JP3836394B2 - Optical waveguide type immunosensor and optical waveguide type immunoassay method - Google Patents

Optical waveguide type immunosensor and optical waveguide type immunoassay method Download PDF

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JP3836394B2
JP3836394B2 JP2002138326A JP2002138326A JP3836394B2 JP 3836394 B2 JP3836394 B2 JP 3836394B2 JP 2002138326 A JP2002138326 A JP 2002138326A JP 2002138326 A JP2002138326 A JP 2002138326A JP 3836394 B2 JP3836394 B2 JP 3836394B2
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optical waveguide
waveguide layer
layer
antibody
refractive index
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JP2003329684A (en
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兼一 内山
一郎 東野
英雄 江藤
美樹 長友
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Toshiba Corp
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Toshiba Corp
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【０００１】
【発明の属する技術分野】
本発明は、免疫センサに関し、特に試料溶液中の極微量の物質を高感度で分析するための光導波路型免疫センサ及び光導波路型免疫測定方法に関する。
【０００２】
【従来の技術】
従来より、抗原と抗体の特異的な反応を利用した微量成分の測定方法として酵素免疫測定法（ＥＬＩＳＡ）が知られている。この方法を応用して光導波路を用いた免疫センサが提案されている。例えば、特開平８−２８５８５１号公報には光導波路型免疫センサとその製造方法が開示されている。この免疫センサは、基板表面に光が入射、放出される一対のグレーティングを形成し、これらグレーティング間に位置する基板表面に単一の光導波路層を形成し、更にこの光導波路層上に抗体固定化膜を形成した構造を有する。
【０００３】
このような構造の免疫センサにおいて、抗体固定化膜に測定対象抗原を含む試料溶液を接触させると抗体と測定対象抗原が結合する。更に、蛍光標識されている抗体を添加すると抗体／測定対象抗原／蛍光標識抗体からなる免疫複合体が基板表面に形成される。このような状態でレーザ光のような光をグレーティングを介して光導波路層に入射させ、エバネッセント波を発生させ、光導波路層上の膜による検体中の生体分子との反応に起因するエバネッセント波の変化量をグレーティングから放出される光を受光する受光素子により検出して検体中の生体分子を分析する。
【０００４】
【発明が解決しようとする課題】
しかしながら、従来の免疫センサは光導波路層が単層で、ここで発生するエバネッセント波の変化量の検出には感度的に限界があり、また光導波路層上の膜構造から試料溶液中の極微量の生体分子分析には不向きであるという問題があった。
【０００５】
そこで、本発明は上記問題に鑑みてなされたもので、試料溶液中の極微量の物質を好感度かつ高精度で分析することが可能な光導波路型免疫センサ及び光導波路型免疫測定方法を提供しようとするものである。
【０００６】
【課題を解決するための手段】
本発明の第１の特徴は、基板と、基板表面に形成された第１光導波路層と、第１光導波路層の両端部付近表面にそれぞれ形成された入力側グレーティング及び出力側グレーティングと、入力側及び出力側グレーティングの間に位置する第１光導波路層上に選択的に形成され、第１光導波路層より高屈折率を有する第２光導波路層と、第２光導波路層上に抗体を固定化した抗体固定化層とを具備する光導波路型免疫センサを提供することである。なお、第１光導波路層の両端部付近表面にそれぞれ形成されるグレーティングは、どちらを入力側グレーティング或いは出力側グレーティングにしてもよい。
【０００７】
本発明の第２の特徴は、第１光導波路層上に形成され、第１光導波路層より高屈折率を有する第２光導波路層上に第１抗体を固定化した抗体固定化層の表面に測定対象となる抗原を含む試料溶液を接触させ、第１抗体と抗原からなる第１免疫複合体を第２光導波路層表面に形成させるステップと、酸化還元酵素標識された第２抗体を試料溶液に添加し、第１免疫複合体と第２抗体からなる第２免疫複合体を第２光導波路層表面に形成させるステップと、過酸化水素水及び荷電性の発色試薬を試料溶液に更に添加し、酸化還元酵素と過酸化水素水との酵素反応により生成されるラジカル酸素原子により発色試薬を発色させるステップと、第２光導波路層の表面側に電圧を印加し、荷電性の発色試薬を第２光導波路層表面に誘引させるステップと、第２光導波路層に裏面側から光を導入し、第２光導波路層表面にエバネッセント波を生じさせ、誘引された発色試薬を励起するステップとを具備する光導波路型免疫測定方法を提供することである。
【０００８】
【発明の実施の形態】
次に、図面を参照して、本発明の実施の形態を説明する。以下の図面の記載において、同一又は類似の部分は同一又は類似の符号を付している。ただし、図面は模式的なものであり、各寸法の比率等は現実のものとは異なることに留意すべきである。従って、具体的な寸法等は以下の説明を参酌して判断すべきものである。また図面相互間においても互いの寸法の関係や比率が異なる部分が含まれていることは勿論である。
【０００９】
（光導波路型免疫センサ）
本発明の実施の形態に係る光導波路型免疫センサは、図１に示すように、例えばガラスからなる基板１と、その表面に基板１より高屈折率の第１光導波路層２が形成されている。また、第１光導波路層２の両端部付近表面には、第１光導波路層２より高い屈折率を有する入力側グレーティング（回折格子）３ａ及び出力側グレーティング３ｂがそれぞれ形成されている。更に、入力側グレーティング３ａ及び出力側グレーティング３ｂの間に位置する第１光導波路層２上には、第１光導波路層２より高い屈折率を有し、外周が傾斜した形状の第２光導波路層４が形成されている。入力側グレーティング３ａ及び出力側グレーティング３ｂを含む第１光導波路層２上には、入力側グレーティング３ａ及び出力側グレーティング３ｂに比べて低屈折率を有する保護膜５が形成されている。第２光導波路層４上面に対応する保護膜５部分には、断面形状が矩状の開口部６が設けられている。保護膜５の表面（開口部６の内面を除く）には、例えば液晶表示装置に用いられるブラックマトリクス（顔料入りレジスト）から作られた迷光トラッピング層７が形成されている。開口部６から露出した第２光導波路層４表面部分には、測定対象となる抗原と結合する抗体が固定化された抗体固定化層８が形成されている。第２光導波路層４の表面側には電源９の陽極が配線され、所望の電圧（例えばパルス状電圧）を印加することが可能な構成となっている。
【００１０】
なお、基板１の屈折率をη1、第１光導波路層２の屈折率をη2、グレーティング３ａ，３ｂの屈折率をη3、第２光導波路層４の屈折率をη4及び保護膜５の屈折率をη5、とすると、それらの屈折率の大小関係は、η4≧η3＞η2＞η1 ＞η5となる。
【００１１】
第１光導波路層２は、例えば、カリウム、ナトリウム等の元素を含む高屈折率のガラスからなる。また、グレーティング３ａ，３ｂは、例えば酸化チタン、酸化亜鉛、ニオブ酸リチウム、ガリウム砒素（ＧａＡｓ）、インジウム錫酸化物（ＩＴＯ）、ポリイミドからなる。更に、第２光導波路層４は、第１光導波路層２より高屈折率の導電性材料、例えば酸化錫（ＳｎＯ２）、ＩＴＯからなる。保護膜５は、例えばフッ素樹脂からなる。抗体固定化層８は、例えば抗体を架橋高分子で固定化した構造である。抗体固定化層８で用いられる架橋高分子としては、例えば光架橋性ポリビニルアルコールのような水素結合性の官能基を含む高分子を挙げることができる。
【００１２】
（光導波路型免疫センサの製造方法）
次に、前述した図１に示す光導波路型免疫センサの製造方法の一例を図２、図３を参照して説明する。
【００１３】
（イ）まず、図２（ａ）に示すように、例えばホウケイ酸ガラスからなる基板１の表面を例えば３８０〜４００℃の硝酸カリウム溶液塩のようなイオン交換溶液に浸漬してカリウム、ナトリウム等の高屈折率元素をイオン交換することにより第１光導波路層２を形成する。その後、この第１光導波路層２上に例えば酸化チタン、酸化亜鉛、ニオブ酸リチウム、ＧａＡｓのような第１光導波路層より屈折率の高い材料からなる第１の高屈折率層１０を例えば化学気相堆積（ＣＶＤ）法等により形成する。
【００１４】
（ロ）次に、図２（ｂ）に示すように、第１の高屈折率層１０をフォトエッチング技術でパターニングすることにより第１光導波路層２の中央付近に外周が傾斜した形状の第２光導波路層４を形成する。つづいて、全面に例えば酸化チタン、酸化亜鉛、ニオブ酸リチウム、ＧａＡｓのような第１光導波路層より屈折率の高い材料からなる第２の高屈折率層を例えばＣＶＤ法等により形成した後、この第２の高屈折率層をフォトエッチング技術でパターニングすることにより図２（ｃ）に示すように、第１光導波路層２の両端部付近表面上に２つのグレーティング３ａ，３ｂを形成する。
【００１５】
（ハ）次に、グレーティング３ａ，３ｂ及び第２光導波路層４を含む第１光導波路層２上に、例えば感光性フッ素系樹脂のようなグレーティング３ａ，３ｂに比べて低屈折率を有する材料の被膜を塗布した後、露光、現像処理を施すことにより図２（ｄ）に示すように第２光導波路層４の表面に対応する箇所に断面形状が矩形の開口部６を有する感光性フッ素系樹脂からなる保護膜５を形成する。つづいて、図３（ｅ）に示すように、保護膜５の表面（開口部６の内面を除く）に例えば液晶表示装置に用いられるブラックマトリクスからなる迷光トラッピング層７をＣＶＤ法や真空蒸着法により形成する。
【００１６】
（ニ）次に、図３（ｆ）に示すように、保護膜５の開口部６から露出した第２光導波路層４表面部分に、測定対象の抗原を特異的に認識する抗体を固定化した抗体固定化層８を形成する。具体的には、抗体を溶媒の存在下で架橋性の高分子（例えば光可溶性のポリビニルアルコール）と混合し、この溶液を保護膜５の開口部６から露出した第２光導波路層４表面部分にインクジェット又はスピンコートにより塗布した後、光照射により光架橋性のポリビニルアルコールを架橋する方法により抗体固定化層８を形成する。
【００１７】
（光導波路型免疫測定方法）
次に、図４を用いて前述した図１に示す光導波路型免疫センサの測定方法について説明する。
【００１８】
（イ）図１に示す光導波路型免疫センサの第２光導波路層４の表面には、図４（ａ）に示すように、タンパク質、遺伝子等の測定対象となる抗原を特異的に認識する第１抗体１１からなる抗体固定化層８が形成されている。この抗体固定化層８の表面に抗原１２を含む試料溶液１３を添加すると、図４（ｂ）に示すように、抗原１２が第１抗体１１と結合し、第１抗体／抗原の免疫複合体を形成する。
【００１９】
（ロ）次に、酵素標識されている第２抗体１４を試料溶液１３に添加すると、第２抗体１４は第１抗体１１とは別の部位で抗原１２に結合する。その結果、図４（ｃ）に示すように、第１抗体／抗原／第２抗体からなる免疫複合体が第２光導波路層４の表面に形成される。なお、第２抗体に標識されている標識酵素として、例えば酸化還元酵素としてペルオキシダーゼ（ＰＯＤ）等を用いることができる。
【００２０】
（ハ）更に、図４（ｄ）に示すように、標識酵素の基質である過酸化水素水（Ｈ２Ｏ２）及び発色試薬１５を試料溶液１３に加えると、過酸化水素からペルオキシダーゼ（ＰＯＤ）等との酸化還元酵素反応によりラジカル酸素原子（Ｏ＊）が生成される。この酵素反応により生成されるラジカル酸素原子により試料溶液１３中の発色試薬を発色させる。ラジカル酸素原子により発色する発色試薬１５として、例えば荷電性のＮ，Ｎ−ビス（２−ヒドロキシ−３−サルフォプロピル）トリジンジカリウム塩等を用いることができる。
【００２１】
この反応を模式すると次式（１）、（２）のようになる：
Ｈ２Ｏ２ ＋ 酸化還元酵素（ＰＯＤ等）→ Ｏ＊ ・・・・（１）
Ｏ＊ ＋ 発色試薬 → 発色 ・・・・（２）
このような状態で、図４（ｄ）に示すように、第２光導波路層４の表面側に陽極が配線された電源９から所望の電圧（例えばパルス状電圧）を印加すると、負電荷を有する発色試薬１５が、第２光導波路層４の表面近傍、すなわちエバネッセント波の発生領域にまで誘引される。この第２光導波路層４に入力側グレーティング３ａを介して光を導入すると、光導波路表面にエバネッセント波が生じ、このエバネッセント波により発色試薬１５が励起されて蛍光を生じる。より詳細には、図１に示すように光源２１（例えば波長６５０ｎｍの半導体レーザ）及び検出器（受光素子）２２をそれぞれ免疫センサの基板１の裏面左側及び右側に配置する。光源２１からレーザ光を偏光フィルタ２３を通して免疫センサの基板１裏面側に入射すると、そのレーザ光は基板１を通して入力側グレーティング３ａと第１光導波路層２の界面で屈折されてその第１光導波路層２を伝播される。第１光導波路層２を伝播されるレーザ光は、この第１光導波路層２より高屈折率の第２光導波路層４との界面で２つのモード（ＴＭモード、ＴＥモード）に分割され、ＴＭモードレーザ光は第１光導波路層２を、ＴＥモードレーザ光は第２光導波路層４を伝播する。このとき、試料溶液１３における発色試薬の発色に基づく変化（例えば吸光度変化）によりこの試料溶液１３直下の第２光導波路層４を伝播する光の強度が変化する。このように第１光導波路層２と第２光導波路層４を伝播した光は、出力側グレーティング３ｂ側端付近において第１光導波路層２と第２光導波路層４の界面で再び結合、干渉し、出力側グレーティング３ｂを介して検出器２２側に入射されるため、第２光導波路層４を伝播する光の強度変化を増幅できる。その結果、試料溶液１３における酵素と過酸化水素水との反応による発色試薬の発色に基づく第２光導波路層４を伝播する光の極微な変化を偏光フィルタ２４を通して検出器２２で検出することが可能になる。
【００２２】
従って、本発明の実施の形態に係る光導波路型免疫センサによれば、抗体を固定化した抗体固定化層８の下方に配置された第２光導波路層４を導電性材料から作り、この第２光導波路層４に外部から所望のパルス状電圧を印加することにより試料溶液中の電荷性の発色試薬を第２光導波路層４表面に効率よく誘引することができ、かつ光導波路を第１光導波路層２と第２光導波路層４により構成して、抗体固定化層８における酵素反応による発色試薬の発色に基づく第２光導波路層４を伝播する光の極微な変化を第１光導波路層２と第２光導波路層４の界面で検出できるため、試料溶液中の極微量の物質を高感度で分析することができる。
【００２３】
また、図１に示すようにグレーティング３ａ，３ｂを含む第１光導波路層２上に保護膜５を形成することによって、第１光導波路層２及びグレーティング３ａ，３ｂを外部圧力が直接加わるのを防止できる。このため、第１光導波路層２及びグレーティング３ａ，３ｂに外部圧力が直接加わることに伴う部材の屈折率変化によって、その第１光導波路層２を伝播する光が外部に漏れるのを防止できる。更に、保護膜５を第１光導波路層２に比べて低屈折率材料で形成することによって、その第１光導波路層２を伝播する光を第１光導波路層２と保護膜５との界面で効果的に全反射させて第１光導波路層２内に封じ込めることができるため、第１光導波路層２から光が外部に漏れるのを防止できる。その結果、試料溶液中の極微量の物質をより高感度で分析することが可能になる。
【００２４】
更に、迷光トラッピング層７を保護膜５の表面（開口部６の内面を除く）に形成することによって、第１の実施の形態で説明したように第１光導波路層２を伝播する光が保護膜５との界面から保護膜５側に漏れた場合、その漏光を迷光トラッピング層７でトラップすることができる。すなわち、第１光導波路層２を伝播する光が保護膜５との界面から保護膜５側に漏れると、保護膜５表面と外界（空気）との屈折率の差により漏光は保護膜５表面で全反射して第２光導波路層４に迷光として入射されるため、前述した検体中のタンパク質の検出感度を低下させる。これに対し、迷光トラッピング層７を保護膜５の表面に形成することによって、漏光が保護膜５表面で全反射することなく迷光トラッピング層７でトラップできるため、その漏光が第２光導波路層４に迷光として入射するのを防止でき、検体中のタンパク質をより高感度で分析することが可能になる。なお、第１光導波路層の両端部付近表面にそれぞれ形成されるグレーティングは、どちらを入力側グレーティング或いは出力側グレーティングにしてもよい。
【００２５】
【発明の効果】
以上説明したように、本発明の光導波路型免疫センサ及び光導波路型免疫測定方法によれば、試料溶液中の極微量の物質を好感度かつ高精度で分析することが可能となる。
【図面の簡単な説明】
【図１】本発明の実施の形態に係る光導波路型免疫センサを示す断面図である。
【図２】図１の光導波路型免疫センサの製造工程を示す工程断面図である（その１）。
【図３】図１の光導波路型免疫センサの製造工程を示す工程断面図である（その２）。
【図４】本発明の実施の形態に係る光導波路型免疫測定方法を説明するための図である。
【符号の説明】
１…基板
２…第１光導波路層
３ａ，３ｂ…グレーティング
４…第２光導波路層
５…保護膜
６…開口部
７…迷光トラッピング層
８…抗体固定化層
９…電源
１０…高屈折率層
１１…第１抗体
１２…抗原
１３…試料溶液
１４…第２抗体
１５…発色試薬
２１…光源
２２…検出器
２３，２４…偏光フィルタ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an immunosensor, and more particularly to an optical waveguide type immunosensor and an optical waveguide type immunoassay method for analyzing a very small amount of a substance in a sample solution with high sensitivity.
[0002]
[Prior art]
Conventionally, an enzyme immunoassay (ELISA) is known as a method for measuring trace components using a specific reaction between an antigen and an antibody. An immunosensor using an optical waveguide has been proposed by applying this method. For example, Japanese Patent Application Laid-Open No. 8-285851 discloses an optical waveguide type immunosensor and a method for manufacturing the same. This immunosensor forms a pair of gratings where light enters and exits the substrate surface, forms a single optical waveguide layer on the substrate surface located between these gratings, and further immobilizes antibodies on this optical waveguide layer It has a structure in which a chemical film is formed.
[0003]
In the immunosensor having such a structure, when the sample solution containing the antigen to be measured is brought into contact with the antibody-immobilized membrane, the antibody and the antigen to be measured are bound. Furthermore, when a fluorescently labeled antibody is added, an immune complex composed of antibody / antigen to be measured / fluorescently labeled antibody is formed on the substrate surface. In such a state, light such as laser light is incident on the optical waveguide layer through the grating to generate an evanescent wave, and the evanescent wave caused by the reaction with the biomolecule in the specimen by the film on the optical waveguide layer is generated. The amount of change is detected by a light receiving element that receives light emitted from the grating to analyze the biomolecule in the specimen.
[0004]
[Problems to be solved by the invention]
However, conventional immunosensors have a single optical waveguide layer, and there is a limit to the sensitivity of detecting the amount of change in the evanescent wave generated here, and the trace amount in the sample solution is determined from the film structure on the optical waveguide layer. There is a problem that it is not suitable for biomolecular analysis.
[0005]
Therefore, the present invention has been made in view of the above problems, and provides an optical waveguide type immunosensor and an optical waveguide type immunoassay method capable of analyzing a very small amount of substance in a sample solution with good sensitivity and high accuracy. It is something to try.
[0006]
[Means for Solving the Problems]
According to a first aspect of the present invention, there are provided a substrate, a first optical waveguide layer formed on the substrate surface, an input side grating and an output side grating formed on surfaces near both ends of the first optical waveguide layer, and an input A second optical waveguide layer selectively formed on the first optical waveguide layer located between the side and output side gratings and having a higher refractive index than the first optical waveguide layer, and an antibody on the second optical waveguide layer An optical waveguide type immunosensor comprising an immobilized antibody immobilization layer is provided. Note that either of the gratings formed on the surfaces near both ends of the first optical waveguide layer may be an input side grating or an output side grating.
[0007]
The second feature of the present invention is the surface of the antibody-immobilized layer formed on the first optical waveguide layer and having the first antibody immobilized on the second optical waveguide layer having a higher refractive index than the first optical waveguide layer. A sample solution containing an antigen to be measured is brought into contact with the sample to form a first immune complex comprising the first antibody and the antigen on the surface of the second optical waveguide layer, and the second antibody labeled with oxidoreductase is sampled Adding to the solution, forming a second immune complex comprising the first immune complex and the second antibody on the surface of the second optical waveguide layer, and further adding a hydrogen peroxide solution and a chargeable coloring reagent to the sample solution And applying a voltage to the surface side of the second optical waveguide layer to form a charged coloring reagent by applying a color to the surface of the second optical waveguide layer by radical oxygen atoms generated by an enzymatic reaction between oxidoreductase and hydrogen peroxide. Attracting to the surface of the second optical waveguide layer; To provide an optical waveguide type immunoassay method comprising the steps of introducing light from the back side into a second optical waveguide layer, generating an evanescent wave on the surface of the second optical waveguide layer, and exciting the attracted coloring reagent. It is.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Next, embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, it should be noted that the drawings are schematic and ratios of dimensions and the like are different from actual ones. Accordingly, specific dimensions and the like should be determined in consideration of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.
[0009]
(Optical waveguide type immunosensor)
As shown in FIG. 1, an optical waveguide type immunosensor according to an embodiment of the present invention has a substrate 1 made of glass, for example, and a first optical waveguide layer 2 having a higher refractive index than that of the substrate 1 formed on the surface thereof. Yes. Further, an input side grating (diffraction grating) 3 a and an output side grating 3 b having a higher refractive index than the first optical waveguide layer 2 are formed on the surfaces near both ends of the first optical waveguide layer 2. Further, the second optical waveguide having a refractive index higher than that of the first optical waveguide layer 2 and having an inclined outer periphery on the first optical waveguide layer 2 positioned between the input side grating 3a and the output side grating 3b. Layer 4 is formed. A protective film 5 having a lower refractive index than the input side grating 3a and the output side grating 3b is formed on the first optical waveguide layer 2 including the input side grating 3a and the output side grating 3b. An opening 6 having a rectangular cross section is provided in the protective film 5 corresponding to the upper surface of the second optical waveguide layer 4. On the surface of the protective film 5 (excluding the inner surface of the opening 6), a stray light trapping layer 7 made of, for example, a black matrix (a pigment-containing resist) used in a liquid crystal display device is formed. An antibody immobilization layer 8 on which an antibody that binds to an antigen to be measured is immobilized is formed on the surface portion of the second optical waveguide layer 4 exposed from the opening 6. The anode of the power source 9 is wired on the surface side of the second optical waveguide layer 4 so that a desired voltage (for example, pulse voltage) can be applied.
[0010]
Incidentally, the refractive index of the substrate 1 .eta.1, 2 the refractive index of the first optical waveguide layer 2 eta, grating 3a, 3 a refractive index of 3b eta, the refractive index of the second optical waveguide layer 4 eta 4 and the protective film 5 If the refractive index of η is η 5 , the magnitude relationship between the refractive indexes is η 4 ≧ η 3 > η 2 > η 1 > η 5 .
[0011]
The first optical waveguide layer 2 is made of a high refractive index glass containing an element such as potassium or sodium. The gratings 3a and 3b are made of, for example, titanium oxide, zinc oxide, lithium niobate, gallium arsenide (GaAs), indium tin oxide (ITO), or polyimide. Further, the second optical waveguide layer 4 is made of a conductive material having a higher refractive index than that of the first optical waveguide layer 2, such as tin oxide (SnO 2 ) or ITO. The protective film 5 is made of, for example, a fluororesin. The antibody immobilization layer 8 has a structure in which, for example, an antibody is immobilized with a crosslinked polymer. Examples of the crosslinked polymer used in the antibody immobilization layer 8 include a polymer containing a hydrogen bonding functional group such as photocrosslinkable polyvinyl alcohol.
[0012]
(Manufacturing method of optical waveguide type immunosensor)
Next, an example of a method for manufacturing the optical waveguide type immunosensor shown in FIG. 1 will be described with reference to FIGS.
[0013]
(A) First, as shown in FIG. 2A, the surface of the substrate 1 made of, for example, borosilicate glass is immersed in an ion exchange solution such as potassium nitrate solution salt at 380 to 400 ° C. The first optical waveguide layer 2 is formed by ion exchange of a high refractive index element. Thereafter, a first high refractive index layer 10 made of a material having a higher refractive index than that of the first optical waveguide layer such as titanium oxide, zinc oxide, lithium niobate, GaAs, or the like is formed on the first optical waveguide layer 2, for example. It is formed by a vapor deposition (CVD) method or the like.
[0014]
(B) Next, as shown in FIG. 2 (b), the first high refractive index layer 10 is patterned by a photo-etching technique, so that the outer periphery of the first optical waveguide layer 2 is inclined near the center. 2 Optical waveguide layer 4 is formed. Subsequently, after forming a second high refractive index layer made of a material having a higher refractive index than the first optical waveguide layer such as titanium oxide, zinc oxide, lithium niobate, and GaAs on the entire surface by, for example, a CVD method, By patterning the second high-refractive index layer by a photoetching technique, two gratings 3a and 3b are formed on the surfaces near both ends of the first optical waveguide layer 2 as shown in FIG.
[0015]
(C) Next, on the first optical waveguide layer 2 including the gratings 3a and 3b and the second optical waveguide layer 4, for example, a material having a lower refractive index than the gratings 3a and 3b such as photosensitive fluorine-based resin. After the coating is applied, exposure and development processes are performed, whereby photosensitive fluorine having an opening 6 having a rectangular cross section at a position corresponding to the surface of the second optical waveguide layer 4 as shown in FIG. A protective film 5 made of a resin is formed. Subsequently, as shown in FIG. 3E, a stray light trapping layer 7 made of, for example, a black matrix used in a liquid crystal display device is formed on the surface of the protective film 5 (excluding the inner surface of the opening 6) by a CVD method or a vacuum evaporation method. To form.
[0016]
(D) Next, as shown in FIG. 3 (f), an antibody that specifically recognizes the antigen to be measured is immobilized on the surface of the second optical waveguide layer 4 exposed from the opening 6 of the protective film 5. The antibody immobilization layer 8 thus formed is formed. Specifically, the antibody is mixed with a crosslinkable polymer (for example, photosoluble polyvinyl alcohol) in the presence of a solvent, and this solution is exposed from the opening 6 of the protective film 5 on the surface portion of the second optical waveguide layer 4. Then, the antibody-immobilized layer 8 is formed by a method in which photocrosslinkable polyvinyl alcohol is crosslinked by light irradiation.
[0017]
(Optical waveguide type immunoassay method)
Next, the measuring method of the optical waveguide type immunosensor shown in FIG. 1 will be described with reference to FIG.
[0018]
(A) On the surface of the second optical waveguide layer 4 of the optical waveguide type immunosensor shown in FIG. 1, as shown in FIG. 4 (a), antigens to be measured such as proteins and genes are specifically recognized. An antibody immobilization layer 8 made of the first antibody 11 is formed. When the sample solution 13 containing the antigen 12 is added to the surface of the antibody immobilization layer 8, as shown in FIG. 4 (b), the antigen 12 binds to the first antibody 11, and the first antibody / antigen immune complex. Form.
[0019]
(B) Next, when the enzyme-labeled second antibody 14 is added to the sample solution 13, the second antibody 14 binds to the antigen 12 at a site different from the first antibody 11. As a result, an immune complex composed of the first antibody / antigen / second antibody is formed on the surface of the second optical waveguide layer 4 as shown in FIG. As the labeling enzyme labeled on the second antibody, for example, peroxidase (POD) can be used as the oxidoreductase.
[0020]
(C) Further, as shown in FIG. 4 (d), when hydrogen peroxide solution (H 2 O 2 ) and a coloring reagent 15 which are substrates for the labeling enzyme are added to the sample solution 13, peroxidase (POD) is converted from hydrogen peroxide. ) And the like generate radical oxygen atoms (O * ). The coloring reagent in the sample solution 13 is colored by radical oxygen atoms generated by this enzymatic reaction. As the coloring reagent 15 that develops color by radical oxygen atoms, for example, charged N, N-bis (2-hydroxy-3-sulfopropyl) tolidine dipotassium salt or the like can be used.
[0021]
This reaction is schematically represented by the following formulas (1) and (2):
H 2 O 2 + oxidoreductase (POD, etc.) → O * (1)
O * + Coloring reagent → Coloring (2)
In this state, as shown in FIG. 4 (d), when a desired voltage (for example, a pulsed voltage) is applied from a power source 9 having an anode wired on the surface side of the second optical waveguide layer 4, negative charges are generated. The coloring reagent 15 is attracted to the vicinity of the surface of the second optical waveguide layer 4, that is, to the region where the evanescent wave is generated. When light is introduced into the second optical waveguide layer 4 via the input side grating 3a, an evanescent wave is generated on the surface of the optical waveguide, and the coloring reagent 15 is excited by the evanescent wave to generate fluorescence. More specifically, as shown in FIG. 1, a light source 21 (for example, a semiconductor laser having a wavelength of 650 nm) and a detector (light receiving element) 22 are arranged on the left side and the right side of the back surface of the substrate 1 of the immunosensor, respectively. When the laser light from the light source 21 is incident on the back surface side of the substrate 1 of the immunosensor through the polarizing filter 23, the laser light is refracted through the substrate 1 at the interface between the input side grating 3a and the first optical waveguide layer 2 and the first optical waveguide. Propagated through layer 2. Laser light propagating through the first optical waveguide layer 2 is divided into two modes (TM mode and TE mode) at the interface with the second optical waveguide layer 4 having a higher refractive index than the first optical waveguide layer 2. The TM mode laser light propagates through the first optical waveguide layer 2 and the TE mode laser light propagates through the second optical waveguide layer 4. At this time, the intensity of light propagating through the second optical waveguide layer 4 immediately below the sample solution 13 changes due to a change (for example, a change in absorbance) based on the color of the coloring reagent in the sample solution 13. The light propagating through the first optical waveguide layer 2 and the second optical waveguide layer 4 in this manner is recombined and interfered at the interface between the first optical waveguide layer 2 and the second optical waveguide layer 4 in the vicinity of the output side grating 3b side end. And since it injects into the detector 22 side via the output side grating 3b, the intensity change of the light which propagates the 2nd optical waveguide layer 4 can be amplified. As a result, a minute change in the light propagating through the second optical waveguide layer 4 based on the coloration of the coloring reagent due to the reaction between the enzyme and the hydrogen peroxide solution in the sample solution 13 can be detected by the detector 22 through the polarizing filter 24. It becomes possible.
[0022]
Therefore, according to the optical waveguide type immunosensor according to the embodiment of the present invention, the second optical waveguide layer 4 disposed below the antibody immobilization layer 8 on which the antibody is immobilized is made of a conductive material. 2 By applying a desired pulsed voltage from the outside to the optical waveguide layer 4, the chargeable coloring reagent in the sample solution can be efficiently attracted to the surface of the second optical waveguide layer 4, and the optical waveguide is the first optical waveguide. The optical waveguide layer 2 and the second optical waveguide layer 4 are configured so that a minute change in light propagating through the second optical waveguide layer 4 based on the color development of the coloring reagent by the enzyme reaction in the antibody immobilization layer 8 is changed to the first optical waveguide. Since detection is possible at the interface between the layer 2 and the second optical waveguide layer 4, a very small amount of substance in the sample solution can be analyzed with high sensitivity.
[0023]
Further, as shown in FIG. 1, external pressure is directly applied to the first optical waveguide layer 2 and the gratings 3a and 3b by forming the protective film 5 on the first optical waveguide layer 2 including the gratings 3a and 3b. Can be prevented. For this reason, it is possible to prevent light propagating through the first optical waveguide layer 2 from leaking to the outside due to a change in the refractive index of the member caused by external pressure being directly applied to the first optical waveguide layer 2 and the gratings 3a and 3b. Further, the protective film 5 is formed of a material having a lower refractive index than that of the first optical waveguide layer 2, so that light propagating through the first optical waveguide layer 2 is transmitted to the interface between the first optical waveguide layer 2 and the protective film 5. Thus, the light can be effectively totally reflected and contained in the first optical waveguide layer 2, so that light can be prevented from leaking outside from the first optical waveguide layer 2. As a result, it becomes possible to analyze a very small amount of substance in the sample solution with higher sensitivity.
[0024]
Further, by forming the stray light trapping layer 7 on the surface of the protective film 5 (excluding the inner surface of the opening 6), the light propagating through the first optical waveguide layer 2 is protected as described in the first embodiment. When leaking from the interface with the film 5 to the protective film 5 side, the leaked light can be trapped by the stray light trapping layer 7. That is, when light propagating through the first optical waveguide layer 2 leaks from the interface with the protective film 5 to the protective film 5 side, the light leakage is caused by the difference in refractive index between the surface of the protective film 5 and the outside (air). Since the light is totally reflected and is incident on the second optical waveguide layer 4 as stray light, the detection sensitivity of the protein in the specimen is reduced. On the other hand, by forming the stray light trapping layer 7 on the surface of the protective film 5, the light leakage can be trapped by the stray light trapping layer 7 without being totally reflected on the surface of the protective film 5. Can be prevented from entering as a stray light, and the protein in the sample can be analyzed with higher sensitivity. Note that either of the gratings formed on the surfaces near both ends of the first optical waveguide layer may be an input side grating or an output side grating.
[0025]
【The invention's effect】
As described above, according to the optical waveguide immunosensor and the optical waveguide immunoassay method of the present invention, it is possible to analyze a very small amount of substance in a sample solution with high sensitivity and high accuracy.
[Brief description of the drawings]
FIG. 1 is a cross-sectional view showing an optical waveguide immunosensor according to an embodiment of the present invention.
FIG. 2 is a process cross-sectional view showing a manufacturing process of the optical waveguide type immunosensor of FIG. 1 (part 1);
3 is a process cross-sectional view showing the manufacturing process of the optical waveguide type immunosensor in FIG. 1 (part 2); FIG.
FIG. 4 is a diagram for explaining an optical waveguide type immunoassay method according to an embodiment of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Board | substrate 2 ... 1st optical waveguide layer 3a, 3b ... Grating 4 ... 2nd optical waveguide layer 5 ... Protective film 6 ... Opening part 7 ... Stray light trapping layer 8 ... Antibody fixed layer 9 ... Power supply 10 ... High refractive index layer DESCRIPTION OF SYMBOLS 11 ... 1st antibody 12 ... Antigen 13 ... Sample solution 14 ... 2nd antibody 15 ... Color developing reagent 21 ... Light source 22 ... Detector 23, 24 ... Polarizing filter

Claims (8)

基板と、
前記基板表面に形成された第１光導波路層と、
前記第１光導波路層の両端部付近表面にそれぞれ形成された入力側グレーティング及び出力側グレーティングと、
前記入力側及び出力側グレーティングの間に位置する前記第１光導波路層上に選択的に形成され、前記第１光導波路層より高屈折率を有する第２光導波路層と、
前記第２光導波路層上に抗体を固定化した抗体固定化層と、
前記第２光導波路表面に配置され、電圧が印可される配線
とを具備することを特徴とする光導波路型免疫センサ。A substrate,
A first optical waveguide layer formed on the substrate surface;
An input side grating and an output side grating respectively formed on surfaces near both ends of the first optical waveguide layer;
A second optical waveguide layer selectively formed on the first optical waveguide layer positioned between the input side and output side gratings and having a higher refractive index than the first optical waveguide layer;
An antibody immobilization layer in which an antibody is immobilized on the second optical waveguide layer;
An optical waveguide type immunosensor comprising: a wiring disposed on a surface of the second optical waveguide and to which a voltage is applied . 前記第２光導波路層が、導電性材料からなることを特徴とする請求項１に記載の光導波路型免疫センサ。 The optical waveguide immunosensor according to claim 1, wherein the second optical waveguide layer is made of a conductive material. 前記抗体固定化層が、前記抗体を架橋高分子で固定化した層であることを特徴とする請求項１又は２に記載の光導波路型免疫センサ。 The optical waveguide immunosensor according to claim 1 or 2, wherein the antibody-immobilized layer is a layer in which the antibody is immobilized with a crosslinked polymer. 前記架橋高分子が、水素結合性の官能基を含むことを特徴とする請求項３に記載の光導波路型免疫センサ。 The optical waveguide type immunosensor according to claim 3, wherein the crosslinked polymer contains a hydrogen bonding functional group. 前記第１光導波路層に比べて低屈折率を有する保護膜が、前記入力側及び出力側グレーティングを含む前記第１光導波路層上に、前記抗体固定化層が露出するように形成されることを特徴とする請求項１乃至４のいずれか１項に記載の光導波路型免疫センサ。 A protective film having a lower refractive index than the first optical waveguide layer is formed on the first optical waveguide layer including the input side and output side gratings so that the antibody immobilization layer is exposed. The optical waveguide type immunosensor according to any one of claims 1 to 4, wherein: 迷光トラッピング層が、前記保護膜上に更に形成されることを特徴とする請求項５に記載の光導波路型免疫センサ。 6. The optical waveguide type immunosensor according to claim 5, wherein a stray light trapping layer is further formed on the protective film. 第１光導波路層上に形成され、前記第１光導波路層より高屈折率を有する第２光導波路層上に第１抗体を固定化した抗体固定化層の表面に測定対象となる抗原を含む試料溶液を接触させ、前記第１抗体と前記抗原からなる第１免疫複合体を前記第２光導波路層表面に形成させるステップと、
酸化還元酵素標識された第２抗体を前記試料溶液に添加し、前記第１免疫複合体と前記第２抗体からなる第２免疫複合体を前記第２光導波路層表面に形成させるステップと、
過酸化水素水及び荷電性の発色試薬を前記試料溶液に更に添加し、前記酸化還元酵素と前記過酸化水素水との酵素反応により生成されるラジカル酸素原子により前記発色試薬を発色させるステップと、
前記第２光導波路層の表面側に電圧を印加し、前記荷電性の発色試薬を前記第２光導波路層表面に誘引させるステップと、
前記第２光導波路層に裏面側から光を導入し、前記第２光導波路層表面にエバネッセント波を生じさせ、前記誘引された発色試薬を励起するステップ
とを具備することを特徴とする光導波路型免疫測定方法。The antigen to be measured is included on the surface of the antibody-immobilized layer formed on the first optical waveguide layer and having the first antibody immobilized on the second optical waveguide layer having a higher refractive index than the first optical waveguide layer. Contacting a sample solution to form a first immune complex comprising the first antibody and the antigen on the surface of the second optical waveguide layer;
Adding a second antibody labeled with oxidoreductase to the sample solution, and forming a second immune complex comprising the first immune complex and the second antibody on the surface of the second optical waveguide layer;
Adding a hydrogen peroxide solution and a chargeable coloring reagent to the sample solution, and coloring the coloring reagent with radical oxygen atoms generated by an enzymatic reaction between the oxidoreductase and the hydrogen peroxide solution;
Applying a voltage to the surface side of the second optical waveguide layer to attract the chargeable coloring reagent to the surface of the second optical waveguide layer;
And a step of introducing light from the back side into the second optical waveguide layer, generating an evanescent wave on the surface of the second optical waveguide layer, and exciting the attracted coloring reagent. Type immunoassay. 前記酸化還元酵素が、ペルオキシダーゼであることを特徴とする請求項７に記載の光導波路型免疫測定方法。 The optical waveguide immunoassay method according to claim 7, wherein the oxidoreductase is peroxidase.
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