WO2016093037A1

WO2016093037A1 - Detection device and detection method

Info

Publication number: WO2016093037A1
Application number: PCT/JP2015/082665
Authority: WO
Inventors: 幸登中村; 剛典永江; 高敏彼谷
Original assignee: コニカミノルタ株式会社
Priority date: 2014-12-09
Filing date: 2015-11-20
Publication date: 2016-06-16
Also published as: JPWO2016093037A1; JP6627778B2

Abstract

The detection device according to the present invention has a holder, an excitation light radiating part, a fluorescence detection part, and a processing part. The holder retains a detection chip having an accommodating part and a metal film which includes a reaction field. The excitation light radiating part radiates excitation light to the metal film retained by the holder. The fluorescence detection part detects, at least twice, fluorescence emitted from a fluorescent substance present on the metal film when the excitation light radiating part radiates excitation light to the metal film. The processing part calculates a signal value indicating the presence or amount of a detected substance on the basis of two or more detection values detected by the fluorescence detection part. The fluorescence detection part detects fluorescence at least once in a state in which the depth of a liquid on the reaction field is a first depth, and detects fluorescence at least once in a state in which the depth of the liquid on the reaction field is a second depth different from the first depth.

Description

Detection apparatus and detection method

The present invention relates to a detection apparatus and a detection method for detecting a substance to be detected using surface plasmon resonance.

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. In SPFS, surface plasmon resonance (hereinafter referred to as “SPR”) generated by irradiating a metal film with light under a predetermined condition is used.

First, in SPFS, a capture body (for example, a primary antibody) that can specifically bind to a substance to be detected is immobilized on a metal film to form a reaction field for specifically capturing the substance to be detected. When a specimen containing a substance to be detected is provided in this reaction field, the substance to be detected binds to the capturing body in the reaction field. Subsequently, when another capture body (for example, secondary antibody) labeled with a fluorescent substance is provided to the reaction field, the target substance bound to the capture body in 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 the emitted 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.

However, at the time of fluorescence detection, if an unreacted fluorescent substance that is not labeled with a substance to be detected is present on the metal film, background noise occurs. For this reason, in order to detect a to-be-detected substance correctly, it is preferable to remove the unreacted fluorescent substance beforehand by washing.

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

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

JP 2011-158369 A

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

Further, in both GC-SPFS and PC-SPFS, there is a possibility that the substance to be detected cannot be detected accurately due to the influence of background noise caused by the unreacted fluorescent substance. When washing is performed to remove the influence of background noise, the binding state between the substance to be detected and the metal film (or primary antibody) changes during the washing process, so the reaction process in real time. There is a problem that it cannot be measured.

An object of the present invention is a detection apparatus and detection method using SPFS, which can accurately detect the presence or amount of a substance to be detected even if an unreacted fluorescent substance is present on a metal film. It is to provide a detection device and a detection method that can be used.

In order to solve the above problems, a detection device according to an embodiment of the present invention is a detection device for detecting a substance to be detected using surface plasmon resonance, and includes a storage unit for storing a liquid. A holder for holding a detection chip having a metal film including a reaction field, which is arranged at the bottom of the housing part and to which a detection target substance labeled with a fluorescent substance is directly or indirectly fixed An excitation light irradiation unit that irradiates the metal film of the detection chip held by the holder with excitation light so that surface plasmon resonance occurs, and the excitation light irradiation in a state where a liquid exists in the storage unit. A fluorescence detection unit that detects fluorescence emitted from the fluorescent material existing on the metal film at least twice when the unit irradiates the metal film with excitation light; and two or more detected by the fluorescence detection unit A processing unit that calculates a signal value indicating the presence or amount of the substance to be detected based on the output value, and the fluorescence detection unit has a first depth of the liquid on the reaction field. In this state, fluorescence is detected at least once, and fluorescence is detected at least once in a state where the depth of the liquid on the reaction field is a second depth different from the first depth.

In order to solve the above-described problem, a detection method according to an embodiment of the present invention is a detection method for detecting a substance to be detected using surface plasmon resonance, and includes a storage unit for storing a liquid, A first step of preparing a detection chip having a metal film including a reaction field, which is arranged at the bottom of the housing part and to which a detection target substance labeled with a fluorescent substance is directly or indirectly fixed; Irradiating excitation light so that surface plasmon resonance occurs in the metal film in a state where the liquid exists in the container so that the depth of the liquid on the reaction field becomes the first depth, and the metal A second step of detecting fluorescence emitted from the fluorescent substance present on the film; and the liquid is arranged such that the depth of the liquid on the reaction field is a second depth different from the first depth. In the state of being present in the housing part, In the third step of irradiating excitation light so that surface plasmon resonance occurs in the genus film and detecting the fluorescence emitted from the fluorescent substance existing on the metal film, in the second step and the third step And a fourth step of calculating a signal value indicating the presence or amount of the substance to be detected based on the obtained detection value.

According to the present invention, a substance to be detected can be detected with high sensitivity and easily in a detection apparatus and a detection method using SPFS. In addition, according to the present invention, a substance to be detected can be detected in real time.

FIG. 1 is a schematic diagram showing the configuration of the SPFS apparatus according to the first embodiment. FIG. 2A is a cross-sectional view of a detection chip used in the SPFS apparatus according to Embodiments 1 to 4, and FIG. 2B is a plan view of the detection chip. FIG. 3 is a perspective view of the diffraction grating. FIG. 4 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus according to the first embodiment. 5A and 5B are schematic diagrams for explaining a part of the detection process of the SPFS device according to the first and fourth embodiments. 6A and 6B are schematic diagrams for explaining the principle of detection of a substance to be detected by the SPFS apparatus. FIG. 7 is a schematic diagram illustrating a configuration of an SPFS apparatus according to a modification. FIG. 8 is a cross-sectional view of a detection chip according to a modification used in the SPFS apparatus. FIG. 9 is a schematic diagram illustrating a configuration of the SPFS apparatus according to the second embodiment. FIG. 10 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus according to the second embodiment. FIG. 11 is a schematic diagram illustrating a configuration of the SPFS apparatus according to the third embodiment. FIG. 12 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus according to the third embodiment. FIG. 13 is a schematic diagram illustrating a configuration of the SPFS apparatus according to the fourth embodiment. FIG. 14 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus according to the fourth embodiment.

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

[Embodiment 1]
In the first embodiment, GC-SPFS that detects first light (for example, p-polarized light) and second light (for example, s-polarized light) included in the fluorescence emitted from the fluorescent material present on the metal film, respectively. The apparatus will be described. The SPFS device according to Embodiment 1 includes a polarizer and switches the rotation angle of the polarizer.

(Configuration of SPFS device and detection chip)
FIG. 1 is a schematic diagram illustrating a configuration of an SPFS apparatus 100 according to the first embodiment. FIG. 2A is a cross-sectional view of the detection chip 10, and FIG. 2B is a plan view of the detection chip 10. The cross-sectional view shown in FIG. 2A shows a cross section taken along line AA in FIG. 2B. FIG. 3 is a perspective view of the diffraction grating 13.

As shown in FIG. 1, the SPFS device 100 includes an excitation light irradiation unit 110, a fluorescence detection unit 120, a transport unit 130, and a control unit 140. The SPFS device 100 is used in a state where two detection chips (the detection chip 10 and the detection chip 10 ′) are mounted on the chip holder (holder) 132 of the transport unit 130. The configuration and operation mechanism of the SPFS device 100 are appropriately designed according to the configuration and number of detection chips 10 to be used. Therefore, the detection chips 10, 10 'will be described first, and then the SPFS device 100 will be described. Note that the detection chip 10 and the detection chip 10 'have the same configuration, and differ only in the depth of the liquid to be stored. The detection chip 10 stores liquid at the first depth h1, and the detection chip 10 'stores liquid at the second depth h2. Hereinafter, in the description of the configuration of the detection chip, the detection chip 10 will be described.

2A and 2B, the detection chip 10 includes a substrate 11, a metal film 12 formed on the substrate 11, and a frame body 14 disposed on the substrate 11. By arranging the frame body 14 on the substrate 11, the accommodating portion 15 for accommodating the liquid is formed. The metal film 12 has a diffraction grating 13, and a capturing body 16 (for example, a primary antibody) is immobilized on the diffraction grating 13. Therefore, the surface of the diffraction grating 13 also functions as a reaction field for binding the capturing body 16 and the substance to be detected.

The substrate 11 is a support member for the metal film 12. The material of the substrate 11 is not particularly limited as long as it has mechanical strength capable of supporting the metal film 12. Examples of the material of the substrate 11 include inorganic materials such as glass, quartz, and silicon; resins such as acrylic resin, polymethyl methacrylate, polycarbonate, polystyrene, and polyolefin.

The metal film 12 is disposed on the substrate 11 at the bottom of the accommodating portion 15. As described above, the metal film 12 has the diffraction grating 13. In the present embodiment, the metal film 12 (diffraction grating 13) is disposed so as to be exposed in the housing portion 15. When the metal film 12 is irradiated with light at a predetermined incident angle, surface plasmons generated in the metal film 12 and evanescent waves generated by the diffraction grating 13 are combined to generate surface plasmon resonance (SPR). The material of the metal film 12 is not particularly limited as long as it is a metal that can generate surface plasmons. Examples of the material of the metal film 12 include gold, silver, aluminum, platinum, copper, and alloys thereof. The method for forming the metal film 12 is not particularly limited. Examples of the method for forming the metal film 12 include sputtering, vapor deposition, and plating. The thickness of the metal film 12 is not particularly limited. The thickness of the metal film 12 is, for example, 30 to 500 nm, and preferably 100 to 300 nm.

The diffraction grating 13 generates an evanescent wave when the metal film 12 is irradiated with light. The shape of the diffraction grating 13 is not particularly limited as long as an evanescent wave can be generated. For example, the diffraction grating 13 may be a one-dimensional diffraction grating or a two-dimensional diffraction grating. In the present embodiment, as shown in FIG. 3, the diffraction grating 13 is a one-dimensional diffraction grating, and a plurality of ridges (and ridges) parallel to each other are formed on the surface of the metal film 12 at a predetermined interval. Is formed. Further, the sectional shape of the diffraction grating 13 is not particularly limited. Examples of the cross-sectional shape of the diffraction grating 13 include a rectangular wave shape, a sine wave shape, a sawtooth shape, and the like. In the present embodiment, the cross-sectional shape of the diffraction grating 13 is a rectangular wave shape. Note that the optical axis of excitation light α described later is parallel to the xz plane. The pitch and depth of the grooves (concave lines) of the diffraction grating 13 are not particularly limited as long as an evanescent wave can be generated, and can be appropriately set according to the wavelength of the irradiated light. For example, the groove pitch of the diffraction grating 13 is preferably in the range of 100 nm to 2000 nm, and the depth of the groove of the diffraction grating 13 is preferably in the range of 10 nm to 1000 nm.

The formation method of the diffraction grating 13 is not particularly limited. For example, after the metal film 12 is formed on the flat substrate 11, an uneven shape may be imparted to the metal film 12. Alternatively, the metal film 12 may be formed on the substrate 11 that has been provided with an uneven shape in advance. In any method, the metal film 12 including the diffraction grating 13 can be formed.

A capturing body 16 for capturing a substance to be detected is fixed to the diffraction grating 13. Here, on the diffraction grating 13 (metal film 12), a region where the capturing body 16 is immobilized is particularly referred to as a “reaction field”. The capturing body 16 specifically binds to the substance to be detected. Thereby, the substance to be detected is indirectly fixed to the metal film 12 (diffraction grating 13). In the present embodiment, the capturing body 16 is fixed substantially uniformly on the surface of the diffraction grating 13.

The type of the capturing body 16 is not particularly limited as long as the target substance can be captured. For example, the capturing body 16 is an antibody or a fragment thereof that can specifically bind to the substance to be detected, an enzyme that can specifically bind to the substance to be detected, and the like.

The method for immobilizing the capturing body 16 is not particularly limited. For example, a self-assembled monomolecular film (hereinafter referred to as “SAM”) or a polymer film to which the capturing body 16 is bonded may be formed on the diffraction grating 13. Examples of SAMs 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 16 (or a functional group that can be converted into a reactive group) may be fixed to the diffraction grating 13, and the capturing body 16 may be bound to the polymer.

As shown in FIG. 1, the diffraction grating 13 (metal film 12) is irradiated with excitation light α at a predetermined incident angle theta _1. In the irradiation region, the surface plasmon generated in the metal film 12 and the evanescent wave generated by the diffraction grating 13 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. In GC-SPFS, unlike PC-SPFS, fluorescence β is emitted with directivity in a specific direction. For example, the emission angle θ ₂ of the fluorescence β is approximated by 2θ ₁ . Note that, under the conditions in which SPR occurs, almost no reflected light γ of the excitation light α is generated.

The frame body 14 is a plate having a through hole, as shown in FIG. 2B. The frame body 14 is disposed on the substrate 11. The inner surface of the through hole becomes the side surface of the accommodating portion 15. The thickness of the frame 14 is not particularly limited, and is designed according to the amount and depth of the liquid stored in the storage unit 15. The method for fixing the frame body 14 on the substrate 11 is not particularly limited. For example, the frame 14 can be fixed on the substrate 11 using a silicon sheet (double-sided seal) having adhesiveness on both sides.

The accommodating part 15 is arrange | positioned on the metal film 12 (or board | substrate 11), and accommodates a liquid. In the present embodiment, liquid is accommodated in the accommodating portion 15 of one detection chip 10 at the first depth h1. Further, the liquid is accommodated in the accommodating portion 15 of the other detection chip 10 'at the second depth h2. The depth of the liquid stored in the storage unit 15 is not particularly limited, but the first depth h1 and the second depth h2 are preferably in the range of 10 μm to 1 cm. When the ratio of the second depth h2 to the first depth h1 is m (h2 / h1), m is in the range of 0.1 to 10, excluding 0.9 to 1.1. Is preferred. The shape and size of the storage portion 15 are not particularly limited as long as a desired amount of liquid can be stored. For example, the storage unit 15 may be a well that stores a liquid, or may be a flow path (flow cell) through which a liquid can be continuously supplied. The detection chip in which the container 15 is a well includes, for example, in addition to general measurement of a substance to be detected (non-real time measurement), mass transfer analysis between the bulk and the surface of the metal film 12 (real time measurement), and enhancement It is also suitable for measuring an electric field space scale (z-axis direction). The detection chip 10 in which the accommodating portion 15 is a flow path has, for example, another measurement for a molecule (captured body) immobilized on the surface of the metal film 12 in addition to a general measurement of a substance to be detected (non-real time measurement). It is also suitable for reaction constant analysis (real-time measurement) of molecules (substances to be detected). In the present embodiment, the accommodating portion 15 is a well. As described above, the accommodating portion 15 is formed by disposing the frame body 14 on the substrate 11, but the method for forming the accommodating portion 15 is not particularly limited. Another example of the method for forming the accommodating portion 15 includes disposing a lid having a recess formed on the lower surface thereof on the substrate 11.

The type of liquid stored in the storage unit 15 is not particularly limited. Examples of the type of liquid include a specimen containing a substance to be detected, a labeling solution containing a fluorescent substance, a buffer solution, and the like. Usually, the refractive index and dielectric constant of a liquid are comparable to the refractive index and dielectric constant of water. There are no particular limitations on the types of the specimen and the substance to be detected. Examples of the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluted solutions thereof. Examples of substances to be detected include nucleic acids (such as DNA and RNA), proteins (such as polypeptides and oligopeptides), amino acids, carbohydrates, lipids, and modified molecules thereof.

Next, each component of the SPFS device 100 will be described. As described above, the SPFS device 100 includes the excitation light irradiation unit 110, the fluorescence detection unit 120, the transport unit 130, and the control unit 140.

The excitation light irradiation unit 110 irradiates the metal film 12 (diffraction grating 13) of the detection chip 10, 10 'with excitation light α having a constant wavelength and light amount. At this time, the excitation light irradiation unit 110 emits p-polarized light with respect to the surface of the metal film 12 so that diffracted light that can be combined with the surface plasmons in the metal film 12 is generated in the metal film 12 (the diffraction grating 13). Irradiate. The optical axis of the excitation light α is along the arrangement direction of the periodic structure in the diffraction grating 13 (the x-axis direction in FIGS. 2A and 2B and FIG. 3). Therefore, when the axis perpendicular to the x axis and parallel to the surface of the metal film 12 is the y axis, and the axis perpendicular to the x axis and perpendicular to the surface of the metal film 12 is the z axis, the optical axis of the excitation light α is xz. It is parallel to the plane (see FIG. 1). Since the excitation light α is p-polarized light with respect to the surface of the metal film 12, the vibration direction of the electric field of the excitation light α is in the xz plane including the optical axis of the excitation light α and the normal to the surface of the metal film 12. Parallel.

The excitation light irradiation unit 110 has at least a light source 111. The excitation light irradiation unit 110 may further include a collimating lens, an excitation light filter, and the like.

The light source 111 emits excitation light α toward the diffraction grating 13 of the

detection chip

10, 10 ′. The type of the light source 111 is not particularly limited. Examples of types of light source 111 include light emitting diodes, mercury lamps, and other laser light sources. In the present embodiment, the light source 111 is a laser diode. For example, the wavelength of the excitation light α emitted from the light source 111 is in the range of 400 nm to 1000 nm.

A collimating lens (not shown) is disposed between the light source 111 and the detection chips 10 and 10 ′, and collimates the excitation light α emitted from the light source 111. The excitation light α emitted from the laser diode (light source 111) has a flat outline shape even when collimated. For this reason, the laser diode is held in a predetermined posture so that the shape of the irradiation spot on the surface of the metal film 12 is substantially circular. The size of the irradiation spot is preferably about 1 mmφ, for example.

The excitation light filter (not shown) is disposed between the light source 111 and the detection chips 10 and 10 ′, and tunes the excitation light α emitted from the light source 111. Examples of types of excitation light filters include bandpass filters and linear polarizing filters. Since the excitation light α from the laser diode (light source 111) has a slight wavelength distribution width, the bandpass filter turns the excitation light α from the laser diode into a narrow band light having only the center wavelength. In addition, since the excitation light α from the laser diode (light source 111) is not completely linearly polarized light, the linear polarization filter converts the excitation light α from the laser diode into completely linearly polarized light. The excitation light filter may include a half-wave plate that adjusts the polarization direction of the excitation light α so that p-polarized light is incident on the metal film 12.

The incident angle θ ₁ (see FIG. 1) of the excitation light α with respect to the metal film 12 is an angle at which the intensity of the enhanced electric field formed by the SPR is the strongest, and as a result, the intensity of the fluorescence β from the fluorescent material is the strongest. Preferably there is. The incident angle θ ₁ of the excitation light α is appropriately selected according to the groove pitch of the diffraction grating 13, the wavelength of the excitation light α, the type of metal constituting the metal film 12, and the like. Since the optimal incident angle θ ₁ of the excitation light α varies depending on various conditions, the SPFS device 100 rotates the optical axis of the excitation light α and the detection chips 10 and 10 ′ relatively to change the incident angle θ. it is preferred to have the first angle adjusting section for adjusting _one (not shown). For example, the first angle adjustment unit may rotate the excitation light irradiation unit 110 or the detection chips 10 and 10 ′ around the intersection between the optical axis of the excitation light α and the metal film 12.

The fluorescence detection unit 120 is arranged so as to pass through the intersection of the optical axis of the excitation light α and the metal film 12 with respect to the excitation light irradiation unit 110 and sandwich the normal to the surface of the metal film 12. The fluorescence detection unit 120 detects the fluorescence β emitted from the fluorescent material on the metal film 12 (diffraction grating 13) at least twice. More specifically, the fluorescence detection unit 120 detects the fluorescence β emitted from the fluorescent material on the reaction field of the detection chip 10 in a state where the liquid depth on the reaction field of the detection chip 10 is the first depth h1. At least once, and at least the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 ′ in the state where the liquid depth on the reaction field of the detection chip 10 ′ is the second depth h2 Detect once.

The fluorescence detection unit 120 includes a polarizer 121, a rotation angle adjustment unit 122, and a light receiving sensor 123. The fluorescence detection unit 120 may further include a condenser lens group, an aperture stop, a fluorescence filter, and the like.

The polarizer 121 is disposed on the optical path of the fluorescence β between the

detection chip

10, 10 ′ and the light receiving sensor 123. The polarizer 121 is the first in the range where the angle of the vibration direction of the electric field from the fluorescence β to the plane (xz plane) including the normal to the surface of the metal film 12 and the optical axis of the excitation light α is 0 ± 30 °. And the second light whose angle in the direction of vibration of the electric field with respect to the plane is in the range of 90 ± 30 ° are extracted. Preferably, the polarizer 121 uses the p-polarized light whose angle of the vibration direction of the electric field with respect to the plane (xz plane) is 0 ° as the first light from the fluorescence β, and the vibration direction of the electric field with respect to the plane (xz). The s-polarized light having an angle of 90 ° is extracted as the second light. The rotation angle of the polarizer 121 is adjusted by the rotation angle adjustment unit 122. The type of the polarizer 121 is not particularly limited as long as light having a predetermined polarization direction can be extracted. Examples of the type of the polarizer 121 include a polarizing plate, a polarizing prism, a liquid crystal filter, and other polarizing filters. In the present embodiment, the polarizer 121 is a polarizing plate.

The rotation angle adjustment unit 122 adjusts the rotation angle of the polarizer 121. The rotation angle adjustment unit 122 includes, for example, a stepping motor.

The light receiving sensor 123 detects the fluorescence β emitted from the fluorescent material on the metal film 12 taken out by the polarizer 121 and detects the fluorescent image on the metal film 12. The type of the light receiving sensor 123 is not particularly limited, and is, for example, a photomultiplier tube having high sensitivity and a high SN ratio, and may be an avalanche photodiode (APD), a photodiode (PD), a CCD image sensor, or the like. .

The condensing lens group (not shown) is arranged between the

detection chip

10, 10 ′ and the light receiving sensor 123, and constitutes a conjugate optical system that is not easily affected by stray light. The condenser lens group forms a fluorescent image on the metal film 12 on the light receiving surface of the light receiving sensor 123.

Fluorescent filter (not shown) is disposed between the

detection chip

10, 10 ′ and the light receiving sensor 123. The fluorescent filter includes, for example, a cut filter and a neutral density (ND) filter, and removes noise components (for example, excitation light α and external light) other than the fluorescent β from the light reaching the light receiving sensor 123, or receives the light sensor. The amount of light reaching 123 is adjusted.

In GC-SPFS, the fluorescence β is emitted from the diffraction grating 13 (reaction field) with directivity in a specific direction. Therefore, the angle of the optical axis of the fluorescence detection unit 120 with respect to the normal of the surface of the metal film 12 is preferably an angle (fluorescence peak angle) at which the intensity of the fluorescence β is maximized. The SPFS device 100 also adjusts the angle of the optical axis of the fluorescence detection unit 120 by relatively rotating the optical axis of the fluorescence detection unit 120 and the detection chips 10 and 10 ′ (illustrated). Preferably omitted). For example, the second angle adjustment unit may rotate the fluorescence detection unit 120 or the

detection chip

10, 10 ′ around the intersection between the optical axis of the fluorescence detection unit 120 and the metal film 12.

The transport unit 130 moves the position of the detection chip 10, 10 '. The transport unit 130 includes a transport stage 131 and a chip holder 132. The chip holder 132 is fixed to the transport stage 131, and holds the detection chips 10, 10 'in a detachable manner. The shape of the chip holder 132 is a shape that can hold the detection chips 10 and 10 ′ and does not obstruct the optical paths of the excitation light α and the fluorescence β. The transfer stage 131 moves the chip holder 132 in one direction and the opposite direction. The shape of the transfer stage 131 is also a shape that does not obstruct the optical paths of the excitation light α and the fluorescence β. The transfer stage 131 is driven by, for example, a stepping motor.

The control unit 140 includes an excitation light irradiation unit 110 (light source 111 and a first angle adjustment unit), a fluorescence detection unit 120 (a rotation angle adjustment unit 122, a light receiving sensor 123, and a second angle adjustment unit), and a conveyance unit 130 (conveyance). The operation of the stage 131) is controlled. The control unit 140 also functions as a processing unit that processes an output signal (detection result) from the fluorescence detection unit 120. Specifically, based on two or more detection values detected by the fluorescence detection unit 120 (light receiving sensor 123), the processing unit calculates a signal value indicating the presence or amount of the substance to be detected, and a noise value as necessary. calculate. The control unit 140 includes, for example, an arithmetic device, a control device, a storage device, an input device, and an output device, and is a computer that executes software.

(Detection operation of SPFS device)
Next, the detection operation (detection method according to the present embodiment) 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. 5A and 5B are schematic diagrams for explaining a part of the detection process of the SPFS device 100. FIG. Here, an example in which a primary antibody is used as the capturing body 16 and the target substance is labeled with the fluorescent substance by binding the secondary antibody labeled with the fluorescent substance to the target substance captured by the primary antibody. explain

First, preparation for detection is performed (step S110). Specifically, two

detection chips

10 and 10 ′ are prepared, and the two

detection chips

10 and 10 ′ are respectively installed in the chip holder 132 of the SPFS apparatus 100. In addition, when a humectant is present on the metal film 12 of the

detection chip

10, 10 ′, the humectant is removed by washing the metal film 12 so that the primary antibody can appropriately capture the substance to be detected. .

Next, the substance to be detected in the specimen is bound to the primary antibody (primary reaction; step S120). Specifically, in each detection chip 10, 10 ', a specimen is provided on the metal film 12, and the specimen and the primary antibody are brought into contact with each other. When a substance to be detected is present in the sample, at least a part of the substance to be detected binds to the primary antibody.

Next, the detection target substance bound to the primary antibody is labeled with a fluorescent substance (secondary reaction; step S130). Specifically, a fluorescent labeling solution containing a secondary antibody labeled with a fluorescent substance is provided on the metal film 12, and the substance to be detected bonded 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. As will be described later, the SPFS device 100 according to the present embodiment can detect the detection target substance without removing the free secondary antibody. However, after labeling with a fluorescent substance, it is preferable to wash the metal film 12 with a buffer or the like to remove free secondary antibodies and the like.

Note that the order of the primary reaction and the secondary reaction is not limited to this. For example, a liquid containing these complexes may be provided on the metal film 12 after the substance to be detected is bound to the secondary antibody. Further, the specimen and the fluorescent labeling solution may be provided on the metal film 12 at the same time.

Next, the first light (for example, p-polarized light) included in the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 while irradiating the reaction field of one detection chip 10 with the excitation light α. ) Is detected (step S140). Specifically, the control unit 140 operates the excitation light irradiation unit 110 to irradiate the diffraction grating 13 of the detection chip 10 with the excitation light α so as to generate SPR, and to detect the detection value A of the light receiving sensor 123. Record. At this time, a liquid (for example, a buffer solution or a fluorescent labeling solution) is present on the reaction field of the detection chip 10 at the first depth h1. 5A, the control unit 140 operates the rotation angle adjustment unit 122 to adjust the rotation angle of the polarizer 121 so that only the first light included in the fluorescence β can be transmitted. .

Next, second light (for example, s-polarized light) included in the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 while irradiating the reaction field of the same detection chip 10 with the excitation light α. Is detected (step S150). Specifically, the control unit 140 operates the excitation light irradiation unit 110 to irradiate the diffraction light 13 of the detection chip 10 with the excitation light α and generate the detection value B of the light receiving sensor 123. Record. At this time, as shown in FIG. 5B, the control unit 140 operates the rotation angle adjustment unit 122 to adjust the rotation angle of the polarizer 121 so that only the second light included in the fluorescence β can be transmitted. To do.

Next, the reaction field to be detected is switched (step S160). Specifically, the control unit 140 operates the transfer stage 131 to move the detection chips 10 and 10 ′. Thereby, the excitation light irradiation unit 110 can irradiate the reaction field of the other detection chip 10 ′ with the excitation light α.

Next, while irradiating the reaction field of the other detection chip 10 ′ with the excitation light α, the first light contained in the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 ′ is detected (step) S170). Specifically, the control unit 140 operates the excitation light irradiation unit 110 to irradiate the diffraction light 13 of the detection chip 10 ′ with the excitation light α so as to generate SPR, and also detects the detection value C of the light receiving sensor 123. Record. At this time, the same liquid as the liquid existing on the reaction field of the detection chip 10 is accommodated at the second depth h2 on the reaction field of the detection chip 10 '. 5A, the control unit 140 operates the rotation angle adjustment unit 122 to adjust the rotation angle of the polarizer 121 so that only the first light included in the fluorescence β can be transmitted. .

Next, the second light contained in the fluorescence β emitted from the fluorescent material on the reaction field of the detection chip 10 ′ is detected while irradiating the reaction field of the same detection chip 10 ′ with the excitation light α (step S180). ). Specifically, the control unit 140 operates the excitation light irradiation unit 110 to irradiate the diffraction light 13 of the detection chip 10 with the excitation light α so as to generate SPR, and to detect the detection value D of the light receiving sensor 123. Record. At this time, as shown in FIG. 5B, the control unit 140 operates the rotation angle adjustment unit 122 to adjust the rotation angle of the polarizer 121 so that only the second light included in the fluorescence β can be transmitted. To do.

Even after the secondary reaction (step S130), the fluorescent labeling solution in the container 15 is replaced with a buffer solution that does not contain a secondary antibody, and the container 15 is washed, so that it binds to the substance to be detected. Part of the secondary antibody is released into the buffer. In the case where no washing is performed after the secondary reaction (step S130), the fluorescent labeling solution is present as it is in the container 15. Therefore, in any case, the detection value A in step S140, the detection value B in step S150, the detection value C in step S170, and the detection value D in step S180 are excited by an enhanced electric field caused by SPR. Component of fluorescent β released from the fluorescent material (fluorescent material that mainly labels the target substance captured by the primary antibody) and light other than the enhanced electric field caused by SPR (excitation light α and external light) ) Excited by the fluorescent substance (mainly a fluorescent substance released in the liquid in the accommodating portion 15).

Finally, the control unit (processing unit) 140 calculates a signal value indicating the presence or amount of the substance to be detected based on the detection value obtained by the fluorescence detection unit 120 in steps S140 to S180 (step S190). Hereinafter, a method for calculating the signal value will be described.

FIG. 6 is a schematic diagram for explaining the principle of detection of a substance to be detected by the SPFS device 100. FIG. 6A shows a state in which liquid exists at the first depth h1 on the reaction field of the diffraction grating 13 (metal film 12) in one detection chip 10, and FIG. 6B shows the other detection chip 10 ′. The liquid is present at the second depth h2 on the reaction field of the diffraction grating 13 (metal film 12) in FIG. Moreover, in FIG. 6A and FIG. 6B, the white star has shown the fluorescent substance.

As shown in FIG. 6A, in the state where the liquid is present at the first depth h1, the fluorescence β emitted from the reaction field includes light generated by the influence of the enhanced electric field caused by SPR (p Polarization component and s-polarization component) and light (p-polarization component and s-polarization component) generated without being affected by the enhanced electric field caused by SPR. That is, the detected value when detecting the fluorescence β is a component by the light (p-polarized component I _p1 and s-polarized component I _s1 ) generated under the influence of the enhanced electric field caused by SPR, and the enhancement caused by SPR. And components generated by the light (p-polarized component I _p2 and s-polarized component I _s2 ) generated without being affected by the electric field. At this time, I _p1 and I _s1 are mainly due to the fluorescence β from the fluorescent substance that labels the substance to be detected captured by the primary antibody, and I _p2 and I _s2 are mainly released to the liquid in the container 15. This is due to the fluorescence β from the fluorescent substance. Therefore, the detection value A of the light receiving sensor 123 in step S140 for detecting only the first light (p-polarized component) is derived from I _p1 and I _p2 , and the step for detecting only the second light (s-polarized component). The detection value B of the light receiving sensor 123 in S150 is derived from I _s1 and I _s2 . That is, the detection values A and B detected by the fluorescence detection unit 120 in the state where the liquid depth on the reaction field is the first depth h1 are expressed by the following equations (1) and (2), respectively. The

In addition, as shown in FIG. 6B, the light generated under the influence of the enhanced electric field due to the SPR is also generated in the fluorescence β emitted from the reaction field in the state where the liquid exists at the second depth h2. (P-polarized component and s-polarized component) and light (p-polarized component and s-polarized component) generated without being affected by the enhanced electric field caused by SPR are included. At this time, the distance from the surface of the diffraction grating 13, which is affected by the enhanced electric field due to SPR, is constant regardless of the depth of the liquid stored in the storage unit 15. For this reason, the magnitudes of I _p1 and I _s1 are the same in the state where the liquid exists at the first depth h1 and the state where the liquid exists at the second depth h2.

On the other hand, when the ratio of the second depth h2 to the first depth h1 is m, there is an m-fold amount of liquid on the reaction field and an m-fold amount of fluorescent material. Become. At this time, compared with the depth of the liquid (several μm to several centimeters), the distance affected by the enhanced electric field due to SPR is 100 nm or less and is negligibly small. For this reason, m is in the liquid accommodated at the second depth h2 with respect to the height of the region not affected by the enhanced electric field due to the SPR in the liquid accommodated at the first depth h1. It can be approximated to be equal to the ratio of the heights of regions not affected by the enhanced electric field due to the SPR. Therefore, the fluorescence β emitted in the state where the liquid exists at the second depth h2 includes I _p2 and I included in the fluorescence β emitted in the state where the liquid exists at the first depth h1. _s2 is included in an amount of m times. The detection values C and D detected by the fluorescence detection unit 120 in the state where the liquid depth on the reaction field is the second depth h2 are expressed by the following equations (3) and (4), respectively.

In GC-SPFS, the fluorescence β emitted from the fluorescent substance that labels the target substance immobilized on the metal film 12 (mainly the target substance captured by the primary antibody) It has been found that the light is p-polarized light with respect to the surface, or light whose polarization angle is close to p-polarized light. On the other hand, it is understood that the fluorescence β emitted from the fluorescent material not fixed on the metal film 12 (mainly the fluorescent material released in the liquid) includes not only p-polarized light but also s-polarized light to some extent. ing. That is, the s-polarized component I _s1 of the fluorescence β generated under the influence of the enhanced electric field due to SPR can be approximated to be almost zero. Therefore, the control unit (processing unit) 140 uses the following values as signal values indicating the presence or amount of the substance to be detected based on the detection values A and C represented by the above formulas (1) and (3), respectively. I _p1 represented by the formula (5) can be calculated.

Furthermore, as necessary, the control unit (processing unit) 140 includes noise values I _p2 , I _s1 , and I that do not indicate the presence or amount of the target substance represented by the following formulas (6) to (8). It is also possible to further calculate _s2 .

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. In the present invention, since the noise can be removed by the above procedure, the measurement of the blank value is not necessarily performed.

However, the blank value may be measured before the secondary reaction (step S130) as necessary. Specifically, blank values A ′ to D ′ are obtained in advance in the same manner as in steps S140 to S180 in the state where the fluorescent substance is not present in the liquid in the storage unit 15. In this case, in step S190, the blank values A ′ to D ′ are subtracted from the detected values A to D, respectively, and then the signal value I _p1 and, if necessary, the noise values I _p2 and I by the above calculation method. _s1 and _Is2 are calculated.

(effect)
As described above, the SPFS device 100 according to the present embodiment removes background noise even if there is an unreacted fluorescent material on the metal film 12, and determines the presence or amount of the detected material. It can be detected accurately. For this reason, the SPFS device 100 according to the present embodiment can detect a substance to be detected with higher sensitivity and easier than the conventional SPFS device.

Further, since the SPFS device 100 according to the present embodiment can remove the noise component contained in the fluorescence β, it is not necessary to remove the free secondary antibody after performing the secondary reaction (step S130). The detection target substance can be detected.

Note that, in this embodiment, the mode in which the first light and the second light are detected at the same time using one polarizer 121 has been described. However, the detection apparatus and the detection method according to this embodiment are described below. However, the present invention is not limited to this embodiment. For example, two polarizers and a light receiving sensor may be used to detect the first light and the second light simultaneously. FIG. 7 is a schematic diagram illustrating a configuration of an SPFS apparatus 100 ′ according to a modification. As shown in FIG. 7, in the SPFS device 100 ′ according to the modification, the rotation angle adjustment unit 122 is unnecessary, and the fluorescence detection unit 120 ′ further includes a half mirror 124 ′, a polarizer 121 ′, and a light receiving sensor 123 ′. Have In this case, one polarizer 121 is adjusted to transmit only the first light, and the other polarizer 121 ′ is adjusted to transmit only the second light. Thereby, half of the fluorescence β emitted from the fluorescent material on the metal film 12 passes through the half mirror 124 ′, and the first light contained in the fluorescence β is detected by the light receiving sensor 123. At the same time, the remaining half of the fluorescence β emitted from the fluorescent material on the metal film 12 is reflected by the half mirror 124 ′, and the second light contained in the fluorescence β is detected by the light receiving sensor 123 ′. Therefore, step S140 and step S150, and step S170 and step S180 of the above embodiment can be performed simultaneously.

In the above-described embodiment, the embodiment using the two detection chips 10, 10 'has been described. However, the detection apparatus and the detection method according to the present invention may use only one detection chip. In this case, the detection chip has a storage section that can store the liquid at the first depth and the second depth when the liquid is stored in the storage section. Examples of the detection chip include a detection chip having a stepped portion or an inclined surface at the bottom of the housing portion. FIG. 8 is a schematic diagram showing the configuration of a detection chip 10 ″ according to a modification. As shown in FIG. 8, the detection chip 10 ″ according to the modification has a step formed on a substrate 11 ″. In addition, the metal film 12 (diffraction grating 13) is disposed on both the upper and lower sides of the step surface. Thus, when the liquid is accommodated in the accommodating portion 15 ″, the depth of the liquid above it Is a first reaction field 17 ″ having a first depth h1 ″ and a second reaction field 18 ″ having a second liquid depth h2 ″ above the first reaction field 17 ″. It is arrange | positioned at the side and the lower stage side, respectively. Other examples of the detection chip include a detection chip that further includes a stepped portion and a lid portion having an inclined surface. In this case, the first reaction field 17 ″ and the second reaction field 18 ″ are disposed on the metal film when the liquid is accommodated in the accommodating part and the lid part is disposed.

Further, in the SPFS device 100 according to the above-described embodiment, the order of the step of detecting the first light (step S140) and the step of detecting the second light (step S150) is not limited to this. Further, the order of the step of detecting the first light (step S170) and the step of detecting the second light (step S180) is not limited to this. That is, the second light may be detected before the step of detecting the first light.

In the present embodiment, the detection apparatus and the detection method using GC-SPFS have been described. However, the detection apparatus and the detection method according to the present embodiment may use PC-SPFS. In this case, the detection chips 10, 10 ′ have a prism made of a dielectric, and the metal film 12 is disposed on the prism. Further, the metal film 12 does not have the diffraction grating 13. The excitation light α is irradiated to the back surface of the metal film 12 corresponding to the reaction field via the prism. Further, in PC-SPFS, the fluorescence β emitted from the fluorescent substance that labels the detection target substance captured by the capturing body 16 includes not only p-polarized light but also s-polarized light to some extent. Therefore, I _s1 derived from light generated under the influence of the enhanced electric field cannot be approximated to be zero. Therefore, the control unit (processing unit) detects the detection value A detected by the fluorescence detection unit as the first light in the state where the liquid depth on the reaction field is the first depth h1, and the liquid level on the reaction field. The detection value B detected as the second light by the fluorescence detector in the state where the depth is the first depth h1, and the fluorescence detector in the state where the depth of the liquid on the reaction field is the second depth h2. Based on the detection value C detected as the first light and the detection value D detected as the second light by the fluorescence detection unit in the state where the depth of the liquid on the reaction field is the second depth h1, A signal value I _p1 + I _s1 indicating the presence or amount of the substance to be detected is calculated by the following formula (9). Furthermore, the control unit (processing unit) is not affected by the enhanced electric field caused by the SPR sound included in the first light, which is represented by the following formula (10) and formula (11) as necessary. Further calculating a noise value I _p2 derived from the generated light and a noise value I _s2 derived from the light that is included in the second light and is not affected by the enhanced electric field due to the SPR Also good.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]

[Embodiment 2]
In the second embodiment, a GC-SPFS apparatus that detects only linearly polarized light (for example, p-polarized light) included in fluorescence emitted from a fluorescent material present on a metal film will be described. Although the SPFS device according to the second embodiment includes a polarizer, it is not necessary to switch the rotation angle of the polarizer.

(Configuration of SPFS device and detection chip)
FIG. 9 is a schematic diagram showing a configuration of the SPFS apparatus 200 according to the present embodiment. The SPFS device 200 includes an excitation light irradiation unit 110, a fluorescence detection unit 220, a transport unit 130, and a control unit 240. In the SPFS device 200, only the fluorescence detection unit 220 and the control unit 240 are different from the SPFS device 100 according to the first embodiment. Therefore, the same components as those of the SPFS device 100 described in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

The fluorescence detection unit 220 is arranged so as to pass through the intersection of the optical axis of the excitation light α and the metal film 12 with respect to the excitation light irradiation unit 110 and sandwich the normal to the surface of the metal film 12. The fluorescence detection unit 220 detects the fluorescence β emitted from the fluorescent material on the metal film 12 (diffraction grating 13) at least twice. More specifically, the fluorescence detection unit 220 is released from the fluorescent material on the reaction field of the detection chip 10 in a state where the depth of the liquid on the reaction field of one detection chip 10 is the first depth h1. Fluorescence β is detected at least once and released from the fluorescent material on the reaction field of the detection chip 10 ′ with the liquid depth on the reaction field of the other detection chip 10 ′ being the second depth h2. Fluorescence β is detected at least once.

The fluorescence detection unit 220 includes a polarizer 221 and a light receiving sensor 123. The fluorescence detection unit 220 may further include a condenser lens group, an aperture stop, a fluorescence filter, and the like.

The polarizer 221 is disposed on the optical path of the fluorescence β between the detection chips 10, 10 ′ and the light receiving sensor 123. The polarizer 221 is linearly polarized light having an angle of the oscillation direction of the electric field within a range of 0 ± 30 ° from the fluorescence β to a plane (xz plane) including the normal to the surface of the metal film 12 and the optical axis of the excitation light α. Take out the light. Preferably, the polarizer 221 extracts, from the fluorescence β, p-polarized light whose angle in the vibration direction of the electric field with respect to the plane (xz plane) is 0 °. The rotation angle of the polarizer 221 is adjusted (or fixed) so as to transmit only the linearly polarized light. The type of the polarizer 221 is not particularly limited as long as linearly polarized light having a predetermined polarization direction can be extracted. An example of the type of the polarizer 221 is the same as the polarizer 121 of the first embodiment, for example.

The control unit 240 operates the excitation light irradiation unit 110 (the light source 111 and the first angle adjustment unit), the fluorescence detection unit 220 (the light receiving sensor 123 and the second angle adjustment unit), and the transport unit 130 (the transport stage 131). Control. The control unit 240 also functions as a processing unit that processes an output signal (detection result) from the fluorescence detection unit 220. Specifically, based on two or more detection values detected by the fluorescence detection unit 220 (light receiving sensor 123), the processing unit obtains a signal value indicating the presence or amount of the target substance, and a noise value as necessary. calculate. The control unit 240 is a computer that includes, for example, an arithmetic device, a control device, a storage device, an input device, and an output device, and executes software.

(Detection operation of SPFS device)
Next, the detection operation (detection method according to the present embodiment) of the SPFS device 200 will be described. FIG. 10 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 200.

First, similarly to step S110, step S120, and step S130 of the first embodiment, a step of preparing for detection (step S210), a primary reaction (step S220), and a secondary reaction (step S230) are performed. Thereby, the detection chips 10 and 10 ′ in a state where the target substance labeled with the fluorescent substance is fixed to the reaction field can be arranged in the chip holder 132 of the SPFS device 200.

Next, linearly polarized light (for example, p-polarized light) included in the fluorescence β emitted from the fluorescent material on the reaction field of the detection chip 10 while irradiating the reaction field of one detection chip 10 with the excitation light α. ) Is detected (step S240). Specifically, the control unit 240 operates the excitation light irradiation unit 110 to irradiate the diffraction grating 13 of the detection chip 10 with the excitation light α so as to generate SPR, and to detect the detection value A of the light receiving sensor 123. Record. At this time, on the reaction field of the detection chip 10, a liquid (for example, a buffer solution or a fluorescent labeling solution) is accommodated at the first depth h1.

Next, the reaction field to be detected is switched (step S250). Specifically, the control unit 240 operates the transfer stage 131 to move the detection chips 10 and 10 ′. Thereby, the excitation light irradiation unit 110 can irradiate the reaction field of the other detection chip 10 ′ with the excitation light α.

Next, while irradiating the reaction field of the other detection chip 10 ′ with the excitation light α, linearly polarized light contained in the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 ′ is detected (step) S260). Specifically, the control unit 240 operates the excitation light irradiation unit 110 to irradiate the diffraction light 13 of the detection chip 10 ′ with the excitation light α so as to generate SPR, and detects the detection value B of the light receiving sensor 123. Record. At this time, the same liquid as the liquid on the reaction field of the detection chip 10 is accommodated on the reaction field of the detection chip 10 'at the second depth h2. Further, the controller 240 does not need to adjust the rotation angle of the polarizer 221.

Finally, the control unit (processing unit) 240 calculates a signal value indicating the presence or amount of the detection target substance based on the detection value obtained by the fluorescence detection unit 220 (step S270). Hereinafter, a method for calculating the signal value will be described.

As described in the first embodiment, the fluorescence β emitted from the reaction field includes light (p-polarized component and s-polarized component) generated under the influence of the enhanced electric field due to SPR, and SPR. And light (p-polarized component and s-polarized component) generated without being affected by the enhanced electric field. In the present embodiment, in steps S240 and S260, only the above-described linearly polarized light included in the fluorescence β is detected, so that the detection values A and B of the light receiving sensor 123 are derived from _Ip1 and _Ip2 . Therefore, the detection values A and B are expressed by the following expressions (12) and (13).

Therefore, in the present embodiment, the control unit (processing unit) 240 indicates the presence or amount of the substance to be detected based on the detection values A and B represented by the above formulas (12) and (13). As a signal value, I _p1 represented by the following formula (14) is calculated.

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.

(effect)
In the detection apparatus and detection method according to Embodiment 2, the same effects as those of the detection apparatus and detection method according to Embodiment 1 can be obtained, and furthermore, the rotation angle of the polarizer 221 does not need to be adjusted. The configuration of the detection device and the detection method are simplified.

[Embodiment 3]
In the third embodiment, a GC-SPFS apparatus that detects fluorescence (including p-polarized light and s-polarized light) contained in fluorescence emitted from a fluorescent substance present on a metal film will be described. The SPFS device according to this embodiment does not have a polarizer.

(Configuration of SPFS device and detection chip)
FIG. 11 is a schematic diagram showing a configuration of the SPFS apparatus 300 according to the present embodiment. The SPFS apparatus 300 includes an excitation light irradiation unit 110, a fluorescence detection unit 320, a transport unit 130, and a control unit 340. In the SPFS apparatus 300, only the fluorescence detection unit 320 and the control unit 340 are different from the SPFS apparatus 100 according to the first embodiment. Therefore, the same components as those of the SPFS device 100 described in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

The fluorescence detection unit 320 is disposed so as to pass through the intersection of the optical axis of the excitation light α and the metal film 12 with respect to the excitation light irradiation unit 110 and sandwich the normal to the surface of the metal film 12. The fluorescence detection unit 320 detects the fluorescence β emitted from the fluorescent material on the metal film 12 (diffraction grating 13) at least twice. More specifically, the fluorescence detection unit 320 is released from the fluorescent material on the reaction field of the detection chip 10 with the liquid depth on the reaction field of one detection chip 10 being the first depth h1. Fluorescence β is detected at least once, and the liquid on the reaction field of the other detection chip 10 ′ is released from the fluorescent material on the reaction field of the detection chip 10 ′ with the second depth h2. Fluorescent β is detected at least once.

The fluorescence detection unit 320 has at least a light receiving sensor 123. The fluorescence detection unit 320 may further include a condenser lens group, an aperture stop, a fluorescence filter, and the like.

The control unit 340 operates the excitation light irradiation unit 110 (the light source 11 and the first angle adjustment unit), the fluorescence detection unit 320 (the light receiving sensor 123 and the second angle adjustment unit), and the conveyance unit 130 (the conveyance stage 131). Control. The control unit 340 also functions as a processing unit that processes an output signal (detection result) from the fluorescence detection unit 320. Specifically, based on two or more detection values detected by the fluorescence detection unit 320 (light receiving sensor 123), the processing unit obtains a signal value indicating the presence or amount of the target substance, and a noise value as necessary. calculate. The control unit 340 is a computer that includes, for example, an arithmetic device, a control device, a storage device, an input device, and an output device, and executes software.

(Detection operation of SPFS device)
Next, the detection operation (detection method according to the present embodiment) of the SPFS device 300 will be described. FIG. 12 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 300.

First, similarly to step S110, step S120, and step S130 of the first embodiment, a step of preparing for detection (step S310), a primary reaction (step S320), and a secondary reaction (step S330) are performed. As a result, the detection chips 10 and 10 ′ in a state where the detection target substance labeled with the fluorescent substance is fixed to the reaction field can be arranged in the chip holder 132 of the SPFS apparatus 300.

Next, while irradiating the reaction field of one detection chip 10 with the excitation light α, the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 is detected (step S340). Specifically, the control unit 340 operates the excitation light irradiation unit 110 to irradiate the diffraction grating 13 of the detection chip 10 with the excitation light α so as to generate SPR, and also detects the detection value A of the light receiving sensor 123. Record. At this time, on the reaction field of the detection chip 10, a liquid (for example, a buffer solution or a fluorescent labeling solution) is accommodated at the first depth h1.

Next, the reaction field to be detected is switched (step S350). Specifically, the control unit 340 operates the transfer stage 131 to move the detection chips 10 and 10 ′. Thereby, the excitation light irradiation unit 110 can irradiate the reaction field of the other detection chip 10 ′ with the excitation light α.

Next, while irradiating the reaction field of the other detection chip 10 ′ with the excitation light α, the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 ′ is detected (step S 360). Specifically, the control unit 340 operates the excitation light irradiation unit 110 to irradiate the diffraction light 13 of the detection chip 10 ′ with the excitation light α so that SPR is generated, and detects the detection value B of the light receiving sensor 123. Record. At this time, the same liquid as the liquid on the reaction field of the detection chip 10 is accommodated on the reaction field of the detection chip 10 'at the second depth h2.

Finally, the control unit (processing unit) 340 calculates a signal value indicating the presence or amount of the substance to be detected based on the detection value obtained by the fluorescence detection unit 320 (step S370). Hereinafter, a method for calculating the signal value will be described.

As described in the first embodiment, the fluorescence β emitted from the reaction field includes light (p-polarized component and s-polarized component) generated under the influence of the enhanced electric field due to SPR, and SPR. And light (p-polarized component and s-polarized component) generated without being affected by the enhanced electric field. In the present embodiment, in steps S340 and S360, the detection values A and B of the light receiving sensor 123 are detected as I _p1 , I _p2 , I _s1 and I in order to detect the fluorescence β including the p-polarized component and the s-polarized component. Derived from _s2 . Therefore, the detection values A and B are expressed by the following equations (15) and (16).

Therefore, the control unit (processing unit) 340 uses the following equation (5) as a signal value indicating the presence of the substance to be detected based on the detection values A and B represented by the equations (15) and (16). _Ip1 represented by 17) is calculated. At this time, the control unit (processing unit) 340 performs calculation with _Is1 being 0.

(effect)
The detection device and the detection method according to Embodiment 3 can obtain the same effects as those of the detection device and detection method according to Embodiment 1, and do not have a polarizer. Configuration and detection methods are simplified.

In the first to third embodiments, the

control units

140, 240, and 340 operate the transfer stage 131 to switch the reaction field that is irradiated with the excitation light α. Is not limited to this. For example, the

control units

140, 240, and 340 move the excitation light irradiation unit 110 and the

fluorescence detection units

120, 220, and 320 to the detection chips 10 and 10 ′ to switch the reaction field irradiated with the excitation light α. May be.

Also, the detection chip 10 ″ according to the modification can be used in the detection apparatus and the detection method according to the second and third embodiments.

[Embodiment 4]
In the fourth embodiment, GC-SPFS that detects first light (for example, p-polarized light) and second light (for example, s-polarized light) included in the fluorescence emitted from the fluorescent material present on the metal film, respectively. The apparatus will be described. The SPFS device according to Embodiment 4 includes a liquid amount adjusting unit for changing the amount of liquid stored in the storage unit.

(Configuration of SPFS device and detection chip)
FIG. 13 is a schematic diagram showing a configuration of the SPFS apparatus 400 according to the present embodiment. The SPFS device 400 includes an excitation light irradiation unit 110, a fluorescence detection unit 120, a transport unit 130, a control unit 440, and a liquid amount adjustment unit 450. Only the control unit 440 and the liquid amount adjustment unit 450 are different from the SPFS apparatus 100 according to the first embodiment. Therefore, the same components as those of the SPFS device 100 described in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

Also, as shown in FIG. 13, only one detection chip 10 is used in the SPFS device 400 according to the present embodiment. Since the configuration of the detection chip 10 is the same as that of the detection chip 10 used in the first embodiment, the description thereof is omitted.

The control unit 440 includes an excitation light irradiation unit 110 (light source 111 and first angle adjustment unit), a fluorescence detection unit 120 (light receiving sensor 123 and second angle adjustment unit), a conveyance unit 130 (conveyance stage 131), and will be described later. It controls the operation of the liquid amount adjustment unit 450 (liquid feed pump drive mechanism 454). The control unit 440 also functions as a processing unit that processes an output signal (detection result) from the fluorescence detection unit 120. Specifically, based on two or more detection values detected by the fluorescence detection unit 120 (light receiving sensor 123), the processing unit calculates a signal value indicating the presence or amount of the substance to be detected, and a noise value as necessary. calculate. The control unit 440 is a computer that includes, for example, an arithmetic device, a control device, a storage device, an input device, and an output device, and executes software.

The liquid amount adjustment unit 450 adjusts the amount of liquid in the storage unit 15 of the detection chip 10 held by the chip holder 132. For example, the liquid amount adjustment unit 450 may increase or decrease the amount of liquid in the storage unit 15. The liquid amount adjusting unit 450 detects when the fluorescence detection unit 120 detects the fluorescence β in a state where the liquid depth on the reaction field is the first depth h1, and the fluorescence detection unit 120 detects the liquid depth on the reaction field. The amount of the liquid in the container 15 is changed between the time when the fluorescence β is detected in the state of the second depth h2. The liquid amount adjustment unit 450 includes, for example, a syringe pump 451 and a liquid feed pump drive mechanism 454.

The syringe pump 451 includes a syringe 452 and a plunger 453 capable of reciprocating within the syringe 452. By the reciprocating motion of the plunger 453, the liquid is sucked and discharged quantitatively.

The liquid feed pump drive mechanism 454 includes a drive device for the plunger 453 and a moving device for the syringe pump 451. The drive device of the syringe pump 451 is a device for reciprocating the plunger 453, and includes, for example, a stepping motor. Since the drive device including the stepping motor can manage the liquid feeding amount and the liquid feeding speed of the syringe pump 451, the liquid amount in the storage unit 15 of the detection chip 10 can be managed. For example, the moving device of the syringe pump 451 freely moves the syringe pump 451 in two directions, ie, an axial direction (for example, a vertical direction) of the syringe 452 and a direction crossing the axial direction (for example, a horizontal direction). The moving device of the syringe pump 451 is configured by, for example, a robot arm, a two-axis stage, or a turntable that can move up and down.

(Detection operation of SPFS device)
Next, the detection operation (detection method according to the present embodiment) of the SPFS device 400 will be described. FIG. 14 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 400.

First, similarly to step S110, step S120, and step S130 of the first embodiment, a step of preparing for detection (step S410), a primary reaction (step S420), and a secondary reaction (step S430) are performed. Thereby, the detection chip 10 in a state where the detection target substance labeled with the fluorescent substance is fixed to the reaction field can be arranged in the chip holder 132 of the SPFS device 400.

Next, the first light (for example, p-polarized light) included in the fluorescence β emitted from the fluorescent substance on the reaction field is detected while irradiating the reaction field of the detection chip 10 with the excitation light α (step S440). ). Specifically, the control unit 440 operates the excitation light irradiation unit 110 to irradiate the diffraction grating 13 of the detection chip 10 with the excitation light α so as to generate SPR, and also detects the detection value A of the light receiving sensor 123. Record. At this time, a liquid (for example, a buffer solution or a fluorescent labeling solution) is accommodated on the reaction field of the detection chip 10 at the first depth h1. 5A, the control unit 440 operates the rotation angle adjustment unit 122 to adjust the rotation angle of the polarizer 121 so that only the first light included in the fluorescence β can be transmitted. .

Next, the second light (for example, s-polarized light) contained in the fluorescence β emitted from the fluorescent material on the reaction field is detected while irradiating the reaction field of the detection chip 10 with the excitation light α (step S450). ). Specifically, the control unit 440 operates the excitation light irradiation unit 110 to irradiate the diffraction light 13 of the detection chip 10 with the excitation light α so as to generate SPR, and to detect the detection value B of the light receiving sensor 123. Record. At this time, as illustrated in FIG. 5B, the control unit 440 operates the rotation angle adjustment unit 122 to adjust the rotation angle of the polarizer 121 so that only the second light included in the fluorescence β can be transmitted. To do.

Next, a liquid is introduced into the container (Step S460). Specifically, the control unit 440 operates the liquid amount adjustment unit 450 to introduce the liquid into the reaction field of the detection chip 10. At this time, the same liquid as the liquid in the container 15 is supplied. Thereby, the depth of the liquid existing on the reaction field in the accommodating portion 15 can be changed from the first depth h1 to the second depth h2.

Next, the first light contained in the fluorescence β emitted from the fluorescent substance on the reaction field is detected while irradiating the reaction field of the detection chip 10 with the excitation light α (step S470). Specifically, the control unit 440 operates the excitation light irradiation unit 110 to irradiate the diffraction light 13 of the detection chip 10 with the excitation light α so as to generate SPR, and to detect the detection value C of the light receiving sensor 123. Record. At this time, a liquid (for example, a buffer solution) is accommodated at the second depth h2 on the reaction field of the detection chip 10. 5A, the control unit 440 operates the rotation angle adjustment unit 122 to adjust the rotation angle of the polarizer 121 so that only the first light included in the fluorescence β can be transmitted. .

Next, the second light contained in the fluorescence β emitted from the fluorescent substance on the reaction field is detected while irradiating the reaction field of the detection chip 10 with the excitation light α (step S480). Specifically, the control unit 440 operates the excitation light irradiation unit 110 to irradiate the diffraction light 13 of the detection chip 10 with the excitation light α so as to generate SPR, and to detect the detection value D of the light receiving sensor 123. Record. At this time, as illustrated in FIG. 5B, the control unit 440 operates the rotation angle adjustment unit 122 to adjust the rotation angle of the polarizer 121 so that only the second light included in the fluorescence β can be transmitted. To do.

Finally, the control unit (processing unit) 440 calculates a signal value indicating the presence or amount of the detection target substance based on the detection value obtained by the fluorescence detection unit 120 (step S490). Hereinafter, a method for calculating the signal value will be described.

As described in the first embodiment, the fluorescence β emitted from the reaction field includes light (p-polarized component and s-polarized component) generated under the influence of the enhanced electric field due to SPR, and SPR. And light (p-polarized component and s-polarized component) generated without being affected by the enhanced electric field. In the present embodiment, in steps S440 and S470, only the p-polarized component contained in the fluorescence β is detected, so that the detection values A and C of the light receiving sensor 123 are derived from I _p1 and I _p2 . Further, in steps S450 and S480, only the s-polarized component contained in the fluorescence β is detected, so that the detection values C and D of the light receiving sensor 123 are derived from I _s1 and I _s2 . Therefore, the detection values A to D are expressed by the following equations (18) to (21).

Therefore, the control unit (processing unit) 440 uses the following equation (1) as a signal value indicating the presence of the substance to be detected based on the detection values A and C represented by the equations (18) and (20). _Ip1 represented by 22) is calculated. The control unit (processing unit) 440 calculates the noise values I _p2 , I _s1 , and I _s2 represented by the above formulas (6) to (8) as necessary, as in the first embodiment.

(effect)
In the detection device and the detection method according to the fourth embodiment, the same effects as those of the detection device and the detection method according to the first embodiment can be obtained. Configuration and detection methods are simplified.

In the fourth embodiment, the case where the liquid is introduced into the storage unit 15 in step S460 has been described. However, in the detection device and the detection method according to the present invention, the liquid may be reduced from the storage unit. Also in this case, the depth of the liquid on the reaction field in the accommodating portion can be changed from the first depth to the second depth.

In each of the above embodiments, the example in which the excitation light α is irradiated onto the detection chips 10 and 10 ′ from the metal film 12 side has been described. However, the

detection chip

10 and 10 ′ is irradiated with the excitation light α from the substrate 11 side. May be.

In the second to fourth embodiments, the detection apparatus and the detection method using GC-SPFS have been described. However, the detection apparatus and the detection method according to the second to fourth embodiments use PC-SPFS. Also good. In this case, the detection chips 10 and 10 ′ have a prism made of a dielectric, and the metal film 12 is disposed on the prism. Further, the metal film 12 does not have the diffraction grating 13. The excitation light α is irradiated to the back surface of the metal film 12 corresponding to the reaction field via the prism.

Furthermore, in the

SPFS devices

100, 200, 300, and 400 according to the above-described embodiments, noise components can be removed from the detected values, and thus unreacted fluorescent substances present in the storage unit 15 are removed by washing. There is no need. For this reason, the detection apparatus according to each of the above embodiments can be used for real-time measurement of a substance to be detected. In this case, the detection apparatus continuously irradiates the metal film (diffraction grating) with excitation light in a state where the liquid depth on the reaction field is the first depth, and is included in the fluorescence emitted from the fluorescent material. Continuously detect linearly polarized light. In parallel with this, the detection apparatus continuously irradiates the metal film (diffraction grating) with excitation light in a state where the liquid depth on the reaction field is the second depth, and the fluorescence emitted from the fluorescent material. The linearly polarized light contained in is continuously detected. Here, “continuation” includes not only continuous operation but also intermittent operation. Thereby, the detection apparatus and the detection method according to the present embodiment can detect a substance to be detected accurately, with time, and easily.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples. In this example, the signal value and the noise value indicating the presence and amount of the substance to be detected were calculated using the SPFS apparatus according to the first embodiment.

1. Preparation of detection chip Two detection chips were prepared in which anti-α-fetoprotein (AFP) antibody was immobilized on a diffraction grating of a metal film via carboxymethyldextran (CMD). The two prepared detection chips were respectively installed in the chip holder of the SPFS apparatus. In the present embodiment, the liquid is accommodated in the accommodating portion of one detection chip so that the depth is 50 μm, and the liquid is accommodated in the accommodating portion of the other detection chip so that the depth is 100 μm. The

2.1 Primary Reaction A specimen containing AFP was provided in the accommodating part of each detection chip, and left for 25 minutes to perform the primary reaction.

3. Measurement of optical blank value After the primary reaction, the specimen in the container was removed, and the container was washed with a phosphate buffer. Thereafter, the rotation angle adjustment unit is operated to adjust the rotation angle of the polarizing plate so that only p-polarized light can be transmitted, and in a state where a buffer solution exists at a depth of 50 μm in the accommodation unit of one detection chip, The diffraction grating was irradiated with excitation light having a wavelength of 637 nm. At the same time, p-polarized light included in the light emitted from the inside of the accommodating portion was detected, and an optical blank value oB _p for p-polarized light was obtained. The oB _p was 150 counts.

Thereafter, the rotation angle adjustment unit is operated to adjust the rotation angle of the polarizing plate so that only s-polarized light can be transmitted. In the state where the same buffer is present at a depth of 50 μm in the same detection chip housing, The diffraction grating was irradiated with excitation light having a wavelength of 637 nm. At the same time, s-polarized light contained in the fluorescence emitted from the inside of the housing was detected, and an optical blank value oB _s for s-polarized light was obtained. The oB _s was 100 count.

For the other detection chip, the optical blank value was measured in the same manner as in the above method in a state where the liquid was present at a depth of 100 nm in the accommodating portion. The optical blank values for p-polarized light and s-polarized light were almost the same as the above values.

4. Secondary reaction A solution containing an anti-AFP antibody labeled with Alexa-Fluor (registered trademark) as a fluorescent dye (hereinafter also referred to as a fluorescent labeling solution) is provided in the receiving part of each detection chip and allowed to stand for 5 minutes. Then, a secondary reaction was performed.

5. Fluorescence detection After the secondary reaction, in the same manner as the measurement of the optical blank value, the diffraction grating is irradiated with excitation light in the state where the fluorescent labeling liquid exists at a depth of 50 μm in the receiving portion of one detection chip, and is emitted. P-polarized light and s-polarized light contained in the fluorescence detected. In addition, in the state where the fluorescent labeling liquid is present at a depth of 100 μm in the housing part of the other detection chip, the diffraction grating is irradiated with excitation light, and p-polarized light and s-polarized light contained in the emitted fluorescence are emitted. Detected.

6). Processing of Detection Value The detection value A when detecting p-polarized light in a state where the depth of the liquid is 50 μm was 2080 count, and the detection value B when detecting s-polarized light was 1260 count. The detection value C when detecting p-polarized light in a state where the depth of the liquid was 100 μm was 3000 counts, and the detection value D when detecting s-polarized light was 2570 counts. The ratio m of the depth of the liquid accommodated in the accommodating part of the other detection chip to the depth of the liquid accommodated in the accommodating part of one detection chip is 2. Using the control unit (processing unit), a signal value I _p1 indicating the amount of the substance to be detected was calculated from the detection values A and C as represented by the following formula (23).

In addition, using the control unit (processing unit), as represented by the following equations (24) to (26) from the above detection values, the influence of the enhanced electric field included in the p-polarized light and caused by SPR The noise value I _p2 derived from the light generated without being received and the noise value I _s1 derived from the light included in the s-polarized light and generated by the influence of the enhanced electric field caused by the SPR, and s A noise value _Is2 derived from the light included in the polarized light and generated under the influence of the enhanced electric field caused by the SPR was calculated.

From the above results, it was found that the detected substance can be detected by removing the background noise without removing the unreacted fluorescent substance by washing. Further, among the signal values indicating the amount of the substance to be detected, it was confirmed that the signal component due to the s-polarized light was almost 0 and was sufficiently small. Furthermore, the noise value derived from the light generated without being influenced by the enhanced electric field caused by SPR could be calculated.

This application claims priority based on Japanese Patent Application No. 2014-249038 filed on Dec. 9, 2014. The contents described in the application specification and the drawings are all incorporated herein.

The detection apparatus and the detection method according to the present invention can measure a substance to be detected with high reliability, and are useful for clinical examinations, for example.

In addition, the detection apparatus and the detection method according to the present invention can detect a substance to be detected with high reliability without cleaning the metal film surface after providing a fluorescent labeling solution or the like. Therefore, not only can the detection time be shortened, but it is also expected to contribute to the development, spread and development of a quantitative immunoassay device that can be miniaturized and a very simple quantitative immunoassay system.

10, 10 ′, 10 ″

detection chip

11, 11 ″ substrate 12 metal film 13 diffraction grating 14

frame

15, 15 ″ receiving portion 16 capture body 17 ″ first reaction field 18 ″

second reaction field

100, 100 ′ , 200, 300, 400 Surface plasmon enhanced fluorescence measuring device (SPFS device)
DESCRIPTION OF SYMBOLS 110 Excitation light irradiation part 111 Light source 120,120 ', 220,320 Fluorescence detection part 121,121', 221 Polarizer 122 Rotation angle adjustment part 123,123 ', Light reception sensor 124' Half mirror 130 Conveyance part 131 Conveyance stage 132

Chip Holder

140, 240, 340, 440 Control unit (processing unit)
450 Liquid volume adjustment unit 451 Syringe pump 452 Syringe 453 Plunger 454 Liquid feeding pump drive mechanism h1, h1 "first depth h2, h2" second depth α excitation light β fluorescence γ reflected light

Claims

A detection device for detecting a substance to be detected using surface plasmon resonance,
A storage unit configured to store a liquid; and a metal film including a reaction field that is disposed at the bottom of the storage unit and to which a detection target substance labeled with a fluorescent substance is directly or indirectly fixed. A holder for holding the detection chip;
An excitation light irradiation unit that irradiates excitation light on the metal film of the detection chip held by the holder so as to generate surface plasmon resonance;
When the excitation light irradiation unit irradiates the metal film with excitation light in a state where the liquid exists in the storage unit, the fluorescence emitted from the fluorescent substance existing on the metal film is detected at least twice. A fluorescence detector;
A processing unit that calculates a signal value indicating the presence or amount of the substance to be detected based on two or more detection values detected by the fluorescence detection unit;
Have
The fluorescence detection unit detects fluorescence at least once in a state where the depth of the liquid on the reaction field is the first depth, and the depth of the liquid on the reaction field is the first depth. Detecting fluorescence at least once at a second depth different from
Detection device.
A liquid amount changing unit that changes the amount of the liquid in the storage unit of the detection chip held by the holder;
The liquid amount changing unit is configured such that when the fluorescence detection unit detects fluorescence in a state where the depth of the liquid on the reaction field is the first depth, the fluorescence detection unit detects the fluorescence on the reaction field. The amount of the liquid in the container is changed between when the fluorescence is detected in a state where the depth of the liquid is the second depth,
The detection device according to claim 1.
The holder holds the two detection chips,
One of the detection chips has a first reaction field in which when the liquid is stored in the storage section, the depth of the liquid on the storage chip becomes the first depth,
The other detection chip has a second reaction field in which when the liquid is stored in the storage portion, the depth of the liquid on the storage chip becomes the second depth.
The detection device according to claim 1.
The metal film of the detection chip has a diffraction grating at a position corresponding to the reaction field,
The excitation light irradiation unit irradiates the diffraction grating with excitation light,
The processing unit includes a detection value A detected by the fluorescence detection unit when the depth of the liquid on the reaction field is the first depth, and the depth of the liquid on the reaction field is the first depth. Based on the detection value B detected by the fluorescence detection unit at a depth of 2, a signal value I p1 indicating the presence or amount of the substance to be detected is calculated by the following equation (1).
The detection device according to any one of claims 1 to 3.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]
The angle of the vibration direction of the electric field with respect to a plane including the normal to the surface of the metal film and the optical axis of the excitation light irradiated by the excitation light irradiation unit is 0 ± 30 ° from the fluorescence emitted from the fluorescent material. A polarizer that takes out the first light in the first light and the second light in the range of 90 ± 30 ° of the vibration direction of the electric field with respect to the plane at the same time or different times,
The metal film of the detection chip has a diffraction grating at a position corresponding to the reaction field,
The excitation light irradiation unit irradiates the diffraction grating with excitation light,
The fluorescence detection unit detects the first light and the second light,
The processing unit includes a detection value A detected as the first light by the fluorescence detection unit in a state where the depth of the liquid on the reaction field is the first depth, and the liquid on the reaction field. Based on the detection value C detected by the fluorescence detection unit as the first light in the state where the depth of the second depth is the second depth, the presence or amount of the substance to be detected is calculated by the following equation (2): A signal value I p1 is calculated,
The detection device according to any one of claims 1 to 3.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]
The processing unit includes the detection value A, the detection value B detected by the fluorescence detection unit as the second light when the depth of the liquid on the reaction field is the first depth, Based on the detection value C and the detection value D detected as the second light by the fluorescence detection unit when the depth of the liquid on the reaction field is the second depth, the following formula According to (3) to (5), the noise value I p2 derived from the light included in the first light and generated without being influenced by the enhanced electric field due to the surface plasmon resonance, and the second The noise value I s1 derived from the light generated by the influence of the enhanced electric field caused by the surface plasmon resonance and the enhanced electric field contained in the second light and caused by the surface plasmon resonance further to calculate a noise value I s2 derived from the effect on the light produced without being The detection device of claim 5.
The angle of the vibration direction of the electric field with respect to a plane including the normal to the surface of the metal film and the optical axis of the excitation light irradiated by the excitation light irradiation unit is 0 ± 30 ° from the fluorescence emitted from the fluorescent material. A polarizer that takes out the first light in the first light and the second light in the range of 90 ± 30 ° of the vibration direction of the electric field with respect to the plane at the same time or different times,
The detection chip includes a prism made of a dielectric, and the metal film disposed on the prism,
The excitation light irradiation unit irradiates the back surface of the metal film corresponding to the reaction field via the prism with excitation light,
The fluorescence detection unit detects the first light and the second light,
The processing unit includes a detection value A detected as the first light by the fluorescence detection unit in a state where the depth of the liquid on the reaction field is the first depth, and the liquid on the reaction field. The detection value B detected as the second light by the fluorescence detection unit when the depth of the liquid is the first depth, and the depth of the liquid on the reaction field is the second depth In the state where the fluorescence detection unit detects the detection light C as the first light and the depth of the liquid on the reaction field is the second depth, the fluorescence detection unit uses the second light as the second light. Based on the detected value D detected, a signal value I p1 + I s1 indicating the presence or amount of the substance to be detected is calculated by the following equation (6).
The detection device according to any one of claims 1 to 3.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]
The processing unit has a noise value derived from light generated without being affected by an enhanced electric field caused by the surface plasmon resonance included in the first light according to the following expressions (7) and (8): The noise value I s2 derived from the light generated without being affected by the enhanced electric field caused by the surface plasmon resonance included in the second light is further calculated as I p2. Detection device.
The angle of the vibration direction of the electric field with respect to a plane including the normal to the surface of the metal film and the optical axis of the excitation light irradiated by the excitation light irradiation unit is 0 ± 30 ° from the fluorescence emitted from the fluorescent material. A polarizer for taking out the linearly polarized light inside,
The metal film of the detection chip has a diffraction grating at a position corresponding to the reaction field,
The excitation light irradiation unit irradiates the diffraction grating with excitation light,
The fluorescence detection unit detects the linearly polarized light,
The processing unit includes a detection value A detected by the fluorescence detection unit when the depth of the liquid on the reaction field is the first depth, and the depth of the liquid on the reaction field is the first depth. Based on the detection value B detected by the fluorescence detection unit at a depth of 2, a signal value I p1 indicating the presence or amount of the substance to be detected is calculated by the following equation (9):
The detection device according to any one of claims 1 to 3.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]
The first light is p-polarized light with respect to the surface of the metal film,
The second light is s-polarized light with respect to the surface of the metal film.
The detection device according to any one of claims 5 to 8.
The detection device according to claim 9, wherein the linearly polarized light is p-polarized light with respect to a surface of the metal film.
A detection method for detecting a substance to be detected using surface plasmon resonance,
Detection having a storage part for storing a liquid and a metal film including a reaction field which is arranged at the bottom of the storage part and to which a detection target substance labeled with a fluorescent substance is directly or indirectly fixed A first step of preparing a chip;
Irradiating excitation light so that surface plasmon resonance occurs in the metal film in a state where the liquid exists in the container so that the depth of the liquid on the reaction field becomes the first depth, and the metal A second step of detecting fluorescence emitted from the fluorescent material present on the film;
Surface plasmon resonance is generated in the metal film in a state where the liquid is present in the container so that the depth of the liquid on the reaction field is a second depth different from the first depth. A third step of irradiating excitation light and detecting fluorescence emitted from the fluorescent material present on the metal film;
A fourth step of calculating a signal value indicating the presence or amount of the substance to be detected based on the detection values obtained in the second step and the third step;
A detection method.
The detection method according to claim 12, further comprising a step of changing an amount of liquid in the housing portion between the second step and the third step.
In the first step, two detection chips are prepared,
One of the detection chips has a first reaction field in which when the liquid is stored in the storage section, the depth of the liquid on the storage chip becomes the first depth,
The other detection chip has a second reaction field in which when the liquid is stored in the storage portion, the depth of the liquid on the storage chip becomes the second depth.
The detection method according to claim 12.
The metal film of the detection chip has a diffraction grating at a position corresponding to the reaction field,
In the second step and the third step, the diffraction grating is irradiated with excitation light,
In the fourth step, based on the detection value A obtained in the second step and the detection value B obtained in the third step, the presence of the detected substance represented by the following formula (1) Or calculating a signal value I p1 indicating the amount,
The detection method according to any one of claims 12 to 14.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]
The metal film of the detection chip has a diffraction grating at a position corresponding to the reaction field,
In each of the second step and the third step, the angle of the vibration direction of the electric field with respect to a plane including the normal to the surface of the metal film and the optical axis of the excitation light out of the fluorescence emitted from the fluorescent material is Detecting first light in a range of 0 ± 30 ° and second light in an angle of an oscillation direction of an electric field with respect to the plane of 90 ± 30 °,
In the fourth step, based on the detection value A detected as the first light in the second step and the detection value C detected as the first light in the third step, the following formula ( 2) to calculate a signal value I p1 indicating the presence or amount of the substance to be detected,
The detection method according to any one of claims 12 to 14.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]
In the fourth step, the detection value A, the detection value B detected as the second light in the second step, the detection value C, and the detection detected as the second light in the fourth step Based on the value D, the following formulas (3) to (5) are derived from light included in the first light and generated without being affected by the enhanced electric field due to the plasmon resonance: Noise value I p2 , noise value I s1 derived from the light generated by the influence of the enhanced electric field caused by the surface plasmon resonance and included in the second light, and included in the second light The detection method according to claim 16, further comprising calculating a noise value Is2 derived from light generated without being influenced by an enhanced electric field caused by the surface plasmon resonance.
The detection chip includes a prism made of a dielectric, and the metal film disposed on the prism,
In the second step and the third step, excitation light is irradiated to the back surface of the metal film corresponding to the reaction field via the prism, and fluorescence of the metal film is emitted from the fluorescence emitted from the fluorescent material. The first light within the range of 0 ± 30 ° in the electric field oscillation direction with respect to the plane including the normal to the surface and the optical axis of the excitation light, and the angle of the electric field oscillation direction with respect to the plane is 90 ± 30 °. Each detecting a second light within the range of
In the fourth step, the detection value A detected as the first light in the second step, the detection value B detected as the second light in the second step, and the first value in the third step A signal value indicating the presence or amount of the substance to be detected according to the following equation (6) based on the detection value C detected as the light of the above and the detection value D detected as the second light in the third step I p1 + I s1 is calculated,
The detection method according to any one of claims 12 to 14.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]
In the fourth step, the noise derived from the light included in the first light and generated without being influenced by the enhanced electric field due to the plasmon resonance, according to the following expressions (7) and (8): The value I p2 and a noise value I s2 derived from light included in the second light and generated without being affected by an enhanced electric field due to the surface plasmon resonance are further calculated in claim 18. The detection method described.
The metal film of the detection chip has a diffraction grating at a position corresponding to the reaction field,
In each of the second step and the third step, the angle of the oscillation direction of the electric field with respect to a plane including the normal to the surface of the metal film and the optical axis of the excitation light is 0 from the fluorescence emitted from the fluorescent material. Detects linearly polarized light within a range of ± 30 °,
In the fourth step, based on the detection value A detected in the second step and the detection value B detected in the third step, the presence or amount of the substance to be detected is expressed by the following equation (9). Calculating the signal value I p1 ,
The detection method according to any one of claims 12 to 14.

[In the above formula, m is the ratio of the second depth to the first depth, and is a positive real number other than 1.] ]
The first light is p-polarized light with respect to the surface of the metal film,
The second light is s-polarized light with respect to the surface of the metal film.
The detection method according to any one of claims 17 to 19.
The detection method according to claim 20, wherein the linearly polarized light is p-polarized light with respect to a surface of the metal film.