JP4035016B2 - Surface plasmon resonance sensor chip, and sample analysis method and analyzer using the same - Google Patents

Description

【０１４９】
なお、領域Ａにおける３箇所の測定ポイントをそれぞれＡ−１，Ａ−２，Ａ−３と表記し、同様に領域Ｂおける３箇所の測定ポイントをそれぞれＢ−１，Ｂ−２，Ｂ−３、領域Ｃおける３箇所の測定ポイントをそれぞれＣ−１，Ｃ−２，Ｃ−３と表記した。
表１に示すように、それぞれの領域の共鳴角の平均は、領域Ａが２１．６４１°、領域Ｂが２０．７９１°、領域Ｃが１９．８６０°となった。
【０１５０】
次に、図１８のような、上記のセンサチップを折り曲げず平板状のままとしたセンサチップを別途作成し、領域Ａ，Ｂ，Ｃと同様に共鳴角検出型のＳＰＲ測定装置FLEX CHIPS^TM Kinetic Analysis Systemで角度スキャンを行いながら反射光の強度を計測した。入射光には波長約８７０ｎｍの光を用いた。試料は精製水を用いた。以下、平板状のセンサチップの測定スポットを領域Ｄという。この計測においては、領域Ｄの４００箇所のポイントで計測を行った。
【０１５１】
計測された共鳴角前後での入射光の入射角と反射光強度との関係を図２１に示す。また、計測結果を表２に示す。
【表２】

図２１にあるように、領域Ｄでは共鳴角は全てのポイントで同じ角度となっていた。また、共鳴角の平均は、表２のように２０．８３２°となった。
【０１５２】
領域Ａ，Ｂ，Ｃ，Ｄにおいて、領域内の回折格子は同じ溝ピッチと溝深さとを有しており、また計測した試料も同じ精製水であるため、各領域の共鳴角は本来同一の角度となるはずである。しかし、領域Ａ，Ｂ，Ｃでは、角度を導入していない領域Ｄに比べて、それぞれ−０．８０９°，０．０４１°，０．９７２°の角度を有するため、共鳴角が見かけ上変化したように測定された。
以上の結果から、回折格子面に傾斜角度を導入することにより、回折格子面に入射する入射光の入射角を変えることができたことが確認された。
【０１５３】
（実施例２）
実施例１と同様に、平板状のポリカーボネート製の基体の表面に、溝ピッチ約８７０ｎｍ，溝深さ約４０ｎｍの凹凸形状を形成し、この凹凸形状を回折格子とし、さらに基体の表面に厚さ約８０ｎｍで金を蒸着して、センサチップを作成した。このセンサチップを折り曲げ、異なる傾斜角度を持つ面を２６面形成した。以下、角度を持つそれぞれの面の領域を、それぞれ水平面に対してなす傾斜角度の小さい順に領域Ｎｏ．１、領域Ｎｏ．２と自然数の番号を付けて呼ぶ。
【０１５４】
こうして２６の異なる傾斜角度を持つ面を形成されたセンサチップを用いて、ＳＰＲ測定装置FLEX CHIPS^TM Kinetic Analysis Systemで角度スキャンを行いながら反射光の強度を計測した。試料は精製水を用いた。入射光としては波長が約８７０ｎｍの光を用いた。共鳴角を特定し、傾斜角度を有していない実施例１の領域Dの測定結果と比較して、それぞれの領域がどれだけの傾斜角度を有しているかを特定した。測定結果を表３に示す。
【表３】

その結果、それぞれの領域は−０．９３６°〜０．９７８°の角度を有していることが判明した。
【０１５５】
次に、これらのセンサチップを用いて、ＳＰＲ測定装置FLEX CHIPS^TM Kinetic Analysis Systemで波長約８７０ｎｍの光を入射光とし、濃度が２．５％，５％，１０％，２０％，３０％，４０％，５０％であるエタノール水溶液をそれぞれ試料として、反射光の強度を計測した。
【０１５６】
それぞれの領域におけるエタノール濃度と、共鳴角がエタノール濃度の増加によって変化した変化量（以下、シフト量という）とを表４に示す。
【表４】

いずれの領域でも、各エタノール濃度に対して変動係数（CV：coefficient of variation）は１．２％〜６．３％と良好であった。また、いずれの領域でも同量の共鳴角のシフトを示した。
以上の結果から、角度を有する回折格子面でも角度を有さない回折格子面と同等の計測精度を持つことが分かった。
【０１５７】
（実施例３）
実施例２で取得した計測データのうち、入射光の入射角が２１．５°であった際の測定データを選択し、その測定データの中でエタノール濃度が０％，２．５％，５％，１０％，２０％，３０％，４０％，５０％であるエタノール水溶液それぞれを試料としたときの、反射光強度が最小となる領域の角度を算出した。具体的には、それぞれの領域が有する角度（表３）と、その領域における反射光強度とを用いて、反射光強度が最小となる角度を、回帰計算によって求めた。
【０１５８】
この結果判明した、エタノール水溶液のエタノール濃度と、反射光強度が最小となる領域の角度、つまり共鳴角のシフト量との関係を図２２に示す。
【０１５９】
一般的に行われている方法、即ち、角度スキャンを行う方法で得られる共鳴角から、エタノール水溶液のエタノール濃度と、領域Ｎｏ．７及び領域Ｎｏ．１５の回折格子面における共鳴角のシフト量との関係を調べた。この結果を図２３に示す。
【０１６０】
図２２と図２３とを比較した結果を表５に示す。
【表５】

比較の結果、図２２と図２３とが各エタノール濃度に対して同様のシフト量を示していることが判明した。したがって、一般的に行われてきた方法の結果（図２３）判明する共鳴角のシフト量が、本発明による回折格子面を保有するセンサチップを用いたことにより、入射光の入射角が一定の測定法で、最小反射強度を与える回折格子面が示す共鳴角の変化という形で得られる事が分かった。
【０１６１】
（実施例４）
実施例２で取得した計測データのうち、入射光の入射角が２１．５°の場合及び入射角が２１．０°の場合のデータについて解析した。
【０１６２】
入射光の入射角が２１．５°の場合では、領域Ｎｏ．２，７，１０，１６，１９の反射光強度とエタノール濃度との関係を解析した。その結果を図２４及び表６に示す。また、入射光の入射角が２１．０°の場合では、領域Ｎｏ．１０，１３，１８，２０，２２の反射光強度とエタノール濃度との関係を解析した。その結果を図２５及び表７に示す。
【０１６３】
【表６】

【表７】

【０１６４】
図２４から、入射光の入射角が２１．５°の場合は、エタノール濃度が０％〜１０％の範囲は領域Ｎｏ．２の反射光強度を、エタノール濃度が１０％〜３０％の範囲は領域Ｎｏ．１０の反射光強度を、エタノール濃度が３０％〜５０％の範囲は領域Ｎｏ．１９の反射光強度をそれぞれ用いることで、入射光の入射角が一定であっても広範囲なエタノール濃度域に対して計測が可能になることが確認された。
【０１６５】
同様に、図２５から、入射光の入射角が２１．０°の場合は、エタノール濃度が０％〜１０％の範囲は領域Ｎｏ．１０の反射光強度を、エタノール濃度が１０％〜３０％の範囲は領域Ｎｏ．１８の反射光強度を、エタノール濃度が３０％〜５０％の範囲は領域Ｎｏ．２２の反射光強度をそれぞれ用いることで、入射光の入射角が一定であっても広範囲なエタノール濃度域に対して計測が可能になることが確認された。
【０１６６】
したがって、従来の角度スキャンを行いながら反射光強度を測定する方法に比べ、入射光の入射角を一定にし、角度スキャンを行わない簡便な計測法でも広範囲な測定が可能であることが分かった。
【０１６７】
（実施例５）
平板状のポリカーボネート製の基体の表面に、溝ピッチ約８７０ｎｍ，溝深さ約４０ｎｍの凹凸形状を形成し、この凹凸形状を回折格子とした。
【０１６８】
さらに、この基体を、図２６に示すように、回折格子と直交する方向の断面が、曲率半径が約１１５０ｍｍの曲面となり、且つ、回折格子が基体の凸側の面にくるように加工した。次いで、この基体の表面に厚さ約８０ｎｍで金を蒸着して、センサチップを作成した。
【０１６９】
このセンサチップを用いて、共鳴角検出型のＳＰＲ測定装置FLEX CHIPS^TM Kinetic Analysis System（HTS Biosystems Inc.）で角度スキャンを行いながら反射光の強度を測定した。測定には波長約８７０ｎｍの光を入射光に用い、試料は精製水を用いた。
【０１７０】
センサチップ表面の、回折格子に直交する方向に約０．３３ｍｍ間隔の領域における反射光の共鳴角を検出した結果を表８に示す。
【表８】

各領域間の接面の角度差の合計が０．１７°となり、本来両端部の接面がなすべき角度である０．３°よりも小さいため、曲率半径が設計よりも大きくなるよう形成されていたことが推定される。
【０１７１】
しかし、０．３３ｍｍ間隔の隣あう領域間の角度は平均０．０１０°と非常に小さい値に制御されており、曲面を形成した構造であってもセンサチップ表面に回折格子面を形成することができることが確認できた。
【０１７２】
【発明の効果】
以上詳述したように、本発明によれば、表面プラズモン共鳴センサチップの回折格子面に光の照射方向に対して角度の分布を持たせることにより、平行光を照射した場合でも回折格子面から得られる反射光の強度に分布が生じるので、センサ面全体について回折格子面から得られる反射光の強度と回折格子面への実質的な入射角度とに基づき共鳴角度をリアルタイムで算出することできるという利点がある。つまり、簡単な光学系の構成により、リアルタイム測定と大面積の同時測定とを同時に実現することができるという利点がある。
【０１７３】
また、本発明によれば、表面プラズモン共鳴センサチップの回折格子面に光の照射方向に対して角度の分布を持たせることにより、ある一方向から光を照射した場合でも回折格子面から得られる反射光の強度に分布が生じるので、反射光の強度の変化量に基づき分析を行う場合において実質的に計測レンジを拡大することができ、広い計測レンジが必要となる濃度域の広い試料についても対応することができるという利点がある。
【図面の簡単な説明】
【図１】本発明の第１実施形態にかかるセンサチップの構成を示す模式的な斜視図である。
【図２】図１のセンサチップの要部の構成を示す模式的な斜視図である。
【図３】図１のセンサチップに結合物質を固定化した状態を示す模式的な斜視図である。
【図４】図１のセンサチップの製造方法の一例を示す模式的な斜視図であり、（ａ）〜（ｃ）の順に製造手順を示している。
【図５】本発明の第１実施形態にかかる分析装置の構成を示す模式的な模式図である。
【図６】本発明の第２実施形態にかかるセンサチップの要部の構成を示す模式的な斜視図である。
【図７】本発明の第３実施形態にかかるセンサチップの要部の構成を示す模式的な斜視図である。
【図８】本発明の第４実施形態にかかるセンサチップに結合物質及び非結合物質を固定化した状態を示す模式的な斜視図である。
【図９】本発明の第５実施形態にかかる分析装置の構成を示す模式的な模式図である。
【図１０】本発明の第７実施形態にかかるセンサチップの要部の構成を示す模式的な斜視図である。
【図１１】本発明の第７，第１０実施形態にかかる分析装置の構成を示す模式的な模式図である。
【図１２】本発明の第８実施形態にかかるセンサチップの構成を示す模式的な斜視図である。
【図１３】本発明の第９実施形態にかかるセンサチップの構成を示す模式的な斜視図である。
【図１４】本発明の第１０実施形態にかかるセンサチップの構成を示す模式的な斜視図である。
【図１５】図１のセンサチップにかかる回折格子面の配置形態の変形例を示す模式的な斜視図である。
【図１６】図１のセンサチップにかかる回折格子面の配置形態の変形例を示す模式的な斜視図である。
【図１７】図１のセンサチップにかかる回折格子面の配置形態の変形例を示す模式的な斜視図である。
【図１８】本発明の第１実施例にかかるセンサチップの模式的な斜視図である。
【図１９】本発明の第１実施例にかかるセンサチップの模式的な斜視図である。
【図２０】本発明の第１実施例にかかる、入射光の入射角と反射光強度とを示すグラフである。
【図２１】本発明の第１実施例にかかる、入射光の入射角と反射光強度とを示すグラフである。
【図２２】本発明の第３実施例にかかる、エタノール水溶液のエタノール濃度と共鳴角のシフト量とを示すグラフである。
【図２３】本発明の第３実施例にかかる、エタノール水溶液のエタノール濃度と共鳴角のシフト量とを示すグラフである。
【図２４】本発明の第４実施例にかかる、エタノール水溶液のエタノール濃度と反射光強度とを示すグラフである。
【図２５】本発明の第４実施例にかかる、エタノール水溶液のエタノール濃度と反射光強度とを示すグラフである。
【図２６】本発明の第５実施例にかかるセンサチップの模式的な斜視図である。
【符号の説明】
１，２１ センサチップ（表面プラズモン共鳴センサチップ）
１ａ，２１ａ センサ面
２ 基体
３，２３ 金属層
４ 凹凸面
５ａ〜５ｉ，１１６ａ〜１１６ｅ，１２６ａ〜１２６ｅ，１３６ａ〜１３６ｋ
回折格子面（平面）
６，２６ 測定スポット
７ 結合物質
１０ 分析装置
１１ ホルダ
１１ａ，７０ 流路
１２ 光源
１３ 光検出器
１４ 分析部
２５ 回折格子面（曲面）
３１ センサチップ
３３ 金属層
３５ａ〜３５ｉ 回折格子面
３６ 測定スポット
３７ａ〜３７ｉ 非回折面
４１ センサチップ
４３ 金属層
４６ 測定スポット
４７ 結合物質
４８ 非結合物質
５９ 分離装置
７２ 蓋
７９ 試料導入装置
７１ センサチップ
Ｓ０ 基準平面
Ｓ１ 特定平面
８１ センサチップ
８３ 金属層
８７ 測定スポット
８８ 非回折領域
９１ センサチップ
９３ 金属層
９６ 測定スポット
９７ 結合物質
９８ 非結合物質
１００ 流路
１０１ センサチップ
１０２ 蓋[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the structure of a diffraction grating type surface plasmon resonance sensor chip, and more particularly to the structure of a sensor chip suitable for use in small clinical instruments and HPLC detectors.
[0002]
[Prior art]
Conventionally, in fields such as biochemistry and medical examination, surface plasmon resonance (SPR) is used as a quantitative and / or qualitative analysis method for sample fluids containing detection species such as chemical species, biochemical species, or biological species. The analysis method used is known. Surface plasmon resonance is a phenomenon in which, when light is incident on a metal layer, a surface plasmon wave induced on the metal surface is excited by resonating with an evanescent wave generated by the incident light. Surface plasmon resonance depends on the wavelength and angle of incident light, and when surface plasmon resonance is excited, the light energy of a light component having a specific incident angle or specific wavelength shifts to a surface plasmon wave, The reflected light having the corresponding incident angle or wavelength is greatly reduced.
[0003]
In order to cause surface plasmon resonance, a metal having a specific surface plasmon wave and an optical structure that induces an evanescent wave that resonates with the surface plasmon wave are required. Two structures are currently known as optical structures for inducing evanescent waves. One is an optical structure using total reflection of a prism, and the other is an optical structure using a diffraction grating. An element in which these optical structures are combined with the above metal is generally called a surface plasmon resonance sensor chip (hereinafter referred to as a sensor chip).
[0004]
Normally, sensor chips have a structure in which a metal layer is laminated on a substrate, and a binding substance (ligand, molecular recognition element) that can specifically bind by interacting with a specific detection species is applied on the metal layer. Fixed. When the sample is brought into contact with the surface of the metal layer on which the binding substance is immobilized, the detection species in the sample is captured by the binding substance. Surface plasmon resonance also depends on the refractive index of the medium on the surface of the metal layer. If the refractive index of the medium changes, the resonance angle changes when the wavelength is constant, and also when the incident angle is constant. Wavelength changes. Therefore, the refractive index of the medium on the surface of the metal layer can be analyzed by examining the resonance angle or the resonance wavelength based on the intensity of the reflected light. In this case, the change in the refractive index of the medium on the surface of the metal layer corresponds to the change in the amount of the detection species trapped by the binding substance, that is, the change in the concentration of the detection species in the sample. By examining the occurring resonance angle or resonance wavelength, the concentration of the detected species in the sample can be analyzed.
[0005]
Among such sensor chips, a prism type sensor chip is generally composed of a sensor chip body (a metal layer laminated on a transparent substrate) and a prism. The sensor chip is basically disposable. However, since the prism is expensive, if not only the sensor chip body but also the prism is disposable, the measurement cost becomes very high. For this reason, in this type of sensor chip, the sensor chip main body and the prism are generally separate, and when used, the prism is brought into close contact with the sensor chip main body, light is incident on the prism, and reflected light is detected and measured. Yes.
[0006]
On the other hand, the diffraction grating type sensor chip has a structure in which a metal layer is laminated on a transparent substrate having a concavo-convex shape (grating) on the surface. By laminating the metal layer on the concavo-convex shape, the concavo-convex shape also appears on the surface of the metal layer, and the concavo-convex shape on the surface of the metal layer functions as a diffraction grating. This type of sensor chip is inexpensive because it does not use an expensive prism such as a prism type, and can be disposable. Further, since the work of bringing the prism and the sensor chip main body into close contact with each other as in the prism type is unnecessary, there is also an advantage that the reproducibility of the measured value is good without a problem such as a variation in the close contact degree.
[0007]
In addition, the prism type sensor chip has a structure in which the prism is used as a path for incident light and reflected light, but there are restrictions on the beam diameter and the region where the beam can be irradiated. However, the diffraction grating type sensor chip has such restrictions. However, a large-diameter beam can be used, and the beam can be irradiated at an arbitrary position. Therefore, the diffraction grating type sensor chip has an advantage that it can inspect a large area at a time as compared with the prism type and can inspect an arbitrary position on the sensor chip. Due to such advantages, there is an increasing expectation for a diffraction grating type sensor chip today.
[0008]
As a sample analysis method using a diffraction grating type sensor chip, an analysis method for detecting a resonance wavelength under a constant incident angle condition and an analysis method for detecting a resonance angle under a constant incident wavelength condition are generally used. . Among these, in the former method, depending on the setting of the incident angle, the resonance wavelength may be outside the measurement range of the measuring instrument, and in this case, it is necessary to readjust the optical system. On the other hand, in the latter method, since the incident wavelength that falls within the measurement range can be selected in advance, there is no problem like the former method.
[0009]
The following four methods are generally known as methods for detecting the resonance angle under the condition of a constant incident wavelength. The first method is a method of detecting a resonance angle by changing the detection angle of a detector that detects reflected light while changing the incident angle of incident light (this is called angle scanning). . In the second method, the angle of incident light is scanned as in the first method, and the reflected light is detected using a fixed angle detector (array detector such as a CCD) to detect the resonance angle. It is a method to do.
[0010]
The third method is a method of irradiating wedge-shaped light as incident light, changing the detection angle of the detector within the reflection angle range of the reflected light, and detecting the resonance angle. And the 4th method is a method of irradiating wedge-shaped light as incident light similarly to the 3rd method, detecting the reflected light using a fixed angle type detector, and detecting a resonance angle.
Furthermore, in addition to the above method, there is also known an analysis method in which the incident angle and the incident wavelength are both fixed. In this method (fifth method), the intensity distribution of the reflected light with respect to the incident angle or the incident wavelength varies depending on the amount of the detection species trapped by the binding substance, that is, the concentration of the detection species in the sample. The intensity of the reflected light is measured under the condition where the incident angle and the incident wavelength are constant, and the concentration and the like are analyzed based on the intensity change amount (change amount from the state not in contact with the sample).
[0011]
[Problems to be solved by the invention]
However, each analysis method has the following problems. First, the first and second methods require a driving mechanism for changing the optical axis angle of the light source in order to perform an angle scan of incident light, resulting in an increase in cost. In addition, since the drive mechanism is required, the apparatus becomes large, and it is difficult to apply to applications such as home use and POC (point of care). In addition, since angle control in mdegree units is necessary, there is a problem that it is difficult to obtain sufficient accuracy. Furthermore, since the angle scan takes time, it cannot follow a very fast reaction and is not suitable for real-time measurement.
[0012]
In the third and fourth methods, although there is no need to scan the angle of the incident light, the wedge-shaped vertex of the incident light is used as the measurement point so that the light enters the measurement point on the sensor chip surface from various angles. Since it is necessary to make contact, it is not suitable for large area measurement or multi-point simultaneous measurement in which a plurality of measurement points are simultaneously measured. For this reason, it cannot cope with integration of protein chips, DNA chips, etc., and the advantages of the above-described diffraction grating type sensor chip cannot be fully utilized.
[0013]
Further, in the fifth method, as in the third and fourth methods, it is not necessary to scan the angle of the incident light, and it is possible to perform a highly accurate analysis that does not require a driving mechanism, but the spectrum of the surface plasmon wave The measurement range (measurement tolerance) is narrow due to the characteristics of the shape, and it is not effective for a very wide concentration range. For this reason, when the measurement range is exceeded, there is a problem that the incident angle must be readjusted to measure the intensity change of the reflected light.
[0014]
The present invention was devised in view of such problems, and a first object thereof is a surface plasmon resonance sensor chip capable of simultaneously realizing real-time measurement and simultaneous measurement of a large area with a simple optical system configuration. Another object is to provide a sample analysis method and an analysis apparatus using the same.
The second object of the present invention is to provide a surface plasmon resonance sensor chip that can handle samples of a wide concentration range by expanding the measurement range when performing analysis based on the amount of change in the intensity of reflected light. It is an object to provide an analysis method and an analysis apparatus for a sample used.
[0015]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the inventor found that a diffraction grating surface on which a diffraction grating that generates an evanescent wave by light irradiation was formed, and a surface provided along the diffraction grating surface by light irradiation on the surface. In a diffraction grating type surface plasmon resonance sensor chip provided with a metal layer capable of inducing surface plasmon waves, the diffraction grating surface has an angular distribution with respect to the direction of light irradiation, whereby the first, The inventors have found that the second object can be achieved together and have completed the present invention.
[0016]
  First, a first surface plasmon resonance sensor chip (first sensor chip) of the present invention is formed in the vicinity of a metal layer capable of inducing a surface plasmon wave on the surface by light irradiation, and near the metal layer. A diffraction grating that generates an evanescent wave by irradiation includes a plurality of diffraction grating surfaces formed with a constant groove direction and groove pitch, and each of the diffraction grating surfaces is perpendicular to a specific plane perpendicular to a predetermined reference plane And with respect to the reference planeDifferentA diffraction grating is formed in a groove direction perpendicular to the specific plane and arranged at a predetermined inclination angle.
[0017]
With such a configuration, when light (parallel light) is irradiated from a certain direction parallel to the specific plane, the incident angle of the irradiation light on each diffraction grating surface according to the inclination angle of each diffraction grating surface with respect to the reference plane Distribution occurs, and distribution also occurs in the intensity of reflected light obtained from each diffraction grating surface. Therefore, the resonance angle can be calculated in real time based on the intensity of the reflected light obtained from each diffraction grating surface and the substantial incident angle on each diffraction grating surface. That is, by using the sensor chip having the above-described configuration, the same effect as that obtained when light is irradiated from a plurality of angles at the same time can be obtained without using angle scanning or wedge-shaped light irradiation.
[0018]
  When a plurality of diffraction grating surfaces are arranged in a direction parallel to the specific plane, each diffraction grating surface is set so that the inclination angle with respect to the reference plane when viewed from one direction parallel to the specific plane is gradually reduced. It is preferable to arrange them in order. As a result, the reflected light from each diffraction grating surface does not cross each other, and the intensity of the reflected light from each diffraction grating surface can be easily analyzed.The
[0019]
  Moreover, it is preferable to arrange | position the said diffraction grating surface continuously in the convex mountain shape on the side irradiated with light. As a result, the reflected light from each position on the diffraction grating surface is not interlaced, and the analysis of the intensity of the reflected light from each position on the diffraction grating surface is facilitated.The
[0020]
  The diffraction grating may be formed to have a minimum width having only one groove, and a convex arcuate curved surface may be formed on the light irradiation side by the set of diffraction grating surfaces.Yes.
[0021]
  In addition, it is also preferable to provide a plurality of diffraction areas where the diffraction grating surfaces are collectively arranged, and to arrange a plurality of diffraction grating surfaces with different inclination angles in each diffraction area.Yes.In this case, the resonance angle in each diffraction region can be detected at the same time simply by irradiating light from a certain direction parallel to a specific plane. Simultaneous point measurement can be easily performed.
[0022]
  In the first sensor chip, each diffraction grating surface is provided along a sensor surface in contact with the sample. When the first sensor chip is used for quantitative and / or qualitative analysis of the sample, it specifically binds to the detection species (chemical species, biochemical species, or biological species) in the sample on the sensor surface. Using a fixed binding substance (substance capable of capturing a detection species by interactions such as antigen-antibody reaction, complementary DNA binding, receptor / ligand interaction, enzyme / substrate interaction)TheIn particular, in the case of a sensor chip for multipoint simultaneous measurement, it is possible to analyze a plurality of types of detection species at the same time by using a plurality of types of binding substances immobilized corresponding to each diffraction region.The
[0023]
  In addition, a plurality of types of the binding substances may be fixed for each diffraction grating surface.Yes.
[0024]
Further, a non-diffractive surface on which no diffraction grating is formed may be provided on the sensor surface in the same plane as each of the diffraction grating surfaces.Yes.
  In addition, a non-diffractive region in which non-diffractive surfaces on which the diffraction grating is not formed is aggregated is provided corresponding to each diffraction region, and each non-diffractive surface constituting the non-diffractive region is configured as a corresponding diffraction region Each diffraction grating surface may be formed to have the same inclination angle distribution with respect to the reference plane as the inclination angle distribution with respect to the reference plane.Yes.
[0025]
  Further, for each diffraction plane, the reaction region in which the binding substance is immobilized and the substance that does not cause specific binding to the detection species in the sample are immobilized, or no substance is immobilized. A non-reactive area may be provided.Yes.
  In addition, a reaction region in which a binding substance that specifically binds to the detection species in the sample is immobilized is provided in a part of each diffraction region, and the detection species in the sample and the specific species are provided in the other diffraction regions. Non-reactive areas may be provided in which substances that do not bind naturally are immobilized, or where no substance is immobilized.Yes.
[0026]
  In addition, the diffraction grating surface is arranged in a direction perpendicular to the groove direction, and a lid that covers the sensor surface is provided, and the gap between the sensor surface and the lid is directed toward the arrangement direction of the diffraction grating surface. Multiple channels may be formed side by sideYes.
  Further, a lid covering the sensor surface may be provided, and a plurality of flow paths may be formed in parallel between the sensor surface and the lid, and the diffraction region may be provided for each of the flow paths.Yes.
[0027]
  There are the following eight methods for analyzing a sample using the first sensor chip. The first analysis method is an analysis method in which a resonance angle is calculated and a sample is analyzed based on the resonance angle. In this analysis method, a sample is brought into contact with a sensor surface and light is emitted at a constant incident angle parallel to a specific plane. Irradiating the reflected light from each diffraction grating surface, measuring the intensity of the reflected light from each received diffraction grating surface, the measured reflected light intensity and the reference plane of each diffraction grating surface A step of calculating a resonance angle based on the tilt angle, and a step of performing a quantitative and / or qualitative analysis of the sample based on the calculated resonance angle.The
[0028]
In this case, since the resonance angle can be calculated instantaneously without using an angle scan, real-time measurement can be performed, and the resonance angle can be calculated without using wedge-shaped light irradiation. Can also be measured. Therefore, when a sensor chip for multipoint simultaneous measurement provided with a plurality of diffraction regions in which diffraction grating surfaces are collectively arranged, multipoint simultaneous measurement can be performed in real time. Further, since the optical axis of the light source may be constant and parallel light may be used, the optical system can be simplified. Each of these steps may be executed in the order of description or may be executed simultaneously. In particular, when each step is executed simultaneously, it is possible to monitor in real time how the detection species in the sample are bound to the binding substance.
[0029]
  This analysis method can be implemented by using an analyzer having the following configuration. That is, the analyzer holds the first sensor chip by the holding unit in a state where the sample is in contact with the sensor surface, and the specific plane toward the sensor surface of the sensor chip held by the holding unit. The device has a configuration in which light is emitted from the light irradiating means at a constant incident angle in parallel, the reflected light from each diffraction grating surface is received by the light receiving means, and the intensity of the received reflected light is measured by the measuring means. is doing. The analyzer further includes a calculating means and an analyzing means as means for analyzing the sample from the reflected light received by the light receiving means. The calculating means is means for calculating the resonance angle based on the intensity of the reflected light from each diffraction grating surface measured by the measuring means and the inclination angle of each diffraction grating surface with respect to the reference plane, and the analyzing means is calculated by the calculating means. A means for quantitative and / or qualitative analysis of a sample based on the calculated resonance angle.The
[0030]
  The second analysis method is an analysis method in which a resonance angle is calculated and a sample is analyzed based on the resonance angle. The sample is brought into contact with a sensor surface, and light is irradiated at a constant incident angle parallel to a specific plane. Step, receiving the reflected light from the sensor surface, measuring the intensity of the reflected light from each diffraction grating surface, taking into account the intensity of the reflected light from the non-diffractive surface, reflected light from each diffraction grating surface The resonance angle at which the resonance phenomenon between the evanescent wave and the surface plasmon wave occurs is determined based on the step of correcting the intensity of the light, the intensity of the reflected light from each diffraction grating surface, and the inclination angle of each diffraction grating surface with respect to the reference plane. Performing a step of performing a quantitative and / or qualitative analysis of the sample based on the calculated resonance angle.The
[0031]
Here, the non-diffractive surface reflecting the reflected light to be considered in the correction does not have the same inclination angle with respect to the reference plane as the diffraction grating surface on which the reflected light to be corrected is reflected. must not. Therefore, if the diffraction grating surface and the non-diffractive surface do not exist on the same plane, specify the non-diffractive surface corresponding to each diffraction grating surface, and correct it by taking into account the reflected light from the non-diffractive surface Need to do.
[0032]
In this case, in addition to the same advantages as the first analysis method, the intensity of the reflected light from each diffraction grating surface is corrected in consideration of the intensity of the reflected light from the non-diffraction surface. An error in the intensity of the reflected light due to the difference can be corrected.
[0033]
The surface characteristics refer to elements that weaken the reflected light intensity from the sensor chip among elements applied to the sensor chip surface other than the resonance phenomenon between the evanescent wave and the surface plasmon wave. For example, turbidity of the sample solution that scatters incident light, incident light absorbing material contained in the sample solution, elements that weaken the reflected light from the sensor chip, and a small amount of ideal flat surfaces such as distortion, deflection, swelling, and contraction It means the displacement from. In addition, when a component in the sample adsorbs nonspecifically on the surface of the sensor chip, incident light is scattered or absorbed by the nonspecifically adsorbed substance, and reflected from the sensor chip. The light intensity decreases.
[0034]
  This analysis method can be implemented by using an analyzer having the following configuration. That is, the analyzer holds the first sensor chip by the holding unit in a state where the sample is in contact with the sensor surface, and the specific plane toward the sensor surface of the sensor chip held by the holding unit. The device has a configuration in which light is emitted from the light irradiating means at a constant incident angle in parallel, the reflected light from each diffraction grating surface is received by the light receiving means, and the intensity of the received reflected light is measured by the measuring means. is doing. The analyzing apparatus further includes a correcting means, a calculating means, and an analyzing means as means for analyzing the sample from the reflected light received by the light receiving means. The correcting means is means for correcting the intensity of the reflected light from each diffraction grating surface in consideration of the intensity of the reflected light from the non-diffraction surface, and the calculating means is based on each diffraction grating surface corrected by the correcting means. Is a means for calculating the resonance angle based on the intensity of the reflected light of each and the inclination angle of each diffraction grating surface with respect to the reference plane, and the analysis means is quantitative and / or qualitative of the sample based on the resonance angle calculated by the calculation means. Is a means of performingThe
[0035]
  The third analysis method is an analysis method in which a resonance angle is calculated and a sample is analyzed based on the resonance angle. The sample is brought into contact with the sensor surface, and light is irradiated at a constant incident angle parallel to a specific plane. Step, receiving reflected light from the sensor surface and measuring the intensity of the reflected light from each diffraction grating surface, with respect to the measured reflected light intensity from each diffraction grating surface and the reference plane of each diffraction grating surface Based on the inclination angle, a step of calculating a resonance angle at which the resonance phenomenon of the evanescent wave and the surface plasmon wave occurs for each of the reaction region and the non-reaction region, and calculating the resonance angle in the reaction region as the resonance angle in the non-reaction region. Performing a quantitative and / or qualitative analysis of the sample based on the resonance angle corrected forThe
[0036]
In this case, in addition to the advantages similar to those of the first analysis method, the resonance angle in the reaction region is corrected by the resonance angle in the non-reaction region, so that the change in the detected species caused by the reaction can be analyzed more accurately.
[0037]
The correction in this method is not a simple correction of the intensity of reflected light, but a correction of the reaction amount that is performed using a shift of the resonance angle due to an element that is not a specific reaction to be detected. Therefore, the resonance angle is obtained. Therefore, the reaction region and the non-reaction region do not necessarily have the same inclination angle with respect to the reference plane.
[0038]
  This analysis method can be implemented by using an analyzer having the following configuration. That is, the analyzer holds the first sensor chip by the holding unit in a state where the sample is in contact with the sensor surface, and the specific plane toward the sensor surface of the sensor chip held by the holding unit. The device has a configuration in which light is emitted from the light irradiating means at a constant incident angle in parallel, the reflected light from each diffraction grating surface is received by the light receiving means, and the intensity of the received reflected light is measured by the measuring means. is doing. The analyzer further includes a calculating means and an analyzing means as means for analyzing the sample from the reflected light received by the light receiving means. The calculation means calculates the resonance angle at which the resonance phenomenon between the evanescent wave and the surface plasmon wave occurs based on the intensity of the reflected light from each diffraction grating surface measured by the measurement means and the inclination angle of each diffraction grating surface with respect to the reference plane. The analysis means is a means for calculating each of the reaction region and the non-reaction region, and the analysis means quantitatively and / or the sample based on the resonance angle obtained by correcting the resonance angle in the reaction region calculated by the calculation means in consideration of the resonance angle in the non-reaction region. Or a means of qualitative analysis.The
[0039]
  The fourth method is an analysis method for calculating a resonance angle and analyzing a sample based on the resonance angle. In this analysis method, a plurality of different samples are assigned to a plurality of flow paths, and assigned to each flow path. A step of irradiating the sensor surface with light at a constant incident angle parallel to a specific plane while flowing the sample, a step of receiving the reflected light from the sensor surface, and measuring the intensity of the reflected light from each diffraction grating surface The sample flowing through each flow path has a resonance angle at which the resonance phenomenon of the evanescent wave and the surface plasmon wave occurs based on the intensity of the reflected light from each diffraction grating surface and the inclination angle of the diffraction grating surface with respect to the reference plane. And a step of performing a quantitative and / or qualitative analysis of a sample flowing through each flow path based on the resonance angle calculated for each flow path.The
[0040]
In this case, in addition to the same advantages as those of the first analysis method, a plurality of samples can be analyzed simultaneously, so that work can be performed efficiently. In addition, since a plurality of samples can be analyzed under the same conditions, the difference between the samples can be clearly analyzed.
[0041]
  This analysis method can be implemented by using an analyzer having the following configuration. That is, the analyzer holds the first sensor chip by the holding unit, and passes a plurality of samples different from each other by the sample introduction unit to the sensor chip held by the holding unit. Each sample is irradiated with light by a light irradiation means at a constant incident angle parallel to a specific plane toward the sensor surface of the sensor chip in which the sample is introduced into each flow path by the sample introduction means. The apparatus has a configuration in which reflected light from the grating surface is received by the light receiving means, and the intensity of the received reflected light is measured by the measuring means. The analyzer further includes a calculating means and an analyzing means as means for analyzing the sample from the reflected light received by the light receiving means. The calculation means calculates the resonance angle at which the resonance phenomenon between the evanescent wave and the surface plasmon wave occurs based on the intensity of the reflected light from each diffraction grating surface measured by the measurement means and the inclination angle of each diffraction grating surface with respect to the reference plane. A means for calculating for each flow path, and the analysis means is a means for performing quantitative and / or qualitative analysis of a sample flowing through each flow path based on the resonance angle in each flow path calculated by the calculation means. AhThe
[0042]
  The fifth analysis method is an analysis method for measuring the amount of change in intensity of reflected light and analyzing the sample based on the amount of change in intensity. In this analysis method, the sample is brought into contact with the sensor surface and parallel to a specific plane. Irradiating light at a fixed incident angle, receiving reflected light from each diffraction grating surface, measuring the intensity of the reflected light from each received diffraction grating surface, from each measured diffraction grating surface Measuring the amount of change in the intensity of the reflected light with respect to the intensity of the reflected light when the sample is not in contact with the sensor surface, and diffraction in which the measured change is within a predetermined measurement tolerance (measurement range) Select a grating plane and execute a step of quantitative and / or qualitative analysis of the sample based on the amount of change in reflected light intensity from the selected diffraction grating plane.The
[0043]
Thereby, even when the concentration range of the sample is wide, it is not necessary to readjust the optical system so that the measurement value falls within the measurement range, and the measurement range can be substantially expanded. In this case, the steps may be executed in the order of description or may be executed simultaneously.
[0044]
  This analysis method can be implemented by using an analyzer having the following configuration. That is, the analyzer holds the first sensor chip by the holding unit in a state where the sample is in contact with the sensor surface, and the specific plane toward the sensor surface of the sensor chip held by the holding unit. The device has a configuration in which light is emitted from the light irradiating means at a constant incident angle in parallel, the reflected light from each diffraction grating surface is received by the light receiving means, and the intensity of the received reflected light is measured by the measuring means. is doing. Further, this analyzer includes a measuring means and an analyzing means as means for analyzing the sample from the reflected light received by the light receiving means. The measuring means is a means for measuring a change amount of the intensity of the reflected light from each diffraction grating surface measured by the measuring device with respect to the intensity of the reflected light when the sample is not in contact with the sensor surface. The analyzing means selects a diffraction grating surface whose measured reflected light intensity change amount is within a predetermined allowable measurement range, and based on the reflected light intensity change amount from the selected diffraction grating surface, quantitative and A means to perform qualitative analysisThe
[0045]
The sixth analysis method is an analysis method in which the amount of change in intensity of reflected light is measured and the sample is analyzed based on the amount of change in intensity. In this analysis method, the sample is brought into contact with the sensor surface and parallel to a specific plane. Irradiating light at a constant incident angle, receiving reflected light from each diffraction grating surface, measuring the intensity of reflected light from each received diffraction grating surface, and measuring each non-diffraction grating surface In consideration of the intensity of the reflected light from the surface, the step of correcting the intensity of the reflected light from each diffraction grating surface, the sample contacting the sensor surface of the intensity of the reflected light from each corrected diffraction grating surface Measuring the amount of change with respect to the intensity of the reflected light in a non-conducting state, selecting a diffraction grating surface whose measured variation is within a predetermined measurement tolerance (measurement range), and reflecting light from the selected diffraction grating surface Based on the amount of intensity change, To perform the steps of performing and / or qualitative analysisThe
[0046]
As in the second analysis method, the non-diffractive surface reflecting the reflected light to be considered at the time of correction is the same as the reference plane, which is the same as the diffraction grating surface reflecting the reflected light to be corrected. Must have an inclination angle with respect to. Therefore, if the diffraction grating surface and the non-diffractive surface do not exist on the same plane, specify the non-diffractive surface corresponding to each diffraction grating surface, and correct it by taking into account the reflected light from the non-diffractive surface Need to do.
[0047]
In this case, in addition to the same advantages as the fifth analysis method, the intensity of the reflected light from each diffraction grating surface is corrected in consideration of the intensity of the reflected light from the non-diffractive surface. An error in the intensity of the reflected light due to the difference can be corrected.
[0048]
  This analysis method can be implemented by using an analyzer having the following configuration. That is, the analyzer holds the first sensor chip by the holding unit in a state where the sample is in contact with the sensor surface, and the specific plane toward the sensor surface of the sensor chip held by the holding unit. The device has a configuration in which light is emitted from the light irradiating means at a constant incident angle in parallel, the reflected light from each diffraction grating surface is received by the light receiving means, and the intensity of the received reflected light is measured by the measuring means. is doing. The analyzing apparatus further includes a correcting means, a measuring means, and an analyzing means as means for analyzing the sample from the reflected light received by the light receiving means. The correcting means is means for correcting the intensity of the reflected light from each diffraction grating surface in consideration of the intensity of the reflected light from the non-diffraction surface, and the measuring means is based on each diffraction grating surface corrected by the correcting means. Is a means for measuring the amount of change in the intensity of reflected light with respect to the intensity of the reflected light when the sample is not in contact with the sensor surface. A means to select a diffraction grating surface within a range and perform quantitative and / or qualitative analysis of a sample based on the amount of change in reflected light intensity from the selected diffraction grating surface.The
[0049]
  The seventh analysis method is an analysis method for measuring the amount of change in intensity of reflected light and analyzing the sample based on the amount of change in intensity. In this analysis method, the sample is brought into contact with the sensor surface and parallel to a specific plane. Irradiating light at a fixed incident angle, receiving reflected light from each diffraction grating surface, measuring the intensity of reflected light from each received diffraction grating surface, and measuring from each measured diffraction grating surface A step of measuring a change amount of the reflected light intensity with respect to the intensity of the reflected light when the sample is not in contact with the sensor surface for each of the reaction region and the non-reaction region, and the measured change amount of the reflected light intensity is predetermined. Select a diffraction grating surface within the allowable measurement range for each of the reaction region and the non-reaction region, and the amount of change in the intensity of the reflected light from the selected reaction region and the intensity of the reflected light from the selected non-reaction region The amount of change and Based, to perform the steps of performing a quantitative and / or qualitative analysis of a sampleThe
[0050]
As in the third method, the correction in this method is not a simple correction of the intensity of the reflected light, but a correction of the reaction amount that is performed using a shift of the resonance angle due to an element that is not a specific reaction to be detected. Therefore, if the resonance angle is obtained, the reaction region and the non-reaction region do not necessarily have the same inclination angle with respect to the reference plane.
[0051]
In this case, in addition to the advantages similar to those of the fifth analysis method, the resonance angle in the reaction region is corrected by the resonance angle in the non-reaction region, so that the change in the detected species caused by the reaction can be analyzed more accurately.
[0052]
  This analysis method can be implemented by using an analyzer having the following configuration. That is, the analyzer holds the first sensor chip by the holding unit in a state where the sample is in contact with the sensor surface, and the specific plane toward the sensor surface of the sensor chip held by the holding unit. The device has a configuration in which light is emitted from the light irradiating means at a constant incident angle in parallel, the reflected light from each diffraction grating surface is received by the light receiving means, and the intensity of the received reflected light is measured by the measuring means. is doing. Further, this analyzer includes a measuring means and an analyzing means as means for analyzing the sample from the reflected light received by the light receiving means. The measuring means is a means for measuring a change amount of the intensity of the reflected light from each diffraction grating surface measured by the measuring device with respect to the intensity of the reflected light when the sample is not in contact with the sensor surface. Selects the diffraction grating surface where the measured reflected light intensity change is within the predetermined measurement tolerance range for each of the reaction region and the non-reactive region, and selects the change in reflected light intensity from the selected diffraction grating surface. Means for quantitative and / or qualitative analysis of the sample based on the amount of change in the intensity of the reflected light from the non-reactive region.The
[0053]
  The eighth analysis method is an analysis method in which a change amount of reflected light intensity is measured and a sample is analyzed based on the intensity change amount. In this analysis method, a plurality of different samples are assigned to a plurality of flow paths, A step of irradiating the sensor surface with a constant incident angle parallel to a specific plane while flowing the sample assigned to each flow path, receiving the reflected light from the sensor surface, and reflecting the reflected light from each diffraction grating surface A step of measuring the intensity, a step of measuring a change amount of the intensity of the reflected light from each measured diffraction grating surface with respect to the intensity of the reflected light when the sample does not flow in each flow path, and the measured reflected light A diffraction grating surface whose intensity change amount is within a predetermined measurement allowable range is selected for each flow path, and each flow path is defined based on the amount of change in the intensity of reflected light from the diffraction grating surface selected for each flow path. Quantitative and / or qualitative analysis of flowing samples To perform the stepThe
[0054]
In this case, in addition to the same advantages as the fifth analysis method, a plurality of samples can be analyzed at the same time, so that work can be performed efficiently. In addition, since a plurality of samples can be analyzed under the same conditions, the difference between the samples can be clearly analyzed.
[0055]
  This analysis method can be implemented by using an analyzer having the following configuration. That is, the analyzer holds the first sensor chip by the holding unit, and passes a plurality of samples different from each other by the sample introduction unit to the sensor chip held by the holding unit. Each sample is irradiated with light by a light irradiation means at a constant incident angle parallel to a specific plane toward the sensor surface of the sensor chip in which the sample is introduced into each flow path by the sample introduction means. The apparatus has a configuration in which reflected light from the grating surface is received by the light receiving means, and the intensity of the received reflected light is measured by the measuring means. Further, this analyzer includes a measuring means and an analyzing means as means for analyzing the sample from the reflected light received by the light receiving means. The measuring means is a means for measuring the amount of change in the intensity of the reflected light from each diffraction grating surface measured by the measuring device with respect to the intensity of the reflected light in a state where the sample does not flow in each of the flow paths. The means selects a diffraction grating surface in which the amount of change in the measured reflected light intensity is within a predetermined allowable measurement range for each flow path, and based on the amount of change in the intensity of the reflected light from the selected diffraction grating surface, A means of performing quantitative and / or qualitative analysisThe
[0056]
  Further, in each analysis method described above, a sample separation step for separating the sample by physical and / or chemical action may be performed prior to introducing the sample onto the surface plasmon resonance sensor chip.Yes.
[0057]
Thereby, even if impurities other than the detection species are mixed in the sample, they can be properly removed before the analysis, and only the pure detection species can be analyzed. Therefore, accurate analysis can be performed.
[0058]
In addition, by combining with the detection methods (absorbance detection, fluorescence detection, chemiluminescence detection, differential refractometer detection, electrochemical detection, etc.) that are usually used in these analytical means, Measurement by the specific reaction measurement of the species to be detected in can be performed simultaneously.
[0059]
  This analysis method is configured to have a sample separation means for separating the sample by physical and / or chemical action prior to introducing the sample onto the surface plasmon resonance sensor chip in addition to the configuration of the analysis apparatus described above. Can be implemented by usingThe
[0060]
  As a sample separation means, it is desirable to separate the sample by any one of liquid chromatography, HPLC, capillary electrophoresis, microchip electrophoresis, flow injection, or a separation method using a microchannel.Yes.
[0061]
  In each of the analysis methods described above, when the detection species is a luminescent substance, it is bound to the binding substance prior to irradiating the sensor surface with light, or after irradiating the sensor surface with light and receiving reflected light. A step of detecting the emitted light of the luminescent material, and in the analyzing step, the sample may be quantitatively and / or qualitatively analyzed together with the detection result of the emitted light.Yes.
[0062]
As a result, not only surface plasmon resonance but also analysis using a light emission phenomenon can be performed. Since luminescence phenomena such as fluorescence and phosphorescence are very sensitive, more delicate reactions can be detected.
[0063]
  In addition to the configuration of the analysis device described above, the analyzing means is configured to detect the emitted light of the luminescent substance bound to the binding substance when the detection species is a luminescent substance. This can be implemented by using an analyzer configured to perform quantitative and / or qualitative analysis of the sample comprehensively together with the detection result of the emitted light by the means.The
[0064]
  The second surface plasmon resonance sensor chip (second sensor chip) of the present invention is formed in the vicinity of the metal layer capable of inducing a surface plasmon wave on the surface by light irradiation, and in the vicinity of the metal layer. And a diffraction grating curved surface formed with a constant groove direction and groove pitch, the diffraction grating curved surface has a curved surface shape on the convex lake toward the light irradiation side, It is arranged perpendicular to a specific plane perpendicular to a predetermined reference plane, and the diffraction grating is formed in a groove direction perpendicular to the specific plane.The
[0065]
With such a configuration, when light (parallel light) is irradiated from a certain direction parallel to the specific plane, the diffraction grating surface is irradiated according to the inclination angle of the tangential plane with respect to the reference plane at the irradiation position of the irradiation light. Distribution occurs in the incident angle of light, and distribution also occurs in the intensity of reflected light obtained from each position on the diffraction grating surface. Therefore, the resonance angle can be calculated in real time based on the intensity of the reflected light obtained from each position on the diffraction grating surface and the substantial incident angle at that position. That is, by using the sensor chip having the above-described configuration, the same effect as that obtained when light having a predetermined divergence angle (or narrowing angle) is irradiated can be obtained without using angle scanning or wedge-shaped light irradiation. .
[0066]
Preferably, the shape of the diffraction grating surface is a curved surface shape convex toward the side irradiated with light. Thereby, the reflected light from each position on the diffraction grating surface does not intersect, and the analysis of the intensity of the reflected light from each position on the diffraction grating surface becomes easy.
It is also preferable to provide a plurality of diffraction grating surfaces. In this case, the resonance angle in a plurality of diffraction grating surfaces can be detected at the same time simply by irradiating light from a certain direction parallel to a specific plane, so that the binding substance is immobilized corresponding to each diffraction grating surface. Thus, simultaneous multipoint measurement can be easily performed.
[0067]
In the second sensor chip, the diffraction grating surface is provided along the sensor surface in contact with the sample. When the second sensor chip is used for quantitative and / or qualitative analysis of a sample, a sensor surface in which a binding substance that specifically binds to a detection species in the sample is immobilized is used. In particular, in the case of a sensor chip for multipoint simultaneous measurement, it is possible to analyze a plurality of types of detection species at the same time by using a plurality of types of binding substances immobilized corresponding to each diffraction grating surface.
[0068]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(A) First embodiment
First, a first embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 1, a sensor chip (surface plasmon resonance sensor chip) 1 according to the present embodiment has a surface (sensor surface) 1 a covered with a metal layer 3, and diffraction gratings are formed at a plurality of locations on the metal layer 3. The diffraction region 6 is partially provided. In the present embodiment, each diffraction region 6 in which the diffraction grating is formed becomes a measurement spot at the time of analysis by multipoint simultaneous measurement.
[0069]
FIG. 2 is an enlarged perspective view showing the measurement spot 6. As shown in FIG. 2, the measurement spot 6 is a plurality of flat surfaces (hereinafter referred to as diffraction grating surfaces) 5a to 5i on which diffraction gratings are formed. Each of the diffraction grating surfaces 5a to 5i is perpendicular to the specific plane S1 perpendicular to the reference plane S0 when the surface outside the measurement spot 6 on the metal layer 3 is the reference plane S0, and to the reference plane S0. Are arranged at predetermined inclination angles αa to αi. In addition, diffraction gratings having the same shape (same groove depth and groove pitch) are formed on the diffraction grating surfaces 5a to 5i in the groove direction perpendicular to the specific plane S1.
[0070]
Here, the central diffraction grating surface 5e is arranged in parallel to the reference plane S0, and the remaining diffraction grating surfaces 5a to 5d and 5f to 5i gradually increase in inclination angle with respect to the reference plane S0 as the distance from the center increases. In other words, they are arranged so that the inclination angle with respect to the reference plane S0 when viewed from one direction A parallel to the specific plane gradually decreases (that is, αa> αb> αc> αd> αe (αe = 0). )> Αf> αg> αh> αi). Further, the diffraction grating surfaces 5a to 5i are arranged so that adjacent diffraction grating surfaces are continuous with each other.
[0071]
According to this structure, when light is irradiated onto the sensor surface 1a of the sensor chip 1, the irradiated light is diffracted at each measurement spot 6 on the sensor surface 1a, and an evanescent wave is generated by this diffraction phenomenon. At this time, the substantial incident angle to the measurement spot 6 differs for each of the diffraction grating surfaces 5a to 5i. When the incident angle of the irradiation light with respect to the reference plane S0 in FIG. The actual incident angles are θ-αa, θ-αb, θ-αc,..., Θ-αi in order from the diffraction grating surface 5a at the end on the irradiation direction side. Since the incident angle of the irradiation light is different for each of the diffraction grating surfaces 5a to 5i as described above, the wave number of the evanescent wave generated by the diffraction phenomenon is also different for each of the diffraction grating surfaces 5a to 5i. As a result, the degree of resonance (SPR) with the surface plasmon wave generated on the surface of the metal layer 3 varies depending on the diffraction grating surfaces 5a to 5i.
[0072]
In this sensor chip 1, first, the surface 4 of the base 2 shown in FIG. 4 (a) is partially provided with uneven surfaces 4 formed with uneven shapes by laser processing or the like as shown in FIG. 4 (b). Then, as shown in FIG. 4C, the metal layer 3 can be manufactured by laminating the entire surface of the substrate 2 by sputtering or vapor deposition. When the metal layer 3 is laminated on the uneven surface 4, an uneven surface also appears on the surface of the metal layer 3, and the uneven surface that appears on the surface of the metal layer 3 functions as a diffraction grating surface to form the uneven surface 4. Each diffraction region thus formed becomes a measurement spot 6.
[0073]
The material of the base 2 is not limited as long as the surface can form the uneven surface 4 and the mechanical strength that can hold the metal layer 3 is sufficient. Resin is preferable from the viewpoint of easy formation of the concavo-convex surface 4, and acrylic resin (polymethyl methacrylate, etc.), polyester resin (polycarbonate, etc.), polyolefin and the like are mentioned as suitable materials.
The metal layer 3 is not limited to any material as long as it can induce surface plasmon waves. For example, gold, silver, copper, aluminum, an alloy containing these, an oxide of silver, copper, or aluminum can be used. Silver is preferable in terms of sensitivity and inexpensiveness, but gold is preferable in terms of stability. The thickness of the metal layer 3 is preferably 20 to 300 nm, more preferably 30 to 160 nm. When the thickness of the metal layer 3 is small, the irradiation light may pass through the metal layer 3 and reach the surface of the substrate 2, and the irradiation light may be diffracted on the uneven surface 4 on the surface of the substrate 2. In this case, the uneven shape of the uneven surface 4 also functions as a diffraction grating.
[0074]
The uneven surface 4 formed on the substrate 2 may be formed by forming a mold having a predetermined uneven shape using an ion beam in addition to the laser processing described above, and molding the substrate 2 together with the substrate 2 by injection molding into the mold. Is possible. Alternatively, a desired uneven surface 4 can be formed by forming only a plane having an inclination angle without an uneven shape on the surface of the substrate 2 and then attaching a transmissive uneven film thereon. In addition, the desired concavo-convex surface 4 can also be formed by processing the concavo-convex shape using a fine cutting technique or by forming the concavo-convex shape by microcontact printing of PDMS (polydimethylsiloxane) on a flat surface. Can do.
[0075]
The concavo-convex shape of the concavo-convex surface 4 is formed in consideration of the thickness of the metal layer 3 so that a desired diffraction grating is obtained on the surface when the metal layer 3 is laminated. The concavo-convex shape may be a rectangular wave shape, a sine wave shape, a sawtooth shape or the like, but preferably the groove depth (from top to bottom) of the diffraction grating is 10 to 200 nm (more preferably 30 to 120 nm), and the pitch. A periodic concavo-convex shape is used such that (period: distance from the concavo-convex protrusion to the adjacent protrusion) is 400 to 1200 nm (related to the wavelength of irradiation light).
[0076]
The number of diffraction grating surfaces formed in one measurement spot 6 is in the range of 2 to 100, more preferably in the range of 5 to 50. The inclination angle of the diffraction grating surface with respect to the reference plane is in the range of -10 degrees to 10 degrees, preferably in the range of -5 degrees to 5 degrees, more preferably in the range of -3 degrees to 3 degrees. Further, the amount of change in the tilt angle between adjacent diffraction grating surfaces is in the range of 0.001 to 1 degree, preferably in the range of 0.01 to 0.5 degree.
[0077]
The size of the diffraction grating surface depends on the number of measurement spots 6, but when the shape is rectangular, the short side is in the range of 5 μm to 20 mm, preferably in the range of 20 μm to 5 mm, and the shape is circular. The diameter is in the range of 5 μm to 20 mm, preferably in the range of 20 μm to 5 mm. The formation density of the measurement spots 6 is 0.1 to 1,000,000 pieces / cm.²1-100,000 pieces / cm²Is more preferable. Thereby, simultaneous measurement at multiple points is possible at 1 to 10,000,000 measurement spots 6 per chip.
[0078]
Next, a method for using the sensor chip 1 according to the present embodiment will be described.
When the sensor chip 1 is used for analyzing a sample, first, a binding substance 7 is immobilized on each measurement spot 6 as shown in FIG. This binding substance 7 is a binding substance having a property capable of specifically binding to a specific substance by an interaction such as antigen-antibody reaction, complementary DNA binding, receptor / ligand interaction, enzyme / substrate interaction, and the like. The binding substance 7 is selected according to the detection species to be detected (chemical species, biochemical species, biological species, etc.). When a plurality of detection species are included in the sample, a binding substance 7 corresponding to each detection species is selected and immobilized on a separate measurement spot 6.
[0079]
Then, the sensor chip 1 on which the binding substance 7 is thus immobilized is set in the analyzer 10 having the configuration shown in FIG. The analyzer 10 mainly includes a holder 11 for fixing the sensor chip 1, a light source 12, a photodetector 13, and an analysis unit 14.
The holder 11 is formed with a flow path 11a through which a sample fluid containing a detection species passes. The sensor chip 1 is arranged and fixed so that the sensor surface 1a is in contact with the sample flowing through the flow path 11a.
[0080]
The light source 12 is arranged with respect to the sensor chip 1 with the flow path 11a interposed therebetween so as to irradiate light toward the sensor surface 1a of the sensor chip 1. The irradiation direction of the light source 12 is set so as to be parallel to the specific plane S1 and have a predetermined incident angle θ with respect to the reference plane S0. The incident angle θ is adjusted so that the intensity of the reflected light from the diffraction grating surface 6a having the minimum incident angle among the intensity of the reflected light from the diffraction grating surfaces 5a to 5i is minimized. preferable. The light source 12 is preferably a laser light source that emits monochromatic light, particularly a semiconductor laser in terms of price and size, and the wavelength is preferably about 350 to 1300 nm. In addition, monochromatic light obtained by spectrally separating white light such as a halogen / tungsten lamp with an interference filter or a spectroscope can be used as a light source.
[0081]
The photodetector 13 is a detector that detects the reflected light from the sensor chip 1, and is arranged with the flow path 11 a interposed between the sensor chip 1 and the light source 12. As the photodetector 13, for example, an integrated CCD element, a silicon photodiode array, or the like is preferable. Although not shown in the figure, only P-polarized light can resonate the surface plasmon wave, and therefore, between the light source 12 and the sensor chip 1 or between the sensor chip 1 and the photodetector 13. A polarizer for polarizing the irradiation light from the light source 12 or the reflected light from the sensor chip 1 is provided.
[0082]
The analysis unit 14 is an apparatus that performs analysis processing based on detection information from the photodetector 13. The analysis unit 14 functions as calculation means or measurement means and analysis means according to the present invention. Hereinafter, with respect to the sample analysis procedure using the sensor chip 1 according to the present embodiment, in addition to the functions of the analysis unit 14, the resonance angle is calculated and the sample is analyzed based on the resonance angle. A specific description will be given separately for the case where the change in intensity is measured and the sample is analyzed based on the change in reflected light intensity.
[0083]
When analyzing the sample based on the resonance angle, first, the sensor chip 1 is set in the holder 11 and the sensor surface 1a of the sensor chip 1 is brought into contact with the sample (step A1). As a result, the detection species in the sample fluid specifically bind to the binding substance 7 fixed to each measurement spot 6 on the sensor surface 1a. Then, the refractive index of the medium in the vicinity of the surface of the metal layer 3 of each measurement spot 6 changes according to the amount of the detection species bound to the binding substance 7, and the resonance condition of the surface plasmon wave in each measurement spot 6 changes.
[0084]
Next, irradiation light is irradiated from the light source 12 toward the sensor surface 1a (step A2). At this time, the thickness of the irradiation light is adjusted so that the irradiation light illuminates all the measurement spots 6. Irradiation light applied to the sensor surface 1a generates diffracted light on each of the diffraction grating surfaces 5a to 5i arranged at each measurement spot 6. Of these, zero-order diffracted light (reflected light) is detected by the photodetector 13, and the intensity of the detected reflected light is measured (step A3). Therefore, the photodetector serves as a light receiving means and a measuring means.
[0085]
Information on the reflected light detected by the photodetector 13 is sent to the analysis unit 14. The analysis unit 14 extracts the information on the intensity of the reflected light from each measurement spot 6 to which the binding substance 7 is fixed from the information on the reflected light from the photodetector 13, and the diffraction grating surfaces 5a to 5a of each measurement 6 spot. The intensity of reflected light is detected every 5i. Then, the resonance angle is calculated for each measurement spot 6 based on the intensity of the reflected light from the diffraction grating surfaces 5a to 5i. Specifically, a diffraction grating surface where the intensity of the reflected light is minimized is detected for each measurement spot 6, and an actual incident angle on the diffraction grating surface (an angle obtained by subtracting the inclination angle from the incident angle on the reference plane S0). ) As a resonance angle, or based on the actual incident angles of a plurality of diffraction grating surfaces in the vicinity of the diffraction grating surface having the smallest reflected light intensity and the reflected light intensity obtained from these diffraction grating surfaces. Interpolation calculation is performed for the resonance angle at which the light intensity is minimized. In this case, the resonance angle can be calculated more accurately by the interpolation calculation (step A4-1).
[0086]
Then, the analysis unit 14 compares the wavelength of the irradiated light and the calculated resonance angle with a calibration curve (or theoretical concentration conversion formula), and analyzes the concentration of the detection species corresponding to each measurement spot 6. The calibration curve is obtained by preliminarily determining the relationship between the concentration of each detection species, the resonance wavelength, and the resonance angle by a test using a sample with a known concentration, and the calculated resonance angle at each measurement spot 6 is collated with this calibration curve. Thus, the concentration of each detection species in the sample fluid can be measured (step A5-1).
[0087]
By performing analysis using such a technique, the resonance angle at each measurement spot 6 can be calculated simultaneously and in real time, and real-time analysis can be performed for various types of detection species.
In addition, the refractive index change of the medium is very large (for example, when a pigment that has the property of being deposited on the surface by an enzyme reaction or when using fine particles such as colloidal gold as a sensitizing method for the binding reaction) ) If the angle of the diffraction grating surfaces 5a to 5i alone cannot cope with the shift of the resonance angle, it can be dealt with by changing the angle of the incident light.
[0088]
On the other hand, when analyzing the sample based on the amount of change in the reflected light intensity, if the reflected light from the sensor surface 1a is detected by the processing from step A1 to step A3, the analyzing unit 14 obtains the reflected light obtained. The information of the reflected light from each measurement spot 6 is extracted from the above information, and the intensity of the reflected light is detected for each of the diffraction grating surfaces 5a to 5i of each measurement 6 spot. Then, the amount of change in the reflected light intensity with respect to the state where the sample is not in contact with the sensor surface 1a is measured for each of the diffraction grating surfaces 5a to 5i (step A4-2).
[0089]
Next, the analysis unit 14 selects, for each measurement spot 6, a diffraction grating surface in which the amount of change in reflected light intensity is within a predetermined measurement range (measurement allowable range), that is, the range does not exceed the range. Then, the amount of change in the reflected light intensity from the selected diffraction grating surface that is not over the range and the inclination angle of the diffraction grating surface are represented by a calibration curve (the angle of incidence on the diffraction grating surface by a test using a sample with a known concentration). The concentration of the detected species corresponding to each spot 6 is analyzed (step A5-2).
[0090]
Since the wave number of the evanescent wave changes depending on the incident angle of the irradiation light on the diffraction grating surface, the amount of change in the reflected light intensity also changes depending on the incident angle, and the amount of change becomes too large depending on the incident angle. The measurement range of the measuring instrument included may be exceeded. In this case, conventionally, the incident angle has to be changed by readjustment of the optical system. In particular, when performing multi-item measurement, if the concentration difference between the detection species is large, the incident angle must be changed for each detection species to be measured. However, since the sensor chip 1 of the present embodiment is provided with a plurality of diffraction grating surfaces 5a to 5i having different inclination angles, it is possible to select another diffraction grating surface without changing the incident angle of the irradiation light. Thus, the incident angle is substantially changed. That is, it is substantially equivalent to extending the measurement range. As a result, it is possible to cope with a sample having a wide concentration range that requires a wide measurement range.
[0091]
As described above, by analyzing a sample using the sensor chip 1 of the present embodiment, real-time analysis can be performed for various types of detection species, and a sample having a wide concentration range can be handled. There are advantages.
Further, the analyzer 10 using the sensor chip 1 does not require a driving mechanism for angle scanning, and an optical system is configured only by the light source (including the polarizer) 12, the sensor chip 1, and the photodetector 13. Therefore, there is an advantage that the apparatus can be simplified, downsized, and reduced in cost.
[0092]
In recent years, especially in the field of clinical examination, POC characterized by small size and simple operation enabling examination at the site of treatment has been emphasized, and surface plasmon resonance sensor chip has been considered to be applied to immunological examinations etc. Conventionally, it has been difficult to expand to POC in terms of size and cost. However, according to this analyzer 10, since size and cost can be suppressed, it can be applied not only to POC but also to an area such as home inspection. Furthermore, the analyzer 10 is also suitable for HPLC, and can be applied to analysis of blood and urine, analysis of nutrients in food, analysis of chemical substances in waste water, and the like.
[0093]
(B) Second embodiment
Next, a second embodiment of the present invention will be described with reference to FIG.
The basic configuration of the sensor chip 21 according to the present embodiment is the same as that of the first embodiment, and the diffraction chip has a surface covered with a metal layer 23 and diffraction gratings are formed at a plurality of locations on the metal layer 23 ( A measurement spot) 26 is partially provided.
[0094]
Here, the measurement spot 26 is composed of a curved surface (hereinafter referred to as a diffraction grating surface) 25 on which a diffraction grating is formed. The diffraction grating surface 25 has a surface outside the measurement spot 26 on the metal layer 3 as a reference plane S0. At this time, they are arranged perpendicular to the specific plane S1 perpendicular to the reference plane S0. The diffraction grating surface 25 is formed in a convex arc shape on the sensor surface 21a side, and the diffraction grating is formed in the groove direction perpendicular to the specific plane S1.
[0095]
According to this structure, the substantial incident angle to the measurement spot 26 differs depending on the position on the diffraction grating surface 25. When the incident angle of the irradiation light with respect to the reference plane S0 in FIG. The actual incident angle to each position is θ−β, where β is the inclination angle of the tangential plane at that position with respect to the reference plane S0. The inclination angle of the tangential plane in contact with the diffraction grating surface 25 continuously changes from β1 (β1> 0) to β2 (β2 <0) as shown in FIG. A continuous distribution from θ-β1 to θ-β2 occurs in the substantial incident angle, and as a result, a continuous distribution is also present in the intensity of the reflected light obtained from each position of the diffraction grating surface 25. Will occur.
[0096]
Due to such characteristics, when analyzing a sample using the sensor chip 21 of the present embodiment, not only the same advantages as in the first embodiment can be obtained, but also the substantial amount of irradiation light to the diffraction grating surface 25 can be obtained. Since the incident angles are continuously distributed, the resonance angle can be directly detected without approximation or interpolation calculation, and there is an advantage that more accurate analysis is possible.
[0097]
(C) Third embodiment
Next, a third embodiment of the present invention will be described with reference to FIG.
The basic configuration of the sensor chip 31 according to the present embodiment is the same as that of the first embodiment, and the diffraction chip has a surface coated with a metal layer 33 and diffraction gratings are formed at a plurality of locations on the metal layer 33 ( Measurement spot) 36 is partially provided.
[0098]
Here, the measurement spot 36 is composed of diffraction grating surfaces 35a to 35i partially formed with diffraction gratings, and a part of the diffraction grating surfaces 35a to 35i where a diffraction grating is not formed (hereinafter referred to as non-diffraction). 37a to 37i are formed. Other configurations are the same as those of the first embodiment.
[0099]
According to this structure, the reflected light of the measurement spot 36 is reflected by the diffraction grating surfaces 35a to 35i and is weakened by surface plasmon resonance (hereinafter referred to as resonance reflected light), and the non-diffracted surfaces 37a to 37a. It is divided into reflected light (hereinafter referred to as reference reflected light) that is reflected by 37i and is not affected by surface plasmon resonance.
[0100]
Due to such characteristics, when the sample is analyzed using the sensor chip 31 of the present embodiment, in addition to the same method as in the first embodiment, the intensity of the resonance reflected light to be measured is determined by the resonance reflected light. The step of correcting by the intensity of the reference reflected light reflected by the non-diffraction surface on the same diffraction grating surface as the reflected diffraction grating surface can be provided. Thereby, in addition to the same advantages as those of the first embodiment, the influence of the error due to the surface characteristics between the diffraction grating surfaces can be corrected by the correction based on the intensity of the reference reflected light, and a more accurate analysis can be performed. There is an advantage that becomes possible.
[0101]
(D) Fourth embodiment
Next, a fourth embodiment of the present invention will be described with reference to FIG.
The basic configuration of the sensor chip 41 according to the present embodiment is the same as that of the first embodiment, and the diffraction chip has a surface coated with a metal layer 43 and diffraction gratings are formed at a plurality of locations on the metal layer 43 ( A measurement spot) 46 is partially provided.
[0102]
Here, in addition to the binding substance 47, a non-binding substance 48 is immobilized on the measurement spot 46. The non-binding substance 48 is a substance that does not have the property of specifically binding to the detection species to be detected. When a plurality of detection species are included in the sample, the binding substance 47 and the non-binding substance 48 corresponding to each detection type may be selected and immobilized on separate measurement spots 46, respectively.
[0103]
Therefore, the region where the binding substance is immobilized becomes a reaction region, and the region where the non-binding substance is immobilized and the region where both the binding material and the non-binding material are not immobilized become non-reaction regions. In addition, when the metal forming the metal layer 43 and the detection species do not specifically bind, the non-binding substance 48 is not fixed, and the binding substance 47 and the non-binding substance 48 are not fixed and the metal layer is not fixed. A region where 43 is exposed may be set as a non-reactive region.
Other configurations are the same as those of the first embodiment.
[0104]
According to this structure, the reflected light reflected from the measurement spot 46 is divided into reflected light reflected by the reaction region and reflected light reflected by the non-reaction region. The reflected light reflected in the reaction region varies depending on the quantitative and / or qualitative factors of the sample, but the reflected light reflected in the non-reactive region is not affected by the sample and has an intensity only by the structure of the diffraction grating surface. It is determined.
[0105]
Due to such characteristics, when analyzing the sample using the sensor chip 41 of the present embodiment, surface plasmon resonance occurs in each of the reaction region and the non-reaction region in addition to the same method as in the first embodiment. And a step of calculating a resonance angle obtained by subtracting a resonance angle in a non-reactive region on the same diffraction grating surface as the reaction region from the resonance angle in the reaction region. As a result, the reflected light when the detection species is bound to the binding substance in the reaction region can be analyzed with reference to the reflected light reflected in the non-reactive region near the reaction region, and thus the same advantages as in the first embodiment. In addition, it is possible to reliably analyze changes caused by the specific binding of the detection species to the binding substance.
[0106]
(E) Fifth embodiment
Next, a fifth embodiment of the present invention will be described with reference to FIG.
This embodiment has a structure in which a separation device 59 for separating the sample fluid is installed upstream of the flow path 11a through which the sample fluid flows in the analysis device 10 of the first embodiment.
[0107]
Examples of the separation device 59 include liquid chromatography and HPLC (high performance liquid chromatography) that perform separation according to the adsorptivity and distribution coefficient of the sample, and capillary electrophoresis and microchip electrophoresis that perform separation according to the electronegativity of the sample. , A flow injection method or a separation method using a microchannel is suitable.
[0108]
A microchannel is a groove on the surface of a chip through which a sample flows. A part of this groove is filled with a column equivalent to an HPLC column, or a functional group is provided on the groove surface. Thus, separation is possible.
[0109]
Flow injection is a technique that causes various reactions while the sample is flowing. For example, complex injection and solvent extraction are performed to remove substances other than the detected species in the sample. Separation can be performed.
[0110]
Of course, a device other than those described above may be attached to the analyzer as a separation device.
[0111]
When analysis is performed using this apparatus, detection species such as enzymes and proteins can be separated in advance for each pure substance by a separation apparatus. For this reason, it is possible to analyze a detection species that has become a pure substance, and to perform a more accurate analysis.
[0112]
In addition, by combining with detection methods commonly used as analytical means (absorbance detection, fluorescence detection, chemiluminescence detection, differential refractometer detection, electrochemical detection, etc.) The measurement by the specific reaction measurement of the species to be detected can be performed simultaneously.
[0113]
(F) Sixth embodiment
Next, a sixth embodiment of the present invention will be described with reference to FIG.
The basic configuration of the present embodiment is the same as that of the first embodiment, and the light emitted from the light source 12 is reflected by the sensor chip and is detected by the light detection unit 13.
[0114]
Here, the detection species is a light-emitting substance that can generate light such as fluorescence or phosphorescence. For example, there is a luminescent substance that emits light by reacting with a binding substance or being excited by light supplied from the light source 12. In the present embodiment, the light detection unit 13 is configured to detect the emitted light (emitted light).
[0115]
With such a configuration, in addition to the advantages of the first embodiment, the present embodiment can perform quantitative and / or qualitative analysis of the sample using the detection result of the emitted light, and thus perform more accurate analysis. be able to. In particular, chemiluminescence such as fluorescence is very sensitive, so that a minute reaction can be detected.
[0116]
(G) Seventh embodiment
Next, a seventh embodiment of the present invention will be described with reference to FIGS.
The basic configuration of the present embodiment is the same as that of the first embodiment, and the light emitted from the light source 12 is reflected by the sensor chip 71 and detected by the light detection unit 13.
[0117]
Here, as shown in FIG. 10, a plurality of flow paths 70 through which the sample flows are provided in a direction orthogonal to the unevenness as the diffraction grating. The channel 70 is provided between the lid 72 that covers the surface of the sensor chip 71 and the sensor chip 71, and is formed so as to pass through each measurement spot 6 on the sensor chip 71 as a set of two. A seal is provided between the flow channel 70 and the flow channel 70 so that the sample is not mixed.
Further, as shown in FIG. 11, a sample introduction device 79 is installed upstream of the flow path 70 of the analyzer 10 so that different sample fluids are assigned to the flow paths 70 and the assigned sample fluid is introduced into the flow paths. Has been.
[0118]
With this configuration, in this embodiment, in addition to the same technique as in the first embodiment, a plurality of different samples are assigned to the plurality of flow paths 70, and the sample assigned to each flow path 70 is flowed to the sensor surface. The step of irradiating light can be provided.
[0119]
For this reason, in addition to the advantages of the first embodiment, this embodiment can analyze a plurality of samples at the same time, so that work can be performed efficiently. In addition, since a plurality of samples can be analyzed under the same conditions, the difference between the samples can be clearly analyzed.
[0120]
(H) Eighth embodiment
Next, an eighth embodiment of the present invention will be described with reference to FIG.
The basic configuration of the sensor chip 81 according to the present embodiment is the same as that of the first embodiment, and the diffraction chip has a surface covered with a metal layer 83 and diffraction gratings are formed at a plurality of locations on the metal layer 83 ( Measurement spot) 87 is partially provided.
[0121]
Here, next to the measurement spot 87, there is a diffraction grating in which the surface on which the diffraction grating on the surface of the measurement spot 87 is formed has the same inclination angle distribution with respect to the reference plane S0 as the distribution of inclination angles with respect to the reference plane S0. A non-diffractive region 88 formed with a plane that is not formed (hereinafter referred to as a non-diffractive surface) is provided. Other configurations are the same as those of the first embodiment.
[0122]
According to this structure, the reflected light of the sensor chip 81 is reflected by the measurement spot 87 and is weakened by the surface plasmon resonance, and the reflected light is reflected by the non-diffracting region 88 and is affected by the surface plasmon resonance. The reference reflected light is not separated.
[0123]
Due to such characteristics, when analyzing a sample using the sensor chip 81 of this embodiment, in addition to the same method as in the first embodiment, the intensity of the resonance reflected light to be measured is determined by the resonance reflected light. A step of correcting with the intensity of the reference reflected light reflected by the non-diffractive region corresponding to the reflected measurement spot can be provided. Thereby, in addition to the same advantages as those of the first embodiment, the influence of the error due to the surface characteristics between the diffraction grating surfaces provided on the surface of each measurement spot can be corrected by the correction based on the intensity of the reference reflected light. There is an advantage that more accurate analysis can be performed.
[0124]
(I) Ninth embodiment
Next, a ninth embodiment of the present invention will be described with reference to FIG.
The basic configuration of the sensor chip 91 according to the present embodiment is the same as that of the first embodiment, and the diffraction chip has a surface covered with a metal layer 93 and diffraction gratings are formed at a plurality of locations on the metal layer 93 ( A measurement spot) 96 is partially provided.
[0125]
Here, the binding substance 97 is immobilized on a part of the surface of the measurement spot 96, and the non-binding substance 98 is immobilized on the surface of another measurement spot 96. The non-binding substance 98 is a substance that does not have the property of specifically binding to the detection species to be detected. When a plurality of detection species are included in the sample, a binding substance 97 and a non-binding substance 98 corresponding to each detection species may be selected and immobilized on separate measurement spots 96, respectively.
[0126]
Therefore, the area where the binding substance is immobilized on the surface of the measurement spot where the binding substance is immobilized becomes the reaction area, the area where the non-binding substance is immobilized on the surface of the measurement spot where the non-binding substance is immobilized, and the binding substance. A region where both the non-binding substance and the non-binding substance are immobilized becomes a non-reactive region.
Further, when the metal forming the metal layer 93 and the detection species do not specifically bind, the non-binding substance 98 is not fixed, and the binding substance 97 and the non-binding substance 98 are not fixed and the metal layer is not fixed. A region where 93 is exposed may be set as a non-reactive region.
Other configurations are the same as those of the first embodiment.
[0127]
According to this structure, the reflected light reflected from the measurement spot 96 is divided into reflected light reflected by the reaction region and reflected light reflected by the non-reaction region. The reflected light reflected in the reaction region varies depending on the quantitative and / or qualitative factors of the sample, but the reflected light reflected in the non-reactive region is not affected by the sample and has an intensity only by the structure of the diffraction grating surface. It is determined.
[0128]
Due to such characteristics, when analyzing a sample using the sensor chip 91 of the present embodiment, surface plasmon resonance occurs in each of the reaction region and the non-reaction region in addition to the same method as in the first embodiment. And a step of calculating a resonance angle obtained by subtracting a resonance angle in a non-reactive region on the same diffraction grating surface as the reaction region from the resonance angle in the reaction region. As a result, the reflected light when the detection species is bound to the binding substance in the reaction region can be analyzed with reference to the reflected light reflected in the non-reactive region near the reaction region, and thus the same advantages as in the first embodiment. In addition, it is possible to reliably analyze changes caused by the specific binding of the detection species to the binding substance.
[0129]
(J) Tenth embodiment
Next, a tenth embodiment of the present invention will be described with reference to FIGS.
The basic configuration of the present embodiment is the same as that of the first embodiment, and the light emitted from the light source 12 is reflected by the sensor chip 101 and detected by the light detection unit 13.
[0130]
Here, as shown in FIG. 14, a plurality of flow paths 100 through which a sample flows are provided in parallel on the surface of the sensor chip 101. The channel 100 is provided between the lid 102 that covers the surface of the sensor chip 101 and the sensor chip 101. A seal is provided between the channel 100 and the channel 100 so as not to mix the sample.
Each channel 100 is provided with a plurality of measurement spots 6 similar to those in the first embodiment.
[0131]
Further, as shown in FIG. 11, a sample introduction device 79 is installed upstream of the flow channel 100 of the analyzer 10 so that different sample fluids are assigned to the flow channels 100 and the assigned sample fluid is introduced into the flow channels. Has been.
[0132]
With this configuration, in this embodiment, in addition to the same technique as in the first embodiment, a plurality of different samples are assigned to the plurality of flow paths 100, and the sample assigned to each flow path 100 is flowed to the sensor surface. The step of irradiating light can be provided.
[0133]
For this reason, in addition to the advantages of the first embodiment, this embodiment can analyze a plurality of samples at the same time, so that work can be performed efficiently. In addition, since a plurality of samples can be analyzed under the same conditions, the difference between the samples can be clearly analyzed.
[0134]
Further, the present embodiment and the fourth embodiment can be combined, or the present embodiment and the ninth embodiment can be combined. As a result, it is possible to form a reaction region and a non-reaction region on each diffraction grating surface on the same channel, and correction by reflected light from the non-reaction region can be performed for each sample flowing through each channel. .
[0135]
(K) Other
Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the spirit of the present invention.
For example, in the first embodiment, the central diffraction grating surface 5e is arranged in parallel to the reference plane S0. However, as in the diffraction grating surfaces 116a to 116e shown in FIG. It may be arranged parallel to S0 and may be arranged such that the inclination angles of the diffraction grating surfaces 116a to 116d are gradually increased toward the light irradiation direction. However, when providing diffraction grating surfaces with the same number of faces, the arrangement in which the inclination angle is increased to the right and left around the central diffraction grating surface 5e as in the first embodiment is the central portion. Therefore, the flow of the sample liquid is not hindered.
[0136]
Further, each diffraction grating surface does not necessarily have to be continuous. As in the diffraction grating surfaces 126a to 126e shown in FIG. 16, one end is arranged at the same level as the reference plane S0, and the other end is raised to make a reference. An inclination angle may be given to the surface S0.
When many diffraction grating surfaces are arranged in one measurement spot, the diffraction grating surfaces 136a to 136k may be arranged in a plurality of rows as shown in FIG. Thereby, it becomes possible to arrange many diffraction grating surfaces 136a-136k compactly.
[0137]
Furthermore, in each embodiment, the planar diffraction grating surfaces 5a to 5i are sequentially arranged so that the inclination angle with respect to the reference plane S0 when viewed from one direction parallel to the specific plane S1 is gradually reduced, A diffraction grating surface 25 having a convex curved surface shape is provided toward the irradiation side, and this is reflected light from each diffraction grating surface 5a to 5i or reflection from each position of the diffraction grating surface 25. It is an arrangement or shape for preventing light from intermingling. However, the arrangement or shape is not necessarily limited as long as the reflected light from each position of the diffraction grating surfaces 5a to 5i or the diffraction grating surface 25 can be distinguished.
[0138]
In each embodiment, the sensor chip of the present invention is configured as a sensor chip for multipoint simultaneous measurement (or for multi-item measurement) in which a plurality of measurement spots are provided on the sensor surface. The present invention can be applied to a sensor chip that is one measurement spot.
Furthermore, in each embodiment, the case where the present invention is applied to a diffraction grating type sensor chip having a conventional general structure in which a diffraction grating is formed on the surface of a metal layer has been described. However, the present invention is not limited to various other structures. This can also be applied to a diffraction grating type sensor chip. That is, a sensor including a diffraction grating surface on which a diffraction grating that generates an evanescent wave by light irradiation is formed, and a metal layer that is provided along the diffraction grating surface and can induce a surface plasmon wave on the surface by light irradiation. The present invention can be applied to a chip.
[0139]
It is also possible to implement the embodiments in combination. For example, the third embodiment and the sixth embodiment may be combined, or the fifth embodiment and the seventh embodiment may be combined.
[0140]
In particular, the third embodiment and / or the eighth embodiment and the fourth embodiment and / or the ninth embodiment are combined to perform both correction by a non-diffractive surface and correction by a non-reactive region. Is preferable because accurate analysis can be performed more reliably. That is, the resonance angle is calculated by correcting the reflected light from the reaction region with the non-diffracting surface, and further, the resonance angle is calculated by correcting the reflected light from the non-reactive region with the non-diffracting surface, The resonance angle shift amount derived from the true specific reaction and the detected species concentration can be calculated from both resonance angles.
[0141]
Further, in the fourth embodiment and the ninth embodiment, the non-binding substance is the same regardless of the detection species when selecting a substance that does not cause a specific reaction with the detection species depending on the detection species. May be used.
[0142]
When a non-binding substance is selected according to the detection species, it is preferable to immobilize both the binding substance and the non-binding substance on the same diffraction grating surface as in the fourth embodiment. However, considering the labor for manufacturing the sensor chip, as in the ninth embodiment, the binding substance is immobilized by immobilizing the non-binding substance in the measurement spot existing next to the measurement spot where the binding substance is immobilized. It is advantageous to make the measurement spot itself adjacent to the measured measurement spot the non-reactive region.
[0143]
On the other hand, when the same substance is used as a non-reacting substance for all detection species, BSA (bovine serum albumin) used as a blocking agent, gelatin or the like can be used. In this case, as in the ninth embodiment, non-reactive substances are immobilized on all diffraction grating surfaces of one or more measurement spots on the sensor chip, and the measurement spots themselves are used as non-reactive areas in terms of manufacturing. It is advantageous.
[0144]
【Example】
The present invention will be described below with reference to examples, but the present invention is not limited to the examples as long as the gist of the present invention is not exceeded.
[0145]
Example 1
An uneven shape with a groove pitch of about 870 nm and a groove depth of about 40 nm is formed on the surface of a flat polycarbonate substrate, and this uneven shape is used as a diffraction grating. A sensor chip was created.
Subsequently, the sensor chip was formed in a 15 mm × 25 mm rectangle so that the diffraction grating was parallel to a side having a length of 25 mm. The formed sensor chip is shown in FIG.
[0146]
Next, a straight line parallel to the side of the sensor chip having a length of 25 mm, including points where the distance from one end of the side of the sensor chip having a length of 15 mm is about 5.5 mm, 4 mm, and 5.5 mm, is taken as a crease, The sensor chip was bent by about 0.8 °. That is, as shown in FIG. 19, a rectangular area of 5.5 mm × 25 mm (hereinafter referred to as area A) and a rectangular area of 4 mm × 25 mm (hereinafter referred to as area B) contact each side having a length of 25 mm. On the opposite side of region A from region A, region B and a rectangular region of 5.5 mm × 25 mm (hereinafter referred to as region C) are in contact with a side having a length of 25 mm. In addition, the region B maintains 0 ° with respect to the reference plane, the region C forms an angle of about 0.8 ° with respect to the region B, and the region A forms an angle of about −0.8 ° with respect to the region B. It was bent as if it were. Therefore, the angle formed by the region A and the region C is about 1.6 °. Further, the diffraction grating is made to be the convex surface of the sensor chip so that the sample can be measured on the convex surface of the sensor chip during measurement.
[0147]
Resonance angle detection type SPR measuring device FLEX CHIPS with a 10 mm × 10 mm area in the center of the sensor chip thus created as a measurement spot^TM The intensity of the reflected light was measured while performing an angle scan with Kinetic Analysis System (HTS Biosystems Inc.). For the measurement, light having a wavelength of about 870 nm was used as incident light, and purified water was used as a sample, and measurement was performed at three arbitrary points in each region.
[0148]
FIG. 20 shows the relationship between the incident angle of the incident light and the reflected light intensity before and after the measured resonance angle. In each graph, the angle at which the reflected light intensity is minimum is the resonance angle. Table 1 shows the measurement results for each region. SD is a standard deviation and CV is a coefficient of variation.
[Table 1]

[0149]
Note that the three measurement points in the region A are denoted as A-1, A-2, and A-3, respectively, and similarly, the three measurement points in the region B are denoted as B-1, B-2, and B-3, respectively. The three measurement points in the region C are denoted as C-1, C-2, and C-3, respectively.
As shown in Table 1, the average resonance angles of the respective regions were 21.441 ° in the region A, 20.791 ° in the region B, and 19.860 ° in the region C.
[0150]
Next, as shown in FIG. 18, a sensor chip in which the above-described sensor chip is not bent and remains in a flat plate shape is separately prepared, and the resonance angle detection type SPR measuring device FLEX CHIPS is similarly formed in the regions A, B, and C.^TM The intensity of reflected light was measured while performing angle scan with Kinetic Analysis System. Light with a wavelength of about 870 nm was used as incident light. The sample used purified water. Hereinafter, the measurement spot of the flat sensor chip is referred to as a region D. In this measurement, measurement was performed at 400 points in the region D.
[0151]
FIG. 21 shows the relationship between the incident angle of the incident light and the reflected light intensity before and after the measured resonance angle. The measurement results are shown in Table 2.
[Table 2]

As shown in FIG. 21, in the region D, the resonance angle is the same at all points. The average resonance angle was 20.832 ° as shown in Table 2.
[0152]
In the regions A, B, C, and D, the diffraction gratings in the regions have the same groove pitch and groove depth, and the measured sample is the same purified water. Should be an angle. However, since the regions A, B, and C have angles of −0.809 °, 0.041 °, and 0.972 °, respectively, compared to the region D in which no angle is introduced, the resonance angle apparently changes. As measured.
From the above results, it was confirmed that the incident angle of incident light incident on the diffraction grating surface could be changed by introducing an inclination angle into the diffraction grating surface.
[0153]
(Example 2)
Similar to Example 1, a concavo-convex shape with a groove pitch of about 870 nm and a groove depth of about 40 nm is formed on the surface of a flat polycarbonate substrate, this concavo-convex shape is used as a diffraction grating, and the thickness is further increased on the surface of the substrate. A sensor chip was prepared by depositing gold at about 80 nm. This sensor chip was bent to form 26 surfaces having different inclination angles. In the following, the areas of the respective surfaces having angles are designated in the order of the smaller inclination angles formed with respect to the horizontal plane. 1, region no. Called with a natural number of 2.
[0154]
Using the sensor chip formed with the surfaces having 26 different inclination angles in this way, the SPR measuring device FLEX CHIPS^TM The intensity of reflected light was measured while performing angle scan with Kinetic Analysis System. The sample used purified water. As incident light, light having a wavelength of about 870 nm was used. The resonance angle was specified and compared with the measurement result of the region D of Example 1 having no inclination angle, the inclination angle of each region was specified. Table 3 shows the measurement results.
[Table 3]

As a result, each region was found to have an angle of -0.936 ° to 0.978 °.
[0155]
Next, using these sensor chips, SPR measurement device FLEX CHIPS^TM In the Kinetic Analysis System, light having a wavelength of about 870 nm is used as incident light, and ethanol aqueous solutions having concentrations of 2.5%, 5%, 10%, 20%, 30%, 40%, and 50% are used as samples, respectively. Intensity was measured.
[0156]
Table 4 shows the ethanol concentration in each region and the amount of change (hereinafter referred to as a shift amount) in which the resonance angle is changed by increasing the ethanol concentration.
[Table 4]

In any region, the coefficient of variation (CV) was as good as 1.2% to 6.3% for each ethanol concentration. Further, the same amount of resonance angle shift was shown in any region.
From the above results, it was found that even a diffraction grating surface having an angle has the same measurement accuracy as a diffraction grating surface having no angle.
[0157]
(Example 3)
Among the measurement data acquired in Example 2, the measurement data when the incident angle of incident light is 21.5 ° is selected, and the ethanol concentration is 0%, 2.5%, 5 in the measurement data. The angle of the region where the reflected light intensity is minimum when the ethanol aqueous solutions of%, 10%, 20%, 30%, 40% and 50% were used as samples was calculated. Specifically, using the angle (Table 3) of each region and the reflected light intensity in that region, the angle at which the reflected light intensity is minimum was determined by regression calculation.
[0158]
FIG. 22 shows the relationship between the ethanol concentration of the aqueous ethanol solution and the angle of the region where the reflected light intensity is minimum, that is, the shift amount of the resonance angle, which is found as a result.
[0159]
From the resonance angle obtained by a generally performed method, that is, a method of performing an angle scan, the ethanol concentration of the aqueous ethanol solution and the region No. 7 and region no. The relationship with the shift amount of the resonance angle on the 15 diffraction grating surfaces was examined. The result is shown in FIG.
[0160]
Table 5 shows the result of comparison between FIG. 22 and FIG.
[Table 5]

As a result of comparison, it was found that FIGS. 22 and 23 show the same shift amount for each ethanol concentration. Therefore, the resonance angle shift amount which is found as a result of a generally performed method (FIG. 23) uses the sensor chip having the diffraction grating surface according to the present invention, so that the incident angle of the incident light is constant. It was found that the measurement method can be obtained in the form of a change in the resonance angle indicated by the diffraction grating surface giving the minimum reflection intensity.
[0161]
Example 4
Among the measurement data acquired in Example 2, data was analyzed when the incident angle of incident light was 21.5 ° and when the incident angle was 21.0 °.
[0162]
When the incident angle of incident light is 21.5 °, the region No. The relationship between the reflected light intensity of 2, 7, 10, 16, and 19 and the ethanol concentration was analyzed. The results are shown in FIG. When the incident angle of incident light is 21.0 °, the region No. The relationship between the reflected light intensity of 10, 13, 18, 20, and 22 and the ethanol concentration was analyzed. The results are shown in FIG.
[0163]
[Table 6]

[Table 7]

[0164]
From FIG. 24, when the incident angle of incident light is 21.5 °, the range of ethanol concentration from 0% to 10% is the region No. 2, the ethanol concentration in the range of 10% to 30% is region No. 2. No. 10 reflected light intensity, the range of ethanol concentration 30% -50% is the region No. It was confirmed that by using each of the 19 reflected light intensities, measurement was possible over a wide range of ethanol concentration even if the incident angle of incident light was constant.
[0165]
Similarly, from FIG. 25, when the incident angle of incident light is 21.0 °, the range where the ethanol concentration is 0% to 10% is the region No. No. 10 reflected light intensity, the range of ethanol concentration from 10% to 30% is region No. 10. No. 18 reflected light intensity, the range of ethanol concentration from 30% to 50% is region No. It was confirmed that by using each of the reflected light intensities of 22, it was possible to measure in a wide ethanol concentration range even if the incident angle of incident light was constant.
[0166]
Therefore, it was found that, compared with the conventional method of measuring the intensity of reflected light while performing angle scanning, a wide range of measurement is possible even with a simple measurement method in which the incident angle of incident light is constant and no angle scanning is performed.
[0167]
(Example 5)
An uneven shape with a groove pitch of about 870 nm and a groove depth of about 40 nm was formed on the surface of a flat polycarbonate substrate, and this uneven shape was used as a diffraction grating.
[0168]
Further, as shown in FIG. 26, the substrate was processed so that the cross section in the direction perpendicular to the diffraction grating was a curved surface having a curvature radius of about 1150 mm, and the diffraction grating was on the convex surface of the substrate. Subsequently, gold was vapor-deposited with a thickness of about 80 nm on the surface of the substrate to produce a sensor chip.
[0169]
Using this sensor chip, the resonance angle detection type SPR measuring device FLEX CHIPS^TM The intensity of reflected light was measured while performing an angle scan with Kinetic Analysis System (HTS Biosystems Inc.). For the measurement, light having a wavelength of about 870 nm was used as incident light, and purified water was used as a sample.
[0170]
Table 8 shows the result of detecting the resonance angle of the reflected light in the region of about 0.33 mm spacing in the direction orthogonal to the diffraction grating on the sensor chip surface.
[Table 8]

The sum of the angle differences of the tangent surfaces between each region is 0.17 °, which is smaller than 0.3 ° which is the angle that the tangent surfaces of both ends should form, so that the radius of curvature is larger than the design. It is estimated that it was.
[0171]
However, the angle between adjacent regions with an interval of 0.33 mm is controlled to an extremely small value of 0.010 ° on average, and a diffraction grating surface should be formed on the sensor chip surface even in a structure with a curved surface. I was able to confirm.
[0172]
【The invention's effect】
As described above in detail, according to the present invention, the diffraction grating surface of the surface plasmon resonance sensor chip has an angular distribution with respect to the light irradiation direction, so that even when parallel light is irradiated, Since the distribution of the intensity of the reflected light is obtained, the resonance angle can be calculated in real time based on the intensity of the reflected light obtained from the diffraction grating surface and the substantial incident angle on the diffraction grating surface for the entire sensor surface. There are advantages. That is, there is an advantage that real-time measurement and simultaneous measurement of a large area can be realized simultaneously with a simple optical system configuration.
[0173]
Further, according to the present invention, the diffraction grating surface of the surface plasmon resonance sensor chip has an angular distribution with respect to the light irradiation direction, so that the light can be obtained from the diffraction grating surface even when light is irradiated from one direction. Since the distribution of reflected light intensity occurs, the measurement range can be substantially expanded when analyzing based on the amount of change in reflected light intensity, and even for samples with a wide concentration range that require a wide measurement range. There is an advantage that it can cope.
[Brief description of the drawings]
FIG. 1 is a schematic perspective view showing a configuration of a sensor chip according to a first embodiment of the present invention.
2 is a schematic perspective view showing a configuration of a main part of the sensor chip of FIG. 1. FIG.
3 is a schematic perspective view showing a state in which a binding substance is immobilized on the sensor chip of FIG. 1. FIG.
4 is a schematic perspective view showing an example of a manufacturing method of the sensor chip of FIG. 1, and shows manufacturing procedures in the order of (a) to (c). FIG.
FIG. 5 is a schematic diagram showing the configuration of the analyzer according to the first embodiment of the present invention.
FIG. 6 is a schematic perspective view showing a configuration of a main part of a sensor chip according to a second embodiment of the present invention.
FIG. 7 is a schematic perspective view showing a configuration of a main part of a sensor chip according to a third embodiment of the present invention.
FIG. 8 is a schematic perspective view showing a state where a binding substance and a non-binding substance are immobilized on a sensor chip according to a fourth embodiment of the present invention.
FIG. 9 is a schematic diagram showing a configuration of an analyzer according to a fifth embodiment of the present invention.
FIG. 10 is a schematic perspective view showing a configuration of a main part of a sensor chip according to a seventh embodiment of the present invention.
FIG. 11 is a schematic diagram showing a configuration of an analyzer according to seventh and tenth embodiments of the present invention.
FIG. 12 is a schematic perspective view showing a configuration of a sensor chip according to an eighth embodiment of the present invention.
FIG. 13 is a schematic perspective view showing a configuration of a sensor chip according to a ninth embodiment of the present invention.
FIG. 14 is a schematic perspective view showing the configuration of a sensor chip according to a tenth embodiment of the present invention.
15 is a schematic perspective view showing a modification of the arrangement form of the diffraction grating surface according to the sensor chip of FIG. 1. FIG.
FIG. 16 is a schematic perspective view showing a modification of the arrangement form of the diffraction grating surface according to the sensor chip of FIG. 1;
FIG. 17 is a schematic perspective view showing a modification of the arrangement form of the diffraction grating surface according to the sensor chip of FIG. 1;
FIG. 18 is a schematic perspective view of the sensor chip according to the first embodiment of the present invention.
FIG. 19 is a schematic perspective view of the sensor chip according to the first embodiment of the present invention.
FIG. 20 is a graph showing an incident angle of incident light and reflected light intensity according to the first embodiment of the present invention.
FIG. 21 is a graph showing an incident angle of incident light and reflected light intensity according to the first example of the present invention.
FIG. 22 is a graph showing the ethanol concentration of the aqueous ethanol solution and the shift amount of the resonance angle according to the third example of the present invention.
FIG. 23 is a graph showing the ethanol concentration of the aqueous ethanol solution and the resonance angle shift amount according to the third embodiment of the present invention.
FIG. 24 is a graph showing the ethanol concentration and reflected light intensity of an aqueous ethanol solution according to the fourth example of the present invention.
FIG. 25 is a graph showing the ethanol concentration and reflected light intensity of an aqueous ethanol solution according to the fourth example of the present invention.
FIG. 26 is a schematic perspective view of a sensor chip according to a fifth embodiment of the present invention.
[Explanation of symbols]
1,21 Sensor chip (surface plasmon resonance sensor chip)
1a, 21a Sensor surface
2 Base
3,23 metal layer
4 Uneven surface
5a-5i, 116a-116e, 126a-126e, 136a-136k
Diffraction grating surface (plane)
6,26 Measurement spot
7 Binding substances
10 Analyzer
11 Holder
11a, 70 flow path
12 Light source
13 Photodetector
14 Analysis Department
25 Diffraction grating surface (curved surface)
31 Sensor chip
33 metal layers
35a-35i Diffraction grating surface
36 Measurement spot
37a-37i Non-diffractive surface
41 Sensor chip
43 Metal layer
46 Measurement spot
47 binding substances
48 Unbound material
59 Separation device
72 lids
79 Sample introduction device
71 Sensor chip
S0 Reference plane
S1 specific plane
81 Sensor chip
83 Metal layer
87 Measurement spot
88 Non-diffractive region
91 Sensor chip
93 Metal layer
96 measuring spot
97 binding substances
98 Unbound material
100 channels
101 Sensor chip
102 lid

Claims (19)

A metal layer capable of inducing surface plasmon waves on the surface by light irradiation,
A diffraction grating formed in the vicinity of the metal layer and generating an evanescent wave by light irradiation includes a plurality of diffraction grating surfaces formed with a constant groove direction and groove pitch,
Each of the diffraction grating surfaces is disposed perpendicular to a specific plane perpendicular to the predetermined reference plane and at a different predetermined inclination angle with respect to the reference plane, and each groove is perpendicular to the specific plane. A surface plasmon resonance sensor chip, wherein the diffraction grating is formed in a direction. A plurality of the diffraction grating surfaces are arranged in a direction parallel to the specific plane, and each diffraction grating surface is sequentially arranged so that an inclination angle with respect to the reference plane is gradually reduced when viewed from one direction parallel to the specific plane. The surface plasmon resonance sensor chip according to claim 1, wherein the surface plasmon resonance sensor chip is formed. 3. The surface plasmon resonance sensor chip according to claim 1, wherein the diffraction grating surface is continuously arranged in a convex mountain shape on the light irradiation side. The diffraction grating surface is formed to have a minimum width having only one groove, and a convex arcuate curved surface is formed on the side irradiated with light by the set of diffraction grating surfaces. The surface plasmon resonance sensor chip described. Each diffraction grating surface is formed along a sensor surface in contact with a sample, and a binding substance that specifically binds to a detection species in the sample is immobilized on each sensor surface on each sensor surface. The surface plasmon resonance sensor chip according to any one of claims 1 to 4, wherein the surface plasmon resonance sensor chip is characterized.   6. The surface plasmon resonance sensor chip according to claim 5, wherein a plurality of types of the binding substances are immobilized for each diffraction grating surface. The diffraction grating surface according to claim 1, wherein a plurality of diffraction regions in which the diffraction grating surfaces are aggregated are provided, and a plurality of diffraction grating surfaces having different inclination angles are arranged in each diffraction region. The surface plasmon resonance sensor chip according to the section.   Each of the diffraction grating surfaces is provided along a sensor surface in contact with the sample, and plural types of binding substances that specifically bind to the detection species in the sample are immobilized on the sensor surface corresponding to the diffraction regions. The surface plasmon resonance sensor chip according to claim 7, wherein: 9. The sensor surface according to claim 5, wherein a non-diffractive surface on which no diffraction grating is formed is provided on the same plane as each of the diffraction grating surfaces. Surface plasmon resonance sensor chip. For each diffraction grating surface, a reaction region in which the binding substance is immobilized and a substance that does not specifically bind to the detection species in the sample are immobilized, or no substance is immobilized. The surface plasmon resonance sensor chip according to claim 5, wherein a reaction region is provided. The diffraction grating surface is arranged side by side in a direction perpendicular to the groove direction, and includes a lid that covers the sensor surface, and the gap between the sensor surface and the lid is directed toward the arrangement direction of the diffraction grating surface. The surface plasmon resonance sensor chip according to claim 5, wherein a plurality of flow paths are formed in parallel. Corresponding to each of the diffraction regions, a non-diffractive region in which non-diffractive surfaces on which the diffraction grating is not formed is aggregated is provided,
Each non-diffractive surface constituting the non-diffractive region has the same inclination angle distribution with respect to the reference plane as that of each diffraction grating surface constituting the corresponding diffraction region with respect to the reference plane. The surface plasmon resonance sensor chip according to claim 7 or 8. A reaction region in which a binding substance that specifically binds to the detection species in the sample is immobilized is provided in a part of each of the diffraction regions,
In other diffraction regions, a substance that does not specifically bind to a detection species in a sample is immobilized, or a non-reactive region where no substance is immobilized is provided. The surface plasmon resonance sensor chip according to claim 7 or 8. A lid that covers the sensor surface is provided, and a plurality of flow paths are formed in parallel between the sensor surface and the lid,
The surface plasmon resonance sensor chip according to claim 7 or 8, wherein the diffraction region is provided for each of the flow paths. A method for quantitative and / or qualitative analysis of a sample using the surface plasmon resonance sensor chip according to any one of claims 5 to 10, 12, and 13,
Irradiating light at a constant incident angle in parallel with the specific plane by bringing a sample into contact with the sensor surface;
Receiving reflected light from the sensor surface and measuring the intensity of the reflected light from each diffraction grating surface;
And a step of performing a quantitative and / or qualitative analysis of the sample based on the intensity of the reflected light. A method for quantitative and / or qualitative analysis of a sample using the surface plasmon resonance sensor chip according to claim 11 or 14,
Assigning a plurality of different samples to the plurality of flow paths, irradiating the sensor surface with light at a constant incident angle parallel to the specific plane while flowing the samples assigned to the flow paths;
Receiving reflected light from the sensor surface and measuring the intensity of the reflected light from each diffraction grating surface;
And a step of performing a quantitative and / or qualitative analysis of the sample flowing through each of the flow paths based on the intensity of the reflected light. An analyzer that performs quantitative and / or qualitative analysis of a sample using the surface plasmon resonance sensor chip according to any one of claims 5 to 10, 12, and 13,
Holding means for holding the surface plasmon resonance sensor chip in a state where the sample is in contact with the sensor surface;
In a state where the surface plasmon resonance sensor chip is held by the holding means, light irradiation means for irradiating light at a constant incident angle parallel to the specific plane toward the sensor surface;
A light receiving means for receiving reflected light from the sensor surface;
Measuring means for measuring the intensity of reflected light from each diffraction grating surface received by the light receiving means;
An analysis apparatus comprising: an analysis unit that performs quantitative and / or qualitative analysis of a sample based on the intensity of the reflected light. An analyzer for performing quantitative and / or qualitative analysis of a sample using the surface plasmon resonance sensor chip according to claim 11 or 14,
Holding means for holding the surface plasmon resonance sensor chip;
In a state where the surface plasmon resonance sensor chip is held by the holding means, a plurality of different samples are assigned to the plurality of flow paths, and sample introduction means for introducing the samples assigned to the flow paths,
A light irradiating means for irradiating the sensor surface with light at a constant incident angle parallel to the specific plane toward the sensor surface in a state where the sample is introduced into each flow path by the sample introducing means;
A light receiving means for receiving reflected light from the sensor surface;
Measuring means for measuring the intensity of reflected light from each diffraction grating surface received by the light receiving means;
An analysis apparatus comprising: an analysis unit that performs quantitative and / or qualitative analysis of a sample flowing through each of the flow paths based on the intensity of the reflected light. A metal layer capable of inducing surface plasmon waves on the surface by light irradiation,
A diffraction grating formed in the vicinity of the metal layer and generating an evanescent wave by light irradiation is provided with a diffraction grating curved surface formed with a constant groove direction and groove pitch,
The diffraction grating curved surface has an arcuate curved surface convex toward the light irradiation side, and is disposed perpendicular to a specific plane perpendicular to a predetermined reference plane and has a groove direction perpendicular to the specific plane. A surface plasmon resonance sensor chip, wherein the diffraction grating is formed.

Images