WO2015068813A1

WO2015068813A1 - Chip, and method for measuring surface plasmon-enhanced fluorescence

Info

Publication number: WO2015068813A1
Application number: PCT/JP2014/079604
Authority: WO
Inventors: 剛典永江; 高敏彼谷; 幸登中村; 平山　博士
Original assignee: コニカミノルタ株式会社
Priority date: 2013-11-07
Filing date: 2014-11-07
Publication date: 2015-05-14
Also published as: JPWO2015068813A1; JP6586884B2

Abstract

　In the present invention, a chip is prepared having a liquid storing part for retaining a liquid, a metal film including a diffraction grating arranged in the bottom part of the liquid storing part, and a capturing body immobilized on the diffraction grating. A sample liquid is introduced to the liquid storing part, and the sample liquid in the liquid storing part is then substituted with another liquid. Excitation light is radiated to the diffraction grating from the direction of an opening in the liquid storing part so as to generate surface plasmon resonance in the diffraction grating, and fluorescence, emitted from a fluorescent substance for labeling the substance to be detected, is detected. The surface area of the liquid in the liquid storing part when fluorescence is detected is greater than the surface area of the sample liquid in the liquid storing part when the sample liquid is introduced.

Description

Chip and surface plasmon enhanced fluorescence measurement method

The present invention relates to a chip used in a surface plasmon enhanced fluorescence measuring apparatus and a surface plasmon enhanced fluorescence measuring method.

In clinical examinations and 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 is 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 for detecting a substance to be detected with high sensitivity, surface plasmon excitation enhanced fluorescence spectroscopy (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 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 of 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 plasmon using a diffraction grating (see Patent Document 2). In GC-SPFS, a metal film formed with a diffraction grating 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 GC-SPFS, when a liquid is provided on a diffraction grating to detect fluorescence, it is preferable that a lid does not exist on the liquid. This is because the background is increased by the autofluorescence of the lid when the excitation light passes through the lid. In particular, when the substance to be detected is at a low concentration, an increase in background may lead to failure in detection of the substance to be detected.

On the other hand, when the upper part of the liquid is opened to the outside without providing a lid, the liquid surface is inclined by the meniscus. 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.

An object of the present invention is a chip used in a measuring apparatus that uses GC-SPFS and a measuring method that uses GC-SPFS, which prevents an increase in background and reduces detection accuracy due to a deviation in the emission angle of fluorescence. It is providing the chip | tip and the measuring method which can suppress this.

In order to solve the above-described problem, a chip according to an embodiment of the present invention detects a fluorescence emitted from a fluorescent substance that labels a target substance by being excited by an electric field based on surface plasmon resonance. A chip used in a surface plasmon enhanced fluorescence measuring apparatus for detecting the presence or amount of a substance, including a liquid reservoir for holding a liquid and a diffraction grating disposed at the bottom of the liquid reservoir And the horizontal size of the liquid reservoir in the cross section in the depth direction of the liquid reservoir increases toward the top.

In addition, in order to solve the above-described problem, the surface plasmon enhanced fluorescence measurement method according to an embodiment of the present invention uses a fluorescent material that labels a target substance to emit fluorescence that is excited by an electric field based on surface plasmon resonance. A surface plasmon enhanced fluorescence measurement method for detecting the presence or amount of the substance to be detected, a liquid reservoir for holding a liquid, and a diffraction grating disposed at the bottom of the liquid reservoir A step of preparing a chip having a metal film including a capture body fixed to the diffraction grating, a step of introducing a sample liquid into the liquid reservoir, and a step of separating the sample liquid in the liquid reservoir From the fluorescent substance that labels the substance to be detected by irradiating the diffraction grating with excitation light from the opening side of the liquid reservoir so that surface plasmon resonance occurs in the diffraction grating. Released Detecting the fluorescence, and the surface area of the liquid in the liquid reservoir in the step of detecting fluorescence is the sample liquid in the liquid reservoir in the step of introducing the sample liquid into the liquid reservoir. Greater than the surface area of

According to the present invention, a substance to be detected can be detected with higher sensitivity and accuracy in a measuring apparatus and a measuring method using GC-SPFS.

It is a schematic diagram showing the structure of the surface plasmon enhanced fluorescence measurement device and the chip according to the first embodiment. 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 flowchart showing the operation of the surface plasmon enhanced fluorescence measurement device according to the first embodiment. FIG. 5A is a schematic cross-sectional view showing the chip after introducing the sample solution, and FIG. 5B is a schematic cross-sectional view showing the chip during fluorescence detection. FIG. 6A is a schematic diagram illustrating optical paths of excitation light and fluorescence when it is assumed that there is no meniscus, and FIG. 6B is a schematic diagram illustrating optical paths of excitation light and fluorescence when there is a meniscus. 7A and 7B are graphs showing the measurement results of meniscus. 8A and 8B are schematic cross-sectional views showing modifications of the chip.

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 sidewall 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 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 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 formation method of 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.

In the diffraction grating 230 (reaction field), a capturing body for capturing a substance to be detected is immobilized. 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 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. As described above, in the GC-SPFS, the fluorescence β is emitted with directivity in a specific direction.

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 a liquid on the metal film 220. Since the opening of the liquid reservoir 250 is not closed with a lid, the surface of the liquid in the liquid reservoir 250 is exposed to the outside. Accordingly, the surface of the liquid in the liquid reservoir 250 forms a meniscus depending on the size of the liquid surface, the hydrophobicity of the inner surface of the side wall 240, the surface tension of the liquid, and the like. As will be described later, in the chip 200 according to the present embodiment, in order to reduce the influence of the meniscus at the time of fluorescence detection, the horizontal size of the liquid reservoir 250 in the cross section in the depth direction of the liquid reservoir 250. The distance (the distance between the side walls 240 facing each other) increases as it goes upward (away from the metal film 220). More specifically, the interval between the side walls 240 in the upper part of the liquid reservoir 250 is larger than the interval between the side walls 240 in the lower part of the liquid reservoir 250, and the inner surface of the side wall 240 in the upper part of the liquid reservoir 250 and the liquid reservoir The inner surface of the side wall 240 at the bottom of the portion 250 is connected in a horizontal plane (see FIG. 1).

The material of the side wall 240 is not particularly limited as long as it can hold a liquid on the metal film 220, and may be appropriately selected according to required properties (for example, hydrophobicity of the surface). 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. Further, 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. 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. The excitation light irradiation unit emits only the P wave for the metal film 220 toward the metal film 220 so that diffracted light that can be combined with surface plasmons in the metal film 220 is generated in the diffraction grating 230. The excitation light irradiation unit irradiates the metal film 220 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 metal film 220 of the chip 200. The type of the light source 110 is not particularly limited as long as it can emit light having a wavelength capable of exciting the fluorescent material (for example, 400 to 1000 nm). 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.

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 (11).

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 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 grating 230 pitches

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 in the liquid reservoir 250), and is defined as the following equation (12). The

ω: angular frequency of excitation light α c: speed of light ε ₁ : dielectric constant of medium (liquid 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 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 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.

Next, the detection operation of the SPFS device 100 will be described. FIG. 4 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 detection 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. At this time, as shown in FIG. 5A, it is preferable to introduce the sample liquid 310 only into the lower part of the liquid reservoir 250. Since the capacity of the lower part of the liquid reservoir 250 is smaller than the capacity of the upper part of the liquid reservoir 250, the amount of the sample liquid used can be reduced by introducing the sample liquid 310 only into the lower part of the liquid reservoir 250. it can. 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 the 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 detection 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. Also at this time, as shown in FIG. 5A, it is preferable to introduce the fluorescent labeling liquid only into the lower part of the liquid reservoir 250. 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 from the metal film 220. The light detection unit 150 outputs the measurement result to the control unit 160. At this time, as shown in FIG. 5B, it is preferable that the liquid 320 is introduced to the upper part of the liquid reservoir 250. Since the interval between the side walls 240 at the upper part of the liquid reservoir 250 is larger than the interval between the side walls 240 at the lower part of the liquid reservoir 250, the liquid 320 is introduced to the upper part of the liquid reservoir 250, thereby It is possible to reduce the degree of bending of the liquid surface due to the above. That is, it is possible to reduce the deviation of the emission angle of the fluorescence β by bringing the liquid surface shape above the metal film 220 close to a flat surface.

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

As described above, the chip 200 according to the present embodiment is larger as the horizontal size of the liquid reservoir 250 in the cross section in the depth direction of the liquid reservoir 250 is higher (away from the metal film 220). . For this reason, by increasing the amount of the liquid in the liquid reservoir 250, the deviation of the emission angle of the fluorescence due to the meniscus can be achieved without placing a lid or the like on the liquid reservoir 250 that may cause an increase in background. Can be reduced. Therefore, the detection target substance can be detected with high sensitivity and high accuracy by using the chip 200 according to the present embodiment.

Further, the chip 200 according to the present embodiment is smaller as the horizontal size of the liquid reservoir 250 in the cross section in the depth direction of the liquid reservoir 250 becomes lower (closer to the metal film 220). For this reason, the usage-amount of the sample liquid when performing a primary reaction and the usage-amount of a reagent when performing a secondary reaction can be reduced. Therefore, the measurement cost can be reduced by using the chip 200 according to the present embodiment.

[Embodiment 2]
In the chip 200 according to the second embodiment, the horizontal size R of the liquid reservoir 250 and the contact angle θ _{c of the} inner surface of the side wall 240 are set corresponding to the numerical aperture of the light detection unit 150 of the SPFS device 100. This is different from the chip 200 according to the first embodiment. Therefore, only this point will be described in the present embodiment.

In order to accurately detect the fluorescence β in the light detection unit 150, it is preferable that the fluorescence β can reach the opening (aperture) of the light detection unit 150 even if the emission angle of the fluorescence β is shifted due to the meniscus. . That is, it is preferable that the deviation width of the emission angle of the fluorescence β is smaller than a predetermined value associated with the numerical aperture of the light detection unit 150.

FIG. 6A is a schematic diagram showing optical paths of excitation light α and fluorescence β when it is assumed that there is no meniscus. Excitation light α on the right side in the figure is reflected light of excitation light α. Here, θ ₁ is an incident angle of the excitation light α when the excitation light α is incident on the liquid 320 (for example, a buffer solution) from a medium (for example, air) on the liquid 320. θ ₁ ′ is a refraction angle of the excitation light α when the excitation light α is incident on the liquid 320 from the medium on the liquid 320. θ ₂ is a refraction angle of the fluorescence β when the fluorescence β enters the medium on the liquid 320 from the liquid 320. h is the depth of the liquid 320 in the liquid reservoir 250. As shown in this figure, the incident angle of the fluorescence β when the fluorescence β enters the medium on the liquid 320 from the liquid 320 can be approximated by 2θ ₁ ′. In the following description, θ ₂ is used as a reference for the emission angle of the fluorescence β.

Note that the wavelength of the excitation light α, the pitch Λ of the diffraction grating 230, the depth h of the liquid 320, the incident angle θ ₁ of the excitation light α, the refractive index n ₁ and the dielectric constant of the medium on the liquid 320, and the refraction of the liquid 320 The rate n _{2, the} dielectric constant, and the like are dependently determined according to the type of liquid 320 used at the time of fluorescence detection, the type of fluorescent material, the atmosphere at the time of fluorescence detection, and the like.

FIG. 6B is a schematic diagram showing optical paths of excitation light α and fluorescence β when there is a meniscus. Here, when the surface direction of the metal film 220 is the x-axis, the height direction of the liquid reservoir 250 is the y-axis, and the intersection of the optical axis of the excitation light α and the diffraction grating 230 is the origin, the liquid reservoir A function representing the shape of the meniscus of the liquid 320 in 250 is represented by the following equation (13). Since the refractive index and dielectric constant of the liquid 320 are considered to be substantially the same as the refractive index and dielectric constant of water, the shape of the meniscus of the liquid 320 is the size of the liquid reservoir 250 at the position of the surface of the liquid 320 in the horizontal direction. And the contact angle θ _c of the inner surface of the side wall 240.

In FIG. 6B, θ ₁ ″ represents the excitation light α in the liquid 320 with respect to a straight line passing through the incident point and perpendicular to the surface of the metal film 220 when the excitation light α is incident on the liquid 320 from the medium on the liquid 320. Δθ ₁ represents the inclination of the surface of the liquid 320 at the incident point when the excitation light α is incident on the liquid 320 from the medium on the liquid 320. By using the above equation (13), the following equation (14) is obtained. X ₁ is the value of the x coordinate of the incident point, and Δθ ₂ ′ is the liquid 320 at the incident point when the fluorescence β is incident on the medium on the liquid 320 from the liquid 320. represents the inclination of the surface, .x ₂ represented as the equation (13) using the following equation (15) is the value of x-coordinate of the point of incidence.

In this case, _{x 1} and _{x 2} are respectively approximated by the following equation (16) and (17).

From Snell's law, the following equations (18) and (19) hold (see FIG. 6A). Similarly, the following formulas (20) and (21) are established from Snell's law (see FIG. 6B). Here, n ₁ is the refractive index of the medium on the liquid 320. n ₂ is the refractive index of the liquid 320.

By substituting each numerical value into the above equations (18) to (21) and solving these equations, the emission angle deviation width Δθ ₂ of the fluorescence β caused by the meniscus of the liquid 320 can be calculated. As described above, in order to accurately detect the fluorescence β in the light detection unit 150, the emission angle deviation width Δθ ₂ of the fluorescence β is larger than a predetermined value associated with the numerical aperture NA of the light detection unit 150. Small is preferable. That is, it is preferable that the following formula (22) is satisfied.

As described above, the meniscus shape of the liquid 320 is determined by the horizontal size R of the liquid reservoir 250 at the surface of the liquid 320 and the contact angle θ _c of the inner surface of the side wall 240. Accordingly, the chip 200 according to the present embodiment, as the above equation (22) is satisfied, R and theta _c is set. From the viewpoint of accurately detecting the fluorescence β, R and θ _c are more preferably set so that the fluorescence β is not totally reflected on the surface of the liquid 320. R and θ _c are more preferably set so that the fluorescence β can reach the light detection unit 150 even when the depth of the liquid 320 varies. Further, it is more preferable that R and θ _c are set so that the fluorescence β can reach the light detection unit 150 even if the chip 200 is displaced.

Note that the shape of the meniscus of the liquid 320 in the liquid reservoir 250 (the function of the above equation (13)) can be measured by, for example, a laser displacement meter. FIG. 7 is a graph showing the measurement results of meniscus of TBST (Tris Buffered Saline with Tween 20) when the side wall 240 is formed of polytetrafluoroethylene. FIG. 7A is a graph showing measurement results when the shape of the horizontal cross section of the liquid reservoir 250 at the liquid level is a circle having a diameter of 18 mm, and FIG. 7B is a graph showing the liquid reservoir 250 at the liquid level. It is a graph which shows a measurement result in case the shape of a horizontal cross section of this is a circle with a diameter of 10 mm. The horizontal axis indicates the distance in the horizontal direction from the center of the liquid level, and the vertical axis indicates the height from the center of the liquid level. From such measurement results, the function of equation (13) can be determined.

As described above, in the chip 200 according to the present embodiment, the horizontal size R of the liquid reservoir 250 and the contact angle of the inner surface of the sidewall 240 correspond to the numerical aperture of the light detection unit 150 of the SPFS device 100. θ _c is set. For this reason, the deviation of the emission angle of the fluorescence β can be made smaller than the numerical aperture of the light detection unit 150. Therefore, the detection target substance can be detected with high sensitivity and high accuracy by using the chip 200 according to the present embodiment.

In each of the above embodiments, the example using the chip 200 shown in FIG. 5 has been described, but the shape of the liquid reservoir 250 of the chip according to the present invention is not limited to this. As described above, the effect of the present invention can be obtained when the horizontal size of the liquid reservoir 250 in the cross section in the depth direction of the liquid reservoir 250 increases as it moves upward (away from the metal film 220). It is done. Therefore, as shown in FIG. 8A, an R surface may be formed on the inner surface of the side wall 240, or a tapered surface (inclined surface) may be formed on the inner surface of the side wall 240 as shown in FIG. 8B. .

This application claims priority based on Japanese Patent Application No. 2013-230969 filed on November 7, 2013. The contents described in the application specification and the drawings are all incorporated herein.

The present invention is useful for, for example, clinical examination because the substance to be detected can be measured with high reliability.

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 Light detection part 160 Control part 200 Chip 210 Substrate 220 Metal film 230 Diffraction grating 240 Side wall 250 Reservoir part 310 Sample liquid 320 Liquid α Excitation light β Fluorescence

Claims

A fluorescent substance that labels a substance to be detected is used in a surface plasmon-enhanced fluorescence measuring device that detects fluorescence emitted by excitation by an electric field based on surface plasmon resonance and detects the presence or amount of the substance to be detected. A chip,
A liquid reservoir for holding liquid;
A metal film including a diffraction grating disposed at the bottom of the liquid reservoir,
The horizontal size of the liquid reservoir in the cross section in the depth direction of the liquid reservoir is larger as it goes up.
Chip.
The surface plasmon enhanced fluorescence measurement device has a light irradiation unit that irradiates the diffraction grating with excitation light, and a light detection unit that detects fluorescence.
In the cross section in the depth direction of the liquid reservoir including the light irradiator and the light detector, the horizontal size R of the liquid reservoir at the liquid level at the time of fluorescence detection and the side wall of the liquid reservoir The contact angle θ c of the inner surface is set so that the following expression (1) is established,
The chip according to claim 1.

[In the above formula (1), Δθ 2 is calculated by the following formulas (2) to (5), and the refraction angle of the fluorescence when the fluorescence enters the medium on the liquid from the liquid is calculated. This is the fluctuation range due to the meniscus. NA is the numerical aperture of the light detection unit. ]

[In the above formulas (2) to (5), n 1 is the refractive index of the medium on the liquid. n 2 is the refractive index of the liquid. θ 1 is an incident angle of the excitation light when the excitation light is incident on the liquid from the medium on the liquid when it is assumed that there is no meniscus. θ 1 ′ is a refraction angle of the excitation light when the excitation light is incident on the liquid from the medium on the liquid when it is assumed that there is no meniscus. θ 2 is a refraction angle of the fluorescence when the fluorescence is incident on the medium on the liquid from the liquid when it is assumed that there is no meniscus. θ 1 ″ is the angle of the excitation light in the liquid with respect to a straight line passing through the incident point and perpendicular to the surface of the metal film when the excitation light in the presence of the meniscus enters the liquid from the medium on the liquid Δθ 1 is expressed by the following equations (6) and (10), and the liquid surface at the incident point when the excitation light in the presence of the meniscus enters the liquid from the medium on the liquid. Δθ 2 ′ is the liquid level at the incident point when the fluorescence when there is a meniscus is incident from the liquid to the medium on the liquid, expressed by the following equations (8) and (10). The slope of

In [the formula (6), x 1 is the x-coordinate of the point of incidence of when excitation light in the case where there is a meniscus is incident to the liquid from a medium on the liquid, is approximated by the following equation (7) The ]

In [the formula (8), x 2 is the x-coordinate of the point of incidence when the fluorescence when there is menisci incident on a medium on the liquid from the liquid, is approximated by the following equation (9) . ]

[Equation (10) in the cross section is that the plane direction of the metal film is the x axis, the height direction of the liquid reservoir is the y axis, and the intersection of the optical axis of the excitation light and the diffraction grating is the origin. Is a function representing the shape of the meniscus of the liquid formed under the influence of R and θ c . ]
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 liquid reservoir for holding a liquid, a metal film including a diffraction grating disposed at the bottom of the liquid reservoir, and a capturing body fixed to the diffraction grating; ,
Introducing a sample liquid into the liquid reservoir;
Replacing the sample liquid in the liquid reservoir with another liquid;
Irradiating the diffraction grating with excitation light from the opening side of the liquid reservoir so as to cause surface plasmon resonance in the diffraction grating, and detecting fluorescence emitted from a fluorescent substance that labels the substance to be detected When,
Including
The surface area of the liquid in the liquid reservoir in the step of detecting fluorescence is larger than the surface area of the sample liquid in the liquid reservoir in the step of introducing the sample liquid into the liquid reservoir.
Surface plasmon enhanced fluorescence measurement method.