JP2015111063A - Surface plasmon-field enhanced fluorescence measurement method and surface plasmon enhanced fluorescence measurement apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measurement method and a measurement apparatus using GC-SPFS and capable of suppressing degradation in detection accuracy due to a shift of an output angle of fluorescent light while preventing an increase of background.SOLUTION: A chip including liquid reservoir surrounded by a light transmission sidewall and holding a liquid, a metal film arranged on a bottom of the liquid reservoir and containing diffraction grating, a capture element immobilized to the diffraction grating, and a detection target substance coupled to the capture element and marked by a fluorescent substance is prepared. The diffraction grating is irradiated with excitation light through an interface between the sidewall and the liquid held in the liquid reservoir. Fluorescent light emitted from the fluorescent substance is detected through the interface between the liquid held in the liquid reservoir and the sidewall, thereby obtaining a fluorescent signal.

Description

  The present invention relates to a surface plasmon enhanced fluorescence measurement method and a surface plasmon enhanced fluorescence measurement apparatus.

  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 a metal film is irradiated with light under a predetermined condition. 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 sample solution containing a substance to be detected is provided in this reaction field, the substance to be detected is bound to the reaction field. Next, when a capture body (for example, 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 (see Patent Document 1). 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.

  On the other hand, GC-SPFS couples excitation light and surface plasmons using a diffraction grating (see Patent Document 2). In GC-SPFS, a metal film on which a diffraction grating is formed is used. In this method, excitation light and surface plasmons are combined by irradiating the diffraction grating with excitation light. In PC-SPFS, it is known that fluorescence is emitted in all directions, whereas in GC-SPFS, fluorescence is emitted in a specific direction with directivity.

JP-A-10-307141 JP 2011-158369 A

  In general, in GC-SPFS, a liquid exists on the diffraction grating when the excitation light is irradiated, and the excitation light is irradiated to the diffraction grating through the liquid. In such a situation, when the upper part of the liquid is opened to the outside, the meniscus causes an inclination on the surface of the liquid. When the liquid surface is tilted in this way, the incident angle of the excitation light and the emission angle of the fluorescence are shifted, and the fluorescence cannot properly reach the light detection unit, and the detection of the target substance fails. There is a risk that. Although it is conceivable to expand the detection range of the light detection unit using a lens or the like in order to cope with the deviation of the emission angle of the fluorescence, the use of the lens or the like is not preferable from the viewpoint of reducing the background.

  The present invention has been made in view of the above points, and is a measurement method and a measurement apparatus using GC-SPFS, which prevents an increase in background and reduces detection accuracy due to a deviation in emission angle of fluorescence. It is an object of the present invention to provide a measurement method and a measurement apparatus that can be suppressed.

  The present inventors have found that the above problem can be solved by irradiating excitation light without passing through the surface of the liquid (interface between liquid and air) and detecting fluorescence, and have further studied to complete the present invention. It was.

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

［上記式（１）において、ｔは、前記液溜部の底部の水平方向の大きさである。θは、前記回折格子に対する励起光の入射角である。］ [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 liquid reservoir that is surrounded by a light-transmitting side wall and holds a liquid; a metal film that includes a diffraction grating disposed at the bottom of the liquid reservoir; and is fixed to the diffraction grating. A step of preparing a chip having a capturing body and a target substance labeled with a fluorescent substance, which is bound to the capturing body; and an interface between the side wall and the liquid held in the liquid reservoir in the diffraction grating Irradiating excitation light through the surface plasmon, and detecting fluorescence emitted from the fluorescent material through an interface between the liquid retained in the liquid reservoir and the side wall to obtain a fluorescent signal, Enhanced fluorescence measurement method
[2] In a cross section in the depth direction of the liquid reservoir including the optical axis of the excitation light, the depth h of the liquid held in the liquid reservoir satisfies the following formula (1): [1] The surface plasmon enhanced fluorescence measurement method described in 1.

[In the above formula (1), t is the horizontal size of the bottom of the liquid reservoir. θ is an incident angle of excitation light to the diffraction grating. ]

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

［上記式（１）において、ｔは、前記液溜部の底部の水平方向の大きさである。θは、前記回折格子に対する励起光の入射角である。］ [3] A liquid reservoir that is surrounded by a light-transmitting side wall and holds a liquid, a metal film including a diffraction grating disposed at the bottom of the liquid reservoir, and fixed to the diffraction grating A chip having a capture body and a target substance labeled with a fluorescent substance bound to the capture body is attached, and the presence or amount of the target substance is detected by irradiating the diffraction grating with excitation light. An apparatus for measuring surface plasmon enhanced fluorescence, wherein the diffraction grating has an interface between the side wall and the liquid held in the liquid reservoir in order to excite the fluorescent material by an enhanced electric field and emit fluorescence. A surface plasmon enhancement comprising: a light irradiating part for irradiating excitation light through the light irradiating part; and a light detecting part for detecting the fluorescence emitted from the fluorescent substance through the interface between the liquid held in the liquid reservoir and the side wall. Fluorescence measuring device.
[4] In the cross section in the depth direction of the liquid reservoir including the light irradiation unit and the light detection unit, the depth h of the liquid held in the liquid reservoir satisfies the following formula (1): [3] The surface plasmon enhanced fluorescence measuring apparatus according to [3].

[In the above formula (1), t is the horizontal size of the bottom of the liquid reservoir. θ is an incident angle of excitation light to the diffraction grating. ]

  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 more highly sensitively and with high precision.

1 is a schematic diagram showing the configuration of a surface plasmon enhanced fluorescence measurement device and a chip according to Embodiment 1. FIG. 2A and 2B are perspective views of the diffraction grating. It is a cross-sectional schematic diagram for demonstrating the incident angle of the excitation light with respect to a metal film. 3 is a schematic cross-sectional view showing excitation light and fluorescence optical paths in the chip according to Embodiment 1. FIG. 3 is a flowchart showing the operation of the surface plasmon enhanced fluorescence measurement device according to the first embodiment. FIG. 4 is a schematic cross-sectional view showing a configuration of a chip according to a second embodiment and optical paths of excitation light and fluorescence. FIG. 7A is a graph showing the relationship (simulation result) between the incident angle of excitation light on the metal film and the reflectance of excitation light. FIG. 7B is a graph showing a relationship (actual measurement value) between the incident angle of the excitation light with respect to the metal film and the intensity of the reflected light of the excitation light when the excitation light is irradiated onto the diffraction grating through the surface of the liquid. FIG. 7C is a graph showing the relationship between the number of measurements and the intensity of fluorescence (actual measurement value) when excitation light is irradiated onto the diffraction grating through the surface of the liquid.

  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 and a chip 200 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 fluorescence filter 140, a light detection unit 150, and a control unit 160. 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, a metal film 220 including a diffraction grating 230, and a side wall 240 that forms a liquid reservoir 250 for holding a liquid. 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 mm to several cm. However, the chip 200 may be a smaller structure or a larger structure not included in the category of “chip”. Good.

  The substrate 210 is a support member for the metal film 220 and the side wall 240. The material of the substrate 210 is not particularly limited as long as it has mechanical strength capable of supporting the metal film 220 and the side wall 240. 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 so as to be positioned at the bottom of the liquid reservoir 250. 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 (SPR). 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. The pitch of the diffraction grating is preferably in the range of 100 to 2000 nm from the viewpoint of generating SPR. In the present specification, the “diffraction grating pitch” refers to the center-to-center distance Λ of the protrusions in the arrangement direction of the protrusions, as shown in FIGS.

  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 a substance to be detected is immobilized on the diffraction grating 230 (reaction field). The capturing body specifically binds to the substance to be detected. For example, 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. 3, the excitation light α is irradiated onto the 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. As described above, in the GC-SPFS, the fluorescence β is emitted with directivity in a specific direction. As shown in FIG. 3, the emission angle of the fluorescence β is approximated by 2θ.

  The side wall 240 is disposed on the substrate 210 so as to surround the metal film 220. The side wall 240 forms a liquid reservoir 250 for holding the liquid 310 on the metal film 220. Since the opening of the liquid reservoir 250 is open, the surface of the liquid 310 in the liquid reservoir 250 is exposed to the outside. Therefore, the surface of the liquid 310 in the liquid reservoir 250 forms a meniscus. As will be described later, the SPFS device 100 according to the present embodiment irradiates the diffraction grating 230 with the excitation light α through the side wall 240 and avoids the fluorescence β through the side wall 240 in order to avoid the influence of meniscus at the time of fluorescence detection. Is detected (see FIG. 1). Therefore, the side wall 240 is formed of a material having optical transparency with respect to the wavelengths of the excitation light α and the fluorescence β.

  The material of the side wall 240 is not particularly limited as long as it has optical transparency with respect to the wavelengths of the excitation light α and the fluorescence β and can hold the liquid 310 on the metal film 220. Examples of the material of the sidewall 240 include inorganic materials such as glass, quartz, and silicon, and resins such as polymethyl methacrylate, polycarbonate, polystyrene, and polyolefin. The material of the side wall 240 is preferably an optical plastic such as an acrylic resin or an optical glass. Note that the side wall 240 may be integrally formed with the substrate 210.

  Next, each component of the SPFS device 100 will be described. As described above, the SPFS device 100 includes the light source 110, the collimating lens 120, the excitation light filter 130, the fluorescence filter 140, the light detection unit 150, and the control unit 160.

  The light source 110, the collimating lens 120, and the excitation light filter 130 constitute an excitation light irradiation unit (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 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. In the SPFS apparatus 100 according to the present embodiment, the excitation light irradiation unit irradiates the diffraction grating 230 with the excitation light α so as to pass through the interface between the side wall 240 of the chip 200 and the liquid 310 in the liquid reservoir 250.

  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 an axis perpendicular to the x-axis and perpendicular to the surface of the metal film 220 (axis in the height direction of the liquid reservoir 250) is the y-axis, the optical axis of the excitation light α is parallel to the xy plane (FIG. 1).

  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 as long as light having a wavelength (for example, 400 to 1000 nm) that can excite the fluorescent material can be emitted. Examples of the light source 110 include laser diodes, light emitting diodes, mercury lamps, and other laser light sources.

  The collimating lens 120 collimates the excitation light α emitted from the light source 110. Even if the excitation light α emitted from the light source 110 is collimated, the contour shape may be flat. Therefore, the light source 110 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 light source 110 has a slight wavelength distribution width, the bandpass filter turns the excitation light α from the light source 110 into narrowband light having only the center wavelength. In addition, since the excitation light α from the light source 110 is not completely linearly polarized light, the linear polarization filter converts the excitation light α from the light source 110 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.

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
ｎ：回折格子２３０上の媒質（液溜部２５０内の液体３１０）の屈折率
θ：励起光αの回折格子２３０に対する入射角
ｍ：整数
Λ：回折格子２３０のピッチ The incident angle θ (see FIG. 3) of the excitation light α with respect to the metal film 220 (diffraction grating 230) has the highest intensity of the enhanced electric field formed by SPR, and as a result, the intensity of the fluorescence β from the fluorescent material is the highest. A stronger angle is preferred. The incident angle θ of the excitation light α is appropriately selected according to the pitch Λ of the diffraction grating 230, the wavelength of the excitation light α, the type of metal constituting the metal film 220, and the like. For example, the incident angle θ of the excitation light α is set so as to satisfy the following formula (2).

k ₀ : Wave number of excitation light α = 2π / (λ ₀ / n)
λ ₀ : wavelength of excitation light α in vacuum n: refractive index of medium (liquid 310 in liquid reservoir 250) on diffraction grating 230 θ: incident angle of excitation light α with respect to diffraction grating 230 m: integer Λ: diffraction Pitch of lattice 230

ω：励起光αの角周波数
ｃ：光速度
ε_１：回折格子２３０上の媒質（液溜部２５０内の液体３１０）の誘電率＝ｎ^２
ε_２：回折格子２３０を構成する媒質（金属）の誘電率 Here, k _sp is the wave number of plasmon excited at the interface between the two types of media (the interface between the metal film 220 and the liquid 310 in the liquid reservoir 250), and is defined as the following equation (3). Is done.

ω: angular frequency of excitation light α c: speed of light ε ₁ : dielectric constant of medium (liquid 310 in liquid reservoir 250) on diffraction grating 230 = n ²
ε ₂ : dielectric constant of medium (metal) constituting diffraction grating 230

  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 fluorescence filter 140 and the light detection unit 150 constitute 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 fluorescence detection unit may further include a condensing lens in order to expand the detection range of the light detection unit 150, but it is preferable not to include the condensing lens from the viewpoint of reducing the background.

  The fluorescence filter 140 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 150, The amount of light reaching the light detection unit 150 is adjusted.

  The light detection unit 150 detects a fluorescent image on the metal film 220. For example, the light detection unit 150 is a photomultiplier tube with high sensitivity and high SN ratio. The light detection unit 150 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 an angle (fluorescence peak angle) at which the intensity of the fluorescence β emitted from the diffraction grating 230 (reaction field) and transmitted through the side wall 240 becomes maximum. preferable. 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 160 includes an excitation light irradiation unit (light source 110), a fluorescence detection unit (light detection unit 150), an excitation light irradiation unit, and angle adjustment units (first angle adjustment unit and second angle adjustment unit) of the fluorescence detection unit. Control the behavior. Further, the control unit 160 analyzes the output signal (fluorescence signal) from the light detection unit 150 to analyze the presence or amount of the detection target substance. The control unit 160 is, for example, a computer that executes software.

  FIG. 4 is a schematic cross-sectional view showing the configuration of the chip 200 according to the first embodiment and the optical paths of the excitation light α and the fluorescence β. Here, it is assumed that the outer surface and the inner surface of the side wall 240 are parallel.

In FIG. 4, θ ₁ is an incident angle of the excitation light α when the excitation light α is incident on the side wall 240 from an external medium (for example, air). θ ₂ is a refraction angle of the excitation light α when the excitation light α is incident on the side wall 240 from an external medium. θ ₂ is also an incident angle of the excitation light α when the excitation light α enters the liquid 310 (for example, a buffer solution) from the side wall 240. θ ₃ is a refraction angle of the excitation light α when the excitation light α enters the liquid 310 (for example, a buffer solution) from the side wall 240. The sum of the incident angle theta and theta ₃ excitation light α with respect to the metal film 220 is 90 °.

θ ₄ is an incident angle of the fluorescence β when the fluorescence β is incident on the side wall 240 from the liquid 310. As described above, the emission angle of the fluorescence β with respect to the metal film 220 can be approximated by 2θ. Therefore, the sum of the theta ₄ and 2θ is 90 °. θ ₅ is the refraction angle of the fluorescence β when the fluorescence β enters the side wall 240 from the liquid 310. θ ₅ is also the incident angle of the fluorescence β when the fluorescence β enters the external medium from the side wall 240. θ ₆ is a refraction angle of the fluorescence β when the fluorescence β enters the external medium from the side wall 240.

Incidentally, and the wavelength of the excitation light alpha, pitch Λ of the diffraction grating 230, the incident angle of the excitation light alpha with respect to the metal film 220 theta, refractive index n ₁ and a dielectric constant of the medium outside, the refractive index of the side wall 240 n ₁ and the dielectric constant The refractive index n ₂ and the dielectric constant of the liquid 310 are dependently determined according to the type of the liquid 310 used at the time of fluorescence detection, the type of fluorescent material, the atmosphere at the time of fluorescence detection, and the like.

From Snell's law, the following formulas (4) to (7) are established. Here, n ₁ is the refractive index of the external medium. n ₂ is the refractive index of the side wall 240. n ₃ is the refractive index of the liquid 310.

In addition, in order to detect the fluorescence β appropriately, it is necessary not to totally reflect at each refraction point. Therefore, the refractive index n _{2 of} the side wall 240, the shape of the side wall 240, and the refractive index n _{3 of the} liquid 310 are selected so that the following expressions (8) and (9) are established.

上記式（１０）および式（１１）において、ｔは、液溜部２５０の底部の水平方向の大きさである。θは、金属膜２２０に対する励起光αの入射角である。 Further, in order for the excitation light α to pass through the interface between the side wall 240 and the liquid 310, the depth h of the liquid 310 is selected so as to satisfy the following formula (10). The left side of Equation (10) corresponds to h ₁ in FIG. That is, Expression (10) specifies that the point at which the excitation light α is incident on the liquid 310 from the side wall 240 is lower than the surface of the liquid 310. Of course, the height of the side wall 240 is higher than the depth h of the liquid 310. On the other hand, in order for the fluorescence β to pass through the interface between the liquid 310 and the side wall 240, the depth of the liquid 310 is selected so as to satisfy the following expression (11). If Expression (10) is satisfied, Expression (11) is also satisfied. Therefore, the depth of the liquid 310 may be selected so as to satisfy the following Expression (10).

In the above equations (10) and (11), t is the horizontal size of the bottom of the liquid reservoir 250. θ is an incident angle of the excitation light α with respect to the metal film 220.

  Next, the detection operation of the SPFS device 100 will be described. FIG. 5 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 100. In this example, the primary antibody is immobilized on the metal film 220 (diffraction grating 230) 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 sample solution is introduced into the liquid reservoir 250 of the chip 200, and the target substance in the sample solution reacts with the primary antibody (primary reaction, step S20). When a substance to be detected is present in the sample solution, at least a part of the substance to be detected binds to the primary antibody. Thereafter, the inside of the liquid reservoir 250 is washed with a buffer solution or the like to remove substances that have not bound to the primary antibody.

  The sample liquid is an aqueous liquid containing a specimen. For example, the sample solution is a liquid sample or a diluted solution obtained by diluting a liquid sample with a liquid such as a buffer solution. The sample solution may contain a surfactant or the like. There are no particular limitations on the types of the specimen and the substance to be detected. Examples of specimens include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva and semen. 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 introduced into the liquid reservoir 250, and the substance to be detected 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. Thereafter, the inside of the liquid reservoir 250 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 introduced into the liquid reservoir 250 after the substance to be detected is bound to the secondary antibody. Further, the sample and the fluorescent labeling solution may be introduced into the liquid reservoir 250 at the same time. In any case, the sample liquid introduced into the liquid reservoir 250 is replaced with another liquid such as a buffer solution.

  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 160 causes the light source 110 to emit the excitation light α. At the same time, the control unit 160 causes the light detection unit 150 to detect the intensity of the fluorescence β. The light detection unit 150 outputs the measurement result to the control unit 160. At this time, as shown in FIG. 1, the excitation light irradiation unit (light irradiation unit) does not pass through the surface (meniscus) of the liquid 310 held in the liquid reservoir 250 and does not pass through the interface between the side wall 240 and the liquid 310. Then, the diffraction grating 230 is irradiated with excitation light α. Further, the light detection unit 150 detects the fluorescence β emitted from the fluorescent material through the interface between the liquid 310 and the side wall 240 without passing through the surface (meniscus) of the liquid 310.

  Finally, the control unit 160 analyzes the output signal (fluorescence signal) from the light detection unit 150, and analyzes the presence of the substance to be detected or the amount of the substance to be detected (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.

  As described above, the SPFS device 100 according to the present embodiment irradiates the excitation light α through the interface between the side wall 240 and the liquid 310 without passing through the surface (meniscus) of the liquid 310, and the liquid 310 and the side wall 240. Fluorescence β is detected through the interface. Since the shape of the interface between the side wall 240 and the liquid 310 does not change, this makes it possible to reduce the deviation of the emission angle of the fluorescence β caused by the meniscus (see also the simulation result of the second embodiment). . Therefore, according to the SPFS apparatus 100 according to the present embodiment, a substance to be detected can be detected with high sensitivity and high accuracy.

[Embodiment 2]
In the first embodiment, the example in which the excitation light α is incident on the side wall 240 on the outer peripheral surface of the side wall 240 has been described. In the second embodiment, an example in which excitation light α is incident on the sidewall 240 on the upper surface of the sidewall 240 will be described.

  FIG. 6 is a schematic cross-sectional view showing the configuration of the chip 200 according to the second embodiment and the optical paths of the excitation light α and the fluorescence β. Here, it is assumed that the outer surface and the inner surface of the side wall 240 are parallel, and the upper surface of the side wall 240 and the upper surface of the substrate 210 are parallel. As shown in FIG. 6, in the present embodiment, excitation light α is incident on the upper surface of the side wall 240 of the chip 200. The incident excitation light α passes through the interface between the side wall 240 and the liquid 310 and reaches the diffraction grating 230.

In FIG. 6, θ ₁ is an incident angle of the excitation light α when the excitation light α is incident on the side wall 240 from an external medium (for example, air). θ ₂ is an incident angle of the excitation light α when the excitation light α enters the liquid 310 (for example, a buffer solution) from the side wall 240. θ is an incident angle of the excitation light α with respect to the metal film 220. As described above, the emission angle of the fluorescence β with respect to the metal film 220 can be approximated by 2θ.

θ ₃ is a refraction angle of the fluorescence β when the fluorescence β enters the side wall 240 from the liquid 310. θ ₃ is also the incident angle of the fluorescence β when the fluorescence β enters the external medium from the side wall 240. θ ₄ is a refraction angle of the fluorescence β when the fluorescence β enters the external medium from the side wall 240.

From Snell's law, the following formulas (12) to (15) hold. Here, n ₁ is the refractive index of the external medium. n ₂ is the refractive index of the side wall 240. n ₃ is the refractive index of the liquid 310.

  FIG. 7A is a graph showing a relationship (simulation result) between the incident angle θ of the excitation light α with respect to the metal film 220 and the reflectance of the excitation light α. This simulation was performed using the RCWA method under the following conditions. The metal film 230 was a gold thin film. A diffraction grating 230 (pitch Λ: 300 nm) having the shape shown in FIG. 2A is formed on the gold thin film. The wavelength of the excitation light α was 637 nm. The liquid 310 was water.

  From the graph shown in FIG. 7A, it can be seen that, under this condition, when the incident angle is about 26 ° (26.2 °), the reflectance of the excitation light α is the lowest and SPR is most efficiently generated.

Here, the refractive index n ₁ of the external medium (air) is 1, the refractive index n _{2 of the} side wall 240 (optical resin) is 1.525, and the refractive index n ₃ of the liquid 310 (water) is 1. Suppose that 33 and θ = 26 °. In this case, it is possible to obtain θ ₁ = 65.1 °, θ ₂ = 57 °, θ ₃ = 32.5 °, and θ ₄ = 55 ° by solving the above equations (12) to (15). it can.

In FIG. 6, it is assumed that the horizontal size t of the bottom of the liquid reservoir 250 is 5 mm. In this case, from the above equation (10), the depth h of the liquid 310 must exceed 8.7 mm. Therefore, the height _{h 2} of the side wall 240 also needs to exceed the 8.7 mm. This numerical value of 8.7 mm corresponds to h ₁ in FIG.

Moreover, the magnitude of t ₂ in FIG. 6 is obtained by the following equation (16). Therefore, the thickness t _{1 of the} side wall 240 is set so as to satisfy the following expression (17).

  FIG. 7B shows that the chip 200 shown in FIG. 6 (pitch Λ of diffraction grating 230: 400 nm, diameter t of liquid reservoir 250: 10 mm) is used to apply excitation light α (wavelength: 633 nm) to the surface of liquid 310 (wavelength: 633 nm). 6 is a graph showing a relationship (actual measurement value) between the incident angle θ of the excitation light α with respect to the metal film 220 and the intensity of the reflected light of the excitation light α when the diffraction grating 230 is irradiated through the meniscus. In this experiment, the intensity of reflected light was measured five times using the same chip. For each measurement, the liquid 310 in the liquid reservoir 250 was removed, and the liquid 310 was provided again in the liquid reservoir 250.

  From the graph shown in FIG. 7B, the incident angle (resonance angle) at which the reflected light intensity of the excitation light α is the lowest decreases from 7.2 ° to 7.7 for each measurement even though the measurement is performed under the same conditions. It can be seen that the angle is 8 °. This is probably because the surface shape (meniscus) of the liquid 310 changes every time the liquid 310 is provided in the liquid reservoir 250, and the incident angle of the excitation light α with respect to the diffraction grating 230 changes.

  7C uses the chip 200 shown in FIG. 6 (pitch Λ of diffraction grating 230: 400 nm, diameter t of liquid reservoir 250: 10 mm) to apply excitation light α (wavelength: 637 nm) to the surface of liquid 310 (wavelength: 637 nm). It is a graph which shows the intensity | strength (actual value) of fluorescence (beta) when irradiating the diffraction grating 230 through a meniscus) and detecting fluorescence (beta) through the interface of the liquid 310 and the side wall 240. FIG. In this experiment, the intensity of fluorescent β was measured 5 times using the same chip. For each measurement, the liquid 310 (TBS-T) in the liquid reservoir 250 was removed, and the liquid 310 (TBS-T) was provided again in the liquid reservoir 250. Further, instead of binding a fluorescently labeled substance to be detected to the surface of the diffraction grating 230, a SAM film was formed on the surface of the diffraction grating 230, and a fluorescent dye (allophycocyanin) was immobilized thereon.

  From the graph shown in FIG. 7C, it can be seen that the intensity of the fluorescence β varies from 1119 to 1191 counts / second for each measurement even though the measurement is performed under the same conditions. This is probably because the surface shape (meniscus) of the liquid 310 changes every time the liquid 310 is provided in the liquid reservoir 250, and the incident angle of the excitation light α with respect to the diffraction grating 230 changes.

  As shown in FIGS. 7B and 7C, when the diffraction grating 230 is irradiated with the excitation light α through the surface (meniscus) of the liquid 310, the incident angle of the excitation light α changes every time the measurement is performed. Since the intensity varies greatly, quantitative detection cannot be performed.

  On the other hand, in the SPFS apparatus 100 according to the first and second embodiments, the excitation light α is irradiated to the diffraction grating 230 without passing through the surface (meniscus) of the liquid 310, and therefore the incident angle of the excitation light α every measurement. Does not change, and the intensity of the detected fluorescence β is also stable. For this reason, the SPFS apparatus 100 according to Embodiments 1 and 2 can perform quantitative detection.

  The SPFS device 100 according to the present embodiment has the same effects as the SPFS device 100 according to the first embodiment.

  Since the present invention can measure a substance to be detected with high reliability, it is useful for clinical examinations, for example.

100 Surface plasmon enhanced fluorescence measuring device (SPFS device)
DESCRIPTION OF SYMBOLS 110 Light source 120 Collimating lens 130 Excitation light filter 140 Fluorescence filter 150 Photodetection part 160 Control part 200 Chip 210 Substrate 220 Metal film 230 Diffraction grating 240 Side wall 250 Liquid reservoir part 310 Liquid α Excitation light β Fluorescence

Claims (4)

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,
A liquid reservoir that is surrounded by a light-transmitting side wall and holds a liquid; a metal film that includes a diffraction grating disposed at the bottom of the liquid reservoir; and a capturing body fixed to the diffraction grating; Preparing a chip having a substance to be detected, which is bound to the capturing body and labeled with a fluorescent substance;
Irradiating the diffraction grating with excitation light through an interface between the side wall and the liquid held in the liquid reservoir;
Detecting fluorescence emitted from the fluorescent substance through an interface between the liquid held in the liquid reservoir and the side wall to obtain a fluorescent signal;
A method for measuring surface plasmon enhanced fluorescence, comprising: 2. The depth h of the liquid held in the liquid reservoir in a cross section in the depth direction of the liquid reservoir including the optical axis of the excitation light satisfies the following formula (1). Surface plasmon enhanced fluorescence measurement method.

[In the above formula (1), t is the horizontal size of the bottom of the liquid reservoir. θ is an incident angle of excitation light to the diffraction grating. ] A liquid reservoir that is surrounded by a light-transmitting side wall and holds a liquid; a metal film that includes a diffraction grating disposed at the bottom of the liquid reservoir; and a capturing body fixed to the diffraction grating; A surface plasmon that is mounted on a chip having a fluorescent substance-labeled target substance bound to the capturing body and detects the presence or amount of the target substance by irradiating the diffraction grating with excitation light An enhanced fluorescence measuring device comprising:
A light irradiation unit configured to irradiate the diffraction grating with excitation light through an interface between the side wall and the liquid held in the liquid reservoir, in order to excite the fluorescent substance by the enhanced electric field and emit fluorescence;
A light detection unit that detects fluorescence emitted from the fluorescent material through an interface between the liquid held in the liquid reservoir and the side wall;
A surface plasmon enhanced fluorescence measuring apparatus. The depth h of the liquid held in the liquid reservoir in a cross section in the depth direction of the liquid reservoir including the light irradiation unit and the light detection unit satisfies the following formula (1). The surface plasmon enhanced fluorescence measuring apparatus described in 1.

[In the above formula (1), t is the horizontal size of the bottom of the liquid reservoir. θ is an incident angle of excitation light to the diffraction grating. ]

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