JP2015021889A - Surface plasmon enhanced fluorescence measuring method and surface plasmon enhanced fluorescence measuring apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface plasmon resonance fluorescence analysis method using GC-SPFS capable of detecting a substance to be detect at a high sensitivity.SOLUTION: A chip is prepared which includes: a metal film having a diffraction grating; a capture body fixed to the diffraction grating; and a substance to be detected that is bound to the capture body and is marked with a fluorescent material. The diffraction grating is irradiated with excitation light so that a standing wave of surface plasmon occurs in the diffraction grating. Fluorescence discharged from the fluorescent material is detected to obtain a fluorescent signal.

Description

  The present invention relates to a surface plasmon enhanced fluorescence measurement method and a surface plasmon enhanced fluorescence measurement apparatus that detect a target substance contained in a specimen using surface plasmon resonance.

  In a clinical test or the like, if a very small amount of a substance to be detected such as protein or DNA can be detected with high sensitivity and quantity, it becomes possible to quickly grasp the patient's condition and perform treatment. For this reason, a method capable of detecting a minute amount of a substance to be detected with high sensitivity and quantity is demanded.

  As a method that can detect a substance to be detected with high sensitivity, surface plasmon-field enhanced fluorescence spectroscopy (hereinafter abbreviated as “SPFS”) is known. SPFS utilizes the fact that surface plasmon resonance (hereinafter abbreviated as “SPR”) occurs when light is irradiated onto a metal film under predetermined conditions. A capture body (for example, a primary antibody) that can specifically bind to the substance to be detected is immobilized on the metal film to form a reaction field for specifically capturing the substance to be detected. When a specimen containing a substance to be detected is provided in this reaction field, the substance to be detected binds to the reaction field. Next, when a secondary antibody labeled with a fluorescent substance is provided to the reaction field, the target substance bound to the reaction field is labeled with the fluorescent substance. When the metal film is irradiated with excitation light in this state, the fluorescent substance that labels the substance to be detected is excited by the electric field enhanced by SPR and emits fluorescence. Therefore, the presence or amount of the substance to be detected can be detected by detecting fluorescence. In SPFS, a fluorescent substance is excited by an electric field enhanced by SPR, so that a substance to be detected can be detected with high sensitivity.

  SPFS is roughly classified into prism coupling (PC) -SPFS and lattice coupling (GC) -SPFS by means for coupling (coupling) excitation light and surface plasmons. In PC-SPFS, a prism having a metal film formed on one surface is used. In this method, the excitation light is totally reflected at the interface between the prism and the metal film, thereby coupling the excitation light and the surface plasmon. PC-SPFS is the mainstream method at present, but because of the use of a prism and the large incident angle of excitation light on the metal film, PC-SPFS is a problem in terms of downsizing the measuring device. have.

  On the other hand, GC-SPFS couples excitation light and surface plasmons using a diffraction grating (see Non-Patent Document 1). Since GC-SPFS does not use a prism and the incident angle of excitation light with respect to the diffraction grating is small, the measuring device can be made smaller than PC-SPFS.

Keiko Tawa, Hironobu Hori, Kenji Kintaka, Kazuyuki Kiyosue, Yoshiro Tatsu, and Junji Nishii, "Optical microscopic observation of fluorescence enhanced by grating-coupled surface plasmon resonance", Optics Express, Vol. 16, pp. 9781-9790.

  As described above, GC-SPFS has the advantage that the measuring device can be downsized compared to PC-SPFS, but research on GC-SPFS has not progressed compared to research on PC-SPFS. Accordingly, there is room for improvement in detection sensitivity in the measurement method and measurement apparatus using GC-SPFS.

  The present invention has been made in view of the above points, and provides a measurement method and a measurement apparatus using GC-SPFS, which can detect a substance to be detected with higher sensitivity. The purpose is to do.

  The present inventors have found that the above problem can be solved by generating a standing wave of surface plasmons in the diffraction grating, and have further studied and completed the present invention.

  That is, the present invention relates to the following surface plasmon enhanced fluorescence measurement method.

［上記式（１）〜（３）において、ε_１は前記回折格子上の媒体の誘電率であり、ε_２は前記回折格子を構成する金属の誘電率であり、ｎは前記媒体の屈折率であり、ＭおよびＮは整数であり、ｌおよびｍは１〜５の範囲内の整数である。］
［３］前記回折格子に励起光を照射する工程では、前記捕捉体に結合した前記被検出物質に所定の周波数で前記金属膜の面方向を含む方向の振動を加え、前記蛍光シグナルを周波数解析することにより被検出物質の存在またはその量を分析する工程をさらに含む、［１］または［２］に記載の表面プラズモン増強蛍光測定方法。
［４］前記被検出物質に加えられる前記振動の周波数は、前記捕捉体に結合した前記被検出物質が共振する周波数未満であるか、または前記捕捉体に結合した前記被検出物質が共振する周波数である、［３］に記載の表面プラズモン増強蛍光測定方法。 [1] A surface plasmon-enhanced fluorescence measurement method in which a fluorescent substance that labels a substance to be detected detects fluorescence emitted by being excited by an electric field based on surface plasmon resonance, and detects the presence or amount of the substance to be detected. A step of preparing a chip having a metal film formed with a diffraction grating, a capture body immobilized on the diffraction grating, and a target substance bound to the capture body and labeled with a fluorescent material; Irradiating the diffraction grating with excitation light so that a standing wave of surface plasmons is generated in the diffraction grating, and detecting fluorescence emitted from the fluorescent material to obtain a fluorescence signal. Surface plasmon enhanced fluorescence measurement method.
[2] The pitch Λ (nm) of the diffraction grating and the wavelength λ ₀ (nm) of the excitation light in vacuum satisfy the following formula (1), and the fill factor f of the diffraction grating is expressed by the following formula ( 2), the incident angle θ (°) of the excitation light with respect to the diffraction grating satisfies the following formulas (3) to (5), or the intensity of the fluorescence from the fluorescent substance is the following formula ( [3] The surface plasmon enhanced fluorescence measurement method according to [1], which is in a range of 50% or more with respect to the intensity of fluorescence from the fluorescent material when the formula (5) is satisfied.

[In the above formulas (1) to (3), ε ₁ is the dielectric constant of the medium on the diffraction grating, ε ₂ is the dielectric constant of the metal constituting the diffraction grating, and n is the refractive index of the medium. M and N are integers, and l and m are integers in the range of 1-5. ]
[3] In the step of irradiating the diffraction grating with excitation light, a vibration in a direction including a plane direction of the metal film is applied to the detected substance coupled to the capturing body at a predetermined frequency, and the fluorescence signal is subjected to frequency analysis. The method for measuring surface plasmon enhanced fluorescence according to [1] or [2], further comprising analyzing the presence or amount of the substance to be detected.
[4] The frequency of the vibration applied to the detected substance is less than the frequency at which the detected substance coupled to the capturing body resonates, or the frequency at which the detected substance coupled to the capturing body resonates. The method for measuring surface plasmon enhanced fluorescence according to [3].

  The present invention also relates to the following surface plasmon enhanced fluorescence measuring apparatus.

［上記式（１）〜（３）において、ε_１は前記回折格子上の媒体の誘電率であり、ε_２は前記回折格子を構成する金属の誘電率であり、ｎは前記媒体の屈折率であり、ＭおよびＮは整数であり、ｌおよびｍは１〜５の範囲内の整数である。］
［７］前記捕捉体に結合した前記被検出物質に、所定の周波数で前記金属膜の面方向を含む方向の振動を加える振動付与部をさらに有し、前記振動付与部により前記被検出物質に振動を加えた状態で、前記光検出部により前記蛍光物質からの蛍光を検出し、前記光検出部からの出力信号を周波数解析することにより被検出物質の存在またはその量を分析する、［５］または［６］に記載の表面プラズモン増強蛍光測定装置。
［８］前記振動付与部が加える前記振動の周波数は、前記捕捉体に結合した前記被検出物質が共振する周波数未満であるか、または前記捕捉体に結合した前記被検出物質が共振する周波数である、［７］に記載の表面プラズモン増強蛍光測定装置。
［９］前記振動付与部は、交番電界を印加することで前記被検出物質に振動を加える、［７］または［８］に記載の表面プラズモン増強蛍光測定装置。
［１０］前記振動付与部は、前記金属膜を１つの壁面とする流路の他の壁面を振動させることで前記被検出物質に振動を加える、［７］または［８］に記載の表面プラズモン増強蛍光測定装置。
［１１］前記振動付与部は、前記金属膜を１つの壁面とする流路内において液体を往復させることで前記被検出物質に振動を加える、［７］または［８］に記載の表面プラズモン増強蛍光測定装置。
［１２］前記振動付与部は、周波数１×１０^５〜１×１０^１３Ｈｚの電磁波を照射することで前記被検出物質に振動を加える、［７］または［８］に記載の表面プラズモン増強蛍光測定装置。 [5] A chip having a metal film on which a diffraction grating is formed, a capture body fixed to the diffraction grating, and a target substance that is bound to the capture body and is labeled with a fluorescent substance is mounted and excited. A surface plasmon-enhanced fluorescence measuring device that detects the presence or amount of a substance to be detected by irradiating light to the diffraction grating, in order to excite the fluorescent substance by an enhanced electric field and emit fluorescence. A light source for irradiating the diffraction grating with the excitation light, and a light detection unit for detecting fluorescence from the fluorescent material, wherein the light source generates a standing wave of surface plasmon in the diffraction grating. And a surface plasmon enhanced fluorescence measuring apparatus that irradiates the diffraction grating with the excitation light.
[6] The pitch Λ (nm) of the diffraction grating and the wavelength λ ₀ (nm) of the excitation light in vacuum satisfy the following formula (1), and the fill factor f of the diffraction grating is expressed by the following formula ( 2), the incident angle θ (°) of the excitation light with respect to the diffraction grating satisfies the following formulas (3) to (5), or the intensity of the fluorescence from the fluorescent substance is the following formula ( [3] The surface plasmon enhanced fluorescence measurement device according to [5], which is in a range of 50% or more with respect to the intensity of fluorescence from the fluorescent material when the formula (5) is satisfied.

[In the above formulas (1) to (3), ε ₁ is the dielectric constant of the medium on the diffraction grating, ε ₂ is the dielectric constant of the metal constituting the diffraction grating, and n is the refractive index of the medium. M and N are integers, and l and m are integers in the range of 1-5. ]
[7] The apparatus further includes a vibration applying unit that applies a vibration in a direction including a surface direction of the metal film to the detected substance coupled to the capturing body at a predetermined frequency, and the vibration applying part applies the vibration to the detected substance. In a state where vibration is applied, fluorescence from the fluorescent material is detected by the light detection unit, and the presence or amount of the detection target material is analyzed by frequency analysis of an output signal from the light detection unit, [5 ] Or the surface plasmon enhanced fluorescence measuring apparatus according to [6].
[8] The frequency of the vibration applied by the vibration applying unit is less than a frequency at which the detected substance coupled to the capturing body resonates, or a frequency at which the detected substance coupled to the capturing body resonates. The surface plasmon enhanced fluorescence measuring apparatus according to [7].
[9] The surface plasmon enhanced fluorescence measurement device according to [7] or [8], wherein the vibration applying unit applies vibration to the target substance by applying an alternating electric field.
[10] The surface plasmon according to [7] or [8], wherein the vibration applying unit applies vibration to the detection target substance by vibrating another wall surface of the channel having the metal film as one wall surface. Enhanced fluorescence measuring device.
[11] The surface plasmon enhancement according to [7] or [8], wherein the vibration applying unit applies vibration to the detected substance by reciprocating a liquid in a flow path having the metal film as one wall surface. Fluorescence measuring device.
[12] The surface plasmon enhanced fluorescence according to [7] or [8], wherein the vibration applying unit applies vibration to the detected substance by irradiating an electromagnetic wave having a frequency of 1 × 10 ⁵ to 1 × 10 ¹³ Hz. measuring device.

  ADVANTAGE OF THE INVENTION According to this invention, in a measuring method and measuring apparatus using GC-SPFS, a to-be-detected substance can be detected with higher sensitivity.

1 is a schematic diagram illustrating a configuration of a surface plasmon enhanced fluorescence measurement device according to Embodiment 1. FIG. 2A and 2B are perspective views of the diffraction grating. 3 is a flowchart showing the operation of the surface plasmon enhanced fluorescence measurement device according to the first embodiment. 4A and 4B are partially enlarged cross sections of the diffraction grating showing the charge distribution of the standing wave of the surface plasmon. 6 is a schematic diagram illustrating a configuration of a surface plasmon enhanced fluorescence measurement device according to Embodiment 2. FIG. 6A to 6C are schematic views for explaining the vibration of the detection target substance bound to the reaction field. It is a figure for demonstrating an inverted pendulum model. It is a graph which shows the example of distribution of the electric field intensity on a diffraction grating. It is a graph which shows the change of the fluorescence intensity of the composite_body | complex couple | bonded by the composite_body | complex couple | bonded by specific binding (SP), and non-specific binding (US). 6 is a flowchart showing the operation of the surface plasmon enhanced fluorescence measurement apparatus according to the second embodiment.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

[Embodiment 1]
FIG. 1 is a schematic diagram showing a configuration of a surface plasmon enhanced fluorescence measurement apparatus (hereinafter referred to as “SPFS apparatus”) 100 according to Embodiment 1 of the present invention. As shown in FIG. 1, the SPFS device 100 includes a light source 110, a collimating lens 120, an excitation light filter 130, a first condenser lens 140, a fluorescent filter 150, a second condenser lens 160, a light detection unit 170, and a control unit. 180. The SPFS device 100 is used in a state where the chip 200 is mounted on a chip holder (not shown). Therefore, the chip 200 will be described first, and then the SPFS device 100 will be described.

  The chip 200 includes a substrate 210 and a metal film 220 formed on the substrate 210. A diffraction grating 230 is formed on the metal film 220. A capture body (for example, a primary antibody) is immobilized on the diffraction grating 230, and the surface of the diffraction grating 230 also functions as a reaction field for binding the capture body and the substance to be detected. In FIG. 1, the capturing body and the substance to be detected are omitted. The chip 200 is preferably a structure in which each piece has a length of several millimeters to several centimeters. However, the chip 200 is a smaller structure or a larger structure not included in the category of “chip”. May be.

  The substrate 210 is a support member for the metal film 220. The material of the substrate 210 is not particularly limited as long as it has mechanical strength capable of supporting the metal film 220. Examples of the material of the substrate 210 include inorganic materials such as glass, quartz, and silicon, and resins such as polymethyl methacrylate, polycarbonate, polystyrene, and polyolefin.

  The metal film 220 is disposed on the substrate 210. As described above, the diffraction grating 230 is formed on the metal film 220. When the metal film 220 is irradiated with light, surface plasmons generated in the metal film 220 and evanescent waves generated by the diffraction grating 230 are combined to generate surface plasmon resonance. The material of the metal film 220 is not particularly limited as long as it is a metal that generates surface plasmons. Examples of the material of the metal film 220 include gold, silver, copper, aluminum, and alloys thereof. A method for forming the metal film 220 is not particularly limited. Examples of the method for forming the metal film 220 include sputtering, vapor deposition, and plating. The thickness of the metal film 220 is not particularly limited. For example, the thickness of the metal film 220 is about 30 to 70 nm.

  The diffraction grating 230 generates an evanescent wave when the metal film 220 is irradiated with light. The shape of the diffraction grating 230 is not particularly limited as long as an evanescent wave can be generated. For example, the diffraction grating 230 may be a one-dimensional diffraction grating as shown in FIG. 2A or a two-dimensional diffraction grating as shown in FIG. 2B. In the one-dimensional diffraction grating shown in FIG. 2A, a plurality of ridges parallel to each other are formed on the surface of the metal film 220 at a predetermined interval. In the two-dimensional diffraction grating shown in FIG. 2B, convex portions having a predetermined shape are periodically arranged on the surface of the metal film 220. Examples of the arrangement of the convex portions include a square lattice, a triangular (hexagonal) lattice, and the like. Examples of the cross-sectional shape of the diffraction grating 230 include a rectangular wave shape, a sine wave shape, a sawtooth shape, and the like. In this specification, the “diffraction grating pitch” refers to the center distance Λ of convex portions in the direction in which the convex portions are arranged (x-axis direction), as shown in FIGS. 2A and 2B. The “fill factor of the diffraction grating” refers to the ratio of the width in the same direction of the protrusions to the center distance Λ of the protrusions (a number in the range of 0 to 1). In the example shown in FIGS. 2A and 2B, the optical axis of the excitation light α described later is parallel to the xz plane.

  The method for forming the diffraction grating 230 is not particularly limited. For example, after the metal film 220 is formed on the flat substrate 210, the metal film 220 may be provided with an uneven shape. Alternatively, the metal film 220 may be formed over the substrate 210 that has been previously provided with an uneven shape. In any method, the metal film 220 including the diffraction grating 230 can be formed.

  A capture body for capturing the substance to be detected is immobilized on the diffraction grating 230 (reaction field) (see the primary antibody 410 in FIG. 6). The capturing body specifically binds to the substance to be detected. In the present embodiment, the capturing body is fixed substantially uniformly on the surface of the diffraction grating 230. The type of capturing body is not particularly limited as long as it can capture the substance to be detected. For example, the capturing body is an antibody (primary antibody) or a fragment thereof specific to the substance to be detected, an enzyme that can specifically bind to the substance to be detected, or the like.

The method for immobilizing the capturing body is not particularly limited. For example, a self-assembled monomolecular film (hereinafter referred to as “SAM film”) or a polymer film to which a capturing body is bonded may be formed on the diffraction grating 230. Examples of SAM films include films formed with substituted aliphatic thiols such as HOOC— (CH ₂ ) ₁₁ —SH. Examples of the material constituting the polymer film include polyethylene glycol and MPC polymer. Alternatively, a polymer having a reactive group that can be bound to the capturing body (or a functional group that can be converted into a reactive group) may be fixed to the diffraction grating 230, and the capturing body may be bound to the polymer.

  As shown in FIG. 1, the excitation light α is applied to the metal film 220 (diffraction grating 230) at a predetermined incident angle θ. In the irradiation region, the surface plasmon generated in the metal film 220 and the evanescent wave generated by the diffraction grating 230 are combined to generate SPR. When a fluorescent substance is present in the irradiation region, the fluorescent substance is excited by the enhanced electric field formed by SPR, and fluorescent β is emitted.

  Next, each component of the SPFS device 100 will be described.

  The light source 110, the collimating lens 120, and the excitation light filter 130 constitute an excitation light irradiation unit. The excitation light irradiation unit emits the collimated excitation light α having a constant wavelength and light amount so that the shape of the irradiation spot on the surface of the metal film 220 (diffraction grating 230) of the chip 200 is substantially circular. Further, the excitation light irradiation unit emits only the P wave for the metal film 220 toward the diffraction grating 230 so that diffracted light that can be combined with the surface plasmons in the metal film 220 is generated in the diffraction grating 230. The excitation light irradiation unit irradiates the diffraction grating 230 with the excitation light α so that the optical axis of the excitation light α is along the arrangement direction of the periodic structure in the diffraction grating 230 (the x-axis direction in FIGS. 2A and 2B). Therefore, when the axis perpendicular to the x axis and parallel to the surface of the metal film 220 is the y axis, and the axis perpendicular to the x axis and perpendicular to the surface of the metal film 220 is the z axis, the optical axis of the excitation light α is xz. It is parallel to the plane (see FIG. 1).

  In the present embodiment, light source 110 is a laser diode. The light source 110 emits excitation light α (single mode laser light) toward the diffraction grating 230 of the chip 200. The type of the light source 110 is not particularly limited, and may not be a laser diode. Examples of the light source 110 include a light emitting diode, a mercury lamp, and other laser light sources.

  The collimating lens 120 collimates the excitation light α emitted from the light source 110. The excitation light α emitted from the laser diode (light source 110) has a flat outline shape even when collimated. Therefore, the laser diode is held in a predetermined posture so that the shape of the irradiation spot on the surface of the metal film 220 is substantially circular.

  The excitation light filter 130 includes, for example, a band pass filter and a linear polarization filter, and tunes the excitation light α emitted from the light source 110. Since the excitation light α from the laser diode (light source 110) has a slight wavelength distribution width, the bandpass filter turns the excitation light α from the laser diode into a narrow band light having only the center wavelength. In addition, since the excitation light α from the laser diode (light source 110) is not completely linearly polarized light, the linear polarization filter converts the excitation light α from the laser diode into completely linearly polarized light. The excitation light filter 130 may include a half-wave plate that adjusts the polarization direction of the excitation light α so that the P-wave component is incident on the metal film 220.

  The incident angle θ (see FIG. 1) of the excitation light α with respect to the metal film 220 is preferably an angle at which the intensity of the enhanced electric field formed by SPR is the strongest and as a result, the intensity of the fluorescent β from the fluorescent material is the strongest. . As will be described later, in the SPFS device 100 of the present embodiment, the intensity of the enhanced electric field formed in the vicinity of the diffraction grating 230 is further increased by generating a standing wave of surface plasmons in the diffraction grating 230. . Therefore, the incident angle θ of the excitation light α is appropriately set according to the pitch of the diffraction grating 230, the wavelength of the excitation light α, the type of metal constituting the metal film 220, etc., in order to generate a standing plasmon wave. Selected. Since the optimum incident angle θ of the excitation light α varies depending on various conditions, the SPFS device 100 first adjusts the incident angle θ by rotating the optical axis of the excitation light α and the chip 200 relatively. It is preferable to have an angle adjustment unit (not shown). For example, the first angle adjustment unit may rotate the excitation light irradiation unit or the chip 200 around the intersection between the optical axis of the excitation light α and the metal film 220.

  The 1st condensing lens 140, the fluorescence filter 150, the 2nd condensing lens 160, and the photon detection part 170 comprise a fluorescence detection unit. The fluorescence detection unit is disposed with respect to the excitation light irradiation unit so as to sandwich a straight line passing through the intersection of the optical axis of the excitation light α and the metal film 220 and perpendicular to the metal film 220. The fluorescence detection unit detects the fluorescence β emitted from the fluorescent material on the diffraction grating 230 (reaction field).

  The first condenser lens 140 and the second condenser lens 160 constitute a conjugate optical system that is not easily affected by stray light. The light traveling between the first condenser lens 140 and the second condenser lens 160 becomes substantially parallel light. The first condenser lens 140 and the second condenser lens 160 form a fluorescent image on the metal film 220 on the light receiving surface of the light detection unit 170.

  The fluorescent filter 150 is disposed between the first condenser lens 140 and the second condenser lens 160. The fluorescence filter 150 includes, for example, a cut filter and a neutral density (ND) filter, and removes noise components other than the fluorescence β (for example, excitation light α and external light) from the light reaching the light detection unit 170, The amount of light reaching the light detection unit 170 is adjusted.

  The light detection unit 170 detects a fluorescent image on the metal film 220. For example, the light detection unit 170 is a photomultiplier tube with high sensitivity and a high SN ratio. The light detection unit 170 may be an avalanche photodiode (APD), a photodiode (PD), a CCD image sensor, or the like.

  The angle of the optical axis of the fluorescence detection unit with respect to the perpendicular of the metal film 220 is preferably an angle (fluorescence peak angle) at which the intensity of the fluorescence β emitted from the diffraction grating 230 (reaction field) is maximized. Therefore, the SPFS device 100 preferably has a second angle adjustment unit (not shown) that adjusts the angle of the optical axis of the fluorescence detection unit by relatively rotating the optical axis of the fluorescence detection unit and the chip 200. . For example, the second angle adjustment unit may rotate the fluorescence detection unit or the chip 200 around the intersection between the optical axis of the fluorescence detection unit and the metal film 220.

  The control unit 180 includes an excitation light irradiation unit (light source 110), a fluorescence detection unit (light detection unit 170), an excitation light irradiation unit, and an angle adjustment unit (first angle adjustment unit and second angle adjustment unit) of the fluorescence detection unit. Control the behavior. Further, the control unit 180 analyzes the output signal (fluorescence signal) from the light detection unit 170 to analyze the presence or amount of the detection target substance. The control unit 180 is, for example, a computer that executes software.

  Next, the detection operation of the SPFS device 100 will be described. FIG. 3 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 100. In this example, a primary antibody is immobilized on the metal film 220 as a capturing body.

  First, preparation for measurement is performed (step S10). Specifically, the chip 200 is installed at a predetermined position of the SPFS device 100. Further, when a humectant is present on the metal film 220 of the chip 200, the humectant is removed by washing the metal film 220 so that the primary antibody can appropriately capture the substance to be detected.

  Next, the substance to be detected in the specimen is reacted with the primary antibody (primary reaction, step S20). Specifically, a specimen is provided on the metal film 220, and the specimen and the primary antibody are brought into contact with each other. When a substance to be detected exists in the sample, at least a part of the substance to be detected binds to the primary antibody. Thereafter, the metal film 220 is washed with a buffer solution or the like to remove substances that have not bound to the primary antibody. There are no particular limitations on the types of the specimen and the substance to be detected. Examples of the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluted solutions thereof. Examples of substances to be detected include nucleic acids (such as DNA and RNA), proteins (such as polypeptides and oligopeptides), amino acids, carbohydrates, lipids, and modified molecules thereof.

  Next, the target substance bound to the primary antibody is labeled with a fluorescent substance (secondary reaction, step S30). Specifically, a fluorescent labeling solution containing a secondary antibody labeled with a fluorescent substance is provided on the metal film 220, and the detection target substance bound to the primary antibody is brought into contact with the fluorescent labeling liquid. The fluorescent labeling solution is, for example, a buffer solution containing a secondary antibody labeled with a fluorescent substance. When the substance to be detected is bound to the primary antibody, at least a part of the substance to be detected is labeled with a fluorescent substance (see FIG. 5A). Thereafter, the metal film 220 is washed with a buffer solution or the like to remove free secondary antibodies and the like. The order of the primary reaction and the secondary reaction is not limited to this. For example, a liquid containing these complexes may be provided on the metal film 220 after the substance to be detected is bound to the secondary antibody. Further, the specimen and the fluorescent labeling solution may be provided on the metal film 220 at the same time.

  Next, the excitation light α is irradiated onto the metal film 220, and the intensity of the fluorescence β emitted from the fluorescent material is measured (step S40). Specifically, the control unit 180 causes the light source 110 to emit the excitation light α. At the same time, the control unit 180 causes the light detection unit 170 to detect the intensity of the fluorescence β from the metal film 220. The light detection unit 170 outputs the measurement result to the control unit 180.

  Finally, the control unit 180 analyzes the output signal (fluorescence signal) from the light detection unit 170, and analyzes the presence of the detection target substance or the amount of the detection target substance (step S50).

  By the above procedure, the presence of the substance to be detected in the sample or the amount of the substance to be detected can be detected.

The SPFS device 100 according to the present embodiment is characterized in that the fluorescent material is efficiently excited using the standing wave of the surface plasmon. Therefore, in the SPFS device 100 of the present embodiment, the pitch Λ (nm) of the diffraction grating 230 is generated so that the standing wave of the surface plasmon is generated in the diffraction grating 230 when the excitation light α is irradiated to the diffraction grating 230. The fill factor f of the diffraction grating 230, the wavelength λ ₀ (nm) of the excitation light α in vacuum, and the incident angle θ (°) of the excitation light α with respect to the diffraction grating 230 are set. A method for determining these parameters will be described next.

ｋ_ｓｐ：表面プラズモンの波数
λ_０：真空中の励起光αの波長（ｎｍ）
ｎ：回折格子２３０上の媒体（誘電体）の屈折率
ε_１：回折格子２３０上の媒体（誘電体）の誘電率＝ｎ^２
ε_２：回折格子２３０を構成する金属の誘電率 When SPR is generated by irradiating the diffraction grating 230 with the excitation light α, the wave number k _{sp of the} surface plasmon is defined as the following formula (6).

k _sp : wave number of surface plasmon λ ₀ : wavelength of excitation light α in vacuum (nm)
n: refractive index of medium (dielectric) on diffraction grating 230 ε ₁ : dielectric constant of medium (dielectric) on diffraction grating 230 = n ²
ε ₂ : dielectric constant of the metal constituting the diffraction grating 230

ｋ_ｓｐ：表面プラズモンの波数
ｋ_０：励起光αの波数＝２π／（λ_０／ｎ）
λ_０：真空中の励起光αの波長（ｎｍ）
ｎ：回折格子２３０上の媒体（誘電体）の屈折率
θ：励起光αの格子面に対する入射角（°）
Ｍ，Ｎ：整数
Ｋ：格子ベクトル＝２π／Λ
Λ：回折格子２３０のピッチ（ｎｍ） Further, when a surface plasmon standing wave is generated in the diffraction grating 230, the condition of the following expression (7) is satisfied. When the condition of Equation (7) is satisfied, the surface plasmon is Bragg reflected by the diffraction grating 230 and propagates in the opposite direction. Thereby, a standing wave is generated.

k _sp : wave number of surface plasmon k ₀ : wave number of excitation light α = 2π / (λ ₀ / n)
λ ₀ : wavelength of excitation light α in vacuum (nm)
n: Refractive index of medium (dielectric material) on diffraction grating 230 θ: Incident angle (°) of excitation light α with respect to grating surface
M, N: integer K: lattice vector = 2π / Λ
Λ: pitch of the diffraction grating 230 (nm)

In the above formula (7), M means the diffraction order of surface plasmons, and N means the order of electromagnetic waves in the diffraction grating 230. M and N can be arbitrarily selected within a range where θ satisfies the following expressions (8) and (9).

When SPR is generated by irradiating the excitation light α to the diffraction grating 230 and a standing wave of surface plasmon is generated in the diffraction grating 230, the following expression (10) is obtained from the above expressions (6) and (7). ) Holds.

The above equation (10) can be rewritten as the following equation (11).

Therefore, when the dielectric constant ε ₁ of the medium (dielectric) on the diffraction grating 230 and the dielectric constant ε _{2 of the} diffraction grating 230 (metal) are determined, the diffraction grating 230 for generating a standing wave of surface plasmons. The pitch Λ can be determined according to the wavelength λ ₀ of the excitation light α by the above equation (11). In other words, the wavelength λ ₀ of the excitation light α for generating the standing wave of the surface plasmon can be determined according to the pitch Λ of the diffraction grating 230 by the above equation (11).

Moreover, the said Formula (7) is rewritten like the following formula | equation (12).

Therefore, the incident angle θ of the excitation light α for generating the standing wave of the surface plasmon is determined according to the wavelength λ ₀ of the excitation light α determined by the above equation (11) and the pitch Λ of the diffraction grating 230. sell. Although it is most preferable that the incident angle θ of the excitation light α satisfies the above formula (12), the incident angle θ may be an angle before and after the angle satisfying the above formula (12). Specifically, the incident angle θ of the excitation light α is 50% or more with respect to the intensity (peak intensity) of the fluorescence from the fluorescent material when the intensity of the fluorescence from the fluorescent material satisfies the above formula (12). It may be within the range.

For example, the medium on the diffraction grating 230 is water (refractive index n = 1.33, dielectric constant ε ₁ ≈1.77), and the diffraction grating 230 is made of gold (dielectric constant ε ₂ ≈−12). Assume that the wavelength λ _{0 of} α is 640 nm. M and N are set to −1 and 3, respectively. The dielectric constant ε ₂ of the diffraction grating 230 is a complex number, but the imaginary part is ignored because the imaginary part is smaller than the real part. Further, although the wavelength of the excitation light α is different between the vacuum and the air, the difference is very small, so the difference is ignored here.

Under these conditions, if each numerical value is substituted into the above equation (11), the pitch Λ (nm) of the diffraction grating 230 for generating the standing wave of the surface plasmon is about 666 nm as shown in the following equation (13). It is required to be.

Further, when each numerical value is substituted into the above equation (12), the following equation (14) is obtained. Therefore, it is required that the most preferable incident angle θ (°) of the excitation light α for generating the standing wave of the surface plasmon is about 21 °.

ｌ，ｍ：１〜５の範囲内の整数 In addition, even if the above formulas (11) and (12) are satisfied, if the fill factor f of the diffraction grating 230 (ratio of the width of the convex portion to Λ) does not satisfy the following formula (15), m The amplitude hm of the next harmonic becomes 0 and a standing wave of surface plasmon cannot be generated. Therefore, the fill factor f of the diffraction grating 230 preferably satisfies the following formula (15).

l, m: an integer in the range of 1 to 5

ｄ：回折格子２３０の凹部の深さ（凸部の高さ）（ｎｍ）
ｆ：回折格子２３０のフィルファクター
Λ：回折格子２３０のピッチ（ｎｍ）
ｍ：表面プラズモンの高調波の次数（整数） The above equation (15) is derived as follows. First, the amplitude hm of the m-th harmonic of the surface plasmon is obtained by the following equation (16).

d: Depth of concave portion of diffraction grating 230 (height of convex portion) (nm)
f: Fill factor of diffraction grating 230 Λ: Pitch of diffraction grating 230 (nm)
m: Order of harmonics of surface plasmon (integer)

ｌ：整数 In order to generate the standing wave of the surface plasmon, it is necessary to satisfy the following formula (17). Therefore, the following equation (18) is derived from the equations (16) and (17). Solving equation (18) yields equation (19) below. Equation (19) can be rewritten as Equation (15) above.

l: integer

  In practice, it is considered that the amplitudes of the first to fifth harmonics should be taken into consideration. The fill factor f of the diffraction grating 230 is a numerical value in the range of 0-1. Therefore, l and m are integers within the range of 1-5.

Thus, in the SPFS apparatus 100 of the present embodiment, the pitch Λ (nm) of the diffraction grating 230, the fill factor f of the diffraction grating 230, the wavelength λ ₀ (nm) of the excitation light α in vacuum, and the diffraction grating 230 The incident angle θ (°) of the excitation light α is set so as to satisfy the above formulas (11), (12), and (15). Therefore, the SPFS device 100 according to the present embodiment can generate standing waves of surface plasmons in the diffraction grating 230 by irradiating the diffraction light 230 with the excitation light α, and as a result, on the diffraction grating 230. The number of photons for exciting the fluorescent material can be significantly increased. For example, the pitch Λ of the diffraction grating is selected from the range of 0.1 to 10 μm, and the fill factor f in the diffraction grating 230 satisfies the above formula (15) from the range of 0.1 to 0.9. The wavelength λ ₀ of the excitation light α is selected from the range of 300 to 800 nm, and the incident angle θ of the excitation light α is selected from the range of 5 to 70 °.

FIG. 4 is a diagram illustrating an example of the charge distribution of the standing wave of the surface plasmon. FIG. 4A is a diagram illustrating a long-distance propagation state, and FIG. 4B is a diagram illustrating a short-distance propagation state. FIG. 4A shows the charge distribution of the standing wave of the surface plasmon at the band gap edge (ω ₊ mode) on the high energy side of the band gap in the dispersion curve of the surface plasmon. On the other hand, FIG. 4B shows the charge distribution of the standing wave of the surface plasmon at the band gap end (ω _- mode) on the low energy side of the band gap.

As shown in FIG. 4A, in the ω ₊ mode, the electric field increases at the concave portion of the diffraction grating 230, and the length of penetration of the electric field into the medium (dielectric) side increases. On the other hand, as shown in FIG. 4B, in the ω _- mode, the electric field increases at the convex portion of the diffraction grating 230, and the penetration depth of the electric field into the medium (dielectric) side decreases. From the viewpoint of exciting a fluorescent substance positioned away from the diffraction grating 230, the ω ₊ mode is more preferable, but the number of photons on the diffraction grating 230 can be remarkably increased even in the ω ₋ mode. Note that the charge distributions shown in FIGS. 4A and 4B are examples, and the charge distribution can take other forms depending on the shape of the diffraction grating 230.

  As described above, the SPFS device 100 according to the present embodiment can efficiently excite the fluorescent substance by using the standing wave of the surface plasmon, so that the coverage is more sensitive than the conventional SPFS device. A detection substance can be detected.

  In the above embodiment, the example in which the chip 200 is irradiated with the excitation light α from the metal film 220 side has been described, but the chip 200 may be irradiated with the excitation light α from the substrate 210 side.

[Embodiment 2]
FIG. 5 is a schematic diagram showing the configuration of the SPFS apparatus 300 according to Embodiment 2 of the present invention. As shown in FIG. 5, the SPFS device 300 includes a light source 110, a collimating lens 120, an excitation light filter 130, a first condenser lens 140, a fluorescent filter 150, a second condenser lens 160, a light detection unit 170, and vibration application. Part 310 and control part 180. The SPFS device 300 according to the second embodiment is different from the SPFS device 100 according to the first embodiment in that it further includes a vibration applying unit 310. Therefore, the same components as those of the SPFS device 100 according to Embodiment 1 are denoted by the same reference numerals, and description thereof is omitted.

  The vibration applying unit 310 applies a vibration in a direction including a plane direction at a predetermined frequency to a detection target substance labeled with a fluorescent substance and coupled to a reaction field (capturing body) on the diffraction grating 230. Here, “vibration in a direction including the plane direction” means that the vibration direction includes a component in the plane direction (horizontal direction). In the present embodiment, the vibration applying unit 310 includes a high voltage power supply 312 and a pair of electrodes 314 connected to the high voltage power supply 312. The pair of electrodes 314 are arranged so as to sandwich the diffraction grating 230 (reaction field). The high-voltage power supply 312 can apply an alternating voltage or a pulsed voltage in which the polarity is alternately switched between the electrodes 314. Therefore, the vibration applying unit 310 can apply an alternating electric field whose polarity changes at a predetermined frequency to the reaction field on the diffraction grating 230. When the detected substance bonded to the reaction field has a positive or negative charge, the detected substance is vibrated by an alternating electric field. The voltage is, for example, several hundred volts or less, and preferably several tens volts or less. The frequency of vibration applied by the vibration applying unit 310 is preferably a frequency at which the detection target substance coupled to the reaction field (capturing body) resonates or a frequency lower than that. For example, the frequency is about several Hz to several tens Hz.

  FIG. 6 is a schematic diagram for explaining the vibration of the detection target substance bound to the reaction field. In this figure, the surface of the diffraction grating 230 (for example, the bottom surface of the groove) is shown enlarged. In addition, the polarity of the voltage applied to the pair of electrodes 314 is also shown. In the example shown in FIG. 6A, the detection target substance 420 is bound to the primary antibody 410 (captured body) immobilized on the metal film 220 (diffraction grating 230). Further, the secondary antibody 430 labeled with the fluorescent substance 440 is bound to the substance to be detected 420. It is assumed that the complex composed of the target substance 420, the secondary antibody 430, and the fluorescent substance 440 has a negative charge. In addition to the complex bound to the metal film 220 by such specific binding SP, there is also a complex bound to the metal film 220 by non-specific binding US. Examples of non-specific binding US include direct binding of the target substance 420 or other substance 450 to the metal film 220, direct binding of the secondary antibody 430 to the metal film 220, and the like.

  As shown in FIGS. 6B and 6C, when an alternating electric field is applied to the reaction field, a substance having a positive or negative charge among the substances specifically or non-specifically bound to the reaction field is It is vibrated in the direction including the surface direction. At this time, the length and mass of the complex bound by the specific binding SP and the complex (substance) bound by the non-specific binding US are different. Therefore, the vibration of each component is different. For example, when the secondary antibody 430 is directly bonded to the metal film 220, the secondary antibody 430 hardly oscillates even when an alternating electric field is applied to the reaction field. In addition, when the detected substance 420 is directly bound to the metal film 220 and the secondary antibody 430 is bound to the detected substance 420, the complex bound by this non-specific binding US. The resonant frequency is greater than the resonant frequency of the complex that is bound by the specific binding SP. Therefore, if the difference between these vibrations can be detected, it is possible to distinguish between the complex bound by the specific binding SP and the complex bound by the non-specific binding US.

  Here, the resonance frequency of the complex bound by the specific binding SP will be described. In order to calculate the resonance frequency of the complex, the equation of motion of the protein is examined using the inverted pendulum model shown in FIG.

As shown in FIG. 7, the mass of the protein is m, the length of the protein is l, the spring constant is k, the length from the base point of the protein to the action point of the spring is h, and the viscosity damping coefficient due to the solvent is C. When the moment of inertia, the overturning moment, the restoring force and restoring moment of the spring are as follows:
Moment of inertia: J = ml ²
Falling moment: mg · l · sinθ ≒ mgl · θ
Spring restoring force: -k · hθ
Restoring moment: −k · hθ · h = −k · h ² · θ

In this case, the equation of motion is expressed as the following equation (20).

The above equation (20) can be rewritten as the following equation (21).

Here, when the resonance angular frequency by gravity and spring restoring force is ωn, the following equations (22) and (23) are established. In Expression (22), ξ is a damping ratio.

Using the above formula (22) and formula (23), the above formula (21) is expressed as the following formula (24).

The overall resonance period T including the viscous damping of the solvent is expressed by the following equation (25).

Here, the length h from the protein base point to the spring action point, the protein length l, the protein mass m, the gravitational acceleration g, and the spring constant k are assumed as follows.
h = l = 1.8 × 10 ⁻⁹ (m)
m = 6.17 × 10 ⁻²² (kg)
g = 9.8 (m / t ² )
k = 2.5 (N / m) = 2.5 (kg / t ₂ )

Then, kt, moment of inertia J, and resonance angular frequency ωn are as follows.
kt = kh ² −mgl≈0.81 × 10 ⁻¹⁵ (kgm ² / t ² )
J = m × l ² = 6.17 × 10 ⁻²² × (18 × 10 ⁻⁹ ) ² = 1.99908 × 10 ⁻³⁷ (kgm ² )
ωn = √ (kt / J) ≈0.636 × 10 ¹¹ (Hz)

The viscosity η of the solvent (water) and the size (radius) r of the protein are assumed as follows.
η = 0.89 (cP) = 0.89 × 10 ⁻³ (kg / (mt))
r = 5 (nm)

Then, the volume V of the protein, the viscous damping coefficient C, the damping ratio ξ, the resonance period T, and the resonance frequency f are as follows.
V = 4 / 3πr ³ = 5.236 × 10 ⁻²⁵ (m ³ )
C = η × V = 0.89 × 10 ⁻³ × 5.236 × 10 ⁻²⁵ ≈4.66004 × 10 ⁻²⁸ (kgm ² / t)
ξ = C / (2ωn × J) = 4.66004 × 10 ⁻²⁸ /(2×0.636×10 ¹¹ × 1.999908 × 10 ⁻³⁷ )
T = 2π / (ωn × √ (1-ξ ² )) ≈9.88 × 10 ⁻¹¹ (t)
f = 1 / T≈10 (GHz)

  By selecting a solvent having a higher viscosity, the resonance frequency f of the complex bound by the specific binding SP can be set to an arbitrary value. The resonance frequency f is preferably about several Hz.

  FIG. 8 is a graph showing an example of the electric field intensity distribution on the diffraction grating 230. As shown in FIG. 8, the electric field strength changes periodically corresponding to the shape of the diffraction grating 230. In such a situation, when an alternating electric field is applied to the reaction field to vibrate a complex specifically or non-specifically bound to the reaction field in a direction including the plane direction of the metal film 220, these complexes The ambient electric field strength changes periodically. Therefore, the fluorescence intensity from the fluorescent material contained in these complexes also periodically changes.

  As described above, the length and mass of the complex bound by the specific binding SP and the complex (substance) bound by the non-specific binding US are different. The resonance frequency of the complex bound by the non-specific binding US is higher than the resonance frequency of the complex bound by the specific binding SP. Therefore, as shown in FIG. 9, the frequency of the fluorescent signal from the complex bound by the non-specific binding US is greater than the frequency of the fluorescent signal from the complex bound by the specific binding SP. Become.

  Returning to the description of the SPFS apparatus 300. The control unit 180 controls operations of the excitation light irradiation unit (light source 110), the fluorescence detection unit (light detection unit 170), the excitation light irradiation unit, the angle adjustment unit of the fluorescence detection unit, and the vibration applying unit 310. In addition, the control unit 180 analyzes the frequency of the output signal (fluorescence signal) from the light detection unit 170, so that the fluorescence signal from the complex bound by the specific binding SP is derived from other fluorescence signals (noise). Separate and analyze the presence or amount of the substance to be detected. The control unit 180 is, for example, a computer that executes software.

  For example, the control unit 180 passes the output signal (fluorescence signal) from the light detection unit 170 through a band-pass filter or a low-pass filter, thereby converting the fluorescence signal from the complex bound by the specific binding SP to other fluorescence. It can be separated from the signal (noise). Further, the control unit 180 performs frequency conversion processing (for example, fast Fourier transform) on the output signal (fluorescence signal) from the light detection unit 170, and the fluorescence at the vibration frequency of the complex bound by the specific binding SP. By calculating the intensity, the presence or amount of the substance to be detected can be analyzed. Further, the control unit 180 performs frequency conversion processing (for example, fast Fourier transform) on the output signal (fluorescence signal) from the light detection unit 170, and passes the obtained signal through a filter that passes a predetermined vibration frequency. To separate fluorescence signals from the complex bound by the specific binding SP from other fluorescence signals (noise) by performing frequency inverse transform processing (for example, fast Fourier inverse transform) on the signal that has passed through You can also.

  Next, the detection operation of the SPFS device 300 will be described. FIG. 10 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 300. In this example, the primary antibody 410 is immobilized on the metal film 220 as a capturing body.

  First, preparation for measurement is performed (step S10). Specifically, the chip 200 is installed at a predetermined position of the SPFS device 300. Next, the detection target substance 420 and the primary antibody 410 in the specimen are reacted (primary reaction, step S20). Next, the target substance 420 bound to the primary antibody 410 is labeled with a fluorescent substance 440 (secondary reaction, step S30). Each of these processes is the same as each process of the SPFS apparatus 100 according to the first embodiment.

  Next, vibration with a predetermined frequency is applied to the target substance 420 that is bound to the primary antibody 410 and labeled with the fluorescent substance 440 (step S40). Specifically, the control unit 180 applies an alternating electric field to the vibration applying unit 310 to apply vibration of a predetermined frequency to the detection target substance 420 (see FIGS. 6B and 6C).

  Next, with the vibration of a predetermined frequency applied to the substance to be detected 420, the excitation light α is irradiated onto the metal film 220, and the intensity of the fluorescence β emitted from the fluorescent substance 440 is measured (step S50). Specifically, the control unit 180 causes the light source 110 to emit the excitation light α. At the same time, the control unit 180 causes the light detection unit 170 to detect the intensity of the fluorescence β from the metal film 220. The light detection unit 170 outputs the measurement result to the control unit 180.

  Finally, the control unit 180 performs frequency analysis on the output signal (fluorescence signal) from the light detection unit 170, separates the fluorescence signal derived from the specific binding SP, and detects the presence or detection of the detected substance. The amount of the substance is analyzed (step S60).

  By the above procedure, the fluorescence signal derived from the specific binding SP can be separated, and the presence of the substance to be detected or the amount of the substance to be detected in the sample can be detected with high accuracy.

  As described above, in the present embodiment, the capturing body is fixed substantially uniformly on the surface of the diffraction grating 230. Therefore, the change in fluorescence intensity from the substance to be detected 420 located on the bottom surface of the concave portion of the diffraction grating 230 and the change in fluorescence intensity from the substance to be detected 420 located on the top surface of the convex part may cancel each other. Conceivable. However, since there is a large difference in the electric field strength between the bottom surface of the concave portion of the diffraction grating 230 and the top surface of the convex portion, the change in fluorescence intensity at the top surface of the convex portion is compared with the change in fluorescence intensity at the bottom surface of the concave portion. Very small. For this reason, this is hardly a problem. Further, a mask is arranged in the fluorescence detection unit to shield fluorescence from a specific region (for example, a region having a low electric field intensity), or a specific region (for example, an electric field strength) in a fluorescent image detected by the light detection unit 170. Such a problem may be avoided more reliably by performing a frequency analysis only for a large area).

  As described above, the SPFS device 300 according to the second embodiment can separate the fluorescence signal derived from the specific binding SP in addition to the effects of the SPFS device 100 according to the first embodiment, and thus is non-specific. Even when adsorption occurs, the detection target substance can be detected with high accuracy.

In the present embodiment, the example in which the vibration applying unit 310 applies an alternating electric field to apply vibration to the substance to be detected has been described, but the means for applying vibration to the substance to be detected is not limited to this. For example, in this case, the vibration applying unit 310 may apply vibration to the detection target substance by vibrating the other wall surface of the flow path having the metal film 220 as one wall surface. Further, the vibration applying unit 310 may apply vibration to the detection target substance by reciprocating the liquid in the flow path having the metal film 220 as one wall surface. Furthermore, the vibration imparting unit 310 may apply vibration to the substance to be detected by irradiating an electromagnetic wave having a frequency of 1 × 10 ⁵ to 1 × 10 ¹³ Hz to vibrate molecules.

  In the present embodiment, the example in which the vibration applying unit 310 vibrates the detection target substance mainly in the surface direction of the metal film 220 has been described. (See Japanese Patent Application Laid-Open No. 2011-80935 by the inventors of the present application). Since the enhanced electric field formed by SPR exists only in the vicinity of the metal film 220, the intensity of the fluorescence emitted from the fluorescent material in response to the vibration is increased even if the detection target is vibrated in the thickness direction of the metal film 220. Change. Also, when the substance to be detected is vibrated in the thickness direction of the metal film 220, the frequency of the fluorescence signal differs between the complex bound by the specific binding SP and the complex bound by the non-specific binding US. Therefore, when the detection target substance is vibrated in the thickness direction of the metal film 220, the fluorescence signal derived from the specific binding SP is separated and the presence or detection of the detection target substance in the specimen is separated as in the above embodiment. The amount of the detection substance can be detected with high accuracy. The vibration applying unit 310 may vibrate the substance to be detected in the surface direction of the metal film 220, may vibrate in the thickness direction, or may vibrate in the surface direction and the thickness direction.

  Since the surface plasmon enhanced fluorescence measurement method and the surface plasmon enhanced fluorescence measurement apparatus according to the present invention can measure a substance to be detected with high reliability, they are useful for clinical examinations, for example.

100,300 Surface plasmon enhanced fluorescence measuring device (SPFS device)
DESCRIPTION OF SYMBOLS 110 Light source 120 Collimating lens 130 Excitation light filter 140 1st condensing lens 150 Fluorescence filter 160 2nd condensing lens 170 Light detection part 180 Control part 200 Chip 210 Substrate 220 Metal film 230 Diffraction grating 310 Vibration imparting part 312 High voltage power supply 314 Electrode 410 Primary antibody 420 Target substance 430 Secondary antibody 440 Fluorescent substance α Excitation light β Fluorescence

Claims (12)

A fluorescent substance for labeling a substance to be detected is a surface plasmon-enhanced fluorescence measuring method for detecting fluorescence emitted by being excited by an electric field based on surface plasmon resonance, and detecting the presence or amount of the substance to be detected,
Preparing a chip having a metal film on which a diffraction grating is formed, a capturing body fixed to the diffraction grating, and a target substance that is bound to the capturing body and is labeled with a fluorescent material;
Irradiating the diffraction grating with excitation light so that a standing wave of surface plasmons is generated in the diffraction grating;
Detecting fluorescence emitted from the fluorescent material to obtain a fluorescent signal;
A method for measuring surface plasmon enhanced fluorescence, comprising: The pitch Λ (nm) of the diffraction grating and the wavelength λ ₀ (nm) of the excitation light in vacuum satisfy the following formula (1):
The fill factor f of the diffraction grating satisfies the following formula (2):
The incident angle θ (°) of the excitation light with respect to the diffraction grating satisfies the following formulas (3) to (5), or the intensity of fluorescence from the fluorescent substance is expressed by the following formulas (3) to ( 5) Within a range of 50% or more with respect to the intensity of fluorescence from the fluorescent material when satisfying
The surface plasmon enhanced fluorescence measurement method according to claim 1.

[In the above formulas (1) to (3), ε ₁ is the dielectric constant of the medium on the diffraction grating, ε ₂ is the dielectric constant of the metal constituting the diffraction grating, and n is the refractive index of the medium. M and N are integers, and l and m are integers in the range of 1-5. ] In the step of irradiating the diffraction grating with excitation light, a vibration in a direction including a surface direction of the metal film is applied to the detected substance bonded to the capturing body at a predetermined frequency,
Further comprising analyzing the presence or amount of the substance to be detected by frequency analysis of the fluorescent signal,
The surface plasmon enhanced fluorescence measurement method according to claim 1 or 2.   The frequency of the vibration applied to the substance to be detected is less than a frequency at which the substance to be detected coupled to the capturing body resonates, or is a frequency at which the substance to be detected coupled to the capturing body resonates. The surface plasmon enhanced fluorescence measurement method according to claim 3. A chip having a metal film on which a diffraction grating is formed, a capturing body fixed on the diffraction grating, and a target substance that is bound to the capturing body and is labeled with a fluorescent material, is attached, and excitation light is transmitted through the chip. A surface plasmon-enhanced fluorescence measurement device that detects the presence or amount of a substance to be detected by irradiating a diffraction grating,
A light source for irradiating the diffraction grating with the excitation light in order to excite the fluorescent material with an enhanced electric field to emit fluorescence;
A light detection unit for detecting fluorescence from the fluorescent material,
The light source irradiates the diffraction grating with the excitation light so that a standing wave of surface plasmon is generated in the diffraction grating;
Surface plasmon enhanced fluorescence measurement device. The pitch Λ (nm) of the diffraction grating and the wavelength λ ₀ (nm) of the excitation light in vacuum satisfy the following formula (1):
The fill factor f of the diffraction grating satisfies the following formula (2):
The incident angle θ (°) of the excitation light with respect to the diffraction grating satisfies the following formulas (3) to (5), or the intensity of fluorescence from the fluorescent substance is expressed by the following formulas (3) to ( 5) Within a range of 50% or more with respect to the intensity of fluorescence from the fluorescent material when satisfying
The surface plasmon enhanced fluorescence measuring apparatus according to claim 5.

[In the above formulas (1) to (3), ε ₁ is the dielectric constant of the medium on the diffraction grating, ε ₂ is the dielectric constant of the metal constituting the diffraction grating, and n is the refractive index of the medium. M and N are integers, and l and m are integers in the range of 1-5. ] A vibration applying unit that applies a vibration in a direction including a surface direction of the metal film at a predetermined frequency to the target substance coupled to the capturing body;
In a state in which the substance to be detected is vibrated by the vibration applying unit, the fluorescence from the fluorescent substance is detected by the light detection unit, and the output signal from the light detection unit is analyzed by frequency analysis. Analyze the presence or quantity,
The surface plasmon enhanced fluorescence measuring apparatus according to claim 5 or 6.   The frequency of the vibration applied by the vibration applying unit is less than a frequency at which the detected substance coupled to the capturing body resonates, or a frequency at which the detected substance coupled to the capturing body resonates. Item 8. The surface plasmon enhanced fluorescence measurement device according to Item 7.   The surface plasmon enhanced fluorescence measurement device according to claim 7 or 8, wherein the vibration applying unit applies vibration to the target substance by applying an alternating electric field.   The surface plasmon enhanced fluorescence measurement according to claim 7 or 8, wherein the vibration applying unit applies vibration to the detection target substance by vibrating another wall surface of the flow path having the metal film as one wall surface. apparatus.   9. The surface plasmon enhanced fluorescence measurement device according to claim 7, wherein the vibration applying unit applies vibration to the detected substance by reciprocating a liquid in a flow path having the metal film as one wall surface. . 9. The surface plasmon enhanced fluorescence measurement device according to claim 7, wherein the vibration applying unit applies vibration to the detected substance by irradiating an electromagnetic wave having a frequency of 1 × 10 ⁵ to 1 × 10 ¹³ Hz.

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