×１０^−４［°］）。
【００７２】
第３図の結果より、ブラシ状構造物のみを担持した、比較例１のチップ表面における角度変化と、実施例１のチップ表面における角度変化が、他と比べると著しく少なかった。なお、この例では、比較例１の角度変化が、実施例１よりも少なかったが、これは、Ａｃｅｔａｌ−ＰＥＧ／ＰＡＭＡよりも、Ａｃｅｔａｌ−ＰＥＧ−ＳＨの方が、分子量が小さいので、ＰＥＧ−ＳＨのチップ表面上での担持密度の方が、ＰＥＧ／ＰＡＭＡの担持密度よりも大きいことに起因しているものと考えられる。
【００７３】
実際に、チップ表面上における、ＰＥＧ／ＰＡＭＡの固定化量が異なる２種類の表面（固定化量：Δθ＝１２００（ｂｌｏｃｋ１２００）または２０００×１０^−４［°］（ｂｌｏｃｋ２０００））を作出し、上記の要領で、ＢＳＡの吸着量をＳＰＲで測定した（第４図）。その結果、ＰＥＧ／ＰＡＭＡの固定化量の増加、すなわち、担持密度の増加に伴い、ＢＳＡのチップ表面への吸着が、さらに抑制されることが判明した。
【００７４】
【発明の効果】
本発明により、非特異的な反応が著しく抑制された表面を有するセンサチップが提供される。
【図面の簡単な説明】
【図１】Ｒｕ錯体の発光強度を指標として、実施例と比較例のチップにおける非特異的な反応の程度について検討した結果を示す図面である。
【図２】実施例と比較例のチップにおける、２−メルカプトエタノール処理を行ったときの、ＳＰＲにおけるレスポンスについて検討した結果を示す図面である。
【図３】チップ表面におけるＢＳＡの吸着の抑制について、ＳＰＲで検討した結果を示す図面である。
【図４】チップ表面におけるＰＥＧ／ＰＡＭＡの固定化量とＢＳＡの吸着量の関係について、ＳＰＲで検討した結果を示す図面である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a sensor chip that can be used in a biosensor.
[0002]
[Prior art]
The biosensor is suitable for detecting a specific analyte, for example, using a biological sample as a specimen, and various biosensors are currently proposed.
[0003]
As one of main biosensors, a biosensor utilizing surface plasmon resonance (SPR) (hereinafter, also referred to as an SPR sensor) is provided. This SPR sensor forms a metal thin film such as gold or silver on the bottom surface of a prism, irradiates light such as a laser from the back surface of the metal thin film while changing the incident angle, measures the reflected light, and measures the metal surface plasmon. A biosensor capable of detecting a reaction, such as an antigen-antibody reaction, occurring on the metal surface using light at a specific incident angle as an index, which is absorbed and resonated with the surface of the metal thin film and its vicinity. Are known to be sensitive to changes in the refractive index (e.g., A. Szabo et al., Curr. Opin. Strnct. Biol. 5 (1995) 699-705).
[0004]
In addition, the SPR sensor enables in-situ observation of a process occurring between the sensor chip surface and a complex biological solution. Therefore, in the SPR sensor, it is easy to obtain data of a test substance over time and continuously without using a label, and is suitable for obtaining kinetic and thermodynamic parameters. ing.
[0005]
Prior art documents related to the present invention described below include, for example, the following documents.
1. JP-A-7-316285 (Patent Document 1)
Patent Document 1 describes a method for producing a polyalkylene oxide derivative that can be used on the surface of a chip of an SPR sensor. However, this document itself discloses the application of a polyalkylene oxide derivative to a biosensor. Not.
[0006]
2. JP-A-11-322916 (Patent Document 2)
Patent Literature 2 discloses a polyoxyethylene derivative having a biotin residue at the ω-terminal which can be more preferably used on the surface of an SPR sensor. There is no disclosure regarding application to biosensors.
[0007]
3. JP 2001-200550 A (Patent Document 3)
Patent Document 3 describes that fine particles of metal such as gold carrying a polyethylene glycol derivative have good dispersibility. However, this document itself discloses that the fine particles of metal are applied to a biosensor. No disclosure is made about the application.
[0008]
4. JP 2002-80903 A (Patent Document 4)
Patent Document 4 discloses a metal fine particle such as gold, which has a block polymer derivative and has a specific methacrylic acid polymer as another block and has polyethylene glycol as a block. There is no disclosure of the application of the metal fine particles to a biosensor per se. The block polymer of Patent Document 4 is a block polymer that can be used in the present invention.
[0009]
5. Japanese Patent No. 2815120 (Patent Document 5)
Patent Document 5 discloses that HS-RY (R is a hydrocarbon group having more than 10 carbon atoms and optionally interrupted by a hetero atom, and Y represents a ligand or a biocompatible porous matrix. An organic molecule represented by the formula (1), which is an active group for covalent bonding), is bonded to the surface of a free metal film such as gold or silver through the thiol group, and the organic molecules are tightly packed. A monolayer is provided, followed by a biocompatible porous matrix with a surface covalently bonded to a hydrogel made of agarose, dextran, polyethylene glycol, or the like.
[0010]
6. Japanese Patent No. 3071823 (Patent Document 6)
Patent Literature 6 describes a surface in which a hydrophilic linker portion and a solid phase reactant such as a biotin derivative residue are sequentially covalently bonded to a spacer molecule bonded to a holding material via a sulfur atom of a thiol group. I have.
[0011]
7. "Roberts et al., J. Am. Chem Soc. 1998, 120, 6548-6555" (Non-Patent Document 1).
Non-Patent Document 1 describes a monolayer self-assembled on a gold surface via a thiol group using a compound based on an HS-spacer molecule-hydrophilic linker. In addition, the surface in which the hydrophilic linker portion is formed from a mixture of a compound having three ethylene oxide units and a compound having six ethylene oxide units promotes ligand-specific binding of cells, but deposits proteins by attached cells. Is also taught.
[0012]
8. "Pavey et al., Biomaterials 20 (1999) 885-890 (Non-Patent Document 2).
Non-Patent Document 2 describes a surface in which various two kinds of combinations of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock polymers are deposited on a metal thin film for detection of SPR. It has also been suggested that polyethylene oxide chains can elongate in the solution on the surface to form a brush-like architecture.
[0013]
[Patent Document 1]
JP-A-7-316285
[Patent Document 2]
JP-A-11-322916
[Patent Document 3]
JP 2001-200050 A
[Patent Document 4]
JP 2002-80903 A
[Patent Document 5]
Japanese Patent No. 2815120
[Patent Document 6]
Japanese Patent No. 3071823
[Non-patent document 1]
Roberts et al. , J. et al. Am. Chem Soc. 1998, 120, 6548-6555
[Non-patent document 2]
Pavey et al. , Biomaterials 20 (1999) 885-890.
[0014]
[Problems to be solved by the invention]
One of the common problems in chips used in biosensors is improvement in detection sensitivity of biosensors.
[0015]
For example, among the above-mentioned conventional technologies, technologies directly related to a chip used in a biosensor (Patent Documents 5 and 6, Non-Patent Documents 1 and 2) all use non-specific reactions on the chip surface as much as possible. It is intended to suppress and improve the detection sensitivity of the biosensor.
[0016]
In addition, some of the present inventors, for the purpose of suppressing the non-specific reaction, by carrying a brush-like structure of polyethylene oxide derivatives having different chain lengths on the surface of the SPR sensor chip, We found that the detection sensitivity was improved, and filed a patent application (Japanese Patent Application No. 2002-65298). Furthermore, it has been found that the detection sensitivity can be improved by supporting metal fine particles treated with a polyethylene oxide derivative on the surface of an SPR sensor chip, and a patent application has been filed (Japanese Patent Application No. 2002-101134).
[0017]
As described above, studies on sensor chips with the aim of improving the detection sensitivity of biosensors have been made with the aim of further improving the detection sensitivity of biosensors. Is important in
[0018]
Therefore, the problem to be solved by the present invention is also to provide a means in a sensor chip for the purpose of improving the detection sensitivity of a biosensor.
[0019]
[Means for Solving the Problems]
The present inventor has further studied the surface structure of the SPR sensor chip which can suppress non-specific reactions in order to solve this problem. As a result, the present inventors thought that by providing a brush structure on the surface of the SPR sensor chip and coating the chip surface with a polymer, it would be possible to further suppress nonspecific reactions. As a specific measure, a block corresponding to the brush structure and a block polymer derivative having a block bonded to the chip surface are used to treat the chip surface to have a bulky brush structure, and It has been conceived that it is possible to obtain a desired sensor chip surface whose surface is coated with a polymer, and furthermore, by using a sensor chip having such a surface in a biosensor, non-specific reaction can be achieved. It has been found that it is possible to further suppress the noise, and the present invention has been completed.
[0020]
That is, the present invention relates to the general formula (1)
[0021]
ALP¹/ P²              (1)
Wherein A represents a hydrogen atom, a functional group, a protected functional group or a ligand; L represents a linking group or a valence bond;¹/ P²Is the block P¹And block P²And the block P¹Is a block of a polymer containing hydrophilic linear monomer units, and the block P²Is a polymer block containing a monomer unit having a functional group capable of binding to the support on the surface of the sensor chip.]
And one or more of the block polymer derivatives represented by²The surface of the sensor chip (hereinafter also referred to as the present chip surface) and the sensor chip having the present chip surface (hereinafter also referred to as the present chip) which are bonded through a functional group capable of bonding to the support on the sensor chip surface. ) And a method of using the present chip (hereinafter, also referred to as the present method of use).
[0022]
BEST MODE FOR CARRYING OUT THE INVENTION
The present chip is preferably used for detection in a biosensor using surface plasmon resonance, but is not limited thereto.A pair of a ligand and a receptor, for example, an antigen or a hapten and an antibody, a sugar and a lectin, Any sensor that can detect any change that occurs on the chip surface by forming a binding pair in a set of a substrate and an enzyme, a hormone and its receptor, an oligonucleotide and its complementary chain, etc. Can also be used. Examples of the change to be detected include radioactivity, contact angle, sedimentation, ultraviolet spectroscopy, fluorescence, chemiluminescence, and electrochemiluminescence, in addition to the surface plasmon resonance described above.
[0023]
As described above, according to the present invention, the test object is brought into contact with the surface of the sensor chip, and the test substance in the test object is converted into a block of the block polymer derivative (1) standing on the surface. P¹The present invention provides a method of using the present chip as a method of using the present invention, wherein the method of using the present chip is detected as the use of the present invention, and the surface of the sensor chip is subjected to surface plasmon resonance. Detection by a device capable of detecting the state is exemplified.
[0024]
Block polymer derivative (1)
On the chip surface, the block polymer derivative (1) is¹/ P²Block P²Is bonded to the surface of the chip, and the block P¹Has a bulky, so-called brush-like structure formed on the chip surface.
[0025]
Block P that constitutes this brush-like structure¹And A connected via L is a group constituting the tip of the brush-like structure, and specifically, corresponds to a hydrogen atom, a functional group, or a protected functional group. The functional group may be any group as long as it can bind a desired ligand. Taking the case where the ligand is a protein or a nucleotide as an example, the functional group or the protected functional group A is a hydrogen atom, a formula (2)
[0026]
Embedded image

[0027]
[Wherein, R¹Are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R²Are, independently of each other, an alkyloxy group having 1 to 6 carbon atoms (ketal group), or two R²Are together an oxy group and an ethylene group which may be substituted by alkyl having 1 to 6 carbon atoms.]
, A carboxyl group, an acetal group, an aldehyde group, a tosyl group, a mercapto group, or a maleimide group.
[0028]
In the formula (2), R¹But independently of each other, examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, a sec-butyl group, Examples thereof include an n-hexyl group, and a methyl group is preferable. Also, R²Can take an alkyloxy group (ketal group) having 1 to 6 carbon atoms independently of each other as described above, and two R²May be combined with each other to form an oxy group (to be an aldehyde group as a whole), and an ethylene group which may be substituted by alkyl having 1 to 6 carbon atoms (to form a cyclic ketal) However, an aldehyde group or a protected aldehyde group (ketal group) is preferable.
[0029]
Further, L is, as shown above, A and block P¹And is a valence bond or an alkylene group having 1 to 6 carbon atoms, as described above, and is preferably a methylene group or an ethylene group.
[0030]
In addition, the block polymer P¹/ P²Occupies the main part of the brush-like structure,¹Is a polymer containing a hydrophilic linear monomer unit (hereinafter, also referred to as a hydrophilic linear polymer). The hydrophilic linear polymer is generally highly mobile and often exhibits good biocompatibility, and has properties suitable as a main part of the brush-like structure. Examples of the hydrophilic linear polymer include polyalkylene oxide, polyacrylamide, polyacrylic acid and the like, and polyalkylene oxide is preferable. When the hydrophilic linear polymer is a polyalkylene oxide, the number of carbon atoms of the alkylene oxide monomer unit constituting the polymer is preferably 2 to 3, that is, polyethylene oxide or polypropylene oxide. is there. Most preferably, the number of carbon atoms is 2, that is, polyethylene oxide. Further, in the polyalkylene oxide, the polymerization number of the alkylene oxide unit is preferably from 2 to 10,000, and more preferably from 10 to 1,000. When this polymerization number exceeds 10,000, the sensitivity of the chip surface using the same tends to decrease.
[0031]
In addition, the block polymer P¹/ P²Of the blocks P connected to the chip surface²Is a polymer block containing a monomer unit having a functional group capable of coordinating with the support on the chip surface. Examples of the functional group capable of coordinating with the support on the chip surface include an amino group, a carboxyl group, a mercapto group, and a sulfide group. Examples of the monomer unit having these functional groups include methacrylic acid (2-diethylaminoethyl), methacrylic acid (2-dimethylaminoethyl), polycysteine, polylysine, polyglutamine, and methacrylic acid (3-trimethoxysilylpropyl). And the like. By subjecting these monomer units to a polymerization reaction in a production process of the block polymer derivative (1) according to a conventional method, a block P having a desired functional group is obtained.²Can be formed.
[0032]
Block P²In one preferred embodiment of the formula (3)
[0033]
Embedded image

[0034]
[Wherein, m represents an integer of 1 to 10, n represents an integer of 2 to 200,³And R⁴Represents, independently of each other, an alkyl group having 1 to 5 carbon atoms, an amino group, a mercapto group, or a sulfide group].
[0035]
This methacrylic acid polymer (3) is²The production process of the block polymer derivative (1) is disclosed in detail in JP-A-2002-80903 (Patent Document 4), and according to this, the desired methacrylic acid polymer (3) is²To produce a block polymer derivative (1).
[0036]
That is, for example, the expression
ALP¹-OH (a)
[A, L, P in the formula¹Are the same as those in the block polymer derivative (1).]
Compound (a) [Method for producing this compound (a) is also known (for example, JP-A-7-316285: Patent Document 1, JP-A-11-322916: Patent Document 2, JP-A-2001) No. -200050: Patent Document 3 etc.)], a block polymer derivative (1) is obtained by a polymerization reaction of poly (dimethylaminoethyl methacrylate) to a desired degree of polymerization. be able to.
[0037]
Here, an example of a production process of acetal-PEG / PAMA is disclosed as the block polymer derivative (1). In this manufacturing process, s and t each mean a positive integer.
[0038]
Embedded image

[0039]
This chip surface, etc.
The material of the surface of the present chip, that is, the material of the support of the surface of the present chip may be the same as the material of the substrate of the present chip (that is, the substrate itself forms the surface of the present chip). It is also possible to form a thin film of a metal or the like on the surface and use the metal or the like as a support. As a material that can be used as the support, for example, a metal such as gold, silver, copper, or aluminum can be used. Glass; semiconductors such as CdS and ZnS; ZrO₂And the like; carbon such as graphite; metal oxides such as titanium oxide and aluminum oxide; alloy oxides such as iridium tin oxide.
[0040]
Block P having an appropriate functional group according to the material of the support on the surface of the present chip²It is preferable to select and use a block polymer derivative (1) having as a segment. That is, a block P having a functional group capable of coordinating with the material of the support on the surface of each chip²By using the block polymer derivative (1) having as a segment, the block polymer derivative (1) can be easily fixed on the chip surface.
[0041]
For example, when the above functional group is an amino group or a mercapto group, the material of the support on the chip surface is a metal such as gold, silver, copper, or aluminum; a semiconductor such as CdS or ZnS; Metal oxides such as titanium and aluminum oxide; When the functional group is a silanol group, the material of the chip support is preferably glass, silicone, titanium oxide, or ceramic. When the functional group is a carboxyl group, the material of the chip support is preferably ceramic or carbon.
[0042]
When producing the chip surface, a polymer having a bondable functional group is coordinated with a support on the chip surface to block a polymer having a bondable functional group.²The chip is immersed in, for example, a solution containing the block polymer derivative (1) to bring the block surface into contact with the block polymer derivative (1).²Is bonded to the chip surface, and the block polymer derivative (1) can be immobilized on the chip surface. As a result, the block P of the block polymer derivative (1)¹The chip surface can be manufactured, the side of which is brushed up on the surface.
[0043]
That is, the block polymer derivative (1) is dissolved in an appropriate buffered aqueous solution, and the polymer solution is brought into contact with the surface of the chip support by immersion or the like at an appropriate temperature, for example, about 20 to 37 ° C. By performing incubation for several tens of minutes to several hours, the surface of the present chip can be manufactured. The concentration of the block polymer derivative (1) in this polymer solution depends on the molecular weight of the block polymer to be used, but is usually 0.1 to 5 mg / ml, preferably about 1 mg / ml of the solution.
[0044]
By adjusting the concentration of the block polymer derivative (1) in the polymer solution, the carrying density of the block polymer derivative (1) on the chip surface can be set. That is, if the concentration of the block polymer derivative (1) in the polymer solution is high, the carrying density of the block polymer derivative (1) on the surface of the present chip increases, and if the concentration is low, the carrying density decreases. On the surface of the chip, the molecules of the block polymer derivative (1) are 0.01 to 10 molecules / nm.²It is preferable that the particles are supported at a density of This loading density is 0.01 molecule / nm.²If it is less than 10, it tends to be difficult to sufficiently suppress the non-specific reaction, which is the object of the present invention, and 10 molecules / nm²If it exceeds, the molecules are not arranged in a single layer but tend to be multilayered, and the detection sensitivity by the chip tends to decrease. The density of the brush-like structure can be confirmed by an atomic force microscope (AFM), and can also be confirmed by SPR or the like.
[0045]
Further, the bulkiness of the brush-like structure on the surface of the present chip may be substantially the same or different.
In particular, when brush-like structures having different bulks are provided on the surface of the present chip, for example, blocks P having different chain lengths may be used.¹It is also possible to carry out the above-mentioned immobilization step with a polymer solution containing a block polymer derivative (1) having¹It is also possible to prepare a polymer solution containing the block polymer derivative (1) having the following, and perform the above-described immobilization step again on a different polymer solution. When the latter re-immobilization step is performed, the polymer solution used in the first immobilization step contains the block P of the block polymer derivative (1).¹In the immobilization process performed later using a longer chain length of the block P,¹Is preferably set short.
[0046]
In addition, when performing one immobilization step, and subsequently performing two or more immobilization steps, washing of the chip surface is performed one or more times after each immobilization step. It is necessary to remove reactants and the like. Washing is not particularly limited as long as it can efficiently remove unreacted substances such as unbound block polymer derivative (1), but a dilute sodium hydroxide solution is preferably used.
[0047]
Masking process
By further performing a masking treatment on the surface of the present chip provided as described above, non-specific reactions can be further suppressed.
[0048]
In this masking treatment, a masking agent is brought into contact with the surface of the present chip, on which the block polymer (1) is obtained, obtained as described above, to immobilize the masking agent on the surface of the present chip. Is performed by
[0049]
Formula (4) as a masking agent
[0050]
Embedded image

[0051]
[Wherein, p is an integer of 1 to 200;⁵Is a hydroxyl group, an alkyl group having 1 to 6 carbon atoms,⁶Is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a pyridylthio group.
And a thio compound represented by Further, as one preferred embodiment of the masking treatment, there can be mentioned an embodiment in which the masking agent is 2-mercaptoethanol.
[0052]
The masking treatment can be performed by bringing the surface of the present chip, which can be produced as described above, into contact with a solution of a masking agent. In this case, the concentration of the masking agent in the masking agent solution varies depending on the type of the masking agent and the like, but is generally about 0.001 to 1000 mM, and preferably about 0.1 to 10 mM. The time of the masking process is preferably about 3 minutes to 1 day. Further, the masking treatment is preferably performed at a temperature of about 20 to 37 ° C.
[0053]
Thus, block P²Is fixed on the plane, and the block P¹The present invention provides a chip surface on which the block polymer derivative (1) is supported and a chip having the chip surface, which is erected to be bulky and constitutes a main part of the brush-like structure. When a biological binding reaction is performed on the surface of the chip, nonspecific reactions can be significantly suppressed, and the detection sensitivity of a sensor using the chip can be improved.
[0054]
EXAMPLES Examples of the present invention will be described below, but the technical scope of the present invention is not limited by these examples.
[Reference Example] Production of block polymer derivative (1) and the like
(1) Synthesis of acetal-PEG-SH (Mn = 5,000)
60 ml of THF as a solvent was placed in a flask at room temperature under argon (Ar) substitution, and 1 mmol of initiator 3,3-diethyl-1-propanol and 1 mmol of K-Naph (0.3168 mol / l) were added thereto with stirring. , Metallized. After sufficient stirring, 112.99 mmol of EO was added, and the mixture was stirred for 2 days while cooling with water, thereby performing polymerization.
[0055]
After stirring for 2 days, 0.5 mmol of K-Naph and 4.5 mmol of triethylamine were added to this solution for the purpose of remetallation. 10 ml of THF solvent and 3.5 mmol of methylsulfonyl chloride as a terminator were dissolved in an eggplant flask under Ar substitution, and the PEG polymerization solution was added dropwise thereto using an equal pressure dropping funnel. After the dropwise addition, the solution was recovered by ether reprecipitation, and then extracted with chloroform and a saturated saline solution.₂SO₄By dehydration and freeze-drying with benzene.
[0056]
To 0.44 mmol of potasium o-ethyldithiocarbonate dried under reduced pressure, 50 ml of THF and 3.6 ml of DMF were added as solvents under Ar and stirred. This solution was added to 0.2 g of acetal-PEG-MS dried under reduced pressure, and reacted at room temperature for 4 hours. After the reaction, extraction was performed with chloroform and saturated saline, and anhydrous Na₂SO₄Dehydrated, purified by ether reprecipitation, and recovered by freeze-drying with benzene.
[0057]
Further, 10 ml of THF as a solvent was added to 0.1 g of acetal-PEG-dithiocarbonate dried under reduced pressure as a solvent under Ar. To this, n-propylamine was added so as to become a 1.4M THF solution, and the mixture was stirred at room temperature for 3 hours and reacted. I let it. After the reaction, extraction was performed with chloroform and saturated saline, and anhydrous Na₂SO₄Dehydrated, purified by ether reprecipitation, and recovered by freeze-drying with benzene.
After collection,¹Structural analysis and GPC measurement were performed by H-NMR.
[0058]
(2) Synthesis of acetal-PEG / PAMA (PEG = 6,000, PAMA = 10,000)
In a reaction vessel under Ar, 45 ml of tetrahydroxyfuran (THF) as a reaction solvent and 1 mmol of 3,3-diethoxy-1-propanol as a reaction starting material are added, and an equimolar amount of a potassium naphthalene (K-Naph) THF solution Was added to metallize the starting material to prepare an initiator. Subsequently, 137 mmol of ethylene oxide (EO) was added using a syringe cooled with liquid nitrogen, and the mixture was stirred at room temperature for 2 days to carry out polymerization. After EO polymerization, a small amount of a sample for GPC measurement was withdrawn with a syringe, 31 mmol of 2-dimethylaminoethyl methacrylate (AMA) was quickly added with the syringe, and the mixture was reacted for 20 minutes under water cooling, and the reaction was stopped with a small amount of acetic acid. . The product was precipitated in 2-propanol cooled to −20 ° C., and the precipitate was separated by centrifugation (−10 ° C., 5000 rpm, 60 min). After removing the solvent by an evaporator, the precipitate was subjected to freeze-drying with benzene. Collected as a white powder. 1 g of the recovered polymer was dissolved in about 100 ml of distilled water, adjusted to pH 5 with hydrochloric acid, and freeze-dried. This polymer was further dissolved in about 100 ml of THF, and the precipitate of the polymer was separated by suction filtration and rinsed several times with THF. The precipitate separated by filtration was dissolved in about 100 ml of distilled water, and adjusted to pH 9 using an aqueous sodium hydroxide solution. Thereafter, the resultant was dissolved in benzene and freeze-dried to recover a white powder. The structure of the product was confirmed by GPC and H-NMR measurements.
[0059]
(3) Synthesis of PAMA (Mn = 22,500)
In a reaction vessel under Ar, 20 ml of tetrahydroxyfuran (THF) as a solvent, 0.5 mmol of diphenylethylene (DPE) as an initiator, and an equimolar amount of butyllithium (BuLi) were added to carry out metallization. Then, a THF solution (0.7 mol / l) of lithium chloride (LiCl) was added in an amount 5 times the molar amount of the initiator, and the mixture was stirred for 5 minutes. 71 mmol of 2- (dimethylamino) ethyl methacrylate (DMAEMA) was added, and the mixture was reacted for 1 hour. The reaction was stopped with a small amount of methanol. All of these reactions were performed at -78 ° C. The product of the synthesized polymer was reprecipitated in hexane, and the precipitate was dried under reduced pressure using a desiccator, and then benzene was freeze-dried to recover a white powder. The structure of the product was confirmed by GPC and H-NMR measurements.
[0060]
[Production Example] Production of this chip
(1) Comparative example
First, so-called piranha cleaning was performed on an SPR sensor chip (SIA kit: BIACORE, hereinafter the same) on the gold surface. The cleaning solution was a special grade concentrated sulfuric acid: special grade hydrogen peroxide solution = 3: 1 (volume ratio), and the cleaning was performed by immersing the chip in the cleaning solution at room temperature for 1 minute. Next, Acetal-PEG-SH produced in the above reference example was dissolved in 50 mM PBS containing 1.0 M NaCl and adjusted to 0.2 mM. Next, the chip was immersed in this Acetal-PEG-SH solution for 20 minutes to fix the Acetal-PEG-SH on the chip surface. This immobilization step was performed three times in total. After completion of each immobilization step, the chip was washed.Washing after each immobilization step was performed once with 50 mM PBS containing 1.0 M NaCl, then once with 50 mM NaCl, The test was performed twice with 50 mM PBS containing 1.0 M NaOH.
[0061]
The chip supporting Acetal-PEG-SH obtained in this manner was used as a chip of Comparative Example 1. When the chip surface of Comparative Example 1 was analyzed by AFM, 0.3 chip / nm was found on the chip surface.²At a moderate density, brush-like structures were observed.
[0062]
Further, the surface of the chip of Comparative Example 1 was further masked using 2-mercaptoethanol as a masking agent. That is, the chip of Comparative Example 1 was immersed in a 1.0 mM 2-mercaptoethanol (MCE) aqueous solution at room temperature for 5 minutes, and further, on the surface on which Acetal-PEG-SH was carried as a brush-like structure, 2-mercaptoethanol was fixed. This masking step with 2-mercaptoethanol was performed once. After the completion of the masking step, the chip was washed twice with ultrapure water at 1,800 vibrations / minute for 20 seconds in order to remove unreacted 2-mercaptoethanol.
[0063]
(2) Example
First, so-called piranha cleaning was performed on the same SPR sensor chip used in the comparative example. The cleaning solution was a special grade concentrated sulfuric acid: special grade hydrogen peroxide solution = 3: 1 (volume ratio), and the cleaning was performed by immersing the chip in the cleaning solution at room temperature for 1 minute. Next, Acetal-PEG / PAMA (PEG: MW6000, PAMA: MW10000) produced in the above reference example was dissolved in a 50 mM Na phosphate buffer solution and adjusted to 0.2 mM. Next, the chip was immersed in this Acetal-PEG / PAMA solution for 4 hours to fix the Acetal-PEG / PAMA on the chip surface. This immobilization step was performed once. After the step of immobilization, the chip was washed. The washing after the immobilization step was performed once with 50 mM NaOH, and then twice with 50 mM Na phosphate buffer.
[0064]
The thus obtained chip supporting Acetal-PEG / PAMA was used as the chip of Example 1. When the chip surface of Example 1 was analyzed by AMF, 0.25 lines / nm were found on the chip surface.²At a moderate density, brush-like structures were observed.
[0065]
Further, the chip surface of Example 1 was further masked using 2-mercaptoethanol as a masking agent. That is, the chip of Example 1 was immersed in a 1.0 mM 2-mercaptoethanol (MCE) aqueous solution at room temperature for 5 minutes, and the PEG side of Acetal-PEG / PAMA was carried as a so-called brush-like structure. Then, 2-mercaptoethanol was further immobilized. This masking step with 2-mercaptoethanol was performed once. After the completion of the masking step, the chip was washed twice with ultrapure water at 1,800 vibrations / minute for 20 seconds in order to remove unreacted 2-mercaptoethanol.
[0066]
[Test example]
(1) Examination of non-specific adsorption suppression effect of ruthenium bipyridine complex on each surface-1
A ruthenium bipyridine complex (hereinafter also referred to as Ru complex) solution (2 μM) was added to the chip surfaces of Examples 1 and 2 and Comparative Examples 1 and 2, and allowed to stand at room temperature for 10 minutes. Thereafter, the Ru complex was removed from each surface, and the HEPES buffer (pH
After washing once in 7.4), the substrate was rinsed with 100 mM of n-tripropyamine (TPA) as an electron supply substance, and the emission intensity of the Ru complex was measured. For comparison, the same measurement was performed on the unmodified chip surface. That is, after the above-mentioned piranha washing, the Ru complex solution was added, and the light emission intensity was measured by a ruthenium chelate type electrochemical light emitting device (custom-made product to Matsushita Electric Industrial).
[0067]
The results are shown in FIG. 1 (the ordinate indicates the emission intensity of the Ru complex. The higher the emission intensity, the stronger the non-specific adsorption, and the weaker the non-specific adsorption, the less the non-specific adsorption. Shown). As shown in FIG. 1, when the chips of Comparative Example 1 and Example 1 are compared, the chip of Example 1 in which the block polymer derivative is supported is more than the chip of Comparative Example 1 in which only the PEG moiety is supported. It can be seen that nonspecific adsorption is suppressed. Further, in both the examples and the comparative examples, the non-specific adsorption was suppressed by performing the masking treatment with 2-mercaptoethanol. In particular, in the chip of Example 2, the non-specific adsorption was suppressed. Is almost completely suppressed.
[0068]
(2) Evaluation of chip surface masking by SPR
The SPR sensor chip having the same gold surface as that used in the above comparative example and the like is subjected to piranha washing having the same contents as described above, and Acetal-PEG-SH (MW. 5000) is added to 50 mM PBS containing 1.0 M NaCl (pH 7.4). ) And adjusted to 0.2 mM. This Acetal-PEG-SH (MW. 5000) solution was set in an SPR device (BiaCore 3000: BIACORE), and flowed at a flow rate of 20 μl / min for 20 minutes at a time. This flow operation was performed three times. PEG-SH (MW. 5000) was immobilized on the chip surface. Thereafter, 1.0 mM 2-mercaptoethanol was flowed at a flow rate of 10 μl / min for 20 minutes, and the change in SPR response was observed.
[0069]
Acetal-PEG / PAMA (PEG: MW6000, PAMA: MW10000) was dissolved in 50 mM Na phosphate buffer and adjusted to 0.2 mM. The Acetal-PEG / PAMA solution was set in the above SPR apparatus, and flowed at a flow rate of 5 μl / min for 1 hour at a time. Immobilization was performed. Thereafter, 1.0 mM 2-mercaptoethanol was flowed at a flow rate of 10 μl / min for 20 minutes, and the change in SPR response was observed. The result is shown in FIG. 2 [vertical axis: angle shift (angle change amount) (Δθ × 10^-4[°]), horizontal axis: elapsed time].
[0070]
The change of the response of SPR due to the 2-mercaptoethanol treatment was compared between the case using Acetal-PEG-SH and the case using Acetal-PEG / PAMA. While the change was negative, Acetal-PEG / PAMA was slightly positive. This is because, in the Acetal-PEG-SH system, an SH exchange reaction between 2-mercaptoethanol and the SH group of PEG-SH occurs, but in the Acetal-PEG / PAMA system, such an exchange reaction does not occur. Rather, it is speculated that 2-mercaptoethanol itself has penetrated between the PEG brush-like structures.
[0071]
From this, it became clear that a more dense coating surface can be formed by treating the chip surface using PEG / PAMA and 2-mercaptoethanol in combination.
(3) Examination of non-specific adsorption suppression effect of ruthenium bipyridine complex on each surface-2
a) gold surface, b) surface of Comparative Example 1, c) surface coated with PAMA manufactured in Reference Example, d) surface of Example 1 were prepared, and 1 mg / ml BSA solution (solvent) was applied to each chip surface. : HEPES buffer), the amount of BSA adsorbed on each surface (flow rate of 20 μl / min, after 10 minutes, the amount of change in angle when running buffer was passed for 3 minutes) was measured by SPR. The result when the Ru complex solution was flown is shown in FIG. 3 (the vertical axis represents the angle change (Δθ × 10^-4[°]).
[0072]
From the results shown in FIG. 3, the change in the angle on the chip surface of Comparative Example 1 carrying only the brush-like structure and the change in the angle on the chip surface of Example 1 were significantly smaller than those of the others. In this example, the angle change of Comparative Example 1 was smaller than that of Example 1. This is because the molecular weight of Acetal-PEG-SH is smaller than that of Acetal-PEG / PAMA, This is considered to be due to the fact that the loading density of SH on the chip surface is higher than the loading density of PEG / PAMA.
[0073]
Actually, two types of surfaces having different amounts of PEG / PAMA immobilization on the chip surface (immobilization amount: Δθ = 1200 (block 1200) or 2000 × 10^-4[°] (block 2000)), and the amount of BSA adsorbed was measured by SPR as described above (FIG. 4). As a result, it was found that with an increase in the amount of PEG / PAMA immobilized, that is, an increase in the loading density, the adsorption of BSA to the chip surface was further suppressed.
[0074]
【The invention's effect】
According to the present invention, there is provided a sensor chip having a surface on which nonspecific reactions are significantly suppressed.
[Brief description of the drawings]
FIG. 1 is a drawing showing the results of examining the degree of nonspecific reaction in the chips of Examples and Comparative Examples using the emission intensity of a Ru complex as an index.
FIG. 2 is a drawing showing the results of examining the response in SPR when 2-mercaptoethanol treatment was performed on the chips of Examples and Comparative Examples.
FIG. 3 is a graph showing the results of an SPR study on the suppression of BSA adsorption on the chip surface.
FIG. 4 is a drawing showing the results of an SPR study of the relationship between the amount of PEG / PAMA immobilized on the chip surface and the amount of BSA adsorbed.

Claims (17)

General formula (1)
^{A-L-P 1 / P} 2 (1)
[In the formula, A represents a hydrogen atom, a functional group, a protected functional group or a ligand, L represents a linking group or a valence bond, and P ¹ / P ² represents a block P ¹ and a block P ² The block polymer is a block polymer, and the block P ¹ is a block of a polymer containing a hydrophilic linear monomer unit. The block P ² has a functional group capable of coordinating with a support on the surface of the sensor chip. Block of a polymer containing a monomer unit having
In one or more block polymers derivative represented is the support of the surface of the sensor chip, via block P ² of the block polymer derivative, the support and the functional group capable of coordinating the sensor chip surface Surface of the sensor chip, which is connected to the sensor chip. A of the block polymer derivative (1) is a hydrogen atom, and the formula (2)

[Wherein, R ¹ , independently of each other, is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ² is, independently of each other, an alkyloxy group having 1 to 6 carbon atoms, or two R ^{2 taken} together and substituted with an oxy group or an alkyl having 1 to 6 carbon atoms. Is a good ethylene group]
The surface of the sensor chip according to claim 1, which is a carboxyl group, an acetal group, an aldehyde group, a tosyl group, a mercapto group, or a maleimide group. 3. The sensor chip surface according to claim 1, wherein L of the block polymer derivative (1) is a valence bond or an alkylene group having 1 to 6 carbon atoms. Block ^{P 1} of the block polymer ^P 1 / ^{P 2} of the block polymer derivative (1) is the number of carbon atoms is 2 to 3 alkylene oxide monomer units, the number of polymerization is a polyalkylene oxide of 2 to 10000, wherein Item 4. A surface of the sensor chip according to any one of Items 1 to 3. The surface of the sensor chip according to claim 4, wherein the polyalkylene oxide is polyethylene oxide. Block P ² of the block polymer P ^{1 /} P ² of the block polymer derivative (1) is an amino group, a carboxyl group, a mercapto group, a sulfide group or a silanol group, and can be coordinated support surface of the sensor chip The surface of the sensor chip according to any one of claims 1 to 5, which is a polymer containing a monomer unit having a functional group. Block ^{P 2} of the block polymer ^P 1 / ^{P 2} of the block polymer derivative (1) is of the formula (3)

[In the formula, m represents an integer of 1 to 10, n represents an integer of 2 to 200, and R ³ and R ⁴ independently represent an alkyl group having 1 to 5 carbon atoms, an amino group, or a mercapto group. Or a sulfide group]. The surface of the sensor chip according to any one of claims 1 to 5, which is a methacrylic acid polymer. Block polymer derivative (1) molecules are carried at a density of 0.01 to 10 molecules / nm ^2, the sensor chip surface according to claim 1. The surface of the sensor chip according to claim 1, further comprising a masking process. The masking agent used for the masking treatment is represented by the formula (4)

[Wherein, p is an integer of 1 to 200, R ⁵ is a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, and R ⁶ is a hydrogen atom, an alkyl having 1 to 6 carbon atoms. Or a pyridylthio group]
The surface of the sensor chip according to claim 9, which is a substance represented by the following formula: The surface of the sensor chip according to claim 10, wherein the masking agent used for the masking treatment is 2-mercaptoethanol. The sensor chip surface according to any one of claims 1 to 11, wherein the support on the sensor chip surface is formed of a material selected from the group consisting of gold, silver, copper, and aluminum. The sensor according to claim 1, wherein the support on the surface of the sensor chip is formed of a material selected from the group consisting of glass, semiconductor, ceramic, carbon, metal oxide, and alloy oxide. Chip surface. A sensor chip having a surface according to claim 1. A test object is brought into contact with the surface of the sensor chip, and the test substance in the test object is bonded to A of the block P ¹ of the block polymer derivative (1) standing on the surface, The method of using a sensor chip according to claim 14, wherein the binding is detected. 16. The method of using a sensor chip according to claim 15, wherein the means for detecting the binding is detection by a device capable of detecting the state of the surface of the sensor chip using surface plasmon resonance. The method of using a sensor chip according to claim 15, wherein the means for detecting the binding is detection by an electrochemiluminescent device.

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