JP2004144477A - Measuring method and instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately calculate a total reflection attenuation angle θ<SB>SP</SB>, and to detect a beam profile with high resolution, in a measuring instrument for measuring intensity of a light beam by a plurality of photoreceiving elements, and for differentiating photoreception signals output from the respective photoreceiving elements in every set of two outputs from the adjacent photoreceiving elements to detect a position of a dark line. <P>SOLUTION: The photoreception signals detected by respective photodiodes of a photodiode array 17 of fine array pitch are added by an adder 50 in every set of three outputs to amplify a dynamic range, adjacent outputs from the adder are differentiated by a differential amplifier array 18, and the total reflection attenuation angle θ<SB>SP</SB>is calculated based on a differentiated signal therein. <P>COPYRIGHT: (C)2004,JPO

Description

すなわち、上記反射光強度が低下する入射角である全反射減衰角θ_ＳＰを知ることにより、被測定物質の誘電率εｓ、つまりは屈折率に関連する特性を求めることができる。
【０００９】
なお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θ_ＳＰ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。
【００１０】
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θ_ＳＰ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。
【００１１】
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。
【００１２】
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。
【００１３】
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。
【００１４】
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θ_ＳＰの角度を測定している。
【００１５】
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θ_ＳＰを測定し、該全反射減衰角θ_ＳＰ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。
【００１６】
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、全反射減衰角θ_ＳＰ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θ_ＳＰ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θ_ＳＰ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、底面に予め形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θ_ＳＰの角度変化量の測定を行っている。
【００１７】
なお本出願人は、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を特開２００１−３３０５６０号公報により提案している。
【００１８】
また、本出願人は、特願２００１−３９７４１１号において、複数個の試料液保持部が設けられた測定チップを用いて測定を行う全反射減衰を利用した測定装置も提案している。このような構成の測定装置を用いれば、測定チップを移動させることなく多数の試料についての測定を同時に行うことができる。
【００１９】
【発明が解決しようとする課題】
以上説明した表面プラズモン測定装置や漏洩モード測定装置等のエバネッセント波を利用した測定装置において、前記界面で反射した光ビームの強度を反射角毎に検出するために、従来は光ビームの偏向に同期移動する光検出器や、広い受光面を有するＣＣＤ等のエリアセンサ等を用いていたが、前者は、全反射解消角の測定範囲に関して比較的大きなダイナミックレンジを確保できるが、その反面、機械的な駆動機構が必要になることから、試料分析を高速で行なうのは困難であり、また、後者は、高速分析が可能である反面、ＣＣＤ等のエリアセンサの分解能、電荷蓄積のダイナミックレンジが低いことから、高い分析精度を確保するのは困難である。
【００２０】
そのため、上記問題を回避するために、本出願人は、上述の特開平１１−３２６１９４号公報に記載されているような、複数の受光素子からなる光検出手段により前記界面で全反射した光ビームの強度を測定し、この光検出手段の各受光素子が出力する光検出信号を、隣接する２つの受光素子の出力毎に微分し、この微分値に基づいて全反射減衰角θ_ＳＰを求める測定装置を提案している。この測定装置によれば、ＣＣＤ等のエリアセンサ等に比べ広いダイナミックレンジで全反射減衰角θ_ＳＰを精度良く検出することができる。
【００２１】
ところで、近年、上述の特開平１１−３２６１９４号公報に記載されているような測定装置においてさらに光検出手段で検出される光ビームのビームプロファイルが知りたいという要望があるが、このような測定装置では、各受光素子の出力信号を大きくするために各受光素子のサイズを大きくしている、すなわち各受光素子の配列ピッチを粗くしているため、ビームプロファイルを生成する際には解像度が低くなるという問題がある。
【００２２】
そのため、ビームプロファイルを高い解像度で検出することができるようにするために、各受光素子の配列ピッチを細かくすると、各受光素子の出力信号が小さくなり、正確に全反射減衰角θ_ＳＰを算出することができなくなる虞がある。また、各受光素子で受光する光量が少ない場合には、ノイズの影響を受けやすくなる虞もある。
【００２３】
本発明は上記の事情に鑑みて、複数の受光素子により光ビームの強度の測定を行い、各受光素子が出力する光検出信号を、隣接する２つの受光素子の出力毎に微分して暗線の位置の検出を行う測定装置において、正確に全反射減衰角θ_ＳＰを算出することが可能であり、かつ高い解像度でビームプロファイルを検出することが可能な測定装置を提供することを目的とする。
【００２４】
【課題を解決するための手段】
本発明による測定方法は、誘電体ブロック、誘電体ブロックの一面に形成された薄膜層、および薄膜層の表面上に試料を保持する試料保持機構を備えてなる測定チップと、光ビームを発生させる光源と、光ビームを誘電体ブロックに対して、誘電体ブロックと薄膜層との界面で全反射条件が得られる入射角で入射させる入射光学系と、複数の受光素子からなり、前記界面で全反射した光ビームの強度を測定する光検出手段とを備えてなる測定装置により試料の分析を行う測定方法において、複数の受光素子をいくつかの受光素子群に分割し、隣接する２つの受光素子群毎にそれぞれの出力の合計を受光素子の並設方向に関して微分し、この微分値に基づいて、前記界面での反射光強度が極小値を取る反射角を求めることにより試料の分析を行うことを特徴とするものである。
【００２５】
本発明による他の測定方法は、誘電体ブロック、該誘電体ブロックの一面に形成された薄膜層、および該薄膜層の表面上に試料を保持する試料保持機構を備えてなる測定チップと、光ビームを発生させる光源と、前記光ビームを前記誘電体ブロックに対して、該誘電体ブロックと前記薄膜層との界面で全反射条件が得られる入射角で入射させる入射光学系と、複数の受光素子からなり、前記界面で全反射した光ビームの強度を測定する光検出手段とを備えてなる測定装置により前記試料の分析を行う測定方法において、隣接する２つ以上の所定数の受光素子それぞれの出力の平均値を、前記受光素子の並設方向に順次算出し、該平均値を前記受光素子の並設方向に関して微分し、該微分値に基づいて、前記界面での反射光強度が極小値を取る反射角を求めることにより前記試料の分析を行うことを特徴とするものである。
【００２６】
また、本発明による測定装置は、誘電体ブロック、誘電体ブロックの一面に形成された薄膜層、および薄膜層の表面上に試料を保持する試料保持機構を備えてなる測定チップと、光ビームを発生させる光源と、光ビームを誘電体ブロックに対して、誘電体ブロックと薄膜層との界面で全反射条件が得られる入射角で入射させる入射光学系と、複数の受光素子からなり、前記界面で全反射した光ビームの強度を測定する光検出手段と、複数の受光素子をいくつかの受光素子群に分割し、隣接する２つの受光素子群毎にそれぞれの出力の合計を受光素子の並設方向に関して微分し、この微分値に基づいて前記界面での反射光強度が極小値を取る反射角を求める演算手段とを備えてなることを特徴とするものである。
【００２７】
本発明による他の測定装置は、誘電体ブロック、該誘電体ブロックの一面に形成された薄膜層、および該薄膜層の表面上に試料を保持する試料保持機構を備えてなる測定チップと、光ビームを発生させる光源と、前記光ビームを前記誘電体ブロックに対して、該誘電体ブロックと前記薄膜層との界面で全反射条件が得られる入射角で入射させる入射光学系と、複数の受光素子からなり、前記界面で全反射した光ビームの強度を測定する光検出手段と、隣接する２つ以上の所定数の受光素子それぞれの出力の平均値を、前記受光素子の並設方向に順次算出し、該平均値を前記受光素子の並設方向に関して微分し、該微分値に基づいて前記界面での反射光強度が極小値を取る反射角を求める演算手段とを備えてなることを特徴とするものである。
【００２８】
上記のような測定装置としては、金属膜を上記薄膜層として用いる前述の表面プラズモン測定装置や、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た光導波層とからなる層を上記薄膜層として用いる前述の漏洩モード測定装置等がある。
【００２９】
本発明による測定装置において、光検出手段により前記界面で全反射した光ビームの強度を測定して試料の分析を行うには種々の方法があり、例えば、光ビームを前記界面で全反射条件が得られる種々の入射角で入射させ、各入射角に対応した位置毎に前記界面で全反射した光ビームの強度を測定して、全反射減衰により発生した暗線の位置（角度）を検出することにより試料分析を行ってもよいし、Ｄ．Ｖ．Ｎｏｏｒｔ，Ｋ．ｊｏｈａｎｓｅｎ，Ｃ．−Ｆ．Ｍａｎｄｅｎｉｕｓ，　Ｐｏｒｏｕｓ　Ｇｏｌｄ　ｉｎ　Ｓｕｒｆａｃｅ　Ｐｌａｓｍｏｎ　Ｒｅｓｏｎａｎｃｅ　Ｍｅａｓｕｒｅｍｅｎｔ，　ＥＵＲＯＳＥＮＳＯＲＳ　ＸＩＩＩ，　１９９９，　ｐｐ．５８５−５８８　に記載されているように、複数の波長の光ビームを前記界面で全反射条件が得られる入射角で入射させ、各波長毎に前記界面で全反射した光ビームの強度を測定して、各波長毎の全反射減衰の程度（暗線の位置および程度）を検出することにより試料分析を行ってもよい。
【００３０】
本発明において、「複数の受光素子をいくつかの受光素子群に分割し、隣接する２つの受光素子群毎にそれぞれの出力の合計を受光素子の並設方向に関して微分する」とは、各受光素子群の出力を合計した後に、隣接する２つの受光素子群毎にそれぞれの出力の合計を受光素子の並設方向に関して微分する場合に限らず、隣接する２つの受光素子群の受光素子をそれぞれ１つずつ受光素子の並設方向に関して微分した後に、この微分値を合計する等、結果的に同じ微分値が得られるのであれば計算の過程はどのようなものであってもよい。
【００３１】
例えば１番の受光素子から６番の受光素子まで順に並んだ６個の受光素子を、１番から３番の受光素子からなる第１の受光素子群と、４番から６番の受光素子からなる第２の受光素子群に分割し、この受光素子群の出力の合計を受光素子の並設方向に関して微分する場合、第１の受光素子群の１番から３番の受光素子のそれぞれの受光素子の出力を合計し、第２の受光素子群の４番から６番の受光素子のそれぞれの受光素子の出力を合計し、第１の受光素子群の出力の合計と第２の受光素子群の出力の合計とを受光素子の並設方向に関して微分してもよいし、１番と４番の受光素子、２番と５番の受光素子、３番と６番の受光素子の出力をそれぞれ受光素子の並設方向に関して微分し、この３つの微分値を合計してもよい。あるいは、１番と６番の受光素子の出力の差分、２番と５番の受光素子の出力の差分、３番と４番の受光素子の出力の差分を合計し、受光素子群間の距離、この場合には受光素子３つ分に相当する距離で除算してもよい。
【００３２】
また、「隣接する２つ以上の所定数の受光素子それぞれの出力の平均値を、前記受光素子の並設方向に順次算出し、該平均値を前記受光素子の並設方向に関して微分する」とは、各所定数の受光素子の出力の平均値を算出した後に、該平均値を前記受光素子の並設方向に関して微分する場合に限らず、例えば、まず隣接する２つの受光素子間の微分値を求め、次に連続した所定数の微分値の平均値を、前記受光素子の並設方向に順次算出する等、結果的に同じ微分値が得られるのであれば計算の過程はどのようなものであってもよい。
【００３３】
例えば１番の受光素子から４番の受光素子まで順に並んだ４個の受光素子から、隣接する３つの受光素子それぞれの出力の平均値を、前記受光素子の並設方向に順次算出し、該平均値を前記受光素子の並設方向に関して微分する場合、１番から３番までの受光素子の出力の平均値と、２番から４番までの受光素子の出力の平均値とを求め、この平均値を受光素子の並設方向に関して微分してもよいし、１番と２番の受光素子、２番と３番の受光素子、３番と４番の受光素子の出力をそれぞれ受光素子の並設方向に関して微分し、これらの微分値の平均値を求めてもよい。なお、「隣接する２つ以上の所定数の受光素子それぞれの出力の平均値」とは、平均値そのものに限定されるものではなく、平均値を反映する値であればよく、例えば所定数の受光素子それぞれの出力の合計値や、この合計値を所望の値で除算した値、あるいは合計値に所望の値を乗算した値等であってもよい。
【００３４】
また、上記各測定方法および測定装置において、「微分値に基づいて前記界面での反射光強度が極小値を取る反射角を求める」とは、極小値を取る反射角そのものを求めることに限定されるものではなく、極小値を取る反射角を反映する値、例えば極小値を取る反射角近傍の微分値などを求める場合も含むものである。なお、「極小値」とは、全反射減衰の状態を反映して生じる極小値を意味し、ノイズ等の影響により生じるものは含まない。
【００３５】
【発明の効果】
複数の受光素子により光ビームの強度の測定を行い、各受光素子が出力する光検出信号を、隣接する２つの受光素子の出力毎に微分して暗線の位置の検出を行う測定装置において、本発明の測定方法および測定装置は、演算手段を、複数の受光素子をいくつかの受光素子群に分割し、隣接する２つの受光素子群毎にそれぞれの出力の合計を受光素子の並設方向に関して微分し、この微分値に基づいて、前記界面での反射光強度が極小値を取る反射角を求めるようにしたので、サイズが小さい受光素子を用いる場合であっても、全反射減衰角θ_ＳＰを算出する際には、複数の受光素子をいくつかの受光素子群に分割し、この受光素子群毎に出力を合計するため、微分する際の信号の出力を大きくすることができる。そのため、配列ピッチが細かい受光素子アレイを用いることができるようになるので、これにより高い解像度でビームプロファイルを検出できるとともに、全反射減衰角θ_ＳＰを正確に算出することができる。
【００３６】
本発明による他の測定方法および測定装置は、複数の受光素子からなり、誘電体ブロックと薄膜層との界面で全反射した光ビームの強度を測定する光検出手段を備えてなる測定装置において、隣接する２つ以上の所定数の受光素子それぞれの出力の平均値を、前記受光素子の並設方向に順次算出し、該平均値を前記受光素子の並設方向に関して微分し、この微分値に基づいて、前記界面での反射光強度が極小値を取る反射角を求めるようにしたので、サイズが小さい受光素子を用いる場合あるいは受光素子が受光する光量が少ない場合であっても、ノイズの影響を受けにくく、精度よく反射光強度が極小値を取る反射角、すなわち全反射減衰角θ_ＳＰを求めることができる。また、受光素子数と同数の平均値を算出することができ、高い解像度で全反射減衰角θ_ＳＰを求めることができる。またこの平均値を用いて、高い解像度のビームプロファイルを得ることができる。
【００３７】
【発明の実施の形態】
以下、本発明の実施の形態を図面を参照して説明する。本発明の第１の実施の形態の測定装置は、複数の誘電体ブロックに光ビームを並列的に入射させることにより複数の試料の分析を同時に行うことが可能な表面プラズモン測定装置であり、図１は本実施の形態の表面プラズモン測定装置の概略構成を示す平面図であり、図２はこの表面プラズモン測定装置の側面形状を示すものである。
【００３８】
上記表面プラズモン測定装置１０１は、同様の構成の複数の表面プラズモン測定ユニット１０１Ａ、１０１Ｂ、１０１Ｃ…により構成されている。
【００３９】
各測定ユニットの構成について、個別の要素を表す符号であるＡ、Ｂ、Ｃ…の符号は省略して説明する。各測定ユニットは、測定チップ９と、光ビーム１３を発生する光源であるレーザ光源１４と、上記光ビーム１３を測定チップ９に対して入射させる入射光学系１５と、測定チップ９で反射された光ビーム１３を平行光化して光検出器１７に向けて射出するコリメーターレンズ１６と、コリメーターレンズ１６より出射された光ビーム１３を受光して光強度を検出する光検出器１７と、光検出器１７に接続された差動アンプアレイ１８と、差動アンプアレイ１８に接続されたドライバ１９と、ドライバ１９に接続されたコンピュータシステム等からなる信号処理部２０と、光検出器１７から出力された信号をデジタル信号へ変換し、信号処理部２０へ出力するＡ／Ｄ変換器２７とからなる。なお、請求項に記載の演算手段は、差動アンプアレイ１８、ドライバ１９および信号処理部２０により構成される。
【００４０】
測定チップ９は、四角錐の４つの稜線が集まる頂角を含む一部分が切り取られ、かつこの四角錐の底面に試料液１１を貯える試料保持機構として機能する凹部１０ｃが形成された形状の誘電体ブロック１０と、この誘電体ブロック１０の凹部１０ｃの底面に形成された、例えば金、銀、銅、アルミニウム等からなる薄膜層である金属膜１２とからなる。この誘電体ブロック１０は、例えば透明樹脂等により形成することができる。なお、金属膜１２の上に後述するセンシング物質３０を設けてもよい。また、測定チップ９の誘電体ブロック１０は、図３に示すように、互いに隣接する複数の表面プラズモン測定ユニットの測定チップの誘電体ブロックと一体的に構成されたものであってもよい。
【００４１】
入射光学系１５は、レーザ光源１４から射出された光ビーム１３を平行光化するコリメーターレンズ１５ａと、この平行光化された光ビーム１３を上記界面１０ｂに向けて収束させる集光レンズ１５ｂとから構成されている。
【００４２】
光ビーム１３は、集光レンズ１５ｂにより上述のように集光されるので、界面１０ｂに対して種々の入射角θで入射する成分を含むことになる。なお、この入射角θは、全反射角以上の角度とされる。そのため、界面１０ｂで全反射した光ビーム１３には、種々の反射角で全反射された成分が含まれることになる。なお、上記入射光学系１５は、光ビーム１３を界面１０ｂ上に点状に集光させずにデフォーカス状態で入射させるように構成してもよい。そのようにすれば、界面１０ｂ上のより広い領域において光ビーム１３が全反射されるので、全反射減衰の状態の検出誤差が平均化されて全反射減衰角の測定精度を高めることができる。
【００４３】
なお光ビーム１３は、界面１０ｂに対してｐ偏光で入射させる。そのようにするためには、予めレーザ光源１４をその偏光方向が上記所定の方向となるように配設すればよい。その他、光ビーム１３を界面１０ｂに対してｐ偏光で入射させるには波長板で光ビーム１３の偏光の向きを制御するようにしてもよい。また、表面プラズモン測定装置１０１は、各測定ユニットの信号処理部２０Ａ、２０Ｂ、２０Ｃ…に接続された１つの表示手段２１を備えている。
【００４４】
以下、上記構成の表面プラズモン測定装置による試料分析について説明する。図２に示す通り、レーザ光源１４から射出された光ビーム１３は、入射光学系１５を通して、誘電体ブロック１０と金属膜１２との界面１０ｂ上に収束される。
【００４５】
界面１０ｂ上に収束され、この界面１０ｂで全反射された光ビーム１３は、コリメーターレンズ１６を通して光検出器１７によって検出される。光検出器１７は、複数の受光素子であるフォトダイオード１７ａ、１７ｂ、１７ｃ…が１列に並設されてなるフォトダイオードアレイであり、フォトダイオードの並設方向が図２の紙面に略平行となるように、かつコリメーターレンズ１６を通して平行光化されて入射される光ビーム１３の伝播方向に対して略直交するように配設されている。したがって、上記界面１０ｂにおいて種々の反射角で全反射された光ビーム１３の各成分を、それぞれ異なるフォトダイオード１７ａ、１７ｂ、１７ｃ…が受光することになる。そして、光検出器１７は、各フォトダイオード１７ａ、１７ｂ、１７ｃ…によって検出された上記光ビーム１３の強度分布を示す信号を差動アンプアレイ１８およびＡ／Ｄ変換器２７に出力する。なおＡ／Ｄ変換器２７でＡ／Ｄ変換された信号は信号処理部２０に入力される。
【００４６】
界面１０ｂに特定入射角θ_ＳＰで入射した上記光ビーム１３の成分は、金属膜１２とこの金属膜１２に接している物質との界面に表面プラズモンを励起させるので、この光については反射光強度が鋭く低下する。つまり上記特定入射角θ_ＳＰが全反射減衰角であり、この角度θ_ＳＰにおいて反射光強度は極小値を示す。この反射光強度が低下する領域は、図２にＤで示すように、界面１０ｂで全反射された光ビーム１３中の暗線として観察される。
【００４７】
次に、光検出器１７から出力された光ビーム１３の強度分布を示す信号の処理について詳細に説明する。図４は、この表面プラズモン測定装置の電気的構成を示すブロック図である。図示の通り差動アンプアレイ１８は、１７ａ、１７ｂ、１７ｃ…毎に設けられたスイッチ５１ａ、５１ｂ、５１ｃ…、これらスイッチ５１ａ、５１ｂ、５１ｃ…の３つの出力毎に設けられた加算機５０ａ、５０ｂ、５０ｃ…、互いに隣接する２つの加算機毎に設けられた差動アンプ１８ａ、１８ｂ、１８ｃ…から構成されている。すなわち、本実施の形態ではそれぞれ３つのフォトダイオードによってフォトダイオード群（受光素子群）が形成されている。なお、スイッチ５１ａ、５１ｂ、５１ｃ…は、信号処理部２０からの指示に基づいて開閉されるものである。
【００４８】
また、ドライバ１９は、差動アンプアレイ１８の各差動アンプ１８ａ、１８ｂ、１８ｃ…の出力をサンプルホールドするサンプルホールド回路２２ａ、２２ｂ、２２ｃ…、これらのサンプルホールド回路２２ａ、２２ｂ、２２ｃ…の各出力が入力されるマルチプレクサ２３、このマルチプレクサ２３の出力をデジタル化して信号処理部２０に入力するＡ／Ｄ変換器２４、マルチプレクサ２３とサンプルホールド回路２２ａ、２２ｂ、２２ｃ…とを駆動する駆動回路２５、および信号処理部２０からの指示に基づいて駆動回路２５の動作を制御するコントローラ２６から構成されている。
【００４９】
上記フォトダイオード１７ａ、１７ｂ、１７ｃ…の各出力は加算機５０ａ、５０ｂ、５０ｃ…により３つの出力毎に加算され、加算機５０ａ、５０ｂ、５０ｃ…の各出力は差動アンプアレイ１８の各差動アンプ１８ａ、１８ｂ、１８ｃ…に入力される。この際、互いに隣接する２つの加算機の出力が、共通の差動アンプに入力される。したがって各差動アンプ１８ａ、１８ｂ、１８ｃ…の出力は、複数のフォトダイオード１７ａ、１７ｂ、１７ｃ…が出力する光検出信号を、３つのフォトダイオードの出力毎にそれらの出力の合計をフォトダイオードアレイ１７並設方向に関して微分したものと考えることができる。
【００５０】
各差動アンプ１８ａ、１８ｂ、１８ｃ…の出力は、それぞれサンプルホールド回路２２ａ、２２ｂ、２２ｃ…により所定のタイミングでサンプルホールドされ、マルチプレクサ２３に入力される。マルチプレクサ２３は、サンプルホールドされた各差動アンプ１８ａ、１８ｂ、１８ｃ…の出力を、所定の順序に従ってＡ／Ｄ変換器２４に入力する。Ａ／Ｄ変換器２４はこれらの出力をデジタル化して信号処理部２０に入力する。
【００５１】
図５は、界面１０ｂで全反射された光ビーム１３の界面１０ｂへの入射角θ毎の光強度と、差動アンプ１８ａ、１８ｂ、１８ｃ…の出力との関係を説明するものである。ここで、光ビーム１３の界面１０ｂへの入射角θと上記反射された光ビーム１３の光強度Ｉとの関係は、同図（１）に示すようなものであるとする。
【００５２】
また図５（２）は、フォトダイオード１７ａ、１７ｂ、１７ｃ…の並設方向を示しており、先に説明した通り、これらのフォトダイオード１７ａ、１７ｂ、１７ｃ…の並設方向位置は上記入射角θと一義的に対応している。
【００５３】
またフォトダイオード１７ａ、１７ｂ、１７ｃ…の並設方向位置、つまりは入射角θと、差動アンプ１８ａ、１８ｂ、１８ｃ…の出力Ｉ’（反射光強度Ｉの微分値）との関係は、同図（３）に示すようなものとなる。
【００５４】
また図５（４）は、フォトダイオード１７ａ、１７ｂ、１７ｃ…により検出され、Ａ／Ｄ変換器２７によりＡ／Ｄ変換されて信号処理部２０に入力された信号により表されるビームプロファイルを示している。
【００５５】
信号処理部２０は、Ａ／Ｄ変換器２４から入力された微分値Ｉ’の値に基づいて、差動アンプ１８ａ、１８ｂ、１８ｃ…の中から、微分値として正の値を有し、かつ全反射減衰角θ_ＳＰに対応する微分値Ｉ’＝０に最も近い出力が得られているもの（図５（３）の例では差動アンプ１８ｋとなる）と、微分値として負の値を有し、かつ全反射減衰角θ_ＳＰに対応する微分値Ｉ’＝０に最も近い出力が得られているもの（図５（３）の例では差動アンプ１８ｊとなる）を選択し、それらの差動アンプが出力する微分値に基づいて、全反射減衰角θ_ＳＰを算出する。なお、場合によっては微分値Ｉ’＝０を出力している差動アンプが存在することもあり、そのときはその差動アンプに基づいて全反射減衰角θ_ＳＰを算出する。以後、所定時間が経過する毎に上記と同様な動作を繰り返し、全反射減衰角θ_ＳＰを算出し、測定開始時からの角度変化量を求め表示手段２１に表示する。
【００５６】
また、信号処理部２０は、上記全反射減衰角θ_ＳＰの算出結果とともに、フォトダイオード１７ａ、１７ｂ、１７ｃ…からＡ／Ｄ変換器２７を介して信号処理部２０に入力された信号により図５（４）に示すようなビームプロファイルを表示手段２１に表示させることができる。この際は、フォトダイオード１７ａ、１７ｂ、１７ｃ…の各出力をＡ／Ｄ変換器２７によりＡ／Ｄ変換し、その信号に基づいてビームプロファイルを生成するので、上記全反射減衰角θ_ＳＰを算出する際の解像度よりも高い解像度でビームプロファイルを生成することができる。
【００５７】
なお、本実施の形態においては、各フォトダイオード群の出力を全て加算して全反射減衰角θ_ＳＰの算出を行ったが、必ずしもそのような態様に限定されるものではなく、各フォトダイオードに接続されたスイッチを適宜開閉させてフォトダイオード群のそれぞれ１つもしくは２つのフォトダイオードの出力を加算して全反射減衰角θ_ＳＰの算出を行ってもよい。
【００５８】
また、フォトダイオード群は、３つのフォトダイオードからなるものに限らず、２つもしくは４つ以上の複数のフォトダイオードにより構成してもよい。
【００５９】
また、差動アンプアレイ１８の構成は図４に示す構成に限らず、例えば図６に示すような構成としてもよい。
【００６０】
この差動アンプアレイ１８’は、２つのフォトダイオード毎に設けられる差動アンプ１８ａ’、１８ｂ’、１８ｃ’…、これら差動アンプ１８ａ’、１８ｂ’、１８ｃ’…毎に設けられたスイッチ５１ａ’、５１ｂ’、５１ｃ’…、これらスイッチ５１ａ’、５１ｂ’、５１ｃ’…の３つの出力毎に設けられた加算機５０ａ’、５０ｂ’、５０ｃ’…から構成されている。
【００６１】
フォトダイオード１７ａとフォトダイオード１７ｆとが差動アンプ１８ａ’に、フォトダイオード１７ｂとフォトダイオード１７ｅとが差動アンプ１８ｂ’に、フォトダイオード１７ｃとフォトダイオード１７ｄとが差動アンプ１８ｃ’にそれぞれ接続されており、これら差動アンプ１８ａ’、１８ｂ’、１８ｃ’の出力は加算機５０ａ’により加算されてサンプルホールド回路２２ａに入力される。以後、フォトダイオード１７ａからフォトダイオードが３つずれる毎に、上記と同様の構成となっている。
【００６２】
すなわち、図４に示す差動アンプアレイ１８は、微分値として、各フォトダイオード群の出力を合計した後に、隣接する２つのフォトダイオード群毎にそれぞれの出力の合計の差分を求めるのに対し、この図６に示す差動アンプアレイ１８’は、隣接する２つのフォトダイオード群のフォトダイオード間の出力の差分を求め、この差分を合計するものであり、この差動アンプアレイ１８’を用いても上記差動アンプアレイ１８を用いた場合と同等の出力を得ることができる。
【００６３】
また、差動アンプアレイ１８を用いた場合と同様に、各フォトダイオード群のそれぞれのフォトダイオード間の出力の差分、すなわち３組のフォトダイオード間の出力の差分を全て合計して全反射減衰角θ_ＳＰの算出を行う態様に限定されるものではなく、各フォトダイオードに接続されたスイッチを適宜開閉させて１組のフォトダイオード間の出力の差分、あるいは２組のフォトダイオード間の出力の差分の合計値から全反射減衰角θ_ＳＰの算出を行ってもよい。例えば各フォトダイオードに入力される光量が十分である場合、あるいは光ビーム１３に生じる暗線の幅が狭い場合等には、隣接する１組のフォトダイオード間の出力の差分を用いて、全反射減衰角θ_ＳＰの算出を行い、各フォトダイオードに入力される光量が小さい場合あるいは光ビーム１３に生じる暗線の幅が広い場合等には、３組のフォトダイオード間の出力の差分を全て合計して全反射減衰角θ_ＳＰの算出を行うようにすれば、より正確に全反射減衰角θ_ＳＰを算出することができる。なお、差動アンプアレイ１８あるいは差動アンプアレイ１８’等のアナログ回路を用いて、微分値を求める場合には、デジタル加算処理によるサンプリングノイズの発生を抑制することができる。
【００６４】
上述のように、測定チップの金属膜１２に接している物質の誘電率つまりは屈折率が変化すると、それに応じて全反射減衰角θ_ＳＰも変化するため、この全反射減衰角θ_ＳＰの角度変化量を時間の経過とともに測定し続けることにより、金属膜１２に接している物質の屈折率変化を調べることができる。
【００６５】
なお金属膜１２の上に、試料液１１の中の特定物質と結合するセンシング物質３０を固定した場合、試料液１１とセンシング物質３０との結合状態に応じてセンシング物質３０の屈折率が変化するので、上記微分値Ｉ’を測定し続けることにより、この結合状態の変化の様子を調べることができる。つまりこの場合は、試料液１１およびセンシング物質３０の双方が、分析対象の試料となる。そのような特定物質とセンシング物質３０との組合せとしては、例えば抗原と抗体等が挙げられる。
【００６６】
上述の表面プラズモン測定装置は、一部の構成を変更することにより漏洩モード測定装置とすることができる。図７は、上述の表面プラズモン測定装置１０１の一部を変更して構成した漏洩モード測定装置の測定ユニットの側面図である。なおこの図７において、図２中の要素と同等の要素には同番号を付してあり、それらについての説明は特に必要の無い限り省略する。
【００６７】
この漏洩モード測定装置も、上述の表面プラズモン測定装置と同様に測定チップ９を用いるように構成されている。この測定チップ９の上面に形成された凹部１０ｃの底面にはクラッド層４０が形成され、さらにその上には光導波層４１が形成されている。これらクラッド層４０と光導波層４１とによって薄膜層が形成されている。
【００６８】
誘電体ブロック１０は、例えば合成樹脂やＢＫ７等の光学ガラスを用いて形成されている。一方クラッド層４０は、誘電体ブロック１０よりも低屈折率の誘電体や、金等の金属を用いて薄膜状に形成されている。また光導波層４１は、クラッド層４０よりも高屈折率の誘電体、例えばＰＭＭＡを用いてこれも薄膜状に形成されている。クラッド層４０の膜厚は、例えば金薄膜から形成する場合で３６．５ｎｍ、光導波層４１の膜厚は、例えばＰＭＭＡから形成する場合で７００ｎｍ程度とされる。
【００６９】
上記構成の漏洩モード測定装置において、レーザ光源１４から射出された光ビーム１３を誘電体ブロック１０を通してクラッド層４０に対して全反射角以上の入射角で入射させると、該光ビーム１３の多くの成分が誘電体ブロック１０とクラッド層４０との界面１０ｂで全反射するが、クラッド層４０を透過して光導波層４１に特定入射角で入射した特定波数の光は、該光導波層４１を導波モードで伝搬されるようになる。こうして導波モードが励起されると、特定入射角で入射した入射光のほとんどが光導波層４１に取り込まれるので、上記界面１０ｂに特定入射角で入射し、全反射された光の強度が鋭く低下する全反射減衰が生じる。
【００７０】
光導波層４１における導波光の波数は、該光導波層４１上の試料液１１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である全反射減衰角を知ることによって、試料液１１の屈折率や、それに関連する試料液１１の特性を分析することができ、上記実施の形態と同様の効果を得ることができる。
【００７１】
次に図８〜図１０を参照して本発明の第２の実施形態について説明する。図８は第２の実施形態である表面プラズモン測定装置の概略構成を示す平面図であり、図９は第２の実施形態である表面プラズモン測定装置の側面形状を示すものであり、図１０は、ビームの界面への入射角と演算処理部の出力との関係を示す図である。なお、図８および図９においては、図１および図２に示す第１の具体的な実施の形態と共通の要素については同番号を付し、特に必要のない限りその説明は省略する。
【００７２】
上記表面プラズモン測定装置２０１は、同様の構成の複数の表面プラズモン測定ユニット２０１Ａ、２０１Ｂ、２０１Ｃ…により構成されている。各測定ユニットの構成について、個別の要素を表す符号であるＡ、Ｂ、Ｃ…の符号は省略して説明する。
【００７３】
各測定ユニットは、測定チップ９と、レーザ光源１４と、入射光学系１５と、コリメーターレンズ１６と、光検出器１７と、Ａ／Ｄ変換器６０と、演算処理部６１と、コンピュータシステム等からなる信号処理部６２とからなる。なお、請求項に記載の演算手段は、演算処理部６１および信号処理部６２により構成される。演算処理部６１および信号処理部６２の動作は後述する。また、表面プラズモン測定装置２０１は、各測定ユニットの信号処理部６２Ａ、６２Ｂ、６２Ｃ…に接続された１つの表示手段２１を備えている。
【００７４】
以下、上記構成の表面プラズモン測定装置による試料分析について説明する。図９に示す通り、レーザ光源１４から射出された光ビーム１３は、入射光学系１５を通して、誘電体ブロック１０と金属膜１２との界面１０ｂ上に収束され、この界面１０ｂで全反射された光ビーム１３は、コリメーターレンズ１６を通して光検出器１７によって検出される。光検出器１７は、上記界面１０ｂにおいて種々の反射角で全反射された光ビーム１３の各成分を、それぞれ異なるフォトダイオード１７ａ、１７ｂ、１７ｃ…で受光する。そして、光検出器１７は、各フォトダイオード１７ａ、１７ｂ、１７ｃ…によって検出された上記光ビーム１３の強度分布を示す信号をＡ／Ｄ変換器６０に出力する。Ａ／Ｄ変換器６０でＡ／Ｄ変換された信号は演算処理部６１に入力される。なお、本実施の形態においては、解像度を向上させるために、第１の実施の形態に比べて、光検出器１７が界面１０ｂから離れた位置に配置されている。
【００７５】
演算処理部６１では、まず上記フォトダイオード１７ａ、１７ｂ、１７ｃ…の各出力から、隣接する３つのフォトダイオードの出力の平均値を、順次フォトダイオードを１つずつずらしながら算出し、その後、前後する平均値の差を求め、信号処理部６２へ出力する。すなわち、まず、フォトダイオード１７ａ、１７ｂ、１７ｃの平均値、フォトダイオード１７ｂ、１７ｃ、１７ｄの平均値、フォトダイオード１７ｃ、１７ｄ、１７ｅの平均値…を順次算出し、その後、フォトダイオード１７ｂ、１７ｃ、１７ｄの平均値−フォトダイオード１７ａ、１７ｂ、１７ｃの平均値、フォトダイオード１７ｃ、１７ｄ、１７ｅの平均値−フォトダイオード１７ｂ、１７ｃ、１７ｄの平均値…を順次求め、信号処理部６２へ出力する。これらの、平均値の差は、複数のフォトダイオード１７ａ、１７ｂ、１７ｃ…が出力する光検出信号を、隣接する３つのフォトダイオードの出力毎にそれらの出力の平均値をフォトダイオードアレイ１７の並設方向に関して微分した微分値Ｉ’と考えることができる。
【００７６】
図１０は、上記の演算処理部６１において演算処理を行う際の信号値を説明するものである。例えば、光ビーム１３の界面１０ｂへの入射角θと上記反射された光ビーム１３の光強度Ｉとの関係は、同図（１）に示すようなものであるとする。図１０（２）に、示すような配置で、フォトダイオード１７ａ、１７ｂ、１７ｃ…が設けられている場合には、同図（３）に示すような出力値が各フォトダイオード１７ａ、１７ｂ、１７ｃ…から出力される。すなわち、同図（３）は、演算処理部６１に入力される信号である。なお、同図（３）は、フォトダイオード１７ａ、１７ｂ、１７ｃ…の配列ピッチが、ビームプロファイルに対して狭いため、各フォトダイオード１７ａ、１７ｂ、１７ｃ…から出力される信号値が小さく、ノイズの影響を受けやすい場合の出力値の一例であり、凹凸の大きいグラフとなっている。同図（４）は、隣接する３つのフォトダイオードの出力の平均値を、順次フォトダイオードを１つずつずらしながら算出した値である。ノイズが相殺されるため、同図（３）に示すグラフに比べ、滑らかなグラフとなり、かつ同図（１）に示す、実際の入射角θと反射された光ビーム１３の光強度Ｉとの関係を示すグラフに近い形状のグラフが得られている。同図（５）は、各平均値の差、すなわち演算処理部６１から出力される微分値Ｉ’を示すものである。
【００７７】
信号処理部６２は、演算処理部６２から入力された微分値Ｉ’の値に基づいて、全反射減衰角θ_ＳＰに対応する微分値Ｉ’＝０に最も近い出力が得られているフォトダイオードの組（図１０（２）の例ではフォトダイオード１７ｈ、１７ｉおよび１７ｊとなる）から全反射減衰角θ_ＳＰを算出する。以後、所定時間が経過する毎に上記と同様な動作を繰り返し、全反射減衰角θ_ＳＰを算出し、測定開始時からの角度変化量を求め表示手段２１に表示する。試料液１１の中の特定物質とセンシング物質３０との結合状態に応じてセンシング物質３０の屈折率が変化するので、上記全反射減衰角θ_ＳＰの角度変化量を測定することにより、この結合状態の変化の様子を調べることができる。
【００７８】
なお、本実施の形態においては、３つのフォトダイオードの平均値から微分値Ｉ’を求め、この微分値Ｉ’に基づいて、全反射減衰角θ_ＳＰの算出を行ったが、必ずしもそのような態様に限定されるものではなく、隣接した２つのフォトダイオードの平均値あるいは隣接した４つ以上のフォトダイオードの平均値を順次求めて、微分値Ｉ’を求めてもよい。
【００７９】
本実施の形態においては、光検出器１７の隣接する３つのフォトダイオードの出力の平均値を、順次フォトダイオードを１つずつずらしながら算出し、その後、前後する平均値の差、すなわち微分値Ｉ’を求め、該微分値Ｉ’に基づいて、全反射減衰角θ_ＳＰの角度を測定するようにしたので、サイズが小さい受光素子を用いる場合あるいは受光素子が受光する光量が少ない場合であっても、ノイズの影響を受けにくく、高い解像度で、かつ正確に全反射減衰角θ_ＳＰの角度を測定することができる。なお、各フォトダイオードの平均値の代わりに合計値を使用してもよく、微分する際の信号値を大きくすることができる。また、平均値の代わりに、合計値を所望の値で除算した値、あるいは合計値に所望の値を乗算した値等を用いてもよい。
【００８０】
なお、演算処理部６１から、微分値に加え各３つのフォトダイオードの平均値を出力し、信号処理部６２において、この平均値に基づいてビームプロファイルを生成すれば、高い解像度で、かつノイズの影響の少ないビームプロファイルを生成することができる。また、第２の実施形態の表面プラズモン測定装置も、第１の実施形態と同様に、一部の構成を変更することにより漏洩モード測定装置とすることができる。
【図面の簡単な説明】
【図１】本発明の第１の実施の形態による表面プラズモン測定装置の概略構成を示す平面図
【図２】表面プラズモン測定装置の側面形状を示す図
【図３】測定チップの概略構成図
【図４】表面プラズモン測定装置の電気的構成を示すブロック図
【図５】光ビームの界面への入射角と差動アンプの出力との関係を示す図
【図６】表面プラズモン測定装置の電気的構成を示すブロック図
【図７】漏洩モード測定装置の一例を示す図
【図８】本発明の第２の実施の形態による表面プラズモン測定装置の概略構成を示す平面図
【図９】表面プラズモン測定装置の側面形状を示す図
【図１０】光ビームの界面への入射角と演算処理部の出力との関係を示す図
【符号の説明】
９　　　測定チップ
１０　　誘電体ブロック
１３　　光ビーム
１４　　レーザ光源
１５　　入射光学系
１６　　コリメータレンズ
１７　　光検出器
１８　　差動アンプアレイ
１９　　ドライバ
２０、６２　　信号処理部
２１　　表示手段
２７、６０　　Ａ／Ｄ変換器
６１　　　演算処理部
１０１、２０１　　表面プラズモン測定装置
１０１Ａ、１０１Ｂ、１０１Ｃ…　　表面プラズモン測定ユニット
２０１Ａ、２０１Ｂ、２０１Ｃ…　　表面プラズモン測定ユニット[0001]
BACKGROUND OF THE INVENTION
In the present invention, the light beam is totally reflected at the interface between the thin film layer in contact with the sample and the dielectric block to generate an evanescent wave, thereby measuring the change in the intensity of the totally reflected light beam and analyzing the sample. The present invention relates to a measurement method and a measurement apparatus using an evanescent wave for performing the above.
[0002]
[Prior art]
In the metal, free electrons collectively vibrate to generate a dense wave called a plasma wave. A quantized version of this dense wave generated on the metal surface is called surface plasmon.
[0003]
Conventionally, various surface plasmon measuring devices for analyzing the characteristics of a substance to be measured have been proposed by utilizing the phenomenon that this surface plasmon is excited by a light wave. Among them, one that uses a system called a Kretschmann arrangement is particularly well known (see, for example, Japanese Patent Laid-Open No. 6-167443).
[0004]
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.
[0005]
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.
[0006]
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.
[0007]
The incident angle at which this total reflection attenuation (ATR) occurs, that is, the total reflection attenuation angle θ _SP If the wave number of the surface plasmon is known, the dielectric constant of the substance to be measured can be obtained. In other words, the wave number of surface plasmon is K _SP , The angular frequency of the surface plasmon is ω, the speed of light in vacuum is c, and the dielectric constant of the metal and the substance to be measured is ε, respectively. _m , Ε _s Then, there is the following relationship.
[0008]
[Expression 1]

That is, the total reflection attenuation angle θ, which is the incident angle at which the reflected light intensity decreases. _SP By knowing, the dielectric constant εs of the substance to be measured, that is, the characteristic related to the refractive index can be obtained.
[0009]
In this type of surface plasmon measurement device, the total reflection attenuation angle θ _SP In order to measure the light intensity with high accuracy and with a large dynamic range, it is considered to use an arrayed light detection means as disclosed in Japanese Patent Application Laid-Open No. 11-326194. 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.
[0010]
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. Total reflection attenuation angle θ _SP In many cases, characteristics relating to the refractive index of the substance to be measured are obtained.
[0011]
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.
[0012]
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.
[0013]
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.
[0014]
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. When the sample solution dissolved in the solvent is added and the predetermined time elapses, the above-mentioned total reflection attenuation angle θ _SP Is measuring the angle.
[0015]
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, the total reflection attenuation angle θ _SP And the total reflection attenuation angle θ _SP By measuring whether or not the angle of the sensor has changed, the binding state between the analyte and the sensing substance is measured, and based on the result, it is determined whether or not the analyte is a specific substance that binds to the sensing substance. be able to. 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, rabbit anti-human IgG antibody can be immobilized on the surface of the thin film layer as a sensing substance, and human IgG antibody can be used as the specific substance.
[0016]
In order to measure the binding state between the analyte and the sensing substance, the total reflection attenuation angle θ _SP It is not always necessary to detect the angle itself. For example, a sample solution is added to the sensing substance, and then the total reflection attenuation angle θ _SP It is also possible to measure the amount of change in angle and measure the coupling state based on the magnitude of the amount of change in angle. If the above-described arrayed light detection means and differentiation means are applied to a measuring device using total reflection attenuation, the amount of change in the differential value is the total reflection attenuation angle θ. _SP 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 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 measurement chip in which a sensing substance is fixed on a thin film layer formed in advance on the bottom surface. The above-mentioned total reflection attenuation angle θ _SP The amount of angle change is measured.
[0017]
In addition, the applicant of the present invention has a measuring device using total reflection attenuation that can measure a large number of samples in a short time by sequentially measuring a plurality of measuring chips mounted on a turntable or the like. This is proposed in Japanese Unexamined Patent Publication No. 2001-330560.
[0018]
In addition, in Japanese Patent Application No. 2001-397411, the present applicant has also proposed a measuring apparatus using total reflection attenuation that performs measurement using a measuring chip provided with a plurality of sample liquid holding units. By using the measuring apparatus having such a configuration, it is possible to simultaneously measure a large number of samples without moving the measuring chip.
[0019]
[Problems to be solved by the invention]
In the measurement apparatus using evanescent waves such as the surface plasmon measurement apparatus and the leakage mode measurement apparatus described above, conventionally, in order to detect the intensity of the light beam reflected at the interface for each reflection angle, it is conventionally synchronized with the deflection of the light beam. Although a moving photodetector and an area sensor such as a CCD having a wide light receiving surface were used, the former can secure a relatively large dynamic range with respect to the measurement range of the total reflection elimination angle. It is difficult to perform sample analysis at high speed because of the need for a simple drive mechanism, and the latter is capable of high-speed analysis, but the resolution of area sensors such as CCDs and the dynamic range of charge accumulation are low. Therefore, it is difficult to ensure high analysis accuracy.
[0020]
For this reason, in order to avoid the above problem, the applicant of the present application has proposed that the light beam totally reflected at the interface by the light detecting means comprising a plurality of light receiving elements as described in the above-mentioned Japanese Patent Application Laid-Open No. 11-326194. The light detection signal output from each light receiving element of this light detecting means is differentiated for each output of two adjacent light receiving elements, and the total reflection attenuation angle θ is based on this differential value. _SP We have proposed a measuring device that calculates According to this measuring apparatus, the total reflection attenuation angle θ has a wider dynamic range than an area sensor such as a CCD. _SP Can be detected with high accuracy.
[0021]
By the way, in recent years, there is a demand for further knowing the beam profile of the light beam detected by the light detecting means in the measuring apparatus as described in the above-mentioned JP-A-11-326194. In order to increase the output signal of each light receiving element, the size of each light receiving element is increased, that is, the arrangement pitch of each light receiving element is increased, so that the resolution is lowered when generating a beam profile. There is a problem.
[0022]
Therefore, if the arrangement pitch of each light receiving element is made fine so that the beam profile can be detected with high resolution, the output signal of each light receiving element becomes small, and the total reflection attenuation angle θ accurately. _SP May not be able to be calculated. In addition, when the amount of light received by each light receiving element is small, there is a possibility that it is easily affected by noise.
[0023]
In view of the above circumstances, the present invention measures the intensity of a light beam by a plurality of light receiving elements, differentiates the light detection signal output from each light receiving element for each output of two adjacent light receiving elements, and generates dark lines. In the measuring device that detects the position, the total reflection attenuation angle θ _SP It is an object of the present invention to provide a measuring apparatus capable of calculating the beam profile and capable of detecting a beam profile with high resolution.
[0024]
[Means for Solving the Problems]
A measuring method according to the present invention generates a light beam with a measuring chip comprising a dielectric block, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer. A light source, an incident optical system that makes a light beam incident on the dielectric block at an incident angle at which the total reflection condition is obtained at the interface between the dielectric block and the thin film layer, and a plurality of light receiving elements. In a measurement method in which a sample is analyzed by a measuring device including a light detection means for measuring the intensity of a reflected light beam, a plurality of light receiving elements are divided into several light receiving element groups, and two adjacent light receiving elements The total output of each group is differentiated with respect to the parallel arrangement direction of the light receiving elements, and the sample is analyzed by obtaining a reflection angle at which the reflected light intensity at the interface takes a minimum value based on this differential value. It is characterized in.
[0025]
Another measurement method according to the present invention includes a dielectric chip, a thin film layer formed on one surface of the dielectric block, a measurement chip including a sample holding mechanism for holding a sample on the surface of the thin film layer, an optical A light source that generates a beam; an incident optical system that causes the light beam to be incident on the dielectric block at an incident angle that provides a total reflection condition at an interface between the dielectric block and the thin film layer; Each of two or more adjacent light receiving elements adjacent to each other in a measurement method for analyzing the sample by a measuring device comprising an element and a light detecting means for measuring the intensity of a light beam totally reflected at the interface Are sequentially calculated in the direction in which the light receiving elements are juxtaposed, the average value is differentiated with respect to the direction in which the light receiving elements are juxtaposed, and the reflected light intensity at the interface is minimized based on the differential value. Take a value It is characterized in that by determining the elevation angle to analyze the sample.
[0026]
In addition, a measuring apparatus according to the present invention includes a dielectric chip, a thin film layer formed on one surface of the dielectric block, a measuring chip including a sample holding mechanism for holding a sample on the surface of the thin film layer, and a light beam. A light source to be generated; an incident optical system that makes a light beam incident on the dielectric block at an incident angle that obtains a total reflection condition at the interface between the dielectric block and the thin film layer; and a plurality of light receiving elements, and the interface The light detection means for measuring the intensity of the light beam totally reflected by the light source and the plurality of light receiving elements are divided into several light receiving element groups, and the total of the outputs of each of the two adjacent light receiving element groups is calculated in parallel with the light receiving elements. And an arithmetic means for differentiating with respect to the installation direction and obtaining a reflection angle at which the reflected light intensity at the interface takes a minimum value based on the differential value.
[0027]
Another measurement apparatus according to the present invention includes a dielectric chip, a thin film layer formed on one surface of the dielectric block, a measurement chip including a sample holding mechanism for holding a sample on the surface of the thin film layer, an optical A light source that generates a beam; an incident optical system that causes the light beam to be incident on the dielectric block at an incident angle that provides a total reflection condition at an interface between the dielectric block and the thin film layer; A light detection means for measuring the intensity of a light beam totally reflected at the interface, and an average value of outputs of two or more adjacent light receiving elements adjacent to each other in order in the direction in which the light receiving elements are arranged side by side. And calculating means for differentiating the average value with respect to the direction in which the light receiving elements are juxtaposed and calculating a reflection angle at which the reflected light intensity at the interface takes a minimum value based on the differential value. It is what.
[0028]
As the above-described measuring apparatus, the above-described surface plasmon measuring apparatus using a metal film as the thin film layer, a clad layer formed on one surface of a dielectric block, and an optical waveguide layer formed on the clad layer And the above-described leaky mode measuring device using the above-mentioned layer as the thin film layer.
[0029]
In the measurement apparatus according to the present invention, there are various methods for analyzing the sample by measuring the intensity of the light beam totally reflected at the interface by the light detection means. For example, the light beam is subjected to the total reflection condition at the interface. Detecting the position (angle) of the dark line generated by the total reflection attenuation by measuring the intensity of the light beam totally reflected at the interface at each position corresponding to each incident angle. Sample analysis may be performed according to D. V. Noort, K .; johansen, C.I. -F. Mandenius, Porous Gold in Surface Plasmon Resonance Measurement, EUROSENSORS XIII, 1999, pp. As described in 585-588, a light beam having a plurality of wavelengths is incident at an incident angle at which the total reflection condition is obtained at the interface, and the intensity of the light beam totally reflected at the interface is measured for each wavelength. Thus, the sample analysis may be performed by detecting the degree of total reflection attenuation (the position and degree of the dark line) for each wavelength.
[0030]
In the present invention, “dividing a plurality of light receiving elements into several light receiving element groups and differentiating the sum of outputs for each of the two adjacent light receiving element groups with respect to the direction in which the light receiving elements are arranged in parallel” means After summing up the outputs of the element groups, not only when the sum of the outputs of each of the two adjacent light receiving element groups is differentiated with respect to the parallel arrangement direction of the light receiving elements, but the light receiving elements of the two adjacent light receiving element groups are respectively Any one of the calculation processes may be used as long as the same differential value is obtained as a result, for example, by summing the differential values after differentiating one by one with respect to the direction in which the light receiving elements are arranged side by side.
[0031]
For example, six light receiving elements arranged in order from the first light receiving element to the sixth light receiving element are divided into a first light receiving element group consisting of the first to third light receiving elements and a fourth light receiving element to the sixth light receiving element. Are divided into second light receiving element groups, and the total of the outputs of the light receiving element groups is differentiated with respect to the parallel arrangement direction of the light receiving elements, the respective light receiving elements of the first to third light receiving elements of the first light receiving element group The outputs of the elements are summed, the outputs of the respective light receiving elements of Nos. 4 to 6 in the second light receiving element group are summed, and the sum of the outputs of the first light receiving element group and the second light receiving element group May be differentiated with respect to the direction in which the light receiving elements are arranged, and the outputs of the first and fourth light receiving elements, the second and fifth light receiving elements, and the third and sixth light receiving elements, respectively. Differentiation may be performed with respect to the direction in which the light receiving elements are arranged, and the three differential values may be summed. Alternatively, the difference between the outputs of the first and sixth light receiving elements, the difference between the outputs of the second and fifth light receiving elements, and the difference between the outputs of the third and fourth light receiving elements are summed, and the distance between the light receiving element groups In this case, it may be divided by a distance corresponding to three light receiving elements.
[0032]
Further, “the average value of the outputs of each of a predetermined number of adjacent two or more light receiving elements is sequentially calculated in the direction in which the light receiving elements are juxtaposed, and the average value is differentiated with respect to the direction in which the light receiving elements are juxtaposed.” Is not limited to the case where, after calculating the average value of the outputs of each predetermined number of light receiving elements, the average value is differentiated with respect to the parallel arrangement direction of the light receiving elements, for example, first, the differential value between two adjacent light receiving elements is calculated. Next, the average value of a predetermined number of consecutive differential values is sequentially calculated in the direction in which the light receiving elements are arranged side by side, and as long as the same differential value is obtained as a result, what is the calculation process? There may be.
[0033]
For example, from four light receiving elements arranged in order from the first light receiving element to the fourth light receiving element, the average value of the output of each of the three adjacent light receiving elements is sequentially calculated in the parallel arrangement direction of the light receiving elements, When differentiating the average value with respect to the direction in which the light receiving elements are arranged side by side, the average value of the outputs of the first to third light receiving elements and the average value of the outputs of the second to fourth light receiving elements are obtained, The average value may be differentiated with respect to the direction in which the light receiving elements are arranged, and the outputs of the first and second light receiving elements, the second and third light receiving elements, and the third and fourth light receiving elements are respectively Differentiation may be performed with respect to the juxtaposed direction, and an average value of these differential values may be obtained. The “average value of the outputs of each of a predetermined number of adjacent two or more light receiving elements” is not limited to the average value itself, and may be any value that reflects the average value. It may be a total value of outputs of the respective light receiving elements, a value obtained by dividing the total value by a desired value, a value obtained by multiplying the total value by a desired value, or the like.
[0034]
In each of the above measuring methods and measuring apparatuses, “determining the reflection angle at which the reflected light intensity at the interface takes a minimum value based on the differential value” is limited to obtaining the reflection angle itself taking the minimum value. This includes not only a case where a value that reflects a reflection angle that takes a minimum value, for example, a differential value in the vicinity of the reflection angle that takes a minimum value. The “minimum value” means a minimum value that reflects the state of total reflection attenuation, and does not include those that are caused by the influence of noise or the like.
[0035]
【The invention's effect】
In a measuring apparatus that measures the intensity of a light beam by a plurality of light receiving elements, differentiates the light detection signal output from each light receiving element for each output of two adjacent light receiving elements, and detects the position of the dark line. In the measuring method and the measuring apparatus of the invention, the calculation means divides the plurality of light receiving elements into several light receiving element groups, and the total of the outputs of each of the two adjacent light receiving element groups is related to the parallel arrangement direction of the light receiving elements. Differentiating, and based on this differential value, the reflection angle at which the reflected light intensity at the interface takes a minimum value is obtained, so even if a light receiving element having a small size is used, the total reflection attenuation angle θ _SP Is calculated, the plurality of light receiving elements are divided into several light receiving element groups, and the outputs are summed for each light receiving element group, so that the output of the signal when differentiating can be increased. As a result, a light receiving element array having a fine arrangement pitch can be used, so that a beam profile can be detected with high resolution and a total reflection attenuation angle θ. _SP Can be calculated accurately.
[0036]
Another measuring method and measuring apparatus according to the present invention is a measuring apparatus comprising a plurality of light receiving elements and comprising a light detecting means for measuring the intensity of a light beam totally reflected at the interface between the dielectric block and the thin film layer. An average value of outputs of two or more adjacent light receiving elements adjacent to each other is sequentially calculated in the direction in which the light receiving elements are arranged in parallel, and the average value is differentiated with respect to the direction in which the light receiving elements are arranged in parallel. Based on this, the reflection angle at which the reflected light intensity at the interface takes a minimum value is obtained, so even if a light receiving element with a small size is used or the amount of light received by the light receiving element is small, the influence of noise The reflection angle at which the reflected light intensity takes a minimum value with high accuracy, that is, the total reflection attenuation angle θ. _SP Can be requested. In addition, an average value equal to the number of light receiving elements can be calculated, and the total reflection attenuation angle θ can be obtained with high resolution. _SP Can be requested. Further, a high resolution beam profile can be obtained by using this average value.
[0037]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings. The measurement apparatus according to the first embodiment of the present invention is a surface plasmon measurement apparatus that can simultaneously analyze a plurality of samples by allowing light beams to enter a plurality of dielectric blocks in parallel. 1 is a plan view showing a schematic configuration of a surface plasmon measuring apparatus according to the present embodiment, and FIG. 2 shows a side shape of the surface plasmon measuring apparatus.
[0038]
The surface plasmon measuring device 101 is composed of a plurality of surface plasmon measuring units 101A, 101B, 101C.
[0039]
The configuration of each measurement unit will be described by omitting the symbols A, B, C,... Each measurement unit is reflected by the measurement chip 9, a laser light source 14 that is a light source that generates a light beam 13, an incident optical system 15 that makes the light beam 13 incident on the measurement chip 9, and the measurement chip 9. A collimator lens 16 that collimates the light beam 13 and emits the light beam 13 toward the light detector 17, a light detector 17 that receives the light beam 13 emitted from the collimator lens 16 and detects light intensity, A differential amplifier array 18 connected to the detector 17, a driver 19 connected to the differential amplifier array 18, a signal processing unit 20 including a computer system connected to the driver 19, and an output from the photodetector 17 The A / D converter 27 converts the converted signal into a digital signal and outputs the digital signal to the signal processing unit 20. Note that the calculation means described in the claims includes a differential amplifier array 18, a driver 19, and a signal processing unit 20.
[0040]
The measurement chip 9 is a dielectric having a shape in which a part including an apex angle where four ridges of a quadrangular pyramid gather is cut out and a concave portion 10c functioning as a sample holding mechanism for storing the sample liquid 11 is formed on the bottom surface of the quadrangular pyramid. The block 10 and a metal film 12 which is a thin film layer made of, for example, gold, silver, copper, aluminum, or the like, formed on the bottom surface of the concave portion 10c of the dielectric block 10. The dielectric block 10 can be formed of, for example, a transparent resin. Note that a sensing substance 30 described later may be provided on the metal film 12. Further, as shown in FIG. 3, the dielectric block 10 of the measurement chip 9 may be configured integrally with the dielectric blocks of the measurement chips of a plurality of adjacent surface plasmon measurement units.
[0041]
The incident optical system 15 includes a collimator lens 15a that collimates the light beam 13 emitted from the laser light source 14, and a condensing lens 15b that converges the collimated light beam 13 toward the interface 10b. It is composed of
[0042]
Since the light beam 13 is condensed by the condenser lens 15b as described above, the light beam 13 includes components incident on the interface 10b at various incident angles θ. In addition, this incident angle (theta) shall be an angle more than a total reflection angle. For this reason, the light beam 13 totally reflected at the interface 10b includes components totally reflected at various reflection angles. The incident optical system 15 may be configured to allow the light beam 13 to be incident in a defocused state without condensing the light beam 13 on the interface 10b in the form of dots. By doing so, since the light beam 13 is totally reflected in a wider area on the interface 10b, the detection error of the total reflection attenuation state is averaged, and the measurement accuracy of the total reflection attenuation angle can be improved.
[0043]
The light beam 13 is incident on the interface 10b as p-polarized light. In order to do so, the laser light source 14 may be arranged in advance so that the polarization direction is the predetermined direction. In addition, in order to make the light beam 13 incident on the interface 10b as p-polarized light, the direction of polarization of the light beam 13 may be controlled by a wave plate. Further, the surface plasmon measuring apparatus 101 includes one display means 21 connected to the signal processing units 20A, 20B, 20C... Of each measuring unit.
[0044]
Hereinafter, sample analysis by the surface plasmon measuring apparatus having the above-described configuration will be described. As shown in FIG. 2, the light beam 13 emitted from the laser light source 14 is converged on the interface 10 b between the dielectric block 10 and the metal film 12 through the incident optical system 15.
[0045]
The light beam 13 converged on the interface 10 b and totally reflected by the interface 10 b is detected by the photodetector 17 through the collimator lens 16. The photodetector 17 is a photodiode array in which a plurality of photodiodes 17a, 17b, 17c,..., Which are a plurality of light receiving elements, are arranged in a line, and the arrangement direction of the photodiodes is substantially parallel to the paper surface of FIG. In this manner, the light beam 13 is collimated through the collimator lens 16 and is arranged so as to be substantially orthogonal to the propagation direction of the incident light beam 13. Therefore, different photodiodes 17a, 17b, 17c,... Receive the components of the light beam 13 totally reflected at various reflection angles at the interface 10b. The photodetector 17 outputs a signal indicating the intensity distribution of the light beam 13 detected by the photodiodes 17a, 17b, 17c... To the differential amplifier array 18 and the A / D converter 27. The signal A / D converted by the A / D converter 27 is input to the signal processing unit 20.
[0046]
The specific incident angle θ at the interface 10b _SP Since the component of the light beam 13 incident on the surface excites surface plasmons at the interface between the metal film 12 and the substance in contact with the metal film 12, the reflected light intensity sharply decreases for this light. That is, the specific incident angle θ _SP Is the total reflection attenuation angle, and this angle θ _SP The reflected light intensity shows a minimum value. The region where the reflected light intensity decreases is observed as a dark line in the light beam 13 totally reflected by the interface 10b, as indicated by D in FIG.
[0047]
Next, processing of a signal indicating the intensity distribution of the light beam 13 output from the photodetector 17 will be described in detail. FIG. 4 is a block diagram showing an electrical configuration of the surface plasmon measuring apparatus. As shown in the figure, the differential amplifier array 18 includes switches 51a, 51b, 51c... Provided for each of 17a, 17b, 17c..., And adders 50a provided for each of the three outputs of these switches 51a, 51b, 51c. 50b, 50c,..., Differential amplifiers 18a, 18b, 18c,... Provided for every two adders adjacent to each other. That is, in this embodiment, a photodiode group (light receiving element group) is formed by three photodiodes. The switches 51a, 51b, 51c... Are opened and closed based on instructions from the signal processing unit 20.
[0048]
The driver 19 samples and holds the outputs of the differential amplifiers 18a, 18b, 18c... Of the differential amplifier array 18, and the sample hold circuits 22a, 22b, 22c. A multiplexer 23 to which each output is input, an A / D converter 24 that digitizes the output of the multiplexer 23 and inputs it to the signal processing unit 20, a drive circuit that drives the multiplexer 23 and the sample hold circuits 22a, 22b, 22c. 25 and a controller 26 that controls the operation of the drive circuit 25 based on an instruction from the signal processing unit 20.
[0049]
The outputs of the photodiodes 17a, 17b, 17c,... Are added every three outputs by the adders 50a, 50b, 50c, and the outputs of the adders 50a, 50b, 50c,. Input to the dynamic amplifiers 18a, 18b, 18c. At this time, the outputs of two adders adjacent to each other are input to a common differential amplifier. Therefore, the outputs of the differential amplifiers 18a, 18b, 18c,... Are the light detection signals output from the plurality of photodiodes 17a, 17b, 17c,. It can be considered that it is differentiated with respect to the 17 juxtaposed direction.
[0050]
The outputs of the differential amplifiers 18a, 18b, 18c... Are sampled and held at predetermined timings by the sample hold circuits 22a, 22b, 22c. The multiplexer 23 inputs the sampled and held outputs of the differential amplifiers 18a, 18b, 18c... To the A / D converter 24 in a predetermined order. The A / D converter 24 digitizes these outputs and inputs them to the signal processing unit 20.
[0051]
FIG. 5 illustrates the relationship between the light intensity for each incident angle θ of the light beam 13 totally reflected at the interface 10b and the output of the differential amplifiers 18a, 18b, 18c. Here, it is assumed that the relationship between the incident angle θ of the light beam 13 on the interface 10b and the light intensity I of the reflected light beam 13 is as shown in FIG.
[0052]
5 (2) shows the direction in which the photodiodes 17a, 17b, 17c,... Are arranged side by side. As described above, the positions of the photodiodes 17a, 17b, 17c,. Corresponds uniquely to θ.
[0053]
Further, the relationship between the positions of the photodiodes 17a, 17b, 17c,..., That is, the incident angle θ, and the output I ′ (differential value of the reflected light intensity I) of the differential amplifiers 18a, 18b, 18c,. The result is as shown in FIG.
[0054]
5 (4) shows a beam profile represented by a signal detected by the photodiodes 17a, 17b, 17c... And A / D converted by the A / D converter 27 and input to the signal processing unit 20. ing.
[0055]
Based on the value of the differential value I ′ input from the A / D converter 24, the signal processing unit 20 has a positive value as a differential value from among the differential amplifiers 18a, 18b, 18c. Total reflection attenuation angle θ _SP The output closest to the differential value I ′ = 0 corresponding to is obtained (in the example of FIG. 5 (3), the differential amplifier 18k), and the differential value has a negative value and is totally reflected. Attenuation angle θ _SP Are selected (the differential amplifier 18j in the example of FIG. 5 (3)), and the differential value output by those differential amplifiers is selected. Based on the total reflection attenuation angle θ _SP Is calculated. In some cases, there may be a differential amplifier that outputs a differential value I ′ = 0. In that case, the total reflection attenuation angle θ is based on the differential amplifier. _SP Is calculated. Thereafter, the same operation as described above is repeated every time a predetermined time elapses, and the total reflection attenuation angle θ _SP Is calculated, and the amount of change in angle from the start of measurement is obtained and displayed on the display means 21.
[0056]
Further, the signal processing unit 20 is configured so that the total reflection attenuation angle θ _SP And a beam profile as shown in FIG. 5 (4) is displayed on the display means 21 by a signal input from the photodiodes 17a, 17b, 17c... To the signal processing unit 20 via the A / D converter 27. Can be made. At this time, each output of the photodiodes 17a, 17b, 17c,... Is A / D converted by the A / D converter 27, and a beam profile is generated based on the signal. _SP It is possible to generate a beam profile at a higher resolution than the resolution used when calculating.
[0057]
In this embodiment, the total reflection attenuation angle θ is obtained by adding all the outputs of each photodiode group. _SP However, the present invention is not necessarily limited to such a mode. The switches connected to the respective photodiodes are appropriately opened and closed, and the outputs of one or two photodiodes in each photodiode group are added. Total reflection attenuation angle θ _SP May be calculated.
[0058]
Further, the photodiode group is not limited to the one composed of three photodiodes, and may be composed of a plurality of two or four or more photodiodes.
[0059]
Further, the configuration of the differential amplifier array 18 is not limited to the configuration illustrated in FIG. 4, and may be configured as illustrated in FIG. 6, for example.
[0060]
This differential amplifier array 18 ′ includes differential amplifiers 18a ′, 18b ′, 18c ′... Provided for every two photodiodes, and a switch 51a provided for each of these differential amplifiers 18a ′, 18b ′, 18c ′. ', 51b', 51c '... and adders 50a', 50b ', 50c' ... provided for each of the three outputs of these switches 51a ', 51b', 51c '....
[0061]
The photodiode 17a and the photodiode 17f are connected to the differential amplifier 18a ′, the photodiode 17b and the photodiode 17e are connected to the differential amplifier 18b ′, and the photodiode 17c and the photodiode 17d are connected to the differential amplifier 18c ′, respectively. The outputs of these differential amplifiers 18a ', 18b', 18c 'are added by an adder 50a' and input to the sample hold circuit 22a. Thereafter, every time three photodiodes are displaced from the photodiode 17a, the configuration is the same as described above.
[0062]
That is, the differential amplifier array 18 shown in FIG. 4 calculates the difference between the outputs of each of the two adjacent photodiode groups after summing up the outputs of the photodiode groups as a differential value. The differential amplifier array 18 ′ shown in FIG. 6 obtains the difference in output between the photodiodes of two adjacent photodiode groups, and sums up the differences. Using this differential amplifier array 18 ′, Also, an output equivalent to that obtained when the differential amplifier array 18 is used can be obtained.
[0063]
Similarly to the case where the differential amplifier array 18 is used, the total output attenuation angle is obtained by adding all the output differences between the photodiodes of each photodiode group, that is, the output differences between the three sets of photodiodes. θ _SP Is not limited to the mode of calculating the above, and the switch connected to each photodiode is appropriately opened and closed so that the difference in output between one set of photodiodes or the sum of the difference in output between two sets of photodiodes is calculated. Total reflection attenuation angle θ _SP May be calculated. For example, when the amount of light input to each photodiode is sufficient, or when the width of the dark line generated in the light beam 13 is narrow, the total reflection attenuation is performed using the difference in output between a pair of adjacent photodiodes. Angle θ _SP When the amount of light input to each photodiode is small, or when the width of the dark line generated in the light beam 13 is wide, the total output attenuation between all three sets of photodiodes is summed up. Angle θ _SP Is calculated more accurately, the total reflection attenuation angle θ is more accurate. _SP Can be calculated. In the case where the differential value is obtained using an analog circuit such as the differential amplifier array 18 or the differential amplifier array 18 ′, it is possible to suppress the occurrence of sampling noise due to digital addition processing.
[0064]
As described above, when the dielectric constant, that is, the refractive index of the substance in contact with the metal film 12 of the measurement chip changes, the total reflection attenuation angle θ is accordingly changed. _SP The total reflection attenuation angle θ _SP By continuously measuring the amount of change in the angle with time, the change in the refractive index of the substance in contact with the metal film 12 can be examined.
[0065]
When a sensing substance 30 that binds to a specific substance in the sample liquid 11 is fixed on the metal film 12, the refractive index of the sensing substance 30 changes according to the binding state between the sample liquid 11 and the sensing substance 30. Therefore, by continuously measuring the differential value I ′, it is possible to examine the state of change in the coupling state. That is, in this case, both the sample liquid 11 and the sensing substance 30 are samples to be analyzed. Examples of the combination of the specific substance and the sensing substance 30 include an antigen and an antibody.
[0066]
The above-described surface plasmon measuring device can be a leakage mode measuring device by changing a part of the configuration. FIG. 7 is a side view of a measurement unit of a leaky mode measurement apparatus configured by changing a part of the surface plasmon measurement apparatus 101 described above. In FIG. 7, the same elements as those in FIG. 2 are denoted by the same reference numerals, and description thereof will be omitted unless particularly necessary.
[0067]
This leakage mode measuring device is also configured to use the measuring chip 9 as in the above-described surface plasmon measuring device. A clad layer 40 is formed on the bottom surface of the recess 10c formed on the upper surface of the measurement chip 9, and an optical waveguide layer 41 is further formed thereon. A thin film layer is formed by the cladding layer 40 and the optical waveguide layer 41.
[0068]
The dielectric block 10 is formed using optical glass, such as a synthetic resin and BK7, for example. On the other hand, the cladding layer 40 is formed in a thin film shape using a dielectric having a lower refractive index than the dielectric block 10 or a metal such as gold. The optical waveguide layer 41 is also formed into a thin film using a dielectric having a higher refractive index than that of the cladding layer 40, such as PMMA. The thickness of the cladding layer 40 is, for example, 36.5 nm when formed from a gold thin film, and the thickness of the optical waveguide layer 41 is, for example, about 700 nm when formed from PMMA.
[0069]
In the leakage mode measuring apparatus having the above configuration, when the light beam 13 emitted from the laser light source 14 is incident on the cladding layer 40 through the dielectric block 10 at an incident angle equal to or greater than the total reflection angle, The component is totally reflected at the interface 10b between the dielectric block 10 and the clad layer 40, but light having a specific wave number that is transmitted through the clad layer 40 and incident on the optical waveguide layer 41 at a specific incident angle passes through the optical waveguide layer 41. Propagated in guided mode. When the waveguide mode is excited in this way, most of the incident light incident at the specific incident angle is taken into the optical waveguide layer 41, so that the intensity of the light incident on the interface 10b at the specific incident angle and totally reflected is sharp. Decreasing total reflection attenuation occurs.
[0070]
Since the wave number of the guided light in the optical waveguide layer 41 depends on the refractive index of the sample liquid 11 on the optical waveguide layer 41, the total reflection attenuation angle that is the specific incident angle at which total reflection attenuation occurs is known. The refractive index of the liquid 11 and the characteristics of the sample liquid 11 associated therewith can be analyzed, and the same effect as in the above embodiment can be obtained.
[0071]
Next, a second embodiment of the present invention will be described with reference to FIGS. FIG. 8 is a plan view showing a schematic configuration of a surface plasmon measuring apparatus according to the second embodiment, FIG. 9 shows a side shape of the surface plasmon measuring apparatus according to the second embodiment, and FIG. It is a figure which shows the relationship between the incident angle to the interface of a beam, and the output of an arithmetic processing part. In FIG. 8 and FIG. 9, elements common to those in the first specific embodiment shown in FIG. 1 and FIG. 2 are denoted by the same reference numerals, and description thereof is omitted unless particularly necessary.
[0072]
The surface plasmon measuring device 201 is composed of a plurality of surface plasmon measuring units 201A, 201B, 201C,. The configuration of each measurement unit will be described by omitting the symbols A, B, C,...
[0073]
Each measurement unit includes a measurement chip 9, a laser light source 14, an incident optical system 15, a collimator lens 16, a photodetector 17, an A / D converter 60, an arithmetic processing unit 61, a computer system, and the like. And a signal processing unit 62. Note that the calculation means described in the claims includes an arithmetic processing unit 61 and a signal processing unit 62. The operations of the arithmetic processing unit 61 and the signal processing unit 62 will be described later. Further, the surface plasmon measuring apparatus 201 includes one display means 21 connected to the signal processing units 62A, 62B, 62C,... Of each measuring unit.
[0074]
Hereinafter, sample analysis by the surface plasmon measuring apparatus having the above-described configuration will be described. As shown in FIG. 9, the light beam 13 emitted from the laser light source 14 is converged on the interface 10b between the dielectric block 10 and the metal film 12 through the incident optical system 15, and is totally reflected by the interface 10b. The beam 13 is detected by a photodetector 17 through a collimator lens 16. The photodetector 17 receives each component of the light beam 13 totally reflected at the interface 10b at various reflection angles by different photodiodes 17a, 17b, 17c. Then, the photodetector 17 outputs a signal indicating the intensity distribution of the light beam 13 detected by each photodiode 17a, 17b, 17c... To the A / D converter 60. The signal A / D converted by the A / D converter 60 is input to the arithmetic processing unit 61. In the present embodiment, in order to improve the resolution, the photodetector 17 is arranged at a position farther from the interface 10b than in the first embodiment.
[0075]
The arithmetic processing unit 61 first calculates the average value of the outputs of the three adjacent photodiodes from the outputs of the photodiodes 17a, 17b, 17c,... The difference between the average values is obtained and output to the signal processing unit 62. That is, first, the average value of the photodiodes 17a, 17b, 17c, the average value of the photodiodes 17b, 17c, 17d, the average value of the photodiodes 17c, 17d, 17e,... The average value of 17d—the average value of the photodiodes 17a, 17b, and 17c, the average value of the photodiodes 17c, 17d, and 17e—the average value of the photodiodes 17b, 17c, and 17d, and the like are sequentially obtained and output to the signal processing unit 62. The difference between the average values is obtained by calculating the photodetection signal output from the plurality of photodiodes 17a, 17b, 17c,... It can be considered as a differential value I ′ differentiated with respect to the installation direction.
[0076]
FIG. 10 illustrates signal values when the arithmetic processing unit 61 performs arithmetic processing. For example, it is assumed that the relationship between the incident angle θ of the light beam 13 on the interface 10b and the light intensity I of the reflected light beam 13 is as shown in FIG. When the photodiodes 17a, 17b, 17c,... Are provided in the arrangement as shown in FIG. 10 (2), the output values as shown in FIG. 10 (3) are the respective photodiodes 17a, 17b, 17c. Is output from…. That is, (3) in the figure is a signal input to the arithmetic processing unit 61. In FIG. 3 (3), since the arrangement pitch of the photodiodes 17a, 17b, 17c... Is narrow with respect to the beam profile, the signal value output from each photodiode 17a, 17b, 17c. It is an example of an output value when it is easily affected, and is a graph with large unevenness. FIG. 4 (4) is a value obtained by calculating the average value of the outputs of three adjacent photodiodes while sequentially shifting the photodiodes one by one. Since the noise is canceled out, the graph becomes smoother than the graph shown in FIG. 3C, and the actual incident angle θ and the light intensity I of the reflected light beam 13 shown in FIG. A graph having a shape close to the graph indicating the relationship is obtained. FIG. 5 (5) shows the difference between the average values, that is, the differential value I ′ output from the arithmetic processing unit 61.
[0077]
Based on the value of the differential value I ′ input from the arithmetic processing unit 62, the signal processing unit 62 performs a total reflection attenuation angle θ. _SP The total reflection attenuation angle θ from the set of photodiodes (in the example of FIG. 10 (2), which are the photodiodes 17h, 17i, and 17j) from which the output closest to the differential value I ′ = 0 corresponding to is obtained. _SP Is calculated. Thereafter, the same operation as described above is repeated every time a predetermined time elapses, and the total reflection attenuation angle θ _SP Is calculated, and the amount of change in angle from the start of measurement is obtained and displayed on the display means 21. Since the refractive index of the sensing substance 30 changes according to the binding state between the specific substance in the sample liquid 11 and the sensing substance 30, the total reflection attenuation angle θ _SP By measuring the amount of change in the angle, it is possible to investigate the state of change in the coupling state.
[0078]
In the present embodiment, the differential value I ′ is obtained from the average value of the three photodiodes, and the total reflection attenuation angle θ is calculated based on the differential value I ′. _SP However, the present invention is not necessarily limited to such an embodiment. The average value of two adjacent photodiodes or the average value of four or more adjacent photodiodes is sequentially obtained to obtain a differential value I ′. You may ask for.
[0079]
In the present embodiment, the average value of the outputs of three adjacent photodiodes of the photodetector 17 is calculated while sequentially shifting the photodiodes one by one, and then the difference between the average values before and after, that is, the differential value I 'Is obtained, and based on the differential value I', the total reflection attenuation angle θ _SP The angle is measured so that even if a light receiving element with a small size is used or the amount of light received by the light receiving element is small, it is not easily affected by noise, and is attenuated in total reflection with high resolution and accuracy. Angle θ _SP Can be measured. The total value may be used instead of the average value of each photodiode, and the signal value at the time of differentiation can be increased. Instead of the average value, a value obtained by dividing the total value by a desired value, a value obtained by multiplying the total value by a desired value, or the like may be used.
[0080]
If the arithmetic processing unit 61 outputs an average value of each of the three photodiodes in addition to the differential value, and the signal processing unit 62 generates a beam profile based on the average value, it is possible to obtain a high resolution and noise level. A beam profile with less influence can be generated. Further, the surface plasmon measuring apparatus of the second embodiment can be a leakage mode measuring apparatus by changing a part of the configuration as in the first embodiment.
[Brief description of the drawings]
FIG. 1 is a plan view showing a schematic configuration of a surface plasmon measuring apparatus according to a first embodiment of the present invention.
FIG. 2 is a view showing a side shape of a surface plasmon measuring device.
FIG. 3 is a schematic configuration diagram of a measurement chip.
FIG. 4 is a block diagram showing an electrical configuration of the surface plasmon measuring apparatus.
FIG. 5 is a diagram showing the relationship between the incident angle of the light beam on the interface and the output of the differential amplifier.
FIG. 6 is a block diagram showing an electrical configuration of the surface plasmon measuring apparatus.
FIG. 7 is a diagram showing an example of a leakage mode measurement device
FIG. 8 is a plan view showing a schematic configuration of a surface plasmon measuring apparatus according to a second embodiment of the present invention.
FIG. 9 is a diagram showing a side shape of a surface plasmon measuring device.
FIG. 10 is a diagram showing the relationship between the incident angle of the light beam on the interface and the output of the arithmetic processing unit.
[Explanation of symbols]
9 Measuring chip
10 Dielectric block
13 Light beam
14 Laser light source
15 Incident optical system
16 Collimator lens
17 Photodetector
18 Differential amplifier array
19 Driver
20, 62 Signal processor
21 Display means
27, 60 A / D converter
61 Arithmetic processing part
101, 201 Surface plasmon measuring device
101A, 101B, 101C ... Surface plasmon measurement unit
201A, 201B, 201C ... Surface plasmon measurement unit

Claims (4)

A measurement chip comprising a dielectric block, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer;
A light source that generates a light beam;
An incident optical system that causes the light beam to be incident on the dielectric block at an incident angle at which a total reflection condition is obtained at an interface between the dielectric block and the thin film layer;
In a measurement method for analyzing the sample by a measurement device comprising a plurality of light receiving elements and comprising a light detection means for measuring the intensity of a light beam totally reflected at the interface,
Dividing the plurality of light receiving elements into several light receiving element groups;
Differentiating the sum of the outputs for each of the two adjacent light receiving element groups with respect to the parallel arrangement direction of the light receiving elements,
A measurement method characterized in that the sample is analyzed by obtaining a reflection angle at which the reflected light intensity at the interface takes a minimum value based on the differential value. A measurement chip comprising a dielectric block, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer;
A light source that generates a light beam;
An incident optical system that causes the light beam to be incident on the dielectric block at an incident angle at which a total reflection condition is obtained at an interface between the dielectric block and the thin film layer;
In a measurement method for analyzing the sample by a measurement device comprising a plurality of light receiving elements and comprising a light detection means for measuring the intensity of a light beam totally reflected at the interface,
An average value of outputs of two or more adjacent light receiving elements adjacent to each other is sequentially calculated in the direction in which the light receiving elements are arranged,
Differentiating the average value with respect to the parallel arrangement direction of the light receiving elements,
A measurement method characterized in that the sample is analyzed by obtaining a reflection angle at which the reflected light intensity at the interface takes a minimum value based on the differential value. A measurement chip comprising a dielectric block, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer;
A light source that generates a light beam;
An incident optical system that causes the light beam to be incident on the dielectric block at an incident angle at which a total reflection condition is obtained at an interface between the dielectric block and the thin film layer;
A light detection means comprising a plurality of light receiving elements and measuring the intensity of the light beam totally reflected at the interface;
The plurality of light receiving elements are divided into several light receiving element groups, and a total of outputs for each of two adjacent light receiving element groups is differentiated with respect to the parallel arrangement direction of the light receiving elements, and the interface is based on the differential value. And a calculating means for obtaining a reflection angle at which the intensity of reflected light takes a minimum value. A measurement chip comprising a dielectric block, a thin film layer formed on one surface of the dielectric block, and a sample holding mechanism for holding a sample on the surface of the thin film layer;
A light source that generates a light beam;
An incident optical system that causes the light beam to be incident on the dielectric block at an incident angle at which a total reflection condition is obtained at an interface between the dielectric block and the thin film layer;
A light detection means comprising a plurality of light receiving elements and measuring the intensity of the light beam totally reflected at the interface;
An average value of outputs of two or more adjacent light receiving elements adjacent to each other is sequentially calculated in the direction in which the light receiving elements are arranged in parallel, and the average value is differentiated with respect to the direction in which the light receiving elements are arranged in parallel. And a calculating means for obtaining a reflection angle at which the reflected light intensity at the interface takes a minimum value.

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