JP4037428B2 - Sensor substrate - Google Patents
Sensor substrate Download PDFInfo
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- JP4037428B2 JP4037428B2 JP2005243819A JP2005243819A JP4037428B2 JP 4037428 B2 JP4037428 B2 JP 4037428B2 JP 2005243819 A JP2005243819 A JP 2005243819A JP 2005243819 A JP2005243819 A JP 2005243819A JP 4037428 B2 JP4037428 B2 JP 4037428B2
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- general formula
- compound
- sensor substrate
- coating
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Description
本発明は、非特異吸着を抑制し、生理活性物質を固定化できるセンサー用基板、特に表面プラズモン共鳴バイオセンサーに用いるためのセンサー用基板、並びにその製造方法に関する。 The present invention relates to a sensor substrate capable of suppressing non-specific adsorption and immobilizing a physiologically active substance, particularly a sensor substrate for use in a surface plasmon resonance biosensor, and a method for producing the same.
現在、臨床検査等で免疫反応など分子間相互作用を利用した測定が数多く行われているが、従来法では煩雑な操作や標識物質を必要とするため、標識物質を必要とすることなく、測定物質の結合量変化を高感度に検出することのできるいくつかの技術が使用されている。例えば、表面プラズモン共鳴（ＳＰＲ）測定技術、水晶発振子マイクロバランス（ＱＣＭ）測定技術、金のコロイド粒子から超微粒子までの機能化表面を使用した測定技術である。ＳＰＲ測定技術はチップの金属膜に接する有機機能膜近傍の屈折率変化を反射光波長のピークシフト又は一定波長における反射光量の変化を測定して求めることにより、表面近傍に起こる吸着及び脱着を検知する方法である。ＱＣＭ測定技術は水晶発振子の金電極（デバイス）上の物質の吸脱着による発振子の振動数変化から、ｎｇレベルで吸脱着質量を検出できる技術である。また、金の超微粒子（ｎｍレベル）表面を機能化させて、その上に生理活性物質を固定して、生理活性物質間の特異認識反応を行わせることによって、金微粒子の沈降、配列から生体関連物質の検出ができる。 Currently, many measurements using intermolecular interactions such as immune reactions are performed in clinical examinations, etc., but conventional methods require complicated operations and labeling substances, so measurement without the need for labeling substances Several techniques that can detect a change in the amount of a substance bound with high sensitivity are used. For example, surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, and measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles. SPR measurement technology detects adsorption and desorption near the surface by measuring the refractive index change in the vicinity of the organic functional film in contact with the metal film of the chip by measuring the peak shift of the reflected light wavelength or the change in the amount of reflected light at a fixed wavelength. It is a method to do. The QCM measurement technique is a technique that can detect the adsorption / desorption mass at the ng level from the change in the oscillation frequency of the oscillator due to the adsorption / desorption of a substance on the gold electrode (device) of the crystal oscillator. In addition, by functionalizing the surface of gold ultrafine particles (nm level), immobilizing a physiologically active substance on the surface, and performing a specific recognition reaction between the physiologically active substances, it is possible to obtain a living body from the sedimentation and arrangement of gold fine particles. Related substances can be detected.
上記した技術においては、いずれの場合も、生理活性物質を固定化する表面が重要である。以下、当技術分野で最も使われている表面プラズモン共鳴（ＳＰＲ）を例として、説明する。 In any of the above techniques, the surface on which the physiologically active substance is immobilized is important. Hereinafter, the surface plasmon resonance (SPR) most used in this technical field will be described as an example.
一般に使用される測定チップは、透明基板（例えば、ガラス）、蒸着された金属膜、及びその上に生理活性物質を固定化できる官能基を有する薄膜からなり、その官能基を介し、金属表面に生理活性物質を固定化する。該生理活性物質と検体物質間の特異的な結合反応を測定することによって、生体分子間の相互作用を分析する。 A commonly used measurement chip is composed of a transparent substrate (eg, glass), a deposited metal film, and a thin film having a functional group capable of immobilizing a physiologically active substance thereon, and the metal surface is interposed through the functional group. Immobilize physiologically active substances. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the analyte substance.
生理活性物質を固定化できる官能基を有する薄膜としては、金属と結合する官能基、鎖長の原子数が１０以上のリンカー、及び生理活性物質と結合できる官能基を有する化合物を用いて、生理活性物質を固定化した測定チップが報告されている（特許文献１を参照）。また、金属膜と、該金属膜の上に形成されたプラズマ重合膜からなる測定チップが報告されている（特許文献２を参照）。 As a thin film having a functional group capable of immobilizing a physiologically active substance, a functional group capable of binding to a metal, a linker having a chain length of 10 or more atoms, and a compound having a functional group capable of binding to a physiologically active substance, A measurement chip in which an active substance is immobilized has been reported (see Patent Document 1). In addition, a measurement chip comprising a metal film and a plasma polymerization film formed on the metal film has been reported (see Patent Document 2).
一方、生理活性物質と検体物質間の特異的な結合反応を測定する場合、検体物質は必ずしも単一成分ではなく、例えば細胞抽出液中などのような不均一系で検体物質を測定することも要求される。その場合、種々の蛋白質、脂質などの夾雑物が検出表面に非特異的な吸着を起こすと、測定検出感度が著しく低下する。上記の検出表面では、非特異吸着が極めて起こりやすく問題があった。 On the other hand, when measuring a specific binding reaction between a physiologically active substance and a sample substance, the sample substance is not necessarily a single component, and for example, the sample substance may be measured in a heterogeneous system such as in a cell extract. Required. In that case, when various kinds of contaminants such as proteins and lipids cause nonspecific adsorption on the detection surface, the measurement and detection sensitivity is remarkably lowered. The above detection surface has a problem that non-specific adsorption is very likely to occur.
この問題を解決するためにいくつかの方法が検討されている。例えば、金属表面にリンカーを介し、親水性のハイドロゲルを固定化することで、物理吸着を抑制する方法も使用されてきた（特許文献１、特許文献３及び特許文献４を参照）。しかしながら、この方法でも非特異吸着の抑制性は十分なレベルではなかった。 Several methods have been investigated to solve this problem. For example, a method of suppressing physical adsorption by immobilizing a hydrophilic hydrogel on a metal surface via a linker has been used (see Patent Document 1, Patent Document 3, and Patent Document 4). However, even with this method, the suppression of nonspecific adsorption was not at a sufficient level.
本発明は上記した従来技術の問題を解消することを解決すべき課題とした。即ち、本発明は、非特異吸着を抑制し、生理活性物質を固定化できるセンサー用基板、並びにその製造方法を提供することを解決すべき課題とした。 The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a sensor substrate capable of suppressing nonspecific adsorption and immobilizing a physiologically active substance, and a method for producing the same.
本発明者らは鋭意検討を重ねた結果、金属から成る基板表面に高分子化合物被膜を有するセンサー用基板において、基板表面に高分子化合物被膜を形成した後に、基板表面と化学結合を形成する化合物で基板表面を処理することによって、上記課題を解決できることを見出し、本発明を完成するに至った。 As a result of intensive studies, the present inventors have found that in a sensor substrate having a polymer compound film on the surface of a metal substrate, a compound that forms a chemical bond with the substrate surface after the polymer compound film is formed on the substrate surface. The present inventors have found that the above problems can be solved by treating the substrate surface with the present invention, and have completed the present invention.
即ち、本発明によれば、金属から成る基板表面に高分子化合物被膜を有するセンサー用基板であって、基板表面に高分子化合物被膜を形成した後に、基板表面と化学結合を形成する化合物で基板表面を処理することにより得られるセンサー用基板が提供される。 That is, according to the present invention, there is provided a sensor substrate having a polymer compound film on a metal substrate surface, wherein the substrate is formed of a compound that forms a chemical bond with the substrate surface after the polymer compound film is formed on the substrate surface. A sensor substrate obtained by treating the surface is provided.
好ましくは、基板は、金、銀、銅、白金又はアルミニウムからなる群より選ばれる自由電子金属からなるものである。
好ましくは、基板表面と化学結合を形成する化合物は、チオール化合物、スルフィド化合物又はジスルフィド化合物である。
好ましくは、基板表面と化学結合を形成する化合物は、親水性官能基を有する化合物である。
Preferably, the substrate is made of a free electron metal selected from the group consisting of gold, silver, copper, platinum or aluminum.
Preferably, the compound that forms a chemical bond with the substrate surface is a thiol compound, a sulfide compound, or a disulfide compound.
Preferably, the compound that forms a chemical bond with the substrate surface is a compound having a hydrophilic functional group.
好ましくは、基板表面に高分子化合物被膜を形成した後に基板表面を処理するために使用する基板表面と化学結合を形成する化合物と同一の化合物を用いて、高分子化合物被膜を形成する前に基板表面が処理されている。 Preferably, after forming the polymer compound film on the substrate surface, the substrate is used before forming the polymer compound film by using the same compound as the compound that forms a chemical bond with the substrate surface used to treat the substrate surface. The surface is treated.
好ましくは、高分子化合物被膜は、生理活性物質を固定化できる官能基を有している。
好ましくは、本発明のセンサー用基板は、バイオセンサー用基板である。
好ましくは、本発明のセンサー用基板は、非電気化学的検出に使用され、さらに好ましくは表面プラズモン共鳴分析に使用される。
Preferably, the polymer compound coating has a functional group capable of immobilizing a physiologically active substance.
Preferably, the sensor substrate of the present invention is a biosensor substrate.
Preferably, the sensor substrate of the present invention is used for non-electrochemical detection, more preferably for surface plasmon resonance analysis.
本発明の別の側面によれば、生理活性物質が共有結合により表面に結合している、上記した本発明のセンサー用基板が提供される。 According to another aspect of the present invention, there is provided the sensor substrate of the present invention described above, wherein a physiologically active substance is bonded to the surface by a covalent bond.
本発明のさらに別の側面によれば、（ａ）金属から成る基板表面に高分子化合物被膜を形成する工程、及び（ｂ）高分子化合物被膜が形成された基板表面を、基板表面と化学結合を形成する化合物で処理する工程を含む、上記した本発明のセンサー用基板の製造方法が提供される。 According to still another aspect of the present invention, (a) a step of forming a polymer compound film on the surface of a substrate made of metal, and (b) a substrate surface on which the polymer compound film is formed are chemically bonded to the substrate surface. There is provided a method for producing the sensor substrate of the present invention described above, which comprises a step of treating with a compound that forms.
本発明のさらに別の側面によれば、（ａ）金属から成る基板表面に高分子化合物被膜を形成する工程、（ｂ）高分子化合物被膜が形成された基板表面を、基板表面と化学結合を形成する化合物で処理する工程、及び（ｃ）前記工程（ｂ）の後に、高分子化合物被膜に生理活性物質を固定化する工程を含む、生理活性物質が共有結合により表面に結合している上記した本発明のセンサー用基板の製造方法が提供される。 According to still another aspect of the present invention, (a) a step of forming a polymer compound film on a substrate surface made of metal, (b) a substrate surface on which the polymer compound film is formed is chemically bonded to the substrate surface. The step in which the physiologically active substance is covalently bonded to the surface, comprising the step of treating with the compound to be formed, and (c) the step of immobilizing the physiologically active substance on the polymer compound film after the step (b) A method for producing the sensor substrate of the present invention is provided.
本発明により、非特異吸着を抑制し、生理活性物質を固定化できるセンサー用基板を提供することが可能になった。 According to the present invention, it has become possible to provide a sensor substrate capable of suppressing nonspecific adsorption and immobilizing a physiologically active substance.
以下、本発明の実施の形態について説明する。
本発明のセンサー用基板は、金属から成る基板表面に高分子化合物被膜を有するセンサー用基板であって、基板表面に高分子化合物被膜を形成した後に、基板表面と化学結合を形成する化合物で基板表面を処理することにより得られることを特徴とする。この特徴により、本発明のセンサー用基板では、非特異吸着を抑制しつつ生理活性物質を固定化できるという効果が達成できる。
Embodiments of the present invention will be described below.
The sensor substrate of the present invention is a sensor substrate having a polymer compound coating on the surface of a metal substrate, and the substrate is made of a compound that forms a chemical bond with the substrate surface after the polymer compound coating is formed on the substrate surface. It is obtained by treating the surface. With this feature, the sensor substrate of the present invention can achieve the effect that the physiologically active substance can be immobilized while suppressing non-specific adsorption.
基板表面と化学結合を形成する化合物としては、特に制限はないが分子内に、
のいずれかの官能基を有するものなどが挙げられる。特に−ＳＨ、−Ｓ−、または−Ｓ−Ｓ−を有する化合物が好ましい。具体的には、ジスルフィド化合物が挙げられる。 And those having any of the functional groups. Particularly preferred are compounds having —SH, —S—, or —S—S—. Specific examples include disulfide compounds.
これら基板表面と化学結合を生成する化合物は、分子内に親水性官能基を有することで基板表面が水溶液中に存在する際に水和層が形成され、水溶液中の物質の非特異的な吸着を防止する効果があり好ましい。親水性官能基としては、−ＯＨ、−ＳＨ、−ＣＯＯＨ、−ＮＨ2、−ＮＲ1Ｒ2（式中、Ｒ1及びＲ2は互いに独立に水素原子又は低級アルキル基を示す）、−ＣＨＯ、ポリエチレンオキシド、ポリプロピレンオキシドなどが挙げられる。 These compounds that form a chemical bond with the substrate surface have a hydrophilic functional group in the molecule, so that a hydrated layer is formed when the substrate surface exists in an aqueous solution, and nonspecific adsorption of substances in the aqueous solution. Is preferable. Examples of the hydrophilic functional group include —OH, —SH, —COOH, —NH 2 , —NR 1 R 2 (wherein R 1 and R 2 independently represent a hydrogen atom or a lower alkyl group), —CHO , Polyethylene oxide, polypropylene oxide and the like.
基板表面と化学結合を形成する化合物の一例としては、式：Ｘ1−Ｒ1−Ｙ1で示される化合物を使用することができる。 As an example of a compound that forms a chemical bond with the substrate surface, a compound represented by the formula: X 1 —R 1 —Y 1 can be used.
Ｘ1は金属膜に対する結合性を有する基である。具体的には、非対称又は対称スルフィド（−SSR11Ｙ11、−SSR1Y1）、スルフィド（−SR11Ｙ11、−SR1Y1）、ジセレニド（−SeSeR11Ｙ11、−SeSeR1Y1）、セレニド（SeR11Ｙ11、−SeR1Y1）、チオール（−SH）、ニトリル（−CN）、イソニトリル、ニトロ（−NO2）、セレノール（−SeH）、３価リン化合物、イソチオシアネート、キサンテート、チオカルバメート、ホスフィン、チオ酸またはジチオ酸（−COSH、−CSSH）が好ましく用いられる。 X 1 is a group having a binding property to the metal film. Specifically, asymmetric or symmetric sulfide (—SSR 11 Y 11 , —SSR 1 Y 1 ), sulfide (—SR 11 Y 11 , —SR 1 Y 1 ), diselenide (—SeSeR 11 Y 11 , —SeSeR 1 Y) 1 ), selenide (SeR 11 Y 11 , —SeR 1 Y 1 ), thiol (—SH), nitrile (—CN), isonitrile, nitro (—NO 2 ), selenol (—SeH), trivalent phosphorus compound, iso Thiocyanate, xanthate, thiocarbamate, phosphine, thioacid or dithioacid (-COSH, -CSSH) is preferably used.
Ｒ1（とＲ11）は場合によりヘテロ原子により中断されており、好ましくは適当に密な詰め込みのため直鎖（枝分かれしていない）であり、場合により二重及び／又は三重結合を含む炭化水素鎖である。鎖の長さは10原子を越えることが好ましい。炭素鎖は場合によりフッ素化されることができる。 R 1 (and R 11 ) is optionally interrupted by a heteroatom, preferably straight-chain (unbranched) for reasonably close packing, and optionally carbon containing double and / or triple bonds It is a hydrogen chain. The chain length is preferably greater than 10 atoms. The carbon chain can optionally be fluorinated.
Ｙ1とＹ11は好ましくは同一であり、具体的にはヒドロキシル、カルボキシル、アミノ、アルデヒド、ヒドラジド、カルボニル、エポキシ、又はビニル基などである。 Y 1 and Y 11 are preferably the same, specifically, hydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, epoxy, vinyl group or the like.
Ｘ1−Ｒ1−Ｙ1で示される化合物の具体例としては、7−カルボキシ−１−ヘプタンチオール、10-カルボキシ-1-デカンチオール、4,4'-ジチオジブチリックアシッド、11-ヒドロキシ-1-ウンデカンチオール、11-アミノ-1-ウンデカンチオールなどが挙げられる。 Specific examples of the compound represented by X 1 -R 1 -Y 1 include 7-carboxy-1-heptanethiol, 10-carboxy-1-decanethiol, 4,4′-dithiodibutyric acid, 11-hydroxy- Examples include 1-undecanethiol, 11-amino-1-undecanethiol, and the like.
本発明で用いられる高分子化合物は親水性高分子化合物でも疎水性高分子化合物でもよい。親水性高分子化合物としては、アルブミン及びカゼインなどの蛋白質；寒天、アルギン酸ナトリウム及びデンプン誘導体などの糖誘導体；カルボキシメチルセルロース及びヒドロキシメチルセルロースなどのセルロース化合物；キチン及びキトサンなどの多糖類；ポリビニルアルコール、ポリ−Ｎ−ビニルピロリドン、ポリアクリルアミド、ポリアクリル酸、ポリエチレングリコール及びポリメチルビニルエーテルなどの合成親水性高分子；デキストラン誘導体及びゼラチン等の天然高分子などを挙げることができる。バイオセンサーへの応用との観点からは、水溶性天然高分子が好ましく、デキストラン誘導体が特に好ましい。 The polymer compound used in the present invention may be a hydrophilic polymer compound or a hydrophobic polymer compound. Examples of hydrophilic polymer compounds include proteins such as albumin and casein; sugar derivatives such as agar, sodium alginate and starch derivatives; cellulose compounds such as carboxymethyl cellulose and hydroxymethyl cellulose; polysaccharides such as chitin and chitosan; polyvinyl alcohol, poly- Examples thereof include synthetic hydrophilic polymers such as N-vinylpyrrolidone, polyacrylamide, polyacrylic acid, polyethylene glycol and polymethyl vinyl ether; natural polymers such as dextran derivatives and gelatin. From the viewpoint of application to a biosensor, a water-soluble natural polymer is preferable, and a dextran derivative is particularly preferable.
親水性高分子化合物の基板へのコーティングは常法によって行うことができ、例えば、スピン塗布、エアナイフ塗布、バー塗布、ブレード塗布、スライド塗布、カーテン塗布、さらにはスプレー法、蒸着法、キャスト法、浸漬法等によって行うことができる。また、本明細書中上記した基板表面と化学結合を形成する化合物を介して化学的に結合してもよい。 Coating of the hydrophilic polymer compound on the substrate can be performed by a conventional method, for example, spin coating, air knife coating, bar coating, blade coating, slide coating, curtain coating, spraying, vapor deposition, casting, It can be performed by an immersion method or the like. Moreover, you may chemically couple | bond through the compound which forms a chemical bond with the board | substrate surface mentioned above in this specification.
本発明で用いることができる疎水性高分子化合物とは、スチレン類、マレイン酸ジエステル類、（メタ）アクリル酸エステル類、ビニルエステル類、クロトン酸エステル類、イタコン酸ジエステル類、フマル酸ジエステル類、アリル化合物類、ビニルエーテル類、ビニルケトン類、アクリルアミド類などに代表されるモノマー類から選ばれる１種類以上のモノマーを重合して成るポリマーであり、且つ、エステル結合を有するモノマー単位が好ましくは１０％以上、より好ましくは３０％以上、さらに好ましくは５０％以上である、非水溶性のポリマーを指称する。本発明で用いる疎水性ポリマーは、好ましくは水への溶解度（２５℃）が１０質量ｇ以下、より好ましくは１質量ｇ以下、最も好ましくは０．１質量ｇ以下である。疎水性の高分子化合物の具体例としては、ポリスチレン、ポリエチレン、ポリプロピレン、ポリエチレンテレフタレート、ポリビニルクロライド、ポリメチルメタクリレート、ポリエステル、ナイロンなどが挙げられる。 Hydrophobic polymer compounds that can be used in the present invention include styrenes, maleic acid diesters, (meth) acrylic acid esters, vinyl esters, crotonic acid esters, itaconic acid diesters, fumaric acid diesters, It is a polymer formed by polymerizing one or more monomers selected from monomers represented by allyl compounds, vinyl ethers, vinyl ketones, acrylamides, etc., and the monomer unit having an ester bond is preferably 10% or more More preferably 30% or more, still more preferably 50% or more, referring to a water-insoluble polymer. The hydrophobic polymer used in the present invention preferably has a water solubility (25 ° C.) of 10 mass g or less, more preferably 1 mass g or less, and most preferably 0.1 mass g or less. Specific examples of the hydrophobic polymer compound include polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, polymethyl methacrylate, polyester, and nylon.
疎水性高分子化合物の被膜の形成方法としては、疎水性高分子化合物を（基板）表面に塗布してもよいし、前記したモノマー類を（基板）表面で重合させてもよい。本発明において、好適な塗布の方法としては、例えば、スピン塗布、エアナイフ塗布、バー塗布、ブレード塗布、スライド塗布、カーテン塗布、さらにはスプレー法、蒸着法、キャスト法、浸漬法などが挙げられる。 As a method for forming a film of the hydrophobic polymer compound, the hydrophobic polymer compound may be applied to the (substrate) surface, or the monomers described above may be polymerized on the (substrate) surface. In the present invention, suitable coating methods include, for example, spin coating, air knife coating, bar coating, blade coating, slide coating, curtain coating, spraying, vapor deposition, casting, and dipping.
疎水性高分子化合物層の厚みは特に限定されないが、センサーの高感度化と非特異吸着の抑制の観点から、０．１ｎｍ以上５００ｎｍ以下が好ましく、１ｎｍ以上５０ｎｍ以下がより好ましい。 The thickness of the hydrophobic polymer compound layer is not particularly limited, but is preferably from 0.1 nm to 500 nm, more preferably from 1 nm to 50 nm, from the viewpoint of increasing the sensitivity of the sensor and suppressing nonspecific adsorption.
上記した高分子材料は、化学薬品、カップリング剤、界面活性剤、表面蒸着などを使用した化学処理、又は加熱、紫外線、放射線、プラズマ、イオンなどを使用した物理的処理によって、表面修飾することが可能である。 The above polymer materials may be surface modified by chemical treatment using chemicals, coupling agents, surfactants, surface deposition, etc., or physical treatment using heating, ultraviolet rays, radiation, plasma, ions, etc. Is possible.
高分子化合物被膜層には生理活性物質と共有結合を生成しうる官能基を有することが好ましい。生理活性物質と共有結合を生成しうる好ましい官能基としては−ＯＨ、−ＳＨ、−ＣＯＯＨ、−ＮＲ1Ｒ2（式中、Ｒ1及びＲ2は互いに独立に水素原子又は低級アルキル基を示す）、−ＣＨＯ、−ＮＲ3ＮＲ1Ｒ2（式中、Ｒ1、Ｒ2及びＲ3は互いに独立に水素原子又は低級アルキル基を示す）、−ＮＣＯ、−ＮＣＳ、エポキシ基、またはビニル基などが挙げられる。ここで、低級アルキル基における炭素数は特に限定されないが、一般的にはＣ１〜Ｃ１０程度であり、好ましくはＣ１〜Ｃ６である。 The polymer compound coating layer preferably has a functional group capable of forming a covalent bond with a physiologically active substance. Preferred functional groups capable of forming a covalent bond with a physiologically active substance include —OH, —SH, —COOH, —NR 1 R 2 (wherein R 1 and R 2 independently represent a hydrogen atom or a lower alkyl group) ), —CHO, —NR 3 NR 1 R 2 (wherein R 1 , R 2 and R 3 independently represent a hydrogen atom or a lower alkyl group), —NCO, —NCS, epoxy group, or vinyl group Etc. Here, the number of carbon atoms in the lower alkyl group is not particularly limited, but is generally about C1 to C10, preferably C1 to C6.
高分子化合物の被膜を形成後、基板表面に生理活性物質を固定化することができるよう化学修飾することで、生体分子間の相互作用を電気的信号等の信号に変換して、対象となる物質を測定・検出することが可能となり有用である。この化学修飾は常法により、水溶液中または、有機溶剤中で基板表面に生理活性物質と共有結合を生成しうる官能基を導入できる。例えば−ＣＯＯＣＨ3基を含有する疎水性高分子化合物であるポリメチルメタクリレートを金属膜上にコーティングした後、その表面をＮａＯＨ水溶液（１Ｎ）に４０℃１６時間接触させると、最表面に−ＣＯＯＨ基が生成する。 After forming the polymer compound coating, the bioactive substance is chemically modified so that it can be immobilized on the substrate surface, thereby converting the interaction between biomolecules into a signal such as an electrical signal. It is useful because it can measure and detect substances. This chemical modification can introduce a functional group capable of forming a covalent bond with a physiologically active substance on the substrate surface in an aqueous solution or an organic solvent by a conventional method. For example, after coating polymethyl methacrylate, which is a hydrophobic polymer compound containing —COOCH 3 groups, on a metal film, when the surface is brought into contact with an aqueous NaOH solution (1N) at 40 ° C. for 16 hours, —COOH groups are formed on the outermost surface. Produces.
本発明のセンサー用基板の好ましい検出手段としては、非電気化学的手法でありさらに好ましくは表面プラズモン共鳴である。これらの検出手段に使用するため、基板を薄膜として形成してもよい。 A preferred detection means for the sensor substrate of the present invention is a non-electrochemical technique, and more preferably surface plasmon resonance. For use in these detection means, the substrate may be formed as a thin film.
本発明において基板表面として用いられる金属は、自由電子を有し、空気中で容易に発火するなどの不安定性が無ければ、いずれの金属でも使用できるが、取り扱い易さの観点から、金、銀、銅、白金、アルミニウムが好ましく、金がより好ましい。これらの金属は単独、または組み合わせて使用してもよい。また、（基板）表面への付着性を上げるために、（基板）表面と金属との間に、クロム等からなる介在層を設けてもよい。 The metal used as the substrate surface in the present invention may be any metal as long as it has free electrons and has no instability such as being easily ignited in air. From the viewpoint of ease of handling, gold, silver Copper, platinum and aluminum are preferable, and gold is more preferable. These metals may be used alone or in combination. In order to improve adhesion to the (substrate) surface, an intervening layer made of chromium or the like may be provided between the (substrate) surface and the metal.
金属膜の膜厚は任意であるが、例えば、表面プラズモン共鳴バイオセンサー用を考えた場合、０．１ｎｍ以上５００ｎｍ以下であるのが好ましく、特に１ｎｍ以上２００ｎｍ以下であるのが好ましい。５００ｎｍを超えると、媒質の表面プラズモン現象を十分検出することができない。また、クロム等からなる介在層を設ける場合、その介在層の厚さは、０．１ｎｍ以上、１０ｎｍ以下であるのが好ましい。 Although the thickness of the metal film is arbitrary, for example, when considering use for a surface plasmon resonance biosensor, the thickness is preferably 0.1 nm to 500 nm, particularly preferably 1 nm to 200 nm. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. Moreover, when providing the intervening layer which consists of chromium etc., it is preferable that the thickness of the intervening layer is 0.1 to 10 nm.
本発明における、好適な金属層の形成方法としては、例えば、スパッタ法、蒸着法、イオンプレーティング法、電気めっき法、無電解めっき法などが挙げられる。 Examples of suitable metal layer formation methods in the present invention include sputtering, vapor deposition, ion plating, electroplating, and electroless plating.
表面プラズモン共鳴を利用したセンサーの場合、金属層は、基板上に配置されていることが好ましい。ここで、「基板上に配置される」とは、金属層が基板上に直接接触するように配置されている場合のほか、介在層を介して配置されている場合も含む。本発明において、好適に使用できる基板としては、例えば、ガラスや合成樹脂などが挙げられる。該合成樹脂の中でも、特に代表的なものとしては、ポリメチルメタクリレート、ポリエチレンテレフタレート、ポリカーボネート、シクロオレフィンポリマーなどの透明性樹脂が挙げられる。 In the case of a sensor using surface plasmon resonance, the metal layer is preferably disposed on the substrate. Here, “disposed on the substrate” includes not only the case where the metal layer is disposed so as to be in direct contact with the substrate, but also the case where the metal layer is disposed via an intervening layer. In the present invention, examples of a substrate that can be suitably used include glass and synthetic resin. Among the synthetic resins, particularly representative ones include transparent resins such as polymethyl methacrylate, polyethylene terephthalate, polycarbonate, and cycloolefin polymer.
通常のバイオセンサーは、検出対象とする化学物質を認識するレセプター部位と、そこに発生する物理的変化又は化学的変化を電気信号に変換するトランスデューサー部位とから構成される。生体内には、互いに親和性のある物質として、酵素／基質、酵素／補酵素、抗原／抗体、ホルモン／レセプターなどがある。バイオセンサーでは、これら互いに親和性のある物質の一方を基板に固定化して分子認識物質として用いることによって、対応させるもう一方の物質を選択的に計測するという原理を利用している。 A normal biosensor is composed of a receptor site for recognizing a chemical substance to be detected and a transducer site for converting a physical change or chemical change generated therein into an electrical signal. In the living body, there are enzymes / substrates, enzymes / coenzymes, antigens / antibodies, hormones / receptors and the like as substances having affinity for each other. Biosensors use the principle that one of these substances having affinity with each other is immobilized on a substrate and used as a molecular recognition substance, thereby selectively measuring the other substance to be matched.
上記のようにして得られたバイオセンサー用表面において、上記の官能基を介して生理活性物質を共有結合させることによって、金属表面又は金属膜に生理活性物質を固定化することができる。 On the biosensor surface obtained as described above, the physiologically active substance can be immobilized on the metal surface or metal film by covalently bonding the physiologically active substance via the functional group.
本発明のバイオセンサー用表面上に固定される生理活性物質としては、測定対象物と相互作用するものであれば特に限定されず、例えば免疫蛋白質、酵素、微生物、核酸、低分子有機化合物、非免疫蛋白質、免疫グロブリン結合性蛋白質、糖結合性蛋白質、糖を認識する糖鎖、脂肪酸もしくは脂肪酸エステル、あるいはリガンド結合能を有するポリペプチドもしくはオリゴペプチドなどが挙げられる。 The physiologically active substance immobilized on the biosensor surface of the present invention is not particularly limited as long as it interacts with the measurement target, and examples thereof include immune proteins, enzymes, microorganisms, nucleic acids, low molecular organic compounds, non-molecular compounds, and the like. Examples include immune proteins, immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand binding ability.
免疫蛋白質としては、測定対象物を抗原とする抗体やハプテンなどを例示することができる。抗体としては、種々の免疫グロブリン、即ちＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ、ＩｇＤを使用することができる。具体的には、測定対象物がヒト血清アルブミンであれば、抗体として抗ヒト血清アルブミン抗体を使用することができる。また、農薬、殺虫剤、メチシリン耐性黄色ブドウ球菌、抗生物質、麻薬、コカイン、ヘロイン、クラック等を抗原とする場合には、例えば抗アトラジン抗体、抗カナマイシン抗体、抗メタンフェタミン抗体、あるいは病原性大腸菌の中でＯ抗原26、86、55、111 、157 などに対する抗体等を使用することができる。 Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, IgD can be used. Specifically, when the measurement target is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O antigens 26, 86, 55, 111, 157 and the like can be used.
酵素としては、測定対象物又は測定対象物から代謝される物質に対して活性を示すものであれば、特に限定されることなく、種々の酵素、例えば酸化還元酵素、加水分解酵素、異性化酵素、脱離酵素、合成酵素等を使用することができる。具体的には、測定対象物がグルコースであれば、グルコースオキシダーゼを、測定対象物がコレステロールであれば、コレステロールオキシダーゼを使用することができる。また、農薬、殺虫剤、メチシリン耐性黄色ブドウ球菌、抗生物質、麻薬、コカイン、ヘロイン、クラック等を測定対象物とする場合には、それらから代謝される物質と特異的反応を示す、例えばアセチルコリンエステラーゼ、カテコールアミンエステラーゼ、ノルアドレナリンエステラーゼ、ドーパミンエステラーゼ等の酵素を使用することができる。 The enzyme is not particularly limited as long as it shows activity against the measurement object or a substance metabolized from the measurement object, and various enzymes such as oxidoreductase, hydrolase, isomerase , A desorbing enzyme, a synthesizing enzyme and the like can be used. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit specific reactions with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.
微生物としては、特に限定されることなく、大腸菌をはじめとする種々の微生物を使用することができる。
核酸としては、測定の対象とする核酸と相補的にハイブリダイズするものを使用することができる。核酸は、ＤＮＡ（ｃＤＮＡを含む）、ＲＮＡのいずれも使用できる。ＤＮＡの種類は特に限定されず、天然由来のＤＮＡ、遺伝子組換え技術により調製した組換えＤＮＡ、又は化学合成ＤＮＡの何れでもよい。
低分子有機化合物としては通常の有機化学合成の方法で合成することができる任意の化合物が挙げられる。
The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.
As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.
Examples of the low molecular weight organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.
非免疫蛋白質としては、特に限定されることなく、例えばアビジン（ストレプトアビジン）、ビオチン又はレセプターなどを使用できる。
免疫グロブリン結合性蛋白質としては、例えばプロテインＡあるいはプロテインＧ、リウマチ因子（ＲＦ）等を使用することができる。
糖結合性蛋白質としては、レクチン等が挙げられる。
脂肪酸あるいは脂肪酸エステルとしては、ステアリン酸、アラキジン酸、ベヘン酸、ステアリン酸エチル、アラキジン酸エチル、ベヘン酸エチル等が挙げられる。
The non-immune protein is not particularly limited, and for example, avidin (streptavidin), biotin or a receptor can be used.
As the immunoglobulin-binding protein, for example, protein A or protein G, rheumatoid factor (RF) and the like can be used.
Examples of sugar-binding proteins include lectins.
Examples of the fatty acid or fatty acid ester include stearic acid, arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, and ethyl behenate.
生理活性物質が抗体や酵素などの蛋白質又は核酸である場合、その固定化は、生理活性物質のアミノ基、チオール基等を利用し、金属表面の官能基に共有結合させることで行うことができる。 When the physiologically active substance is a protein or nucleic acid such as an antibody or an enzyme, the immobilization can be performed by covalently bonding to a functional group on the metal surface using the amino group, thiol group or the like of the physiologically active substance. .
上記のようにして生理活性物質を固定化したバイオセンサーは、当該生理活性物質と相互作用する物質の検出及び／又は測定のために使用することができる。 The biosensor on which a physiologically active substance is immobilized as described above can be used for detection and / or measurement of a substance that interacts with the physiologically active substance.
即ち、本発明によれば、生理活性物質が固定化された本発明のバイオセンサーを用いて、これに被験物質を接触させることにより、該バイオセンサーに固定化されている生理活性物質と相互作用する物質を検出及び／又は測定する方法が提供される。
被験物質としては例えば、上記した生理活性物質と相互作用する物質を含む試料などを使用することができる。
That is, according to the present invention, the biosensor of the present invention on which a physiologically active substance is immobilized is brought into contact with a test substance to thereby interact with the physiologically active substance immobilized on the biosensor. A method of detecting and / or measuring a substance to be provided is provided.
As the test substance, for example, a sample containing a substance that interacts with the above physiologically active substance can be used.
本発明では、バイオセンサー用表面に固定化されている生理活性物質と被験物質との相互作用を非電気化学的方法により検出及び／又は測定することが好ましい。非電気化学的方法としては、表面プラズモン共鳴（ＳＰＲ）測定技術、水晶発振子マイクロバランス（ＱＣＭ）測定技術、金のコロイド粒子から超微粒子までの機能化表面を使用した測定技術などが挙げられる。 In the present invention, it is preferable to detect and / or measure the interaction between the physiologically active substance immobilized on the biosensor surface and the test substance by a non-electrochemical method. Non-electrochemical methods include surface plasmon resonance (SPR) measurement technology, quartz crystal microbalance (QCM) measurement technology, measurement technology using functionalized surfaces from gold colloidal particles to ultrafine particles.
本発明の好ましい態様によれば、本発明のバイオセンサーは、例えば、透明基板上に配置される金属膜を備えていることを特徴とする表面プラズモン共鳴用バイオセンサーとして用いることができる。 According to a preferred aspect of the present invention, the biosensor of the present invention can be used as a surface plasmon resonance biosensor characterized by including a metal film disposed on a transparent substrate, for example.
表面プラズモン共鳴用バイオセンサーとは、表面プラズモン共鳴バイオセンサーに使用されるバイオセンサーであって、該センサーより照射された光を透過及び反射する部分、並びに生理活性物質を固定する部分とを含む部材を言い、該センサーの本体に固着されるものであってもよく、また脱着可能なものであってもよい。 The surface plasmon resonance biosensor is a biosensor used in the surface plasmon resonance biosensor, and includes a part that transmits and reflects light emitted from the sensor, and a part that fixes a physiologically active substance. And may be fixed to the main body of the sensor or may be removable.
表面プラズモン共鳴の現象は、ガラス等の光学的に透明な物質と金属薄膜層との境界から反射された単色光の強度が、金属の出射側にある試料の屈折率に依存することによるものであり、従って、反射された単色光の強度を測定することにより、試料を分析することができる。 The phenomenon of surface plasmon resonance is due to the fact that the intensity of monochromatic light reflected from the boundary between an optically transparent substance such as glass and the metal thin film layer depends on the refractive index of the sample on the metal exit side. Yes, so the sample can be analyzed by measuring the intensity of the reflected monochromatic light.
表面プラズモンが光波によって励起される現象を利用して、被測定物質の特性を分析する表面プラズモン測定装置としては、Kretschmann配置と称される系を用いるものが挙げられる（例えば特開平６−１６７４４３号公報参照）。上記の系を用いる表面プラズモン測定装置は基本的に、例えばプリズム状に形成された誘電体ブロックと、この誘電体ブロックの一面に形成されて試料液などの被測定物質に接触させられる金属膜と、光ビームを発生させる光源と、上記光ビームを誘電体ブロックに対して、該誘電体ブロックと金属膜との界面で全反射条件が得られるように種々の角度で入射させる光学系と、上記界面で全反射した光ビームの強度を測定して表面プラズモン共鳴の状態、つまり全反射減衰の状態を検出する光検出手段とを備えてなるものである。 As a surface plasmon measuring device for analyzing the characteristics of a substance to be measured using a phenomenon in which surface plasmons are excited by light waves, there is an apparatus using a system called Kretschmann arrangement (for example, JP-A-6-167443). See the official gazette). A surface plasmon measuring apparatus using the above system basically includes a dielectric block formed in a prism shape, for example, and a metal film formed on one surface of the dielectric block and brought into contact with a substance to be measured such as a sample liquid. A light source that generates a light beam; an optical system that causes the light beam to enter the dielectric block at various angles so that a total reflection condition is obtained at an interface between the dielectric block and the metal film; and It comprises light detecting means for detecting the surface plasmon resonance state, that is, the state of total reflection attenuation by measuring the intensity of the light beam totally reflected at the interface.
なお上述のように種々の入射角を得るためには、比較的細い光ビームを入射角を変化させて上記界面に入射させてもよいし、あるいは光ビームに種々の角度で入射する成分が含まれるように、比較的太い光ビームを上記界面に収束光状態であるいは発散光状態で入射させてもよい。前者の場合は、入射した光ビームの入射角の変化に従って、反射角が変化する光ビームを、上記反射角の変化に同期して移動する小さな光検出器によって検出したり、反射角の変化方向に沿って延びるエリアセンサによって検出することができる。一方後者の場合は、種々の反射角で反射した各光ビームを全て受光できる方向に延びるエリアセンサによって検出することができる。 In order to obtain various incident angles as described above, a relatively thin light beam may be incident on the interface by changing the incident angle, or a component incident on the light beam at various angles is included. As described above, a relatively thick light beam may be incident on the interface in a convergent light state or a divergent light state. In the former case, a light beam whose reflection angle changes according to the change in the incident angle of the incident light beam is detected by a small photodetector that moves in synchronization with the change in the reflection angle, or the direction in which the reflection angle changes Can be detected by an area sensor extending along the line. On the other hand, in the latter case, it can be detected by an area sensor extending in a direction in which each light beam reflected at various reflection angles can be received.
上記構成の表面プラズモン測定装置において、光ビームを金属膜に対して全反射角以上の特定入射角で入射させると、該金属膜に接している被測定物質中に電界分布をもつエバネッセント波が生じ、このエバネッセント波によって金属膜と被測定物質との界面に表面プラズモンが励起される。エバネッセント光の波数ベクトルが表面プラズモンの波数と等しくて波数整合が成立しているとき、両者は共鳴状態となり、光のエネルギーが表面プラズモンに移行するので、誘電体ブロックと金属膜との界面で全反射した光の強度が鋭く低下する。この光強度の低下は、一般に上記光検出手段により暗線として検出される。なお上記の共鳴は、入射ビームがｐ偏光のときにだけ生じる。したがって、光ビームがｐ偏光で入射するように予め設定しておく必要がある。 In the surface plasmon measuring apparatus having the above configuration, when a light beam is incident on a metal film at a specific incident angle that is greater than the total reflection angle, an evanescent wave having an electric field distribution is generated in the measured substance in contact with the metal film. The evanescent wave excites surface plasmons at the interface between the metal film and the substance to be measured. When the wave number vector of the evanescent light is equal to the wave number of the surface plasmon and the wave number matching is established, both are in a resonance state and the energy of the light is transferred to the surface plasmon, so that the entire energy is transferred to the interface between the dielectric block and the metal film. The intensity of the reflected light decreases sharply. This decrease in light intensity is generally detected as a dark line by the light detection means. The resonance described above occurs only when the incident beam is p-polarized light. Therefore, it is necessary to set in advance so that the light beam is incident as p-polarized light.
この全反射減衰（ＡＴＲ）が生じる入射角、すなわち全反射減衰角（θＳＰ）より表面プラズモンの波数が分かると、被測定物質の誘電率が求められる。この種の表面プラズモン測定装置においては、全反射減衰角（θＳＰ）を精度良く、しかも大きなダイナミックレンジで測定することを目的として、特開平１１−３２６１９４号公報に示されるように、アレイ状の光検出手段を用いることが考えられている。この光検出手段は、複数の受光素子が所定方向に配設されてなり、前記界面において種々の反射角で全反射した光ビームの成分をそれぞれ異なる受光素子が受光する向きにして配設されたものである。 If the wave number of the surface plasmon is known from the incident angle at which this total reflection attenuation (ATR) occurs, that is, the total reflection attenuation angle (θSP), the dielectric constant of the substance to be measured can be obtained. In this type of surface plasmon measurement apparatus, as shown in Japanese Patent Application Laid-Open No. 11-326194, in order to measure the total reflection attenuation angle (θSP) with high accuracy and a large dynamic range, It is considered to use detection means. This light detection means is provided with a plurality of light receiving elements arranged in a predetermined direction, and arranged so that different light receiving elements receive light beam components totally reflected at various reflection angles at the interface. Is.
そしてその場合は、上記アレイ状の光検出手段の各受光素子が出力する光検出信号を、該受光素子の配設方向に関して微分する微分手段が設けられ、この微分手段が出力する微分値に基づいて全反射減衰角（θＳＰ）を特定し、被測定物質の屈折率に関連する特性を求めることが多い。 In that case, there is provided differential means for differentiating the light detection signals output from the light receiving elements of the arrayed light detection means with respect to the arrangement direction of the light receiving elements, and based on the differential value output by the differential means. In many cases, the total reflection attenuation angle (θSP) is specified to obtain a characteristic related to the refractive index of the substance to be measured.
また、全反射減衰（ＡＴＲ）を利用する類似の測定装置として、例えば「分光研究」第４７巻 第１号（１９９８）の第２１〜２３頁および第２６〜２７頁に記載がある漏洩モード測定装置も知られている。この漏洩モード測定装置は基本的に、例えばプリズム状に形成された誘電体ブロックと、この誘電体ブロックの一面に形成されたクラッド層と、このクラッド層の上に形成されて、試料液に接触させられる光導波層と、光ビームを発生させる光源と、上記光ビームを上記誘電体ブロックに対して、該誘電体ブロックとクラッド層との界面で全反射条件が得られるように種々の角度で入射させる光学系と、上記界面で全反射した光ビームの強度を測定して導波モードの励起状態、つまり全反射減衰状態を検出する光検出手段とを備えてなるものである。 Moreover, as a similar measuring device using total reflection attenuation (ATR), for example, “Spectroscopic Research” Vol. 47, No. 1, (1998), pages 21 to 23 and pages 26 to 27 are described. Devices are also known. This leakage mode measuring device is basically a dielectric block formed in a prism shape, for example, a clad layer formed on one surface of the dielectric block, and formed on the clad layer to be in contact with the sample liquid. Optical waveguide layer to be generated, a light source for generating a light beam, and the light beam to the dielectric block at various angles so that a total reflection condition is obtained at the interface between the dielectric block and the cladding layer. The optical system includes an incident optical system and light detection means for detecting the excitation state of the waveguide mode, that is, the total reflection attenuation state by measuring the intensity of the light beam totally reflected at the interface.
上記構成の漏洩モード測定装置において、光ビームを誘電体ブロックを通してクラッド層に対して全反射角以上の入射角で入射させると、このクラッド層を透過した後に光導波層においては、ある特定の波数を有する特定入射角の光のみが導波モードで伝搬するようになる。こうして導波モードが励起されると、入射光のほとんどが光導波層に取り込まれるので、上記界面で全反射する光の強度が鋭く低下する全反射減衰が生じる。そして導波光の波数は光導波層の上の被測定物質の屈折率に依存するので、全反射減衰が生じる上記特定入射角を知ることによって、被測定物質の屈折率や、それに関連する被測定物質の特性を分析することができる。 In the leakage mode measuring apparatus having the above-described configuration, when a light beam is incident on the cladding layer through the dielectric block at an incident angle greater than the total reflection angle, the light waveguide layer transmits a specific wave number after passing through the cladding layer. Only light having a specific incident angle having a wave length propagates in the waveguide mode. When the waveguide mode is excited in this way, most of the incident light is taken into the optical waveguide layer, resulting in total reflection attenuation in which the intensity of light totally reflected at the interface is sharply reduced. Since the wave number of guided light depends on the refractive index of the substance to be measured on the optical waveguide layer, knowing the specific incident angle at which total reflection attenuation occurs, the refractive index of the substance to be measured and the measurement object related thereto The properties of the substance can be analyzed.
なおこの漏洩モード測定装置においても、全反射減衰によって反射光に生じる暗線の位置を検出するために、前述したアレイ状の光検出手段を用いることができ、またそれと併せて前述の微分手段が適用されることも多い。 In this leakage mode measuring apparatus, the above-mentioned array-shaped light detecting means can be used to detect the position of the dark line generated in the reflected light due to the total reflection attenuation, and the above-described differentiating means is applied in conjunction therewith. Often done.
また、上述した表面プラズモン測定装置や漏洩モード測定装置は、創薬研究分野等において、所望のセンシング物質に結合する特定物質を見いだすランダムスクリーニングへ使用されることがあり、この場合には前記薄膜層（表面プラズモン測定装置の場合は金属膜であり、漏洩モード測定装置の場合はクラッド層および光導波層）上に上記被測定物質としてセンシング物質を固定し、該センシング物質上に種々の被検体が溶媒に溶かされた試料液を添加し、所定時間が経過する毎に前述の全反射減衰角（θＳＰ）の角度を測定している。 In addition, the surface plasmon measurement device and the leakage mode measurement device described above may be used for random screening to find a specific substance that binds to a desired sensing substance in the field of drug discovery research. In this case, the thin film layer A sensing substance is fixed on the sensing substance on the sensing substance (a metal film in the case of a surface plasmon measuring apparatus, a clad layer and an optical waveguide layer in the case of a leakage mode measuring apparatus), and various analytes are placed on the sensing substance. A sample solution dissolved in a solvent is added, and the total reflection attenuation angle (θSP) is measured every time a predetermined time elapses.
試料液中の被検体が、センシング物質と結合するものであれば、この結合によりセンシング物質の屈折率が時間経過に伴って変化する。したがって、所定時間経過毎に上記全反射減衰角（θＳＰ）を測定し、該全反射減衰角（θＳＰ）の角度に変化が生じているか否か測定することにより、被検体とセンシング物質の結合状態を測定し、その結果に基づいて被検体がセンシング物質と結合する特定物質であるか否かを判定することができる。このような特定物質とセンシング物質との組み合わせとしては、例えば抗原と抗体、あるいは抗体と抗体が挙げられる。具体的には、ウサギ抗ヒトＩｇＧ抗体をセンシング物質として薄膜層の表面に固定し、ヒトＩgＧ抗体を特定物質として用いることができる。 If the analyte in the sample liquid binds to the sensing substance, the refractive index of the sensing substance changes with time due to this binding. Therefore, by measuring the total reflection attenuation angle (θSP) every predetermined time and measuring whether or not the total reflection attenuation angle (θSP) has changed, the binding state of the analyte and the sensing substance is determined. It is possible to determine whether or not the analyte is a specific substance that binds to the sensing substance based on the result. Examples of the combination of the specific substance and the sensing substance include an antigen and an antibody, or an antibody and an antibody. Specifically, a rabbit anti-human IgG antibody can be immobilized on the surface of the thin film layer as a sensing substance, and a human IgG antibody can be used as the specific substance.
なお、被検体とセンシング物質の結合状態を測定するためには、全反射減衰（θＳＰ）の角度そのものを必ずしも検出する必要はない。例えばセンシング物質に試料液を添加し、その後の全反射減衰角（θＳＰ）の角度変化量を測定して、その角度変化量の大小に基づいて結合状態を測定することもできる。前述したアレイ状の光検出手段と微分手段を全反射減衰を利用した測定装置に適用する場合であれば、微分値の変化量は、全反射減衰角（θＳＰ）の角度変化量を反映しているため、微分値の変化量に基づいて、センシング物質と被検体との結合状態を測定することができる（本出願人による特願２０００−３９８３０９号参照）。このような全反射減衰を利用した測定方法および装置においては、底面に予め成された薄膜層上にセンシング物質が固定されたカップ状あるいはシャーレ状の測定チップに、溶媒と被検体からなる試料液を滴下供給して、上述した全反射減衰角（θＳＰ）の角度変化量の測定を行っている。 Note that in order to measure the binding state between the subject and the sensing substance, it is not always necessary to detect the angle of total reflection attenuation (θSP) itself. For example, a sample solution can be added to the sensing substance, and the amount of change in the total reflection attenuation angle (θSP) thereafter can be measured, and the binding state can be measured based on the magnitude of the angle change. If the above-described arrayed light detecting means and differentiating means are applied to a measuring device using total reflection attenuation, the change amount of the differential value reflects the angle change amount of the total reflection attenuation angle (θSP). Therefore, the binding state between the sensing substance and the analyte can be measured based on the amount of change in the differential value (see Japanese Patent Application No. 2000-398309 by the present applicant). In such a measurement method and apparatus using total reflection attenuation, a sample liquid consisting of a solvent and an analyte is placed on a cup-shaped or petri-shaped measuring chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The amount of change in angle of the total reflection attenuation angle (θSP) described above is measured.
さらに、ターンテーブル等に搭載された複数個の測定チップの測定を順次行うことにより、多数の試料についての測定を短時間で行うことができる全反射減衰を利用した測定装置が、特開２００１−３３０５６０号公報に記載されている。 Furthermore, a measuring apparatus using total reflection attenuation capable of measuring a large number of samples in a short time by sequentially measuring a plurality of measuring chips mounted on a turntable or the like is disclosed in JP-A-2001-2001. No. 330560.
本発明のバイオセンサーを表面プラズモン共鳴分析に使用する場合、上記したような各種の表面プラズモン測定装置の一部として適用することができる。
以下の実施例により本発明を更に具体的に説明するが、本発明の範囲はこれらの実施例に限定されるものではない。
When the biosensor of the present invention is used for surface plasmon resonance analysis, it can be applied as a part of various surface plasmon measurement devices as described above.
The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.
実施例１：本発明の表面プラズモン共鳴基板
ゼオネックス（日本ゼオン社製）のペレットを２４０℃で溶解し、この溶融物を射出成型器で縦８ｍｍ×横１２０ｍｍ×１．５ｍｍの基板を成型した。この基板に平行平板型６インチ用スパッタ装置（ＳＨ−５５０、アルバック（株）社製）を用いて基板上に金の
厚さが５０ｎｍになるようにスパッタ製膜を行った。エタノール／水（80/20）中11-ヒドロキシ-1-ウンデカンチオールの5.0mM溶液を基板の金膜に接触するように添加し、25℃で18時間表面処理を行った。その後、エタノールで5回、エタノール/水混合溶媒で1回、水で5回洗浄を行った。11-ヒドロキシ-1-ウンデカンチオールで被覆した表面を１０質量％のエピクロロヒドリン溶液（溶媒：0.4M水酸化ナトリウム及びジエチレングリコールジメチルエーテルの1：1混合溶液）に接触させ、25℃の振盪インキュベーター中で４時間反応を進行させた。表面をエタノールで2回、水で5回洗浄した。
Example 1 Surface Plasmon Resonance Substrate of the Present Invention A pellet of ZEONEX (manufactured by ZEON Corporation) was melted at 240 ° C., and this melt was molded into a substrate of 8 mm length × 120 mm × 1.5 mm width by an injection molding machine. Using this parallel plate type 6-inch sputtering apparatus (SH-550, manufactured by ULVAC, Inc.), sputtering was performed on the substrate so that the gold thickness was 50 nm. A 5.0 mM solution of 11-hydroxy-1-undecanthiol in ethanol / water (80/20) was added in contact with the gold film of the substrate, and surface treatment was performed at 25 ° C. for 18 hours. Thereafter, washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water. The surface coated with 11-hydroxy-1-undecanthiol was brought into contact with a 10% by mass epichlorohydrin solution (solvent: 1: 1 mixed solution of 0.4M sodium hydroxide and diethylene glycol dimethyl ether) in a shaking incubator at 25 ° C. The reaction was allowed to proceed for 4 hours. The surface was washed twice with ethanol and five times with water.
次に、25質量％のデキストラン（T500,Pharmacia）水溶液40.5mlに4.5mlの1M水酸化ナトリウムを添加し、その溶液をエピクロロヒドリン処理表面上に接触させた。次に振とうインキュベーター中で25℃で20時間インキュベートした。表面を50℃の水で10回洗浄した。続いて、ブロモ酢酸3.5gを27gの2M水酸化ナトリウム溶液に溶解した混合物を上記デキストラン処理表面に接触させて、28℃の振盪インキュベーターで16時間インキュベートした。表面を水で洗浄し、その後上述の手順を１回繰り返した。このように作製した基板をデキストラン固定化基板と呼ぶ。 Next, 4.5 ml of 1 M sodium hydroxide was added to 40.5 ml of 25% by weight dextran (T500, Pharmacia) aqueous solution, and the solution was brought into contact with the epichlorohydrin treated surface. It was then incubated for 20 hours at 25 ° C. in a shaking incubator. The surface was washed 10 times with 50 ° C. water. Subsequently, a mixture of 3.5 g of bromoacetic acid dissolved in 27 g of 2M sodium hydroxide solution was brought into contact with the dextran-treated surface and incubated for 16 hours in a shaking incubator at 28 ° C. The surface was washed with water and then the above procedure was repeated once. The substrate thus prepared is called a dextran-immobilized substrate.
次に、デキストラン固定化基板をエタノール／水（80/20）中11-ヒドロキシ-1-ウンデカンチオールの5.0mM溶液を基板の金膜に接触するように添加し、25℃で18時間表面処理を行った。その後、エタノールで5回、エタノール/水混合溶媒で1回、水で5回洗浄を行い、本発明の表面プラズモン共鳴基板を作成した。 Next, a dextran-immobilized substrate was added with a 5.0 mM solution of 11-hydroxy-1-undecanthiol in ethanol / water (80/20) so as to contact the gold film of the substrate, and surface treatment was performed at 25 ° C. for 18 hours. went. Thereafter, the surface plasmon resonance substrate of the present invention was prepared by washing 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water.
バイオセンサー表面に対する非特異的な蛋白質の吸着はノイズの原因となるため、極力少ないほうが好ましい。作製した表面プラズモン共鳴基板を用いて、CGP-74514A Hydrochloride（シグマ社製）の非特異吸着性を測定した。基板を特開２００１−３３０５６０号公報の図２２に記載の装置（以下、本発明の表面プラズモン共鳴装置と呼ぶ）に設置した。ＨＢＳ−Ｎバッファー（ビアコア社製）を基板に添加し２０分間静置後、CGP-74514A Hydrochloride溶液（１０μＭ、ＨＢＳ−Ｎバッファー）を基板に添加し１０分間静置した。なお、HBS-Nバッファーの組成は、ＨＥＰＥＳ（N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonicAcid）0.01mol/l（ｐＨ7.4）、ＮａＣｌ0.15mol/lである。１０間静置後の共鳴シグナル（RU値）変化量をCGP-74514A Hydrochlorideの非特異吸着量とした。測定結果を表１に示した。 Since nonspecific protein adsorption to the biosensor surface causes noise, it is preferable that the amount be as small as possible. Using the prepared surface plasmon resonance substrate, the nonspecific adsorption property of CGP-74514A Hydrochloride (manufactured by Sigma) was measured. The substrate was placed in the apparatus shown in FIG. 22 of Japanese Patent Laid-Open No. 2001-330560 (hereinafter referred to as the surface plasmon resonance apparatus of the present invention). HBS-N buffer (Biacore) was added to the substrate and allowed to stand for 20 minutes, and then CGP-74514A Hydrochloride solution (10 μM, HBS-N buffer) was added to the substrate and allowed to stand for 10 minutes. The composition of the HBS-N buffer is HEPES (N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic Acid) 0.01 mol / l (pH 7.4) and NaCl 0.15 mol / l. The amount of change in resonance signal (RU value) after standing for 10 minutes was defined as the amount of nonspecific adsorption of CGP-74514A Hydrochloride. The measurement results are shown in Table 1.
比較例１：
実施例１のデキストラン固定化基板に後処理を行わないものを比較例１の基板とした。この基板の実施例１と同じ操作を行いCGP-74514A Hydrochlorideの非特異吸着量を測定した。測定結果を表１に示した。
Comparative Example 1:
A substrate in which the dextran-immobilized substrate of Example 1 was not subjected to post-processing was used as the substrate of Comparative Example 1. The same operation as in Example 1 was performed on this substrate, and the amount of nonspecific adsorption of CGP-74514A Hydrochloride was measured. The measurement results are shown in Table 1.
表１に示した結果から、本発明の基板を用いたバイオセンサーは、低分子化合物の非特異吸着が非常に少ないことが、表面プラズモン共鳴で確認できた。 From the results shown in Table 1, it was confirmed by surface plasmon resonance that the biosensor using the substrate of the present invention has very little nonspecific adsorption of low molecular weight compounds.
実施例２：本発明のＱＣＭ基板
ポリ(3,3,4,4,5,5,6,6,7,7,8,8,8-トリデカフルオロオクチルアクリレート)-ポリ(ベンジルメタクリレート)コポリマー（モノマー質量比４／６、質量平均分子量３万、以降ポリマーＡと略記）０.２ｇをメチルイソブチルケトンに溶解し、液量が１００ｍＬになるようにメチルイソブチルケトンを添加した。このポリマーＡ溶液を０.４５μｍフィルターで濾過し塗布液Ａを調製した。水晶発振子マイクロバランス発振子（ユーエスアイ・システム社製、以降、ＱＣＭと略記）を、スピンコート機（MODEL ASS-303、ABLE製）の回転盤の中心に発振子面が水平になるように設置し、1000rpmにて回転させながらポリマー溶液Ａ２０μＬを発振子の金面上に滴下し、2分後に回転を止めた。次いで、発振子のもう一方の面を上向きに設置し、同じ操作でポリマーＡ溶液をスピンコート塗布した。
Example 2: QCM substrate of the present invention Poly (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl acrylate) -poly (benzyl methacrylate) copolymer (Monomer mass ratio 4/6, mass average molecular weight 30,000, hereinafter abbreviated as polymer A) 0.2 g was dissolved in methyl isobutyl ketone, and methyl isobutyl ketone was added so that the liquid volume became 100 mL. The polymer A solution was filtered through a 0.45 μm filter to prepare a coating solution A. Crystal oscillator micro balance oscillator (manufactured by USA System Co., Ltd., hereinafter abbreviated as QCM) so that the surface of the oscillator is horizontal at the center of the spin coater (MODEL ASS-303, manufactured by ABLE) Then, 20 μL of the polymer solution A was dropped on the gold surface of the oscillator while rotating at 1000 rpm, and the rotation was stopped after 2 minutes. Next, the other surface of the oscillator was placed upward, and the polymer A solution was spin-coated by the same operation.
ポリマーＡコーティングした発振子を６０℃に保温した1N NaOH水溶液に１６時間浸漬した後、純水の流水中で洗浄した。この発振子を、１−エチル−２，３−ジメチルアミノプロピルカルボジイミド（２００ｍＭ）と、Ｎ−ヒドロキシスクシンイミド（５０ｍＭ）との混合液を３０分接触させ、次に５−アミノ吉草酸（１Ｍ 塩酸でｐＨ８.５に調製）水溶液に９０分浸漬した後、純水の流水中で洗浄した。 The polymer A-coated oscillator was immersed in a 1N NaOH aqueous solution kept at 60 ° C. for 16 hours, and then washed in flowing pure water. This oscillator was brought into contact with a mixed solution of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (200 mM) and N-hydroxysuccinimide (50 mM) for 30 minutes, and then 5-aminovaleric acid (1M hydrochloric acid with (Adjusted to pH 8.5) After immersing in an aqueous solution for 90 minutes, it was washed in running pure water.
この発振子をエタノール／水（80/20）の３−カルボキシプロピルジスルフィドの5.0mM溶液に２５℃１６時間浸漬した後、エタノールで5回、エタノール/水混合溶媒で1回、水で5回洗浄を行い、本発明のＱＣＭ発振子を作成した。 This oscillator was immersed in a 5.0 mM solution of 3-carboxypropyl disulfide in ethanol / water (80/20) at 25 ° C. for 16 hours, then washed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water. The QCM oscillator of the present invention was created.
作成したＱＣＭ発振子を水晶振動子微小秤量装置ＵＱ−２００（ユーエスアイ社製）に取り付け、本体周波数９ＭＨｚにて共振振動数を大気中で測定した。その後ＰｒｏｔｅｉｎＡ（ナカライテスク社製）溶液（１００μｇ／ｍＬ、５０ｍＭ酢酸バッファー、ｐＨ４.５）を３０分間接触させ、その後５０ｍＭ酢酸バッファー（ｐＨ４.５）、次いで純水で洗浄、乾燥した。再び感動後のＱＣＭ発振子の共振振動数を大気中で測定し、ＰｒｉｔｅｉｎＡ溶液接触前後の共振振動数の差を算出した。共振振動数の差を表２に示した。 The prepared QCM oscillator was attached to a quartz crystal microweighing device UQ-200 (manufactured by USAI), and the resonance frequency was measured in the atmosphere at a main body frequency of 9 MHz. Thereafter, a Protein A (manufactured by Nacalai Tesque) solution (100 μg / mL, 50 mM acetate buffer, pH 4.5) was contacted for 30 minutes, and then washed with 50 mM acetate buffer (pH 4.5) and then purified water and dried. The resonance frequency of the QCM oscillator after the impression was measured again in the atmosphere, and the difference between the resonance frequencies before and after contacting the Pritine A solution was calculated. The difference in resonance frequency is shown in Table 2.
比較例２：
３−カルボキシプロピルジスルフィドの5.0mM溶液に浸漬しなかったこと以外、実施例２と同じ方法でＱＣＭ発振子を作成した。このＱＣＭ発振子を実施例２と同じ方法で共振振動数の差を測定した。共振振動数の差を表２に示した。
Comparative Example 2:
A QCM oscillator was prepared in the same manner as in Example 2 except that it was not immersed in a 5.0 mM solution of 3-carboxypropyl disulfide. The difference in resonance frequency of this QCM oscillator was measured in the same manner as in Example 2. The difference in resonance frequency is shown in Table 2.
表２に示した結果から、本発明の基板を用いたバイオセンサーは、タンパク質の非特異吸着が非常に少ないことが水晶振動子で確認できた。 From the results shown in Table 2, it was confirmed that the biosensor using the substrate of the present invention had very little nonspecific adsorption of protein with a crystal resonator.
Claims (16)
一般式（１）：Ｘ １ -Ｒ １ -Ｙ １ （Ｘ １ は非対称又は対称スルフィド、スルフィド、ジセレニド、セレニド、チオール、ニトリル、イソニトリル、ニトロ、セレノール、３価リン化合物、イソチオシアネート、キサンテート、チオカルバメート、ホスフィン、チオ酸またはジチオ酸を示し、Ｒ １ は炭化水素鎖を示し、Ｙ １ はヒドロキシル基、カルボキシル基、アミノ基、アルデヒド基、ヒドラジド基、カルボニル基、エポキシ基、又はビニル基を示す。） A sensor substrate having a polymer compound coating on a metal substrate surface , the step of coating the metal substrate surface with a compound represented by the following general formula (1), and forming a polymer compound coating A sensor substrate obtained by a process including a process and a process of treating a substrate surface with a compound represented by the following general formula (1) in this order .
General formula (1): X 1 -R 1 -Y 1 (X 1 is asymmetric or symmetric sulfide, sulfide, diselenide, selenide, thiol, nitrile, isonitrile, nitro, selenol, trivalent phosphorus compound, isothiocyanate, xanthate, thiol Carbamate, phosphine, thioic acid or dithioic acid, R 1 represents a hydrocarbon chain, Y 1 represents a hydroxyl group, carboxyl group, amino group, aldehyde group, hydrazide group, carbonyl group, epoxy group, or vinyl group .)
一般式（１）：Ｘ １ -Ｒ １ -Ｙ １ （Ｘ １ は非対称又は対称スルフィド、スルフィド、ジセレニド、セレニド、チオール、ニトリル、イソニトリル、ニトロ、セレノール、３価リン化合物、イソチオシアネート、キサンテート、チオカルバメート、ホスフィン、チオ酸またはジチオ酸を示し、Ｒ １ は炭化水素鎖を示し、Ｙ １ はヒドロキシル基、カルボキシル基、アミノ基、アルデヒド基、ヒドラジド基、カルボニル基、エポキシ基、又はビニル基を示す。） (A) a step of coating the surface of the substrate made of metal with a compound represented by the following general formula (1), (b) a step of forming a polymer compound coating, and (c) a polymer compound coating was formed. The manufacturing method of the substrate for sensors in any one of Claims 1-12 including the process of processing the board | substrate surface with the compound represented by following General formula (1) in this order .
General formula (1): X 1 -R 1 -Y 1 (X 1 is asymmetric or symmetric sulfide, sulfide, diselenide, selenide, thiol, nitrile, isonitrile, nitro, selenol, trivalent phosphorus compound, isothiocyanate, xanthate, thiol Carbamate, phosphine, thioic acid or dithioic acid, R 1 represents a hydrocarbon chain, Y 1 represents a hydroxyl group, carboxyl group, amino group, aldehyde group, hydrazide group, carbonyl group, epoxy group, or vinyl group .)
一般式（１）：Ｘ １ -Ｒ １ -Ｙ １ （Ｘ １ は非対称又は対称スルフィド、スルフィド、ジセレニド、セレニド、チオール、ニトリル、イソニトリル、ニトロ、セレノール、３価リン化合物、イソチオシアネート、キサンテート、チオカルバメート、ホスフィン、チオ酸またはジチオ酸を示し、Ｒ １ は炭化水素鎖を示し、Ｙ １ はヒドロキシル基、カルボキシル基、アミノ基、アルデヒド基、ヒドラジド基、カルボニル基、エポキシ基、又はビニル基を示す。） (A) a step of coating a metal substrate surface with a compound represented by the following general formula (1), (b) a step of forming a polymer compound coating, (c) a substrate on which a polymer compound coating is formed A physiological treatment comprising, in this order, a step of treating the surface with a compound represented by the following general formula (1), and (d) a step of immobilizing a physiologically active substance on the polymer compound film after the step (c). The method for producing a sensor substrate according to any one of claims 1 to 12, wherein the active substance is bound to the surface by a covalent bond.
General formula (1): X 1 -R 1 -Y 1 (X 1 is asymmetric or symmetric sulfide, sulfide, diselenide, selenide, thiol, nitrile, isonitrile, nitro, selenol, trivalent phosphorus compound, isothiocyanate, xanthate, thiol Carbamate, phosphine, thioic acid or dithioic acid, R 1 represents a hydrocarbon chain, Y 1 represents a hydroxyl group, carboxyl group, amino group, aldehyde group, hydrazide group, carbonyl group, epoxy group, or vinyl group .)
一般式（１）：ＸGeneral formula (1): X １1 -Ｒ-R １1 -Ｙ-Y １1 （Ｘ(X １1 は非対称又は対称スルフィド、スルフィド、ジセレニド、セレニド、チオール、ニトリル、イソニトリル、ニトロ、セレノール、３価リン化合物、イソチオシアネート、キサンテート、チオカルバメート、ホスフィン、チオ酸またはジチオ酸を示し、ＲRepresents an asymmetric or symmetric sulfide, sulfide, diselenide, selenide, thiol, nitrile, isonitrile, nitro, selenol, trivalent phosphorus compound, isothiocyanate, xanthate, thiocarbamate, phosphine, thioacid or dithioacid, １1 は炭化水素鎖を示し、ＹRepresents a hydrocarbon chain, Y １1 はヒドロキシル基、カルボキシル基、アミノ基、アルデヒド基、ヒドラジド基、カルボニル基、エポキシ基、又はビニル基を示す。）Represents a hydroxyl group, a carboxyl group, an amino group, an aldehyde group, a hydrazide group, a carbonyl group, an epoxy group, or a vinyl group. )
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