JP2007147311A - Sample measuring method and sample measuring instrument - Google Patents
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本発明は、表面プラズモン共鳴現象を利用して測定試料の性状を評価する試料測定方法及び試料測定装置に関する。 The present invention relates to a sample measurement method and a sample measurement apparatus for evaluating the properties of a measurement sample using a surface plasmon resonance phenomenon.
光を金属薄膜に入射させるとき、入射角がある角度以上になると全反射する。全反射する場合、金属薄膜中に光がまったく入射しないわけではなく、金属薄膜にそって進むエバネッセント波が生じるという性質がある。このエバネッセント波の波数は光の入射角に依存している。一方、金属薄膜は、表面に誘電体があると素励起により表面プラズモンというエネルギー波が生じる。表面プラズモンの波数は金属薄膜に接する物質の屈折率に依存している。
このエバネッセント波と表面プラズモン波の波数が一致するとき、エバネッセント波は表面プラズモンの励起に使われ、反射光として計測される光量が減少する。この現象が表面プラズモン共鳴（ＳＰＲ）現象である。
When light is incident on the metal thin film, it is totally reflected when the incident angle exceeds a certain angle. In the case of total reflection, light does not enter the metal thin film at all, and evanescent waves traveling along the metal thin film are generated. The wave number of this evanescent wave depends on the incident angle of light. On the other hand, when a metal thin film has a dielectric on the surface, an energy wave called surface plasmon is generated by elementary excitation. The wave number of surface plasmon depends on the refractive index of the substance in contact with the metal thin film.
When the wave numbers of the evanescent wave and the surface plasmon wave coincide with each other, the evanescent wave is used for excitation of the surface plasmon, and the amount of light measured as reflected light decreases. This phenomenon is a surface plasmon resonance (SPR) phenomenon.
上述のように、表面プラズモンの波数は金属薄膜に接する物質の屈折率に依存しているので、金属薄膜に接する物質の性状変化により屈折率が変化すると、表面プラズモンの波数が変化し、表面プラズモン共鳴現象の起こるエバネッセント波の波数が変化する。つまり、反射光強度の減少するエバネッセント波の波数が変化する。エバネッセント波の波数は光の入射角に依存するため、反射光強度の減少する入射角、すなわち表面プラズモン共鳴現象の生じる入射光の入射角（共鳴角）が変化する。したがって、この入射角すなわち共鳴角を読みとることにより金属薄膜に接する物質の性状（直接的には屈折率）を知ることができる。金属薄膜の表面に接する物質の屈折率が大きくなるほど共鳴角は大きくなる。また、屈折率は一般的に分子量が大きいほど大きい。 As described above, the wave number of surface plasmon depends on the refractive index of the substance in contact with the metal thin film. Therefore, when the refractive index changes due to the property change of the substance in contact with the metal thin film, the wave number of the surface plasmon changes and the surface plasmon changes. The wave number of the evanescent wave where the resonance phenomenon occurs changes. That is, the wave number of the evanescent wave whose reflected light intensity decreases changes. Since the wave number of the evanescent wave depends on the incident angle of light, the incident angle at which the reflected light intensity decreases, that is, the incident angle (resonance angle) of incident light that causes the surface plasmon resonance phenomenon changes. Therefore, by reading the incident angle, that is, the resonance angle, the property (direct refractive index) of the substance in contact with the metal thin film can be known. The resonance angle increases as the refractive index of the substance in contact with the surface of the metal thin film increases. The refractive index is generally larger as the molecular weight is larger.
このように、エバネッセント波に共鳴する現象がセンシング界面に接する物質の屈折率に依存して発生することから、試料そのものの屈折率変化や、金属薄膜に固定化した物質と試料中の検出対象成分や試薬成分とを反応させることによる金属薄膜界面の屈折率変化等を利用した試料の性状評価が可能である。そのため、ＳＰＲ現象を利用した種々のＳＰＲ測定装置が提案されている（特許文献１、特許文献２）。 In this way, since the phenomenon that resonates with the evanescent wave occurs depending on the refractive index of the substance in contact with the sensing interface, the refractive index change of the sample itself, the substance immobilized on the metal thin film, and the detection target component in the sample It is possible to evaluate the properties of the sample using the refractive index change at the interface of the metal thin film by reacting with a reagent component. For this reason, various SPR measurement devices using the SPR phenomenon have been proposed (Patent Documents 1 and 2).
ＳＰＲ測定装置を利用した具体的な分析例としては、例えば抗体を金属薄膜等に予め固定化してその抗体と特異的に結合する抗原である薬物を測定することが試みられている。すなわち、抗体を固定化した金属薄膜等に薬物を含む試料液を接触させると、試料液中の薬物が抗体に結合して屈折率が変化する。従って、抗体が固定化されているが未だ薬物が結合していない時の共鳴角と、薬物が結合した後の共鳴角を比較すると、その変化から薬物の量を測定できる。逆に測定対象としての抗原である薬物を金属薄膜等に予め固定化してその薬物と特異的に結合する抗体との結合を試料液中の薬物と競合的に行わせ、試料液中の薬物を測定することも提案されている（特許文献３）。
しかし、金属薄膜に接する物質の屈折率変化によってもたらされる共鳴角の変化はごく僅かである。そのため、金属薄膜に接する物質の性状を正確に評価しようとすると、０．０１°程度の角度分解能で共鳴角、すなわち、反射率が極小となる入射角（極小角度）を捉えることが必要である。
ところが、ＳＰＲ現象を利用した従来の測定装置では、光学系のレンズの汚れやキズ、金薄膜厚さのムラなどにより、ＳＰＲカーブ（入射角を横軸とし、反射率を縦軸にとったカーブ）にノイズが生じ、微少な極小角度の変化を測定することが困難であった。
見かけ上ノイズが低減されたスムーズなＳＰＲカーブを得ることは、移動平均を計算する等のデータ処理により可能であるが、その場合、却って誤差をもたらす場合もあり、精度の向上は困難であった。
本発明は、上記事情に鑑みてなされたものであって、測定試料の性状を正確に評価することが可能な試料測定方法及び試料測定装置を提供することを課題とする。
However, the change in the resonance angle caused by the change in the refractive index of the material in contact with the metal thin film is negligible. Therefore, to accurately evaluate the property of the substance in contact with the metal thin film, it is necessary to capture the resonance angle, that is, the incident angle (minimum angle) at which the reflectance is minimized with an angular resolution of about 0.01 °. .
However, in the conventional measuring apparatus using the SPR phenomenon, the SPR curve (curve with the incident angle as the horizontal axis and the reflectance as the vertical axis is caused by dirt and scratches on the lens of the optical system and unevenness of the gold thin film thickness. ) Was generated, and it was difficult to measure a slight change in the minimum angle.
It is possible to obtain a smooth SPR curve with apparently reduced noise by data processing such as calculating a moving average, but in that case, an error may be caused on the contrary, and it is difficult to improve accuracy. .
This invention is made | formed in view of the said situation, Comprising: It aims at providing the sample measuring method and sample measuring apparatus which can evaluate the property of a measurement sample correctly.
本発明者は上記の課題を達成するために鋭意検討した結果、共鳴角の変化と共鳴角付近の反射率の変化に相関関係があることを見いだした。さらに、光学系のレンズの汚れやキズ、金薄膜厚さのムラなどに基づくノイズは、入射光の入射角が同じであれば、金属薄膜に接する試料の性状にかかわらず同程度に出る傾向があり、同一の入射角において得られる反射率の変化は、ノイズの影響を受けにくいことを見いだした。
その結果、本発明者は以下の本発明に想到した。
As a result of intensive studies to achieve the above-mentioned problems, the present inventor has found that there is a correlation between the change in the resonance angle and the change in the reflectance near the resonance angle. Furthermore, noise based on dirt and scratches on the lens of the optical system and unevenness in the thickness of the gold thin film tends to appear to the same extent regardless of the properties of the sample in contact with the metal thin film, provided that the incident angle of incident light is the same. It was found that the change in reflectance obtained at the same incident angle is less susceptible to noise.
As a result, the inventor has conceived the following present invention.
［１］一方の面に試料が接触する金属薄膜の他方の面に対して入射光を入射して全反射させた際の、表面プラズモン共鳴による反射光の減衰を観察する試料測定方法であって、
前記金属薄膜に対する入射光の入射角を同一の入射角θとした条件下で、前記試料が基準試料であるときの反射率Ｃと、前記試料が測定試料であるときの反射率Ｓとを各々測定し、ＳからＣを差し引いた差Ｄを用いて前記測定試料の性状を評価することを特徴とする試料測定方法。
[1] A sample measurement method for observing attenuation of reflected light due to surface plasmon resonance when incident light is incident and totally reflected on the other surface of the metal thin film with which the sample contacts one surface. ,
The reflectance C when the sample is a reference sample and the reflectance S when the sample is a measurement sample are set under the condition that the incident angle of incident light with respect to the metal thin film is the same incident angle θ. A sample measurement method comprising measuring and evaluating a property of the measurement sample using a difference D obtained by subtracting C from S.
［２］入射角θが、入射角を変化させたときにＤの極大値Ｄｍａｘが得られる入射角θｍａｘ及び／又はＤの極小値Ｄｍｉｎが得られる入射角θｍｉｎである［１］に記載の試料測定方法。
［３］ｎ（ｎは２以上の正数）個の入射角θ１〜θｎにおけるＤであるＤ１〜Ｄｎを用いて、前記測定試料の性状を評価する［１］に記載の試料測定方法。
［４］所定の範囲の入射角θα〜θβにおけるＤの絶対値を積算した値を用いて、前記測定試料の性状を評価する［１］に記載の試料測定方法。
[2] the incident angle theta is the minimum value D min of the maximum value D max of incidence angle can be obtained theta max and / or D of the D is the incident angle theta min obtained when changing the angle of incidence [1] 2. The sample measuring method according to 1.
[3] The sample according to [1], wherein the properties of the measurement sample are evaluated using D 1 to D n which are D at n (n is a positive number of 2 or more) incident angles θ 1 to θ n Measuring method.
[4] The sample measurement method according to [1], wherein the property of the measurement sample is evaluated using a value obtained by integrating the absolute values of D at incident angles θ α to θ β in a predetermined range.
［５］一方の面に試料が接触する金属薄膜と、該金属薄膜の他方の面側に配置された高屈折プリズムと、前記高屈折プリズムを通して前記金属薄膜に入射光を供給する光源と、前記金属薄膜からの反射光を前記高屈折プリズムを通して検出する検出器と、演算部とを備え、
前記演算部は、前記金属薄膜に対する入射光の入射角を同一の入射角θとした条件下で、前記試料が基準試料であるときに得られる反射率Ｃと、前記試料が測定試料であるときに得られる反射率Ｓとを各々記憶すると共に、ＳからＣを差し引いた差Ｄを用いて前記測定試料の性状を評価することを特徴とする試料測定装置。
［６］前記金属薄膜の前記一方の面に、生化学物質が固定化されている［５］に記載の試料測定装置。
[5] A metal thin film in contact with the sample on one surface, a high refraction prism disposed on the other surface side of the metal thin film, a light source for supplying incident light to the metal thin film through the high refraction prism, A detector for detecting reflected light from the metal thin film through the high refractive prism, and an arithmetic unit;
When the sample is a reference sample and the sample is a measurement sample under the condition that the incident angle of incident light on the metal thin film is the same incident angle θ, And a reflectance D obtained by subtracting C from S, and evaluating the properties of the measurement sample using the difference D obtained by subtracting C from S.
[6] The sample measuring apparatus according to [5], wherein a biochemical substance is immobilized on the one surface of the metal thin film.
本発明の試料測定方法及び試料測定装置によれば、光学系のレンズの汚れやキズ、金薄膜厚さのムラなどに基づくノイズの影響を受けにくく、ＳＰＲ現象を利用して測定試料の性状を正確に評価することができる。 According to the sample measuring method and sample measuring apparatus of the present invention, it is difficult to be affected by noise due to dirt and scratches on the lens of the optical system, unevenness in the thickness of the gold thin film, etc. Accurate evaluation is possible.
図１は、本発明に係る試料測定装置の一実施形態を示す構成図である。本実施形態に係る装置はクレッチマン配置と呼ばれる構成を取り、高屈折のプリズム１と、その一面に装着され金属薄膜が設けられている検出素子２と、プリズム１を通して検出素子２の金属薄膜に向けて光を照射する光源３と、プリズム１を通して金属薄膜で反射された反射光を検出する検出器４と、検出器４の信号が入力される演算部５と、金属薄膜に試料が接触する場としてのフローセル６とから基本的に構成されている。また、光源３と金属薄膜との間に偏光板７が配置されている。
FIG. 1 is a configuration diagram showing an embodiment of a sample measuring apparatus according to the present invention. The apparatus according to the present embodiment has a configuration called a Kretschmann arrangement, a highly refractive prism 1, a detection element 2 mounted on one surface thereof and provided with a metal thin film, and a prism 1 through which the metal film of the detection element 2 is directed. A light source 3 for irradiating light, a detector 4 for detecting reflected light reflected by the metal thin film through the prism 1, a calculation unit 5 to which a signal of the detector 4 is input, and a case where the sample contacts the metal thin film The flow cell 6 is basically configured. A polarizing
高屈折のプリズム１には、高屈折のものであれば特に限定されることはなく、各種のプリズム用材を使用することができる。このような高屈折プリズム用材としては、例えば、ＢＫ７（ｎd＝1.5163）、ＳＦＬ６（ｎd＝1.80518）等が好適に使用されるが、その材質はガラスに限定されるものではなくプラスチック等であってもよい。また、その形状も特に限定はなく、半球状の他、三角形等のものが使用できる。 The high refraction prism 1 is not particularly limited as long as it is a high refraction prism, and various prism materials can be used. As such a material for a high refractive prism, for example, BK7 (nd = 1.5163), SFL6 (nd = 1.80518), etc. are preferably used, but the material is not limited to glass but plastic or the like. Also good. The shape is not particularly limited, and a hemispherical shape or a triangular shape can be used.
検出素子２は、薄膜基板の一方の面に金属薄膜が設けられたものである。薄膜基板としては、カバーグラスと呼ばれるガラス製の薄膜やプラスチック製の薄膜等を使用することができる。金属薄膜としては薄膜化できるものであれば各種の金属を使用することができる。例えば、白金、金、銀、銅、ニッケル、鉄、アルミニウム、ステンレス等が含まれる。特に好ましい金属薄膜としては、金からなる金属薄膜が挙げられる。金属薄膜の厚みは、通常、２５〜９０nm、好ましくは４０〜６０nmである。金属薄膜は、薄膜基板の一面側に蒸着、コーティング等の手法により形成することができる。 The detection element 2 has a metal thin film provided on one surface of a thin film substrate. As the thin film substrate, a glass thin film called a cover glass, a plastic thin film, or the like can be used. Various metals can be used as the metal thin film as long as it can be thinned. For example, platinum, gold, silver, copper, nickel, iron, aluminum, stainless steel and the like are included. A particularly preferable metal thin film is a metal thin film made of gold. The thickness of the metal thin film is usually 25 to 90 nm, preferably 40 to 60 nm. The metal thin film can be formed on one surface side of the thin film substrate by a technique such as vapor deposition or coating.
検出素子２は、金属薄膜が形成された面を外側としてプリズム１に装着される。装着の方法に特に限定はなく、マッチングオイルを用いて表面張力を利用して装着する他、接着による方法やアダプターを用いて圧接させる方法等種々の方法が採用できる。 The detection element 2 is attached to the prism 1 with the surface on which the metal thin film is formed facing outside. There is no particular limitation on the mounting method, and various methods such as a method using adhesion and a method using pressure bonding using an adapter can be employed in addition to mounting using matching oil and utilizing surface tension.
検出素子２の金属薄膜の試料と接する側（プリズム１と反対側）には、検出対象物質等に応じて適宜の生化学物質を固定してもよい。生化学物質としては、タンパク質、ＤＮＡ、糖タンパク、糖脂質、細胞、ウイルス、細菌等が挙げられる。特に試料中の検出対象成分や試薬成分と抗原抗体反応が可能な生化学物質であることが好ましい。抗原抗体反応を利用することにより、選択性の高い定性、定量分析が可能となる。
生化学物質を金属薄膜に固定化する方法としては、抗原に蛋白質等を結合させて物理吸着させる方法や、抗原に金属表面に親和性を持つ官能基、例えばチオール基やジスルフィド基等を結合させてから金属に化学結合させる方法等が挙げられる。
An appropriate biochemical substance may be fixed to the side of the detection element 2 in contact with the sample of the metal thin film (the side opposite to the prism 1) according to the detection target substance or the like. Examples of biochemical substances include proteins, DNA, glycoproteins, glycolipids, cells, viruses, bacteria, and the like. In particular, a biochemical substance capable of antigen-antibody reaction with a detection target component or reagent component in a sample is preferable. By using the antigen-antibody reaction, qualitative and quantitative analysis with high selectivity becomes possible.
As a method for immobilizing biochemical substances on a metal thin film, a protein or the like is bound to an antigen for physical adsorption, or a functional group having affinity for the metal surface such as a thiol group or a disulfide group is bound to the antigen. And then chemically bonding to the metal.
光源３としては、波長が２００〜１，３００nm、好ましくは４００〜８００nmの各種光源を使用することができる。例えば、６５０〜８００nmの光を発光する発光ダイオードを好適に使用することができる。検出器４としては、例えば、フォトダイオード、リニアアレイ、ＣＣＤカメラ等を好適に使用できる。
偏光板７は、ｐ偏光の光波を通過させるｐ偏光板である。表面プラズモンはｐ偏光の光波のみと結合するので、ｓ偏光の光波の全部又は一部を除くことにより、ＳＰＲ現象を高感度で捉えることができる。
As the light source 3, various light sources having a wavelength of 200 to 1,300 nm, preferably 400 to 800 nm can be used. For example, a light emitting diode that emits light of 650 to 800 nm can be suitably used. As the detector 4, for example, a photodiode, a linear array, a CCD camera, or the like can be suitably used.
The
フローセル６は、試料を受け入れる容器であり、その内面には金属薄膜が露出している。フローセル６には入口と出口が設けられ、測定試料と基準試料の何れかが図示しないポンプ等によって循環ないし通過しながら金属薄膜に接するようになっている。
試料は液体でも気体でもよい。基準試料は、評価対象によって異なるが、例えば水溶液中の特定成分の濃度を測定する場合、純水等を用いることができる。また、抗原抗体反応のように、反応に時間を要する反応を利用する場合は、反応開始前（又は反応開始時）の試料を基準試料として用いることができる。
The flow cell 6 is a container for receiving a sample, and a metal thin film is exposed on the inner surface thereof. The flow cell 6 is provided with an inlet and an outlet, and either the measurement sample or the reference sample is in contact with the metal thin film while being circulated or passed by a pump or the like (not shown).
The sample may be liquid or gas. Although the reference sample varies depending on the evaluation target, for example, when measuring the concentration of a specific component in an aqueous solution, pure water or the like can be used. In addition, when using a reaction that takes time for the reaction, such as an antigen-antibody reaction, a sample before the start of the reaction (or at the start of the reaction) can be used as a reference sample.
演算部５は、検出器４から得られた反射率の情報に基づき、後述する具体的手順等により測定試料の性状を評価するものである。
なお、本発明において、反射率とは、文字通り反射率だけでなく、入射光強度が一定の場合の反射光強度も反射率であるとみなす。入射光強度が一定であれば、反射光強度は反射率に比例し、実質的に区別する意味がないからである。
また、本発明において評価とは、測定試料中の特定成分の濃度を求めるものの他、測定試料中の特定成分がある濃度を超えているか否かの判定のように二者択一的な判断等も含む概念である。また、評価すべき性状には、濃度だけでなく屈折率、分子量等の物性や反応の進行具合等も含まれる。
The calculation unit 5 evaluates the properties of the measurement sample based on the reflectance information obtained from the detector 4 according to a specific procedure described later.
In the present invention, the reflectance is not only literally the reflectance, but also the reflected light intensity when the incident light intensity is constant is regarded as the reflectance. This is because if the incident light intensity is constant, the reflected light intensity is proportional to the reflectivity, and there is no substantial difference.
In addition, in the present invention, evaluation refers to alternative determinations such as determination of whether or not a specific component in a measurement sample exceeds a certain concentration in addition to obtaining the concentration of the specific component in the measurement sample. It is a concept that also includes The properties to be evaluated include not only the concentration but also physical properties such as refractive index and molecular weight, the progress of reaction, and the like.
なお、本発明の試料測定装置では、金属薄膜を薄膜基板上に設けたが、プリズムに直接蒸着してもよいし、プリズムに直接表面張力を利用する等の手段で装着してもよい。また、本実施形態におけるフローセルに変えて、バッチ式のセルを備えてもよい。この場合、金属薄膜がセルの底面位置で露出するようにして、これを構試料液等の入った容器に浸けて使用することが考えられる。
また、偏光板７は、光源３と金属薄膜との間に配置することに代えて、金属薄膜と検出器４との間に配置してもよい。さらに、ｓ偏光の光波の全部又は一部が除去された光源を用いれば、偏光板７は不要である。
In the sample measuring apparatus of the present invention, the metal thin film is provided on the thin film substrate. However, the metal thin film may be directly deposited on the prism, or may be mounted on the prism by means such as directly using surface tension. Further, instead of the flow cell in the present embodiment, a batch type cell may be provided. In this case, it is conceivable that the metal thin film is exposed at the bottom surface position of the cell and is immersed in a container containing a sample solution.
Further, the
また、本発明の試料測定方法には、クレッチマン配置に代えて、オットーの光学配置を利用した装置、回折格子を利用した装置、光ファィバーを利用した装置等、公知の種々の形態の表面プラズモン測定装置を用いることができる。
オットーの光学配置では、測定対象の溶液を挟んでプリズムと金属薄膜が配置される。回折格子を利用する場合には、回折格子の表面に金属薄膜が配置される。光ファィバーを利用する場合には、コア表面にコーティングした金属に試料を接触させることによって測定できる。
In addition, the sample measurement method of the present invention includes various known forms of surface plasmon measurement, such as an apparatus using an optical arrangement of Otto instead of a Kretschmann arrangement, an apparatus using a diffraction grating, an apparatus using an optical fiber, etc. An apparatus can be used.
In the Otto optical arrangement, a prism and a metal thin film are arranged with a solution to be measured interposed therebetween. When a diffraction grating is used, a metal thin film is disposed on the surface of the diffraction grating. When an optical fiber is used, the measurement can be performed by bringing the sample into contact with the metal coated on the core surface.
［水溶液中の特定成分の濃度測定］
以下、図１の装置を用いて水溶液中の特定成分の濃度を求める種々の手順を、図２を参照しつつ説明する。
（第１の手順）
まず、フローセル６に基準試料である純水を導入し、入射角θを種々変化させながら反射率を測定し、基準試料の反射率Ｃのカーブを得、演算部５に記憶させる。次に、フローセル６に測定試料である水溶液（濃度未知）を導入し、入射角θを種々変化させながら反射率を測定し、水溶液の反射率Ｓのカーブを得、演算部５に記憶させる。
そして、演算部５において、入射角θ毎にＳからＣを差し引いた差Ｄを計算し、差Ｄの中で極大値Ｄｍａｘが得られる入射角θｍａｘを、微分演算等の手段により求め、そのときのＤｍａｘをデータとして取得する。
次に、予め濃度既知の測定試料（標準溶液）で、上記と同様の手順で作成しておいた検量線（濃度と極大値Ｄｍａｘとの関係式）に照らして、Ｄｍａｘから当該測定試料中の特定成分の濃度を求める。
なお、入射角θｍａｘは、特定成分の濃度が変化しても通常殆ど変化しないが、若干でも変化する場合は、何れかの濃度の標準溶液で得た入射角θｍａｘを、その後の他の測定試料（他の濃度の標準試料、濃度未知の測定試料）についても入射角θｍａｘとみなして使用する。
[Measurement of concentration of specific components in aqueous solution]
Hereinafter, various procedures for determining the concentration of a specific component in an aqueous solution using the apparatus of FIG. 1 will be described with reference to FIG.
(First procedure)
First, pure water as a reference sample is introduced into the flow cell 6, the reflectance is measured while variously changing the incident angle θ, a curve of the reflectance C of the reference sample is obtained, and stored in the calculation unit 5. Next, an aqueous solution (concentration unknown) as a measurement sample is introduced into the flow cell 6, the reflectance is measured while variously changing the incident angle θ, and a curve of the reflectance S of the aqueous solution is obtained and stored in the calculation unit 5.
Then, the calculation unit 5 calculates a difference D obtained by subtracting C from S for each incident angle θ, and obtains an incident angle θ max from which the maximum value D max is obtained in the difference D by means such as a differential calculation, D max at that time is acquired as data.
Next, using a measurement sample (standard solution) with a known concentration in advance, in light of a calibration curve (relational expression between the concentration and the maximum value D max ) prepared in the same procedure as described above, the measurement sample is measured from D max. Find the concentration of the specific component in it.
The incident angle θ max usually hardly changes even if the concentration of the specific component changes. However, if it changes slightly, the incident angle θ max obtained with the standard solution of any concentration is changed to the other angle thereafter. Measurement samples (standard samples of other concentrations, measurement samples whose concentration is unknown) are also regarded as the incident angle θ max and used.
（第２の手順）
まず、フローセル６に基準試料である純水を導入し、入射角θを種々変化させながら反射率を測定し、基準試料の反射率Ｃのカーブを得、演算部５に記憶させる。次に、フローセル６に測定試料である水溶液（濃度未知）を導入し、入射角θを種々変化させながら反射率を測定し、水溶液の反射率Ｓのカーブを得、演算部５に記憶させる。
そして、演算部５において、入射角θ毎にＳからＣを差し引いた差Ｄを計算し、差Ｄの中で極小値Ｄｍｉｎが得られる入射角θｍｉｎを、微分演算等の手段により求め、そのときのＤｍｉｎをデータとして取得する。
次に、予め濃度既知の測定試料（標準溶液）で、上記と同様の手順で作成しておいた検量線（濃度と極小値Ｄｍｉｎとの関係式）に照らして、Ｄｍｉｎから当該測定試料中の特定成分の濃度を求める。
なお、入射角θｍｉｎは、特定成分の濃度が変化しても通常殆ど変化しないが、若干でも変化する場合は、何れかの濃度の標準溶液で得た入射角θｍｉｎを、その後の他の測定試料（他の濃度の標準試料、濃度未知の測定試料）についても入射角θｍｉｎとみなして使用する。
(Second procedure)
First, pure water as a reference sample is introduced into the flow cell 6, the reflectance is measured while variously changing the incident angle θ, a curve of the reflectance C of the reference sample is obtained, and stored in the calculation unit 5. Next, an aqueous solution (concentration unknown) as a measurement sample is introduced into the flow cell 6, the reflectance is measured while variously changing the incident angle θ, and a curve of the reflectance S of the aqueous solution is obtained and stored in the calculation unit 5.
Then, the calculation unit 5 calculates the difference D obtained by subtracting C from S for each incident angle θ, and obtains the incident angle θ min from which the minimum value D min is obtained in the difference D by means such as differential calculation. D min at that time is acquired as data.
Next, using a measurement sample (standard solution) with a known concentration in advance, in light of a calibration curve (relational expression between the concentration and the minimum value Dmin ) prepared in the same procedure as described above, the measurement sample is measured from Dmin. Find the concentration of the specific component in it.
The incident angle θ min usually does not substantially change even if the concentration of the specific component changes, but if it changes slightly, the incident angle θ min obtained with the standard solution of any concentration is changed to the other angle thereafter. Measurement samples (standard samples of other concentrations, measurement samples of unknown concentration) are also used with an incident angle θ min .
（第３の手順）
水溶液（濃度未知）について、第１の手順と同様にして得たＤｍａｘと、第２の手順と同様にして得たＤｍｉｎの絶対値とを合計したＤｔを求め、予め濃度既知の測定試料（標準溶液）で、同様の手順で作成しておいた検量線（濃度とＤｔとの関係式）に照らして、Ｄｔから当該測定試料中の特定成分の濃度を求める。
(Third procedure)
For an aqueous solution (concentration unknown), D t obtained by summing the D max obtained in the same manner as in the first procedure and the absolute value of D min obtained in the same manner as in the second procedure was obtained, and the concentration known in advance was measured. in the sample (standard solution), in light of the similar procedure calibration curve which had been prepared in (relational expression between concentration and D t), determine the concentration of a specific component of the measurement sample from D t.
（第４の手順）
まず、フローセル６に基準試料である純水を導入し、入射角θを種々変化させながら反射率を測定し、基準試料の反射率Ｃのカーブを得、演算部５に記憶させる。次に、フローセル６に測定試料である水溶液（濃度未知）を導入し、入射角θを種々変化させながら反射率を測定し、水溶液の反射率Ｓのカーブを得、演算部５に記憶させる。
そして、演算部５において、入射角θα〜θβにおけるＳからＣを差し引いた差Ｄを計算し、これらのＤの絶対値を積算した値Ｄｉ（入射角θα〜θβの範囲におけるＣのカーブとＳのカーブに挟まれた領域の面積に相当する。）を求める。
次に、予め濃度既知の測定試料（標準溶液）で、上記と同様の手順で作成しておいた検量線（濃度とＤｉとの関係式）に照らして、Ｄｉから当該測定試料中の特定成分の濃度を求める。
(Fourth procedure)
First, pure water as a reference sample is introduced into the flow cell 6, the reflectance is measured while variously changing the incident angle θ, a curve of the reflectance C of the reference sample is obtained, and stored in the calculation unit 5. Next, an aqueous solution (concentration unknown) as a measurement sample is introduced into the flow cell 6, the reflectance is measured while variously changing the incident angle θ, and a curve of the reflectance S of the aqueous solution is obtained and stored in the calculation unit 5.
Then, the calculation unit 5 calculates a difference D obtained by subtracting C from S at the incident angles θ α to θ β, and a value D i (in the range of incident angles θ α to θ β ) obtained by integrating the absolute values of these Ds. This corresponds to the area of the region sandwiched between the C curve and the S curve).
Next, using a measurement sample (standard solution) with a known concentration in advance, in light of a calibration curve (relational expression between the concentration and D i ) prepared in the same procedure as described above, from D i to the measurement sample in the measurement sample Obtain the concentration of a specific component.
（その他の手順）
上記第１〜第３の手順においては、入射角θｍａｘ及び／又は入射角θｍｉｎにおけるＤを用いたが、ＣのカーブからＳのカーブが乖離する範囲であれば、何れの入射角θにおけるＤを用いてもよい。例えば、入射角θが基準試料の共鳴角である場合のＤを用いることができる。
また、上記第２の手順においては、入射角θｍａｘ及び入射角θｍｉｎにおける２つのＤを用いたが、３以上の入射角θにおけるＤを用いてもよい。
また、上記第４の手順においては、入射角θαと入射角θβを、各々ＣのカーブからＳのカーブが乖離する範囲の下限と上限の入射角としたが、入射角θα〜θβの範囲は、より狭くてもよいし、より広くてもよい。
(Other steps)
In the first to third procedures, D at the incident angle θ max and / or the incident angle θ min is used. However, as long as the S curve deviates from the C curve, at any incident angle θ. D may be used. For example, D when the incident angle θ is the resonance angle of the reference sample can be used.
In the second procedure, two Ds at the incident angle θ max and the incident angle θ min are used, but Ds at three or more incident angles θ may be used.
In the fourth procedure, the incident angle θ α and the incident angle θ β are set as the lower limit and the upper limit incident angle, respectively, in which the S curve deviates from the C curve, but the incident angles θ α to θ The range of β may be narrower or wider.
［遅い反応を利用した濃度測定］
抗原抗体反応のように、反応に時間を要する反応を利用する場合は、反応開始前又は反応開始時の試料を基準試料とすることができる。
例えば、抗原を金属薄膜に固定しておき試料中の抗体濃度を測定する場合、試料をフローセルに循環させ、循環当初のＳＰＲカーブを基準試料の反射率Ｃのカーブとし、その後、抗原抗体反応が終了してＳＰＲカーブの変化が見られなくなったとき（定常状態）のＳＰＲカーブを測定試料の反射率Ｓのカーブとすることができる。なお、循環開始から定常状態になるまでの時間は、抗原抗体の種類などにより一概には言えないが、一般的には１〜３０分である。
得られた反射率Ｃのカーブと反射率Ｓのカーブから試料中の抗体濃度を求める手順は、上記［水溶液中の特定成分の濃度測定］において説明したのと同様であり、差Ｄの極大値Ｄｍａｘ、差Ｄの極小値Ｄｍｉｎ、ＤｍａｘとＤｍｉｎの絶対値とを合計したＤｔ、入射角θα〜θβにおけるＤの絶対値を積算した値Ｄｉ、その他種々の態様の差Ｄを用いて、試料中の抗体濃度を得ることができる。
[Concentration measurement using slow reaction]
When using a reaction that requires a long time for the reaction, such as an antigen-antibody reaction, a sample before or at the start of the reaction can be used as a reference sample.
For example, when measuring the antibody concentration in a sample while fixing the antigen to a metal thin film, the sample is circulated in the flow cell, the SPR curve at the beginning of the circulation is used as the reflectance C curve of the reference sample, and then the antigen-antibody reaction is performed. The SPR curve when the change of the SPR curve is no longer seen after completion (steady state) can be used as the reflectance S curve of the measurement sample. Note that the time from the start of circulation to the steady state cannot be generally described depending on the type of antigen antibody, but is generally 1 to 30 minutes.
The procedure for obtaining the antibody concentration in the sample from the obtained curve of reflectance C and reflectance S is the same as that described in the above [Measurement of concentration of specific component in aqueous solution], and the maximum value of difference D D max , the minimum value D min of the difference D, D t summing the D max and the absolute value of D min , the value D i integrating the absolute values of D at the incident angles θ α to θ β , and other various aspects The difference D can be used to obtain the antibody concentration in the sample.
［反応の経時解析］
抗原抗体反応の進行具合を経時的に解析する場合などにも、反応開始前又は反応開始時の試料を基準試料とすることができる。
例えば、抗原を金属薄膜に固定しておき試料中の抗体との反応の進行具合を経時的に解析する場合、試料をフローセルに循環させ、循環当初のＳＰＲカーブを基準試料の反射率Ｃのカーブとし、その後、所定時間毎（例えば１秒毎、３０秒毎等）のＳＰＲカーブを測定試料の反射率Ｓのカーブとすることができる。
得られた反射率Ｃのカーブと反射率Ｓのカーブから反応の進行具合を評価する手順は、上記［水溶液中の特定成分の濃度測定］において説明したのと同様であり、差Ｄの極大値Ｄｍａｘ、差Ｄの極小値Ｄｍｉｎ、ＤｍａｘとＤｍｉｎの絶対値とを合計したＤｔ、入射角θα〜θβにおけるＤの絶対値を積算した値Ｄｉ、その他種々の態様の差Ｄを用いて、所定時間毎における反応の進行具合を評価することができる。
[Analysis of reaction over time]
When analyzing the progress of the antigen-antibody reaction over time, a sample before or at the start of the reaction can be used as a reference sample.
For example, when an antigen is fixed on a metal thin film and the progress of the reaction with the antibody in the sample is analyzed over time, the sample is circulated through the flow cell, and the SPR curve at the beginning of the circulation is the curve of the reflectance C of the reference sample. Then, the SPR curve at every predetermined time (for example, every 1 second, every 30 seconds, etc.) can be used as the curve of the reflectance S of the measurement sample.
The procedure for evaluating the progress of the reaction from the obtained curve of the reflectance C and the curve of the reflectance S is the same as described in the above [Measurement of concentration of specific component in aqueous solution], and the maximum value of the difference D D max , the minimum value D min of the difference D, D t summing the D max and the absolute value of D min , the value D i integrating the absolute values of D at the incident angles θ α to θ β , and other various aspects Using the difference D, the progress of the reaction at every predetermined time can be evaluated.
［その他の用途］
上記は、特定成分の濃度測定や反応の経時解析を例にとって説明したが、その他屈折率や分子量の測定等の場合も、測定試料と基準試料との性状が乖離するほどカーブＣとカーブＳが乖離する。したがって、特定成分の濃度測定や反応の経時解析以外の場合にも、上記と同様の手順により測定試料の性状を評価することができる。
[Other uses]
The above has been described by taking the concentration measurement of a specific component and the analysis of the reaction over time as an example. However, in other cases such as the measurement of refractive index and molecular weight, the curves C and S change as the properties of the measurement sample and the reference sample differ. Deviation. Therefore, the properties of the measurement sample can be evaluated by the same procedure as described above even in cases other than the measurement of the concentration of the specific component and the analysis of the reaction over time.
１…プリズム、２…検出素子、３…光源、４…検出器、
５…演算部、６…フローセル、７…偏光板
DESCRIPTION OF SYMBOLS 1 ... Prism, 2 ... Detection element, 3 ... Light source, 4 ... Detector,
5 ... Calculation unit, 6 ... Flow cell, 7 ... Polarizing plate
Claims (6)
前記金属薄膜に対する入射光の入射角を同一の入射角θとした条件下で、前記試料が基準試料であるときの反射率Ｃと、前記試料が測定試料であるときの反射率Ｓとを各々測定し、ＳからＣを差し引いた差Ｄを用いて前記測定試料の性状を評価することを特徴とする試料測定方法。 A sample measurement method for observing the attenuation of reflected light due to surface plasmon resonance when incident light is incident on the other surface of the metal thin film that is in contact with one surface and totally reflected.
The reflectance C when the sample is a reference sample and the reflectance S when the sample is a measurement sample are set under the condition that the incident angle of incident light with respect to the metal thin film is the same incident angle θ. A sample measurement method comprising measuring and evaluating a property of the measurement sample using a difference D obtained by subtracting C from S.
前記演算部は、前記金属薄膜に対する入射光の入射角を同一の入射角θとした条件下で、前記試料が基準試料であるときに得られる反射率Ｃと、前記試料が測定試料であるときに得られる反射率Ｓとを各々記憶すると共に、ＳからＣを差し引いた差Ｄを用いて前記測定試料の性状を評価することを特徴とする試料測定装置。 A metal thin film in contact with a sample on one surface, a high refraction prism disposed on the other surface side of the metal thin film, a light source for supplying incident light to the metal thin film through the high refraction prism, and the metal thin film A detector that detects the reflected light of the light through the high refraction prism, and an arithmetic unit,
When the sample is a reference sample and the sample is a measurement sample under the condition that the incident angle of incident light on the metal thin film is the same incident angle θ, And a reflectance D obtained by subtracting C from S, and evaluating the properties of the measurement sample using the difference D obtained by subtracting C from S.
The sample measuring apparatus according to claim 5, wherein a biochemical substance is immobilized on the one surface of the metal thin film.
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