JP6627778B2 - Detection device and detection method - Google Patents

Description

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

  In clinical examinations and the like, if a trace amount of a substance to be detected, such as a protein or DNA, can be detected with high sensitivity and quantitatively, it is possible to quickly grasp the condition of a patient and perform treatment. For this reason, a method capable of detecting a trace amount of a substance to be detected with high sensitivity and quantitatively is required.

  Surface Plasmon-field enhanced Fluorescence Spectroscopy (hereinafter abbreviated as "SPFS") is known as a method capable of detecting a target substance with high sensitivity. SPFS utilizes Surface Plasmon Resonance (hereinafter abbreviated as “SPR”) generated by irradiating a metal film with light under predetermined conditions.

  First, in SPFS, a capturing body (for example, a primary antibody) capable of specifically binding 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 the substance to be detected is provided to the reaction field, the substance to be detected binds to the capturing body in the reaction field. Next, when another capture substance (for example, a secondary antibody) labeled with a fluorescent substance is provided to the reaction field, the substance to be detected bound to the capture substance 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, by detecting the emitted fluorescence, the presence or amount of the substance to be detected can be detected. In the SPFS, the fluorescent substance is excited by the electric field enhanced by the SPR, so that the substance to be detected can be detected with high sensitivity.

  However, at the time of fluorescence detection, if an unreacted fluorescent substance which does not label the substance to be detected exists on the metal film, it becomes background noise. Therefore, in order to accurately detect the target substance, it is preferable that unreacted fluorescent substance be removed in advance 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 plasmon. In PC-SPFS, a prism having a metal film formed on one surface is used. In this method, the excitation light and the surface plasmon are coupled by totally reflecting the excitation light at the interface between the prism and the metal film. PC-SPFS is currently the mainstream method, but has a problem in terms of miniaturization of the detection device due to the use of a prism and the large angle of incidence of the excitation light on the metal film. .

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

JP 2011-158369 A

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

  As described above, GC-SPFS has an advantage that the detection device can be downsized as compared with PC-SPFS, but the research on GC-SPFS is more advanced than the research on PC-SPFS. Not in. Therefore, there is room for improvement in the detection sensitivity of the detection device and the detection method using GC-SPFS.

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

  An object of the present invention is to provide a detection device and a 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 the metal film. It is an object of the present invention 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 target substance using surface plasmon resonance, and a storage unit for storing a liquid. A holder for holding a detection chip having a metal film including a reaction field in which a substance to be detected, which is disposed at the bottom of the housing portion and is labeled with a fluorescent substance, is directly or indirectly fixed. An excitation light irradiating unit that irradiates the metal film of the detection chip held by the holder with excitation light so as to generate surface plasmon resonance, and irradiating the excitation light with a liquid present in the storage unit. A fluorescence detecting unit that detects fluorescence emitted from the fluorescent substance present on the metal film at least twice when the unit irradiates the metal film with excitation light; and two or more fluorescence detection units that are detected by the fluorescence detecting unit. Inspection A processing unit that calculates a signal value indicating the presence or amount of the substance to be detected based on the value, wherein the fluorescence detection unit is configured such that a depth of the liquid on the reaction field is a first depth. Fluorescence is detected at least once in the state, and the fluorescence is detected at least once in a state where the depth of the liquid on the reaction field is different from the first depth.

  In order to solve the above problems, 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 a storage unit for storing a liquid. A first step of preparing a detection chip having a metal film including a reaction field in which a substance to be detected, which is labeled with a fluorescent substance, is directly or indirectly fixed, which is disposed at the bottom of the housing section; In a state where the liquid is present in the container such that the depth of the liquid on the reaction field is the first depth, the metal film is irradiated with excitation light so that surface plasmon resonance is generated, A second step of detecting fluorescence emitted from the fluorescent substance present on the membrane, and the step of detecting the liquid so that a depth of the liquid on the reaction field becomes a second depth different from the first depth. In the state where it is present in the accommodation section, the gold A third step of irradiating the film with excitation light so that surface plasmon resonance is generated, and detecting fluorescence emitted from the fluorescent substance existing on the metal film; and a second step and a third step. A fourth step of calculating a signal value indicating the presence or amount of the target substance based on the detected value.

  ADVANTAGE OF THE INVENTION According to this invention, in a detection apparatus and detection method using SPFS, a to-be-detected substance can be easily detected with high sensitivity. Further, according to the present invention, a substance to be detected can be detected in real time.

FIG. 1 is a schematic diagram illustrating a configuration of the SPFS device according to the first embodiment. FIG. 2A is a cross-sectional view of a detection chip used in the SPFS device 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 device 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 detecting a substance to be detected by the SPFS device. FIG. 7 is a schematic diagram illustrating a configuration of an SPFS device according to a modification. FIG. 8 is a cross-sectional view of a detection chip according to a modification used in the SPFS device. FIG. 9 is a schematic diagram illustrating a configuration of the SPFS device according to the second embodiment. FIG. 10 is a flowchart illustrating an example of an operation procedure of the SPFS device according to the second embodiment. FIG. 11 is a schematic diagram illustrating a configuration of the SPFS device according to the third embodiment. FIG. 12 is a flowchart illustrating an example of an operation procedure of the SPFS device according to the third embodiment. FIG. 13 is a schematic diagram illustrating a configuration of the SPFS device according to the fourth embodiment. FIG. 14 is a flowchart illustrating an example of an operation procedure of the SPFS device according to Embodiment 4.

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

[Embodiment 1]
In the first embodiment, GC-SPFS for detecting first light (for example, p-polarized light) and second light (for example, s-polarized light) contained in fluorescence emitted from a fluorescent substance present on a metal film, respectively. The device will be described. The SPFS device according to the first embodiment has 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 the SPFS device 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 has an excitation light irradiation unit 110, a fluorescence detection unit 120, a transport unit 130, and a control unit 140. The SPFS apparatus 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 depending on the configuration and the number of the 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 contained liquid. The detection chip 10 contains a liquid at a first depth h1, and the detection chip 10 'contains a liquid at a second depth h2. Hereinafter, in the description of the configuration of the detection chip, the detection chip 10 will be described.

  As shown in FIGS. 2A and 2B, the detection chip 10 has a substrate 11, a metal film 12 formed on the substrate 11, and a frame 14 arranged on the substrate 11. By arranging the frame body 14 on the substrate 11, an accommodating portion 15 for accommodating the liquid is formed. Further, 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 a 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; and 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 housing 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 arranged 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 combine 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 of 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 parallel ridges (and ridges) are formed on the surface of the metal film 12 at predetermined intervals. 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, and a sawtooth shape. In the present embodiment, the cross-sectional shape of the diffraction grating 13 is a rectangular wave shape. Note that the optical axis of the excitation light α described later is parallel to the xz plane. The pitch and depth of the grooves (concave stripes) 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 pitch of the grooves of the diffraction grating 13 is preferably in the range of 100 nm to 2000 nm, and the depth of the grooves of the diffraction grating 13 is preferably in the range of 10 nm to 1000 nm.

  The method for forming the diffraction grating 13 is not particularly limited. For example, after the metal film 12 is formed on the flat substrate 11, the metal film 12 may be provided with an uneven shape. Further, the metal film 12 may be formed on the substrate 11 which has been previously provided with an uneven shape. In any case, the metal film 12 including the diffraction grating 13 can be formed.

  A capturing body 16 for capturing a substance to be detected is immobilized on the diffraction grating 13. Here, a region on the diffraction grating 13 (metal film 12) where the capturing body 16 is immobilized is particularly called a “reaction field”. The capturing body 16 specifically binds to the substance to be detected. As a result, 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 substance to be detected can be captured. For example, the capture body 16 is an antibody or a fragment thereof capable of specifically binding to the analyte, an enzyme capable of specifically binding to the analyte, and the like.

The method of immobilizing the capturing body 16 is not particularly limited. For example, a self-assembled monolayer (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 SAM include HOOC- _{(CH 2)} 11 film formed by substituted aliphatic thiols such as -SH. Examples of the material constituting the polymer film include polyethylene glycol and MPC polymer. Alternatively, a polymer having a reactive group (or a functional group that can be converted into a reactive group) capable of binding to the capturing body 16 may be immobilized on the diffraction grating 13 and the capturing body 16 may be bonded 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 area, the surface plasmon generated by the metal film 12 and the evanescent wave generated by the diffraction grating 13 combine to generate SPR. When a fluorescent substance exists in the irradiation region, the fluorescent substance is excited by the enhanced electric field formed by the SPR, and the fluorescent light β is emitted. In the GC-SPFS, unlike the PC-SPFS, the fluorescence β is emitted with directivity in a specific direction. For example, the emission angle theta ₂ of fluorescent β is approximated by 2 [Theta] _1. Under the condition that SPR occurs, the reflected light γ of the excitation light α hardly occurs.

  The frame body 14 is a plate having a through hole as shown in FIG. 2B. The frame 14 is disposed on the substrate 11. The inner surface of the through hole is the side surface of the housing 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 body 14 can be fixed on the substrate 11 using a silicon sheet (double-sided seal) having adhesive properties on both sides.

  The storage unit 15 is disposed on the metal film 12 (or the substrate 11) and stores a liquid. In the present embodiment, the liquid is stored in the storage portion 15 of one detection chip 10 at the first depth h1. In addition, the liquid is stored at the second depth h2 in the storage portion 15 of the other detection chip 10 '. The depth of the liquid stored in the storage part 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. Assuming that 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 section 15 are not particularly limited as long as a desired amount of liquid can be stored. For example, the storage section 15 may be a well for storing a liquid, or may be a flow path (flow cell) to which a liquid can be continuously supplied. The detection chip in which the housing portion 15 is a well includes, for example, mass transfer analysis between the bulk and the surface of the metal film 12 (real-time measurement) and enhancement in addition to general measurement of a substance to be detected (non-real-time measurement). It is also suitable for the measurement of the electric field space scale (z-axis direction). The detection chip 10 in which the housing portion 15 is a flow path is, for example, capable of performing another measurement (non-real-time measurement) of a substance to be detected and another molecule (capture body) immobilized on the surface of the metal film 12. It is also suitable for reaction constant analysis (real-time measurement) of molecules (substances to be detected). In the present embodiment, the housing 15 is a well. As described above, the housing portion 15 is formed by disposing the frame body 14 on the substrate 11, but the method of forming the housing portion 15 is not particularly limited. Another example of a method for forming the housing portion 15 includes disposing a lid having a concave portion formed on the lower surface thereof on the substrate 11.

  The type of the 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 liquid containing a fluorescent substance, a buffer, and the like. Usually, the refractive index and the dielectric constant of a liquid are comparable to those of water. The types of the sample and the substance to be detected are not particularly limited. Examples of specimens include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluents thereof. Examples of the substance 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 irradiating unit 110 irradiates the metal film 12 (diffraction grating 13) of the detection chips 10, 10 'with excitation light α having a constant wavelength and light amount. At this time, the excitation light irradiating unit 110 converts the p-polarized light with respect to the surface of the metal film 12 to the metal film 12 (diffraction grating 13) so that the diffraction grating 13 generates diffracted light that can be combined with the surface plasmon in the metal film 12. Irradiation. The optical axis of the excitation light α is along the arrangement direction of the periodic structure in the diffraction grating 13 (x-axis direction in FIGS. 2A, 2B, and 3). Therefore, when an axis perpendicular to the x axis and parallel to the surface of the metal film 12 is set to the y axis, and an axis perpendicular to the x axis and perpendicular to the surface of the metal film 12 is set to 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 on 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 section 110 has at least a light source 111. The excitation light irradiation section 110 may further include a collimating lens, an excitation light filter, and the like.

  The light source 111 emits the excitation light α toward the diffraction grating 13 of the detection chips 10, 10 '. The type of the light source 111 is not particularly limited. Examples of the type of the light source 111 include a light emitting diode, a mercury lamp, 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.

  The collimating lens (not shown) is arranged 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 profile 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 becomes substantially circular. The size of the irradiation spot is preferably, for example, about 1 mmφ.

  The excitation light filter (not shown) is arranged 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 the type of the excitation light filter include a band pass filter and a linear polarization filter. Since the excitation light α from the laser diode (light source 111) has a slight wavelength distribution width, the band-pass filter converts 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 light with 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 the p-polarized light enters the metal film 12.

The incident angle θ ₁ of the excitation light α to the metal film 12 (see FIG. 1) is an angle at which the intensity of the enhanced electric field formed by the SPR becomes strongest, and as a result, the intensity of the fluorescence β from the fluorescent material becomes strongest. Preferably, there is. The incident angle θ ₁ of the excitation light α is appropriately selected according to the pitch of the grooves of the diffraction grating 13, the wavelength of the excitation light α, the type of metal forming the metal film 12, and the like. Since the optimum incident angle θ ₁ of the excitation light α changes depending on changes in various conditions, the SPFS apparatus 100 rotates the optical axis of the excitation light α and the detection chips 10 and 10 ′ to rotate the incident angle θ 1. 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 irradiating unit 110 or the detection chips 10 and 10 ′ about the intersection between the optical axis of the excitation light α and the metal film 12.

  The fluorescence detection unit 120 is disposed with respect to the excitation light irradiation unit 110 so as to pass through an intersection between the optical axis of the excitation light α and the metal film 12 and sandwich a normal to the surface of the metal film 12. The fluorescence detector 120 detects the fluorescence β emitted from the fluorescent substance 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 substance on the reaction field of the detection chip 10 when the depth of the liquid on the reaction field of the detection chip 10 is the first depth h1. Is detected at least once, and at least the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 ′ in a state where the depth of the liquid 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 arranged on the optical path of the fluorescence β between the detection chips 10 and 10 ′ and the light receiving sensor 123. The polarizer 121 has a first direction in which the angle of the electric field in the vibration direction with respect 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 ° from the fluorescence β. And the second light in which the angle of the vibration direction of the electric field with respect to the plane is in the range of 90 ± 30 °. Preferably, the polarizer 121 uses the p-polarized light having an angle of 0 ° of the vibration direction of the electric field with respect to the plane (xz plane) as the first light from the fluorescence β as the first light, and the vibration direction of the electric field with respect to the plane (xz). S-polarized light having an angle of 90 ° is extracted as 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 of 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 substance on the metal film 12 extracted 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 a 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 condenser lens group (not shown) is arranged between the detection chips 10, 10 'and the light receiving sensor 123, and forms a conjugate optical system which is hardly 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.

  The fluorescent filter (not shown) is arranged between the detection chips 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 fluorescence β from the light reaching the light receiving sensor 123, For example, the light amount of the light reaching 123 is adjusted.

  In the GC-SPFS, the fluorescence β is emitted from the diffraction grating 13 (reaction field) in a specific direction with directivity. Therefore, the angle of the optical axis of the fluorescence detection unit 120 with respect to the normal to the surface of the metal film 12 is preferably an angle at which the intensity of the fluorescence β is maximized (fluorescence peak angle). Further, the SPFS device 100 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 ′ (shown in FIG. (Omitted). For example, the second angle adjustment unit may rotate the fluorescence detection unit 120 or the detection chips 10 and 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 positions of the detection chips 10, 10 '. The transfer section 130 has a transfer stage 131 and a chip holder 132. The chip holder 132 is fixed to the transport stage 131, and detachably holds the detection chips 10, 10 '. The shape of the chip holder 132 is a shape that can hold the detection chips 10 and 10 ′ and does not hinder 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 hinder 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 (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, the processing unit converts a signal value indicating the presence or amount of the substance to be detected, and a noise value as necessary, based on two or more detection values detected by the fluorescence detection unit 120 (the light receiving sensor 123). calculate. The control unit 140 is, for example, a computer that includes 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, a detection operation of the SPFS device 100 (a detection method according to the present embodiment) will be described. FIG. 4 is a flowchart illustrating an example of an operation procedure of the SPFS device 100. 5A and 5B are schematic diagrams for explaining a part of the detection process of the SPFS device 100. Here, an example in which a primary antibody is used as the capturing body 16 and the target substance is labeled with a fluorescent substance by binding a secondary antibody labeled with a 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 set in the chip holder 132 of the SPFS device 100. When a humectant is present on the metal film 12 of the detection chips 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 sample and the primary antibody are bound (primary reaction; step S120). Specifically, in each of the detection chips 10, 10 ', a sample is provided on the metal film 12, and the sample is brought into contact with the primary antibody. When the analyte is present in the sample, at least a part of the analyte binds to the primary antibody.

  Next, the substance to be detected 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 bound to the primary antibody is brought into contact with the fluorescent labeling liquid. The fluorescent labeling solution is, for example, a buffer containing a secondary antibody labeled with a fluorescent substance. When the target substance is bound to the primary antibody, at least a part of the target substance is labeled with a fluorescent substance. As will be described later, the SPFS apparatus 100 according to the present embodiment can detect a target substance without removing free secondary antibodies. 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, after the target substance is bound to the secondary antibody, a liquid containing these complexes may be provided on the metal film 12. Further, the specimen and the fluorescent labeling liquid may be simultaneously provided on the metal film 12.

  Next, while irradiating the reaction field of one detection chip 10 with the excitation light α, the first light (for example, p-polarized light) contained in the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 ) Is detected (step S140). Specifically, the control unit 140 operates the excitation light irradiating 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) exists 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, while irradiating the same reaction field of the detection chip 10 with the excitation light α, the 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 Is detected (step S150). Specifically, the control unit 140 operates the excitation light irradiating 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 change 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. I do.

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

  Next, while irradiating the excitation light α to the reaction field of the other detection chip 10 ′, 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 irradiating 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 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 stored at the second depth h2 in 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, while irradiating the reaction field of the same detection chip 10 'with the excitation light α, the second light contained in the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10' is detected (step S180). ). Specifically, the control unit 140 operates the excitation light irradiating unit 110 to irradiate the diffraction grating 13 of the detection chip 10 with the excitation light α so that the SPR is generated, and to change 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. I do.

  Even after the secondary reaction (Step S130), the fluorescent labeling solution in the storage section 15 was replaced with a buffer solution containing no secondary antibody to wash the inside of the storage section 15, the fluorescent labeling solution bound to the substance to be detected. Part of the secondary antibody is released into the buffer. If the washing is not performed after the secondary reaction (step S130), the fluorescent labeling liquid is still present in the container 15. Therefore, in any case, the detection value A in step S140 and the detection value B in step S150, and the detection value C in step S170 and the detection value D in step S180 are excited by the enhanced electric field caused by SPR. Of the fluorescent β released from the emitted fluorescent substance (primarily a fluorescent substance labeling the substance to be detected captured by the primary antibody) and light (excitation light α and external light) other than the enhanced electric field caused by the SPR ), And a component of the fluorescence β emitted from the fluorescent substance (mainly, the fluorescent substance released in the liquid in the storage section 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 values 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 where a liquid exists at a first depth h1 on a reaction field of a diffraction grating 13 (metal film 12) in one detection chip 10, and FIG. 6B shows another detection chip 10 '. 5 shows a state in which a liquid exists at the second depth h2 on the reaction field of the diffraction grating 13 (metal film 12). 6A and 6B, a white star indicates a fluorescent substance.

As shown in FIG. 6A, in a state where the liquid is present at the first depth h1, the fluorescence β emitted from the reaction field has a light (p) generated under the influence of the enhanced electric field caused by the SPR. (Polarized light component and s-polarized light component) and light (p-polarized light component and s-polarized light component) generated without being affected by the enhanced electric field caused by the SPR. That is, the detection value when the fluorescence β is detected includes the component due to the light (the p-polarized component I _p1 and the s-polarized component I _s1 ) generated under the influence of the enhancement electric field due to the SPR, and the enhancement due to the SPR. And components generated by light (p-polarized component I _p2 and s-polarized component I _s2 ) generated without being affected by an 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 into the liquid in the container 15. Due to the fluorescence β from the fluorescent substance. Therefore, the detection value A of the light receiving sensor 123 in the step S140 of detecting only the first light (p-polarized component) is derived from I _p1 and I _p2 and detecting only the second light (s-polarized component). The detection value B of the light receiving sensor 123 in S150 is derived from _Is1 and _Is2 . That is, when the depth of the liquid on the reaction field is the first depth h1, the detection values A and B detected by the fluorescence detection unit 120 are expressed by the following equations (1) and (2), respectively. You.

In addition, as shown in FIG. 6B, in a state where the liquid is present at the second depth h2, the fluorescence β emitted from the reaction field also generates the light generated under the influence of the enhanced electric field caused by the SPR. (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 the SPR. At this time, the distance from the surface of the diffraction grating 13 that is affected by the enhanced electric field caused by the SPR is constant irrespective of the depth of the liquid stored in the storage unit 15. Therefore, 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, the reaction field contains m times as much liquid and m times as much fluorescent material. Become. At this time, compared with the depth of the liquid (several μm to several cm), the distance affected by the enhanced electric field due to SPR is 100 nm or less, which is negligibly small. For this reason, m is equal to the height of the liquid stored at the second depth h2 with respect to the height of the region not affected by the enhanced electric field caused by the SPR in the liquid stored at the first depth h1. Can be approximated to be equal to the height ratio of the region not affected by the enhancement electric field caused by the SPR. Therefore, the fluorescence β emitted in the state where the liquid is present at the second depth h2 includes _Ip2 and _Ip included in the fluorescence β emitted in the state where the liquid is present at the first depth h1. _s2 is included in an amount of m times. When the depth of the liquid on the reaction field is the second depth h2, the detection values C and D detected by the fluorescence detection unit 120 are expressed by the following equations (3) and (4), respectively.

In the GC-SPFS, the fluorescence β emitted from the fluorescent substance labeling the substance to be detected (mainly the substance to be detected captured by the primary antibody) immobilized on the metal film 12, It has been found that the light is p-polarized light with respect to the surface, or the light has a polarization angle close to the p-polarized light. On the other hand, it is understood that the fluorescence β emitted from the fluorescent substance not fixed on the metal film 12 (mainly, the fluorescent substance released in the liquid) contains not only p-polarized light but also s-polarized light to some extent. ing. That is, the s-polarized light component _Is1 of the fluorescence β generated under the influence of the enhanced electric field due to the SPR can be approximated to be substantially zero. Accordingly, the control unit (processing unit) 140 determines the following signal value indicating the presence or amount of the substance to be detected based on the detection values A and C expressed by the above equations (1) and (3). It is possible to calculate I _p1 represented by Expression (5).

Further, if necessary, the control unit (processing unit) 140 may control the noise values I _p2 , I _s1 , and I not indicating the presence or amount of the substance to be detected, which are represented by the following equations (6) to (8). _s2 can be further calculated.

  According to the above procedure, the presence of the target substance or the amount of the target substance in the sample can be detected. In the present invention, since the noise can be removed by the above procedure, it is not always necessary to measure the blank value.

However, if necessary, the blank value may be measured before the secondary reaction (Step S130). Specifically, blank values A ′ to D ′ are obtained in advance in the same procedure as in steps S140 to S180 in a state where no fluorescent substance is present in the liquid in the storage section 15. In this case, in step S190, after subtracting each blank value A ′ to D ′ from each detection value A to D, the signal value I _p1 and, if necessary, the noise values I _p2 and I _p according to the above calculation method. _s1 and _Is2 are calculated.

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

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

  Note that, in the present embodiment, the mode in which the first light and the second light are detected at different times using one polarizer 121 has been described. However, the detection device and the detection method according to the present embodiment are not described. 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 device 100 ′ according to a modification. As shown in FIG. 7, the SPFS device 100 'according to the modification does not require the rotation angle adjustment unit 122, and the fluorescence detection unit 120' further includes a half mirror 124 ', a polarizer 121', and a light receiving sensor 123 '. Having. 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. Accordingly, half of the fluorescence β emitted from the fluorescent substance on the metal film 12 passes through the half mirror 124 ′, and the first light included in the fluorescence β is detected by the light receiving sensor 123. At the same time, the remaining half of the fluorescent light β emitted from the fluorescent substance on the metal film 12 is reflected by the half mirror 124 ′, and the second light included in the fluorescent light 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.

  Further, in the above embodiment, the mode using two detection chips 10, 10 'has been described, but the detection device and the detection method according to the present invention may use only one detection chip. In this case, the detection chip has a storage unit that can store the liquid at the first depth and the second depth when the liquid is stored in the storage unit. Examples of the detection chip include a detection chip having a step or an inclined surface at the bottom of the housing. FIG. 8 is a schematic diagram illustrating a configuration of a detection chip 10 ″ according to a modification. As illustrated in FIG. 8, in the detection chip 10 ″ according to the modification, a step is formed on the substrate 11 ″. In addition, the metal film 12 (diffraction grating 13) is disposed on both the upper side and the lower side of the step surface. Thus, when the liquid is stored in the storage portion 15 ″, the depth of the liquid on the liquid is increased. Is a first depth h1 ″, and a second reaction field 18 ″ where the depth of the liquid is a second depth h2 ″ is an upper level of the step surface. Side and the lower side, respectively. Another example of the detection chip includes a detection chip further including a lid having a step portion and an inclined surface. In this case, the first reaction field 17 "and the second reaction field 18" are arranged on the metal film when the liquid is accommodated in the accommodation section and the lid is arranged.

  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.

［上記式において、ｍは第１の深さに対する第２の深さの比であり、１以外の正の実数である。］Further, in the present embodiment, the detection device and the detection method using GC-SPFS have been described, but the detection device 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 arranged on the prism. Further, the metal film 12 does not have the diffraction grating 13. The excitation light α is applied to the back surface of the metal film 12 corresponding to the reaction field via the prism. Further, in the PC-SPFS, the fluorescence β emitted from the fluorescent substance that labels the substance to be detected captured by the capturing body 16 includes not only p-polarized light but also s-polarized light to some extent. Therefore, _Is1 derived from light generated under the influence of the enhanced electric field cannot be approximated to be zero. Therefore, the control unit (processing unit) determines the detection value A detected as the first light by the fluorescence detection unit in a state where the liquid on the reaction field has the first depth h1 and the liquid value on the reaction field. The detection value B detected by the fluorescence detection unit as the second light when the depth is the first depth h1, and the detection value B when 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 a state where the depth of the liquid on the reaction field is the second depth h1, The signal value I _p1 + I _s1 indicating the presence or amount of the substance to be detected is calculated by the following equation (9). Further, the control unit (processing unit) can be controlled by the enhanced electric field caused by the SPR sound included in the first light, which is expressed by the following equations (10) and (11) as necessary. a noise value I _p2 derived from the generated light, included in the second light, and further calculates a noise value I _s2 derived from the light generated without being affected by the enhanced electric field due to the SPR Is also good.

[In the above equation, m is a 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 device that detects only linearly polarized light (for example, p-polarized light) included in fluorescence emitted from a fluorescent substance existing on a metal film will be described. The SPFS device according to Embodiment 2 has a polarizer, but does not need to switch the rotation angle of the polarizer.

(Configuration of SPFS device and detection chip)
FIG. 9 is a schematic diagram illustrating a configuration of the SPFS device 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 description thereof is omitted.

  The fluorescence detecting section 220 is arranged with respect to the excitation light irradiating section 110 so as to pass through an intersection between the optical axis of the excitation light α and the metal film 12 and sandwich a normal to the surface of the metal film 12. The fluorescence detector 220 detects the fluorescence β emitted from the fluorescent substance on the metal film 12 (diffraction grating 13) at least twice. More specifically, the fluorescence detector 220 emits from the fluorescent substance 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 substance on the reaction field of the detection chip 10 ′ in a state where the depth of the liquid on the reaction field of the other detection chip 10 ′ is the second depth h 2. The detected fluorescence β is detected at least once.

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

  The polarizer 221 is arranged on the optical path of the fluorescence β between the detection chips 10 and 10 ′ and the light receiving sensor 123. The polarizer 221 converts, from the fluorescence β, a linearly polarized light in which the angle of the oscillation direction of the electric field with respect 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 is 0 ± 30 °. Take out the light. Preferably, the polarizer 221 extracts, from the fluorescence β, p-polarized light whose vibration direction angle 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. Examples of the type of the polarizer 221 are the same as, for example, the polarizer 121 of the first embodiment.

  The control unit 240 controls the operations of 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 transfer unit 130 (the transfer stage 131). Control. Further, 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 (the light receiving sensor 123), the processing unit converts 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 executes software, including, for example, an arithmetic device, a control device, a storage device, an input device, and an output device.

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

  First, similarly to steps S110, S120, and 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 detection target substance labeled with the fluorescent substance is fixed in the reaction field can be disposed on the chip holder 132 of the SPFS device 200.

  Next, while irradiating the reaction field of one detection chip 10 with the excitation light α, linearly polarized light (for example, p-polarized light) contained in the fluorescence β emitted from the fluorescent substance on the reaction field of the detection chip 10 ) Is detected (step S240). Specifically, the control unit 240 operates the excitation light irradiating 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 stored on the reaction field of the detection chip 10 at the first depth h1.

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

  Next, while irradiating the excitation light α to the reaction field of the other detection chip 10 ′, 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 irradiating unit 110 to irradiate the diffraction grating 13 of the detection chip 10 ′ with the excitation light α so that SPR is generated, and also 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 stored in the reaction field of the detection chip 10 'at the second depth h2. Further, the control unit 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 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.

［上記式において、ｍは第１の深さに対する第２の深さの比であり、１以外の正の実数である。］As described in the first embodiment, the fluorescence β emitted from the reaction field includes the light (p-polarized component and the s-polarized component) generated under the influence of the enhanced electric field due to the SPR and the fluorescent β emitted from the 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, since only the above-described linearly polarized light included in the fluorescence β is detected, 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 represented by the following equations (12) and (13).

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

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 expressed by Expressions (12) and (13). As a signal value, _Ip1 represented by the following equation (14) is calculated.

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

(effect)
In the detection device and the detection method according to the second embodiment, the same effects as those of the detection device and the detection method according to the first embodiment can be obtained, and 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 device that detects fluorescence (including p-polarized light and s-polarized light) included in the fluorescent light emitted from the fluorescent substance existing on the metal film will be described. The SPFS device according to the present embodiment has no polarizer.

(Configuration of SPFS device and detection chip)
FIG. 11 is a schematic diagram illustrating a configuration of SPFS device 300 according to the present embodiment. The SPFS device 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 device 300, only the fluorescence detection unit 320 and the control unit 340 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 description thereof is omitted.

  The fluorescence detection unit 320 is disposed with respect to the excitation light irradiation unit 110 so as to pass through an intersection between the optical axis of the excitation light α and the metal film 12 and sandwich a normal to the surface of the metal film 12. The fluorescence detector 320 detects the fluorescence β emitted from the fluorescent substance on the metal film 12 (diffraction grating 13) at least twice. More specifically, the fluorescence detection unit 320 is emitted from the fluorescent substance on the reaction field of the detection chip 10 in a state where the depth of the liquid on the reaction field of the one detection chip 10 is the first depth h1. Fluorescence β is detected at least once, and is released from the fluorescent substance on the reaction field of the detection chip 10 ′ in a state where the liquid on the reaction field of the other detection chip 10 ′ has the second depth h 2. Fluorescent β is detected at least once.

  The fluorescence detector 320 has at least the 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 controls the operations of 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 transfer unit 130 (the transfer 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 (the light receiving sensor 123), the processing unit converts 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 executes software, including, for example, an arithmetic device, a control device, a storage device, an input device, and an output device.

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

  First, similarly to steps S110, S120, and 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. Thereby, 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 on the chip holder 132 of the SPFS device 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 irradiating 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 stored on the reaction field of the detection chip 10 at the first depth h1.

  Next, the reaction field to be detected is switched (step S350). Specifically, the control unit 340 operates the transport stage 131 to move the detection chips 10, 10 '. Thereby, the excitation light irradiating 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 irradiating 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 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 stored in 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.

［上記式において、ｍは第１の深さに対する第２の深さの比であり、１以外の正の実数である。］As described in the first embodiment, the fluorescence β emitted from the reaction field includes the light (p-polarized component and the s-polarized component) generated under the influence of the enhanced electric field due to the SPR and the fluorescent β emitted from the 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, since the fluorescence β containing the p-polarized component and the s-polarized component is detected, the detection values A and B of the light receiving sensor 123 are I _p1 , I _p2 , I _s1 and I _s2 . Therefore, the detection values A and B are represented by the following equations (15) and (16).

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

Therefore, based on the detection values A and B expressed by the above equations (15) and (16), the control unit (processing unit) 340 determines the signal value indicating the presence of the substance to be detected as the following equation ( 17) _Ip1 represented by is calculated. At this time, the control unit (processing unit) 340 performs the calculation with _{Is1 set} to 0.

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

(effect)
The detection device and the detection method according to the third embodiment can obtain the same effects as those of the detection device and the detection method according to the first embodiment, and need not have a polarizer. The configuration and detection method 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 for irradiating the excitation light α. However, the method for switching the reaction field to be detected is used. 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 with respect to the detection chips 10, 10 ', and switch the reaction field to which the excitation light α is irradiated. May be.

  Further, the detection chip 10 ″ according to the modified example can be used also in the detection devices and the detection methods according to Embodiments 2 and 3.

[Embodiment 4]
In the fourth embodiment, GC-SPFS for detecting first light (for example, p-polarized light) and second light (for example, s-polarized light) contained in fluorescence emitted from a fluorescent substance present on a metal film, respectively The device will be described. The SPFS device according to the fourth embodiment has a liquid amount adjustment 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 device 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 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 description thereof is omitted.

  Further, as shown in FIG. 13, in SPFS apparatus 400 according to the present embodiment, only one detection chip 10 is used. 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 is omitted.

  The control unit 440 includes an excitation light irradiating unit 110 (the light source 111 and the first angle adjusting unit), a fluorescence detecting unit 120 (the light receiving sensor 123 and the second angle adjusting unit), a transporting unit 130 (the transporting stage 131), and a description which will be described later. The operation of the liquid amount adjusting section 450 (liquid supply pump driving mechanism 454) is controlled. The control unit 440 also functions as a processing unit that processes an output signal (detection result) from the fluorescence detection unit 120. Specifically, the processing unit converts a signal value indicating the presence or amount of the substance to be detected, and a noise value as necessary, based on two or more detection values detected by the fluorescence detection unit 120 (the light receiving sensor 123). calculate. The control unit 440 is a computer that executes software, including, for example, an arithmetic device, a control device, a storage device, an input device, and an output device.

  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 the liquid in the storage unit 15. The liquid amount adjustment unit 450 determines when the fluorescence detection unit 120 detects the fluorescence β when the depth of the liquid on the reaction field is the first depth h1, and when the fluorescence detection unit 120 detects the depth of the liquid on the reaction field. The amount of liquid in the container 15 is changed between the time when the fluorescence β is detected at the second depth h2. The liquid amount adjustment unit 450 includes, for example, a syringe pump 451 and a liquid feed pump driving mechanism 454.

  The syringe pump 451 includes a syringe 452 and a plunger 453 that can reciprocate in the syringe 452. The reciprocating motion of the plunger 453 quantitatively sucks and discharges the liquid.

  The liquid feeding pump driving mechanism 454 includes a driving device for the plunger 453 and a moving device for the syringe pump 451. The driving device of the syringe pump 451 is a device for reciprocating the plunger 453, and includes, for example, a stepping motor. Since the driving device including the stepping motor can manage the amount and speed of liquid supply of the syringe pump 451, the amount of liquid in the storage portion 15 of the detection chip 10 can be managed. The moving device of the syringe pump 451, for example, freely moves the syringe pump 451 in two directions: an axial direction (for example, a vertical direction) of the syringe 452 and a direction (for example, a horizontal direction) transverse to the axial direction. The moving device of the syringe pump 451 includes, for example, a robot arm, a two-axis stage, or a vertically movable turntable.

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

  First, similarly to steps S110, S120, and 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. Accordingly, the detection chip 10 in a state where the detection target substance labeled with the fluorescent substance is fixed in the reaction field can be disposed on the chip holder 132 of the SPFS device 400.

  Next, while irradiating the reaction field of the detection chip 10 with the excitation light α, the first light (for example, p-polarized light) contained in the fluorescence β emitted from the fluorescent substance on the reaction field is detected (step S440). ). Specifically, the control unit 440 operates the excitation light irradiating 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 stored 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, while irradiating the reaction field of the detection chip 10 with the excitation light α, the second light (for example, s-polarized light) included in the fluorescence β emitted from the fluorescent substance on the reaction field is detected (step S450). ). Specifically, the control unit 440 operates the excitation light irradiating 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 change the detection value B of the light receiving sensor 123. Record. At this time, as shown 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. I do.

  Next, a liquid is introduced into the storage section (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 storage section 15 is supplied. Thereby, the depth of the liquid existing on the reaction field in the storage section 15 can be changed from the first depth h1 to the second depth h2.

  Next, while irradiating the reaction field of the 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 is detected (Step S470). Specifically, the control unit 440 operates the excitation light irradiating 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 change the detection value C 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) is accommodated at the second depth h2. 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, while irradiating the reaction field of the detection chip 10 with the excitation light α, the second light included in the fluorescence β emitted from the fluorescent substance on the reaction field is detected (Step S480). Specifically, the control unit 440 operates the excitation light irradiating 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 change the detection value D of the light receiving sensor 123. Record. At this time, as shown 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. I do.

  Finally, the control unit (processing unit) 440 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 (Step S490). Hereinafter, a method for calculating the signal value will be described.

［上記式において、ｍは第１の深さに対する第２の深さの比であり、１以外の正の実数である。］As described in the first embodiment, the fluorescence β emitted from the reaction field includes the light (p-polarized component and the s-polarized component) generated under the influence of the enhanced electric field due to the SPR and the fluorescent β emitted from the 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, since only the p-polarized light component contained in the fluorescence β is detected, the detection values A and C of the light receiving sensor 123 are derived from _Ip1 and _Ip2 . In Steps S450 and S480, only the s-polarized light component contained in the fluorescence β is detected, and thus the detection values C and D of the light receiving sensor 123 are derived from _Is1 and _Is2 . Therefore, the detection values A to D are represented by the following equations (18) to (21).

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

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

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

(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. Further, the detection device and the detection method need not include a polarizer. The configuration and detection method 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, the detection device and the detection method according to the present invention may reduce the liquid from the storage unit. Also in this case, the depth of the liquid on the reaction field in the storage section can be changed from the first depth to the second depth.

  In each of the above embodiments, an example was described in which the detection chips 10 and 10 ′ were irradiated with the excitation light α from the metal film 12 side. However, the detection chips 10 and 10 ′ were irradiated with the excitation light α from the substrate 11 side. May be.

  In the above second to fourth embodiments, the detection device and the detection method using the GC-SPFS have been described. However, the detection device and the detection method according to the second to fourth embodiments use the PC-SPFS. Is also good. In this case, the detection chips 10, 10 'have a prism made of a dielectric, and the metal film 12 is arranged on the prism. Further, the metal film 12 does not have the diffraction grating 13. The excitation light α is applied 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 each of the above-described embodiments, since a noise component can be removed from the detection value, the unreacted fluorescent substance existing in the storage unit 15 is removed by washing. No need. Therefore, the detection device 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 device continuously irradiates the metal film (diffraction grating) with the excitation light in a state where the depth of the liquid on the reaction field is the first depth, and is included in the fluorescence emitted from the fluorescent substance. The linearly polarized light is continuously detected. In parallel with this, the detection device continuously irradiates the metal film (diffraction grating) with excitation light in a state where the liquid on the reaction field has the second depth, and emits the fluorescent light emitted from the fluorescent substance. Is continuously detected. Here, “continuation” includes not only continuous operation but also intermittent operation. Thus, the detection device and the detection method according to the present embodiment can accurately, temporally, and easily detect the target substance.

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

1. Preparation of Detection Chip Two detection chips on which an anti-α-fetoprotein (AFP) antibody was immobilized via carboxymethyl dextran (CMD) on a metal film diffraction grating were prepared. The two prepared detection chips were respectively set in the chip holder of the SPFS device. In the present embodiment, the liquid is accommodated in the accommodation part of one detection chip so as to have a depth of 50 μm, and the liquid is accommodated in the accommodation part of the other detection chip so as to have a depth of 100 μm. You.

2.1 Primary Reaction A specimen containing AFP was provided to the accommodation section of each detection chip, and left standing for 25 minutes to perform a primary reaction.

3. Measurement of Optical Blank Value After the primary reaction, the sample in the container was removed, and the container was washed with a phosphate buffer. Then, by operating the rotation angle adjustment unit, the rotation angle of the polarizing plate is adjusted so that only the p-polarized light can be transmitted, and in a state where the buffer is present at a depth of 50 μm in the housing of one of the detection chips, The diffraction grating was irradiated with excitation light having a wavelength of 637 nm. At the same time, the p-polarized light contained in the light emitted from the storage section was detected, and an optical blank value oB _p for the p-polarized light was obtained. oB _p was 150 counts.

Then, by operating the rotation angle adjustment unit, the rotation angle of the polarizing plate is adjusted so that only s-polarized light can be transmitted, and in a state where the same buffer solution is present at a depth of 50 μm in the accommodation unit of the same detection chip, The diffraction grating was irradiated with excitation light having a wavelength of 637 nm. At the same time, the s-polarized light contained in the fluorescent light emitted from the storage section was detected, and an optical blank value oB _s for the s-polarized light was obtained. oB _s was 100cоunt.

  With respect to the other detection chip, the optical blank value was measured in the same manner as described above in a state where the liquid was present at a depth of 100 nm in the storage section. The optical blank values for p-polarized and s-polarized light were approximately the same as above.

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

5. Fluorescence detection After the secondary reaction, in the same manner as in the measurement of the optical blank value, the excitation light is irradiated on the diffraction grating in a state where the fluorescent labeling liquid is present at a depth of 50 μm in the accommodation part of one of the detection chips and emitted. The p-polarized light and the s-polarized light contained in the fluorescence to be detected were detected. Further, in a state where the fluorescent labeling liquid is present at a depth of 100 μm in the accommodation portion of the other detection chip, the excitation light is irradiated on the diffraction grating to emit p-polarized light and s-polarized light contained in the emitted fluorescence. Detected.

6. Processing of Detected Value When the depth of the liquid was 50 μm, the detected value A when detecting p-polarized light was 2080 count, and the detected 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 count, and the detection value D when detecting s-polarized light was 2570 count. The ratio m of the depth of the liquid stored in the storage part of the other detection chip to the depth of the liquid stored in the storage part of one detection chip is 2. Using the control unit (processing unit), a signal value _Ip1 indicating the amount of the substance to be detected was calculated from the detected values A and C as represented by the following equation (23).

Further, using the control unit (processing unit), the influence of the enhanced electric field contained in the p-polarized light and represented by the following formulas (24) to (26) based on the above detected values and caused by the SPR is obtained. A noise value I _p2 derived from light generated without being affected by the SPR and a noise value I _s1 derived from light included in the s-polarized light and generated under the influence of the enhanced electric field due to the SPR, and s The 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 background noise could be removed and the target substance could be detected without removing the unreacted fluorescent substance by washing. Further, among the signal values indicating the amount of the substance to be detected, the signal component due to s-polarized light was almost 0, and it was confirmed that the signal component was sufficiently small. Further, a noise value derived from light generated without being affected by the enhanced electric field due to SPR could be calculated.

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

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

  Further, the detection device and the detection method according to the present invention can detect a substance to be detected with high reliability without having to wash the surface of the metal film after providing a fluorescent labeling solution or the like. Therefore, it is expected that not only the detection time can be shortened, but also that it contributes to the development, spread, and development of a quantitative immunoassay apparatus 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 "housing 16 capturing body 17" first reaction field 18 "second reaction field 100, 100' , 200, 300, 400 Surface plasmon enhanced fluorescence measurement device (SPFS device)
110 Excitation light irradiation unit 111 Light source 120, 120 ', 220, 320 Fluorescence detection unit 121, 121', 221 Polarizer 122 Rotation angle adjustment unit 123, 123 ', Light receiving sensor 124' Half mirror 130 Transport unit 131 Transport stage 132 Chip Holder 140, 240, 340, 440 Control unit (processing unit)
450 Liquid volume adjusting unit 451 Syringe pump 452 Syringe 453 Plunger 454 Liquid feed pump drive mechanism h1, h1 ″ First depth h2, h2 ″ Second depth α Excitation light β Fluorescence γ Reflected light

Claims (22)

A detection device for detecting a target substance using surface plasmon resonance,
It has a storage section for storing a liquid, and a metal film including a reaction field disposed at the bottom of the storage section and directly or indirectly fixing a substance to be detected which is labeled with a fluorescent substance. A holder for holding the detection chip,
An excitation light irradiator that irradiates the metal film of the detection chip held by the holder with excitation light such that surface plasmon resonance is generated,
When the excitation light irradiating unit irradiates the metal film with excitation light in a state where the liquid is present in the storage unit, fluorescence emitted from the fluorescent substance existing on the metal film is detected at least twice. A fluorescence detector,
A processing unit that removes background noise based on two or more detection values detected by the fluorescence detection unit and calculates a signal value indicating the presence or amount of the target substance,
Has,
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 an amount of the liquid in the storage unit of the detection chip held by the holder;
The liquid amount changing unit is configured to detect when the fluorescence detection unit detects fluorescence in a state where the depth of the liquid on the reaction field is the first depth, and when the fluorescence detection unit detects the fluorescence on the reaction field. Between the time when the depth of the liquid is to detect fluorescence in the state of the second depth, and the amount of the liquid in the container is changed;
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 thereon is 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 thereon is 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 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. A signal value I _p1 indicating the presence or amount of the substance to be detected is calculated by the following equation (1) based on the detection value B detected by the fluorescence detection unit at a depth of 2;
The detection device according to claim 1.

[In the above equation, m is a ratio of the second depth to the first depth, and is a positive real number other than 1. ] From the fluorescence emitted from the fluorescent substance, 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 within a range of 0 ± 30 °. And a polarizer that simultaneously or at a different time takes out the first light within and the second light within the range of 90 ± 30 ° in the direction of oscillation of the electric field with respect to the plane,
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, respectively,
The processing unit includes: a detection value A detected by the fluorescence detection unit as the first light 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 presence or amount of the substance to be detected is determined by the following equation (2) based on the detection value C detected by the fluorescence detection unit as the first light in a state where the depth is the second depth. Calculating the indicated signal value I _p1 ,
The detection device according to claim 1.

[In the above equation, m is a ratio of the second depth to the first depth, and is a positive real number other than 1. ] The processing unit is configured to detect the detection value A, a detection value B detected by the fluorescence detection unit as the second light in a state where the depth of the liquid on the reaction field is the first depth, Based on a detection value C and a detection value D detected by the fluorescence detection unit as the second light in a state where the depth of the liquid on the reaction field is the second depth, the following expression is used. According to (3) to (5), a noise value _Ip2 derived from light included in the first light and generated without being affected by an enhanced electric field due to the surface plasmon resonance, and the second value A noise value _Is1 derived from light generated by being affected by the enhanced electric field caused by the surface plasmon resonance included in the light; and an enhanced electric field caused by the surface plasmon resonance included in the second light. further to calculate a noise value I _s2 derived from the effect on the light produced without being The detection device of claim 5.

From the fluorescence emitted from the fluorescent substance, 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 within a range of 0 ± 30 °. And a polarizer that simultaneously or at a different time takes out the first light within and the second light within the range of 90 ± 30 ° in the direction of oscillation of the electric field with respect to the plane,
The detection chip has a prism made of a dielectric, and the metal film disposed on the prism,
The excitation light irradiator irradiates excitation light to the back surface of the metal film corresponding to the reaction field via the prism,
The fluorescence detection unit detects the first light and the second light, respectively,
The processing unit includes: a detection value A detected by the fluorescence detection unit as the first light 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 by the fluorescence detection unit as the second light when the depth of the liquid is the first depth, and the state where the depth of the liquid on the reaction field is the second depth The detection value C detected by the fluorescence detector as the first light and the fluorescence detector detects the second light when the depth of the liquid on the reaction field is the second depth. Based on the detected value D, 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 claim 1.

[In the above equation, m is a ratio of the second depth to the first depth, and is a positive real number other than 1. ] The processing unit calculates the noise value derived from the light generated without being affected by the enhanced electric field due to the surface plasmon resonance included in the first light according to the following Expressions (7) and (8). and I _p2, further calculates a noise value I _s2 derived from the light generated without being affected by the enhanced electric field due to the surface plasmon resonance contained in said second light, according to claim 7 Detection device.

From the fluorescence emitted from the fluorescent substance, 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 within a range of 0 ± 30 °. Further comprising a polarizer for extracting linearly polarized light within,
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 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. A signal value I _p1 indicating the presence or amount of the substance to be detected is calculated by the following equation (9) based on the detection value B detected by the fluorescence detection unit at a depth of 2;
The detection device according to claim 1.

[In the above equation, m is a 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 claim 5.   The detection device according to claim 9, wherein the linearly polarized light is p-polarized light with respect to the surface of the metal film. A detection method for detecting a target substance using surface plasmon resonance,
Detection having a storage section for storing a liquid, and a metal film including a reaction field disposed at the bottom of the storage section and directly or indirectly fixing a substance to be detected labeled with a fluorescent substance A first step of preparing chips;
In a state where the liquid is present in the container such that the depth of the liquid on the reaction field is the first depth, the metal film is irradiated with excitation light so that surface plasmon resonance is generated, A second step of detecting fluorescence emitted from the fluorescent substance present on the membrane;
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, surface plasmon resonance is generated in the metal film. A third step of irradiating with excitation light and detecting fluorescence emitted from the fluorescent substance present on the metal film;
A fourth step of removing background noise based on the detection values obtained in the second step and the third step, and calculating a signal value indicating the presence or amount of the target substance;
A detection method.   The detection method according to claim 12, further comprising a step of changing an amount of the liquid in the storage section 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 thereon is 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 thereon is 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 substance to be detected represented by the following formula (1) is determined. 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 equation, m is a 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 the second step and the third step, of the fluorescence emitted from the fluorescent substance, the angle of the vibration direction of the electric field with respect to the plane including the normal to the surface of the metal film and the optical axis of the excitation light is changed. A first light within a range of 0 ± 30 °, and a second light within a range of 90 ± 30 ° of an angle of the vibration direction of the electric field with respect to the plane,
In the fourth step, the following expression (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): 2) calculating 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 equation, m is a 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 value detected as the second light in the fourth step. Based on the value D and the following formulas (3) to (5), the light is included in the first light and is derived from light generated without being affected by the enhanced electric field caused by the plasmon resonance. A noise value I _p2 , a noise value I _s1 included in the second light, and a noise value I _s1 derived from light generated under the influence of an enhanced electric field caused by the surface plasmon resonance, and a noise value I _p2 included in the second light. _17. The detection method according to claim 16, further comprising calculating a noise value _Is2 derived from light generated without being affected by an enhanced electric field caused by the surface plasmon resonance.

The detection chip has a prism made of a dielectric, and the metal film disposed on the prism,
In the second step and the third step, the back surface of the metal film corresponding to the reaction field is irradiated with excitation light via the prism, and the fluorescence of the metal film A first light in which the angle of the vibration direction of the electric field with respect to the plane including the normal to the surface and the optical axis of the excitation light is within a range of 0 ± 30 °, and the angle of the vibration direction of the electric field with respect to the plane is 90 ± 30 ° And the 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 A in the third step. A signal value indicating the presence or amount of the substance to be detected by the following equation (6) based on the detection value C detected as the light of the following formula and the detection value D detected as the second light in the third step. Calculating I _p1 + I _s1 ,
The detection method according to any one of claims 12 to 14.

[In the above equation, m is a 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 affected by the enhanced electric field caused by the plasmon resonance is calculated by the following equations (7) and (8). the value I _p2, included in the second light, and calculates a noise value I _s2 derived from the light generated without being affected by the enhanced electric field due to the surface plasmon resonance, to claim 18 Detection method as described.

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 angle of the vibration direction of the electric field with respect to the plane including the normal to the surface of the metal film and the optical axis of the excitation light is set to 0 based on the fluorescence emitted from the fluorescent substance. Detects linearly polarized light within the 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 equation, m is a 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.   21. The detection method according to claim 20, wherein the linearly polarized light is p-polarized light with respect to the surface of the metal film.

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