JP6658752B2 - Detection chip, detection method, detection device and detection system - Google Patents

Description

  The present invention provides a detection chip for detecting the presence or amount of a substance to be detected using surface plasmon resonance, and a detection method for detecting the presence or amount of the substance to be detected using the detection chip. , A detection device and a detection system.

  In clinical examinations and the like, if a trace amount of a substance to be detected such as protein or DNA can be detected with high sensitivity and quantitatively, it becomes possible to quickly grasp the condition of a patient and perform treatment. For this reason, there is a demand for a detection device that can detect a trace amount of a substance to be detected with high sensitivity and quantitatively.

  As a detection device capable of detecting a target substance with high sensitivity, a device using surface plasmon resonance fluorescence spectroscopy (Surface Plasmon-field enhanced Fluorescence Spectroscopy: hereinafter abbreviated as “SPFS”) is known. It is known (for example, see Patent Document 1).

  In the detection device described in Patent Document 1, a substrate having a periodic structure on the surface, a metal film (metal layer) formed on the periodic structure of the substrate, an extinction suppressing layer formed on the metal film, A detection chip (biochip) having a capture body (bispecific antibody) bound on the quenching suppression layer is used. In this detection device, in a state where a substance to be detected, which is labeled with a fluorescent substance, is captured by a capturing body, the metal film is irradiated with excitation light at an angle at which surface plasmon resonance occurs, and a local light is applied to the surface of the metal film. Generates field light. This local field light selectively excites a fluorescent substance that labels the substance to be detected captured on the metal film, and emits fluorescence from the fluorescent substance. The detection device detects the presence or amount of the target substance by detecting the fluorescence.

  At the time of fluorescence detection, it is preferable to irradiate the metal film with excitation light at an appropriate incident angle in order to generate surface plasmon resonance on the metal film. In the detection device described in Patent Literature 1, the incident angle dependence of the reflectance of the excitation light on the metal film is measured, and an appropriate incident angle is determined.

JP 2011-158369 A

  However, the detection device described in Patent Literature 1 can detect a target substance with high accuracy by performing a process for determining an appropriate incident angle, but has a problem in that the detection time is long. . Further, in order to measure the incident angle dependence of the reflectance of the excitation light on the metal film, the incident angle of the excitation light on the metal film must be scanned, and a scanning mechanism is required. For this reason, there is also a problem that the size and cost of the detection device are increased.

  A first object of the present invention is a detection chip for detecting the presence or amount of a substance to be detected by utilizing surface plasmon resonance, wherein the detection chip does not scan the incident angle of the excitation light with respect to the metal film. An object of the present invention is to provide a detection chip that enables highly accurate detection of a detection substance. A second object of the present invention is to provide a detection method, a detection device, and a detection system for detecting the presence or amount of a substance to be detected using the detection chip.

  In order to solve the above problem, a detection chip according to an embodiment of the present invention emits light from a fluorescent substance on the metal film when the metal film of the detection chip is irradiated with light so that surface plasmon resonance occurs. A detection chip used to detect the presence or amount of a substance to be detected by detecting the fluorescence to be detected, the support and a metal film including a diffraction grating disposed on the support Wherein the diffraction grating includes a plurality of types of divided regions having different grating pitches, respectively, and the divided regions are irradiated with light on the metal film in order to emit fluorescent light from a fluorescent substance. Are arranged so that a plurality of types of the divided regions are simultaneously included in the light irradiation spot.

  In order to solve the above problems, a detection method according to an embodiment of the present invention is characterized in that when a metal film of a detection chip is irradiated with light so that surface plasmon resonance occurs, the metal film is emitted from a fluorescent substance on the metal film. A detection method for detecting the presence or amount of a substance to be detected by detecting the fluorescence to be detected, comprising a support, and a metal film including a diffraction grating disposed on the support. A step of directly or indirectly bonding a substance to be detected, which is labeled with a fluorescent substance on the metal film, of the detection chip, and wherein the substance to be detected, which is labeled with the fluorescent substance on the metal film, When the metal film is irradiated with light so that surface plasmon resonance is generated in a state where it is directly or indirectly coupled, a step of detecting fluorescence emitted from the fluorescent substance on the metal film, Including the times The lattice includes a plurality of types of divided regions each having a different lattice pitch, and in the step of detecting the fluorescence, the metal film is irradiated with light so that the plurality of types of the divided regions are simultaneously included in an irradiation spot. .

  In order to solve the above problems, a detection device according to an embodiment of the present invention emits light from a fluorescent substance on the metal film when the metal film of the detection chip is irradiated with light so that surface plasmon resonance occurs. A detection device for detecting the presence or amount of a substance to be detected by detecting the fluorescence to be detected, comprising a support and a metal film including a diffraction grating disposed on the support. A holder for holding a detection chip, a light irradiating unit for irradiating the metal film of the detection chip held by the holder with light, and a substance to be detected that is labeled with a fluorescent substance on the metal film directly. When the light irradiating unit irradiates the metal film with light so that surface plasmon resonance occurs on the metal film in a state where the fluorescent material is coupled to the fluorescent material on the metal film, Emitted fluorescence And a light detection unit for detecting, wherein the diffraction grating includes a plurality of types of divided regions each having a different grating pitch, and the light irradiation unit includes a plurality of types of the divided regions simultaneously in an irradiation spot. The metal film is irradiated with light as described above.

  In order to solve the above problem, a detection system according to an embodiment of the present invention emits light from a fluorescent substance on the metal film when the metal film of the detection chip is irradiated with light so that surface plasmon resonance occurs. By detecting the fluorescence to be detected, a detection system for detecting the presence or amount of the substance to be detected, a support, and disposed on the support, a metal film including a diffraction grating, The diffraction grating has a detection chip including a plurality of divided regions each having a different grating pitch, a holder for holding the detection chip, and the metal film of the detection chip held by the holder. A light irradiating unit that irradiates light, and a substance to be detected, which is labeled with a fluorescent substance on the metal film, is directly or indirectly coupled to the light irradiating unit, and the light irradiating unit has a surface on the metal film. When irradiating the metal film with light such that plasmon resonance occurs, a light detection unit that detects fluorescence emitted from the fluorescent substance on the metal film, and The light irradiator irradiates the metal film with light so as to simultaneously include a plurality of types of the divided regions in the irradiation spot.

  ADVANTAGE OF THE INVENTION According to this invention, a to-be-detected substance can be detected with high precision, without scanning the incident angle of the excitation light with respect to a metal film.

FIG. 1 is a diagram illustrating a configuration of a detection system according to Embodiments 1 to 3. 2A and 2B are diagrams illustrating a configuration of the detection chip according to the first embodiment. FIG. 3 is a perspective view of a diffraction grating in the detection chip according to the first embodiment. FIG. 4 is a schematic diagram illustrating a relationship between a diffraction grating and an irradiation spot of excitation light in the detection chip according to the first embodiment. FIG. 5 is a schematic cross-sectional view illustrating a detection chip according to a modification. FIG. 6 is a flowchart illustrating an example of an operation procedure of the surface plasmon excitation enhanced fluorescence detection device according to the first embodiment. FIGS. 7A and 7B are graphs showing the relationship between the angle of incidence of the excitation light on the metal film and the amount of reflected light of the excitation light. FIG. 8 is a plan view showing a configuration of the detection chip according to the second embodiment. FIG. 9 is a schematic diagram illustrating a relationship between a diffraction grating and an irradiation spot of excitation light in the detection chip according to the second embodiment. 10A and 10B are diagrams illustrating a configuration of a detection chip according to the third embodiment. FIG. 11 is a schematic diagram showing a relationship between a diffraction grating and an irradiation spot of excitation light in the detection chip according to the third embodiment. FIGS. 12A and 12B are graphs showing the relationship between the incident angle of the excitation light on the metal film and the amount of reflected light of the excitation light.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the following description, a surface plasmon excitation-enhanced fluorescence detection device (hereinafter, also referred to as an “SPFS device”) that detects a substance to be detected using surface plasmon resonance (SPR) will be described as a representative example of the detection device.

[Embodiment 1]
(Detection system)
FIG. 1 is a diagram illustrating a configuration of a detection system 1 according to the first embodiment. The detection system 1 according to the present embodiment includes a detection chip 10 and an SPFS device 100. Hereinafter, the detection chip 10 and the SPFS device 100 according to the present embodiment will be described.

(Configuration of SPFS device and detection chip)
As shown in FIG. 1, the SPFS device 100 includes a light irradiation unit (light irradiation unit) 110 for irradiating the detection chip 10 with light, and a light receiving unit (light detection unit) for detecting the fluorescence β emitted from the detection chip 10. ) 120, a liquid sending unit 130 for sending a liquid, a transport unit 140 for transporting the detection chip 10, and a control unit (processing unit) 150 for controlling these. The SPFS apparatus 100 is used with the detection chip 10 mounted on the chip holder 142 of the transport unit 140. Therefore, the detection chip 10 will be described first, and then each component of the SPFS device 100 will be described.

  2A and 2B are diagrams illustrating a configuration of the detection chip 10 according to the first embodiment. FIG. 2A is a plan view of the detection chip 10, and FIG. 2B is a cross-sectional view taken along line BB in FIG. 2A. FIG. 3 is a perspective view of the diffraction grating 35 in the detection chip 10 according to the first embodiment. Hereinafter, as shown in FIGS. 2A, 2B, and 3, the height direction of the detection chip 10 is defined as the z direction, the arrangement direction of the periodic structure of the diffraction grating 35 is defined as the x direction, and the direction perpendicular to the z direction and the x direction is defined. Let the direction be the y direction. FIG. 4 is a schematic diagram illustrating a relationship between the diffraction grating 35 and the irradiation spot 60 when the metal film 30 of the detection chip 10 according to the present embodiment is irradiated with the excitation light α. In the present embodiment, the shape of the irradiation spot 60 is a circular shape.

  As shown in FIGS. 2A and 2B, the detection chip 10 includes a substrate (support) 20, a metal film 30 including a diffraction grating 35 disposed on the substrate 20, and a frame disposed on the substrate 20. 40. By arranging the frame body 40 on the substrate 20, an accommodating portion for accommodating the liquid is formed.

  The substrate 20 is a support member for the metal film 30. The shape of the substrate 20 is not particularly limited. The material of the substrate 20 is not particularly limited as long as it has a mechanical strength capable of supporting the metal film 30. Examples of the material of the substrate 20 include inorganic materials such as glass, quartz, and silicon; and resins such as acrylic resin, polymethyl methacrylate, polycarbonate, polystyrene, and polyolefin.

  The metal film 30 is disposed on the substrate 20 with one surface facing the inside of the housing. As described above, the metal film 30 includes the diffraction grating 35. Thus, when the metal film 30 is irradiated with light at a predetermined incident angle, the surface plasmon generated in the metal film 30 and the evanescent wave generated by the diffraction grating 35 combine to generate surface plasmon resonance (SPR). The thickness of the metal film 30 is not particularly limited, and is, for example, 30 to 500 nm, and preferably 100 to 300 nm. The material of the metal film 30 is not particularly limited as long as it is a metal that can generate surface plasmons. Examples of the material of the metal film 30 include gold, silver, aluminum, platinum, copper, and alloys thereof.

  The diffraction grating 35 generates an evanescent wave when the metal film 30 is irradiated with the excitation light α. The diffraction grating 35 includes a plurality of types of divided regions having different grating pitches. Further, the plurality of types of divided regions each include a plurality of regions. As will be described later in detail, as shown in FIG. 4, when the diffraction grating 35 of the detection chip 10 is irradiated with the excitation light α, the divided area has a similar area in the irradiation spot 60 of the excitation light α. It is preferable that the divided areas are arranged so as to include a plurality of types of divided areas in proportion. For example, in the irradiation spot 60, the ratio of the total area of the types of divided regions having the largest total area to the total area of the types of divided regions having the smallest total area in the irradiation spot 60 is as follows. Preferably, they are arranged so as to be in the range of 4: 6 to 6: 4. Further, even if the position of the irradiation spot 60 of the excitation light α on the metal film 30 is shifted, it is preferable that the area ratio of the plurality of types of divided regions included in the irradiation spot 60 does not substantially change. For example, when the irradiation spot 60 of the excitation light α is moved along the surface direction of the metal film 30, the change rate of the area ratio of the plurality of types of division areas included in the irradiation spot 60 is different from each other. Preferably, they are arranged so as to be 30% or less. Here, the change rate of the ratio of the area of each divided region is the ratio of the area of each divided region included in the irradiation spot 60 before the movement of the irradiation spot 60 to the ratio of the area of the irradiation spot 60 after the movement of the irradiation spot 60. Means the magnitude of the change rate of the ratio of the area of each divided region included in.

  The number of types of divided regions is not particularly limited. However, even if the position of the irradiation spot 60 on the metal film 30 is shifted, from the viewpoint of suppressing the fluctuation of the ratio of the plurality of types of divided regions included in the irradiation spot 60, the number of types of the divided regions is It may be an even number. As shown in FIGS. 2 to 4, in the present embodiment, the number of types of divided regions is two. That is, the diffraction grating 35 includes the first divided region 36 and the second divided region 37. As shown in FIG. 3, the grating pitches of the diffraction grating 35 in the first divided region 36 and the second divided region 37 are different from each other. 2A, 2B, and 4, the first divided region 36 is shown in black, and the second divided region 37 is shown in white. The first divided region 36 and the second divided region 37 each include a plurality of regions.

  The external shape of the divided regions (the first divided region 36 and the second divided region 37) in plan view is not particularly limited. Examples of the planar view shape of the outer shape of the divided region include a square shape, a polygonal shape, a circular shape, and an elliptical shape. In the present embodiment, the outer shapes of the first divided region 36 and the second divided region 37 in plan view are quadrangular. As shown in FIG. 2A, the divided region is not adjacent to a different type of divided region in a first direction (y direction) along a plane direction (x direction and y direction) of the metal film 30, and In a second direction (x direction) along a plane direction (x direction and y direction) of the metal film 30, which is orthogonal to one direction (y direction), the metal film 30 is arranged to be adjacent to different types of divided regions.

  The lattice shape in each of the divided regions (the first divided region 36 and the second divided region 37) is not particularly limited as long as an evanescent wave can be generated. As shown in FIG. 3, in the present embodiment, each of the divided regions (the first divided region 36 and the second divided region 37) is a one-dimensional diffraction grating, and a plurality of grooves (concave stripes) parallel to each other. ) Are formed at predetermined intervals. The distance between the centers of adjacent grooves and the depth of the grooves are not particularly limited as long as an evanescent wave can be generated, and can be appropriately set according to the wavelength of light to be irradiated. For example, the distance between the centers of adjacent grooves is preferably in the range of 100 to 2000 nm, and the depth of the grooves is preferably in the range of 10 to 1000 nm. Further, the sectional shape of the groove is not particularly limited. Examples of the cross-sectional shape of the groove include a rectangular wave shape, a sine wave shape, and a sawtooth shape. In the present embodiment, the cross-sectional shape of the groove is a rectangular wave shape. Note that the optical axis of the excitation light α described later is parallel to the xz plane.

  A capturing body 50 for capturing a substance to be detected is fixed to the diffraction grating 35. The region where the capturing body 50 is fixed on the diffraction grating 35 (metal film 30) is particularly called a “reaction field”. In the present embodiment, the capturing body 50 is fixed substantially uniformly over the first divided region 36 and the second divided region 37, and the reaction field is applied to the first divided region 36 and the second divided region 37. It is formed over. Further, the capturing body 50 can specifically bind to the target substance. Therefore, the substance to be detected can be bound onto the metal film 30 (diffraction grating 35) via the capturing body 50.

  The type of the capturing body 50 is not particularly limited as long as the substance to be detected can be captured. For example, the capture body 50 is an antibody (primary antibody) or a fragment thereof capable of specifically binding to the analyte, an enzyme capable of specifically binding to the analyte, and the like.

  When the detection chip 10 is used, the diffraction grating 35 comes into contact with a liquid such as a buffer solution for operations such as reaction and washing. Therefore, usually, the diffraction grating 35 is arranged in a space that can accommodate the liquid. In the present embodiment, the diffraction grating 35 is disposed at the bottom of the housing. FIG. 5 is a schematic sectional view illustrating a detection chip 10 ′ according to a modification. The diffraction grating 35 may be disposed on the bottom surface of the housing (well) as in the present embodiment, or as shown in FIG. 5, a flow cell (flow cell) to which liquid can be continuously supplied. It may be arranged on the inner surface (for example, the bottom surface).

  The frame body 40 is a member having a through-hole, as shown in FIG. 2A, and is disposed on the substrate 20. The inner surface of the through hole is the side surface of the housing. The thickness of the frame body 40 is not particularly limited, and can be appropriately designed according to the amount of liquid stored in the storage section.

  The storage unit stores a liquid such as a specimen. The shape and size of the storage section are not particularly limited as long as a desired amount of liquid can be stored, and can be appropriately designed according to the application. As described above, the housing portion is formed by arranging the frame body 40 on the substrate 20, but the method of forming the housing portion is not limited to this. Another example of a method of forming the accommodation portion includes disposing a lid having a concave portion formed on the lower surface thereof on the substrate 20.

  The type of liquid stored in the storage section is not particularly limited. Examples of the type of liquid include a specimen containing a substance to be detected, a fluorescent 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 the refractive index and the dielectric constant 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.

(Method of manufacturing detection chip)
Next, an example of a method for manufacturing the detection chip 10 according to the present embodiment will be described. The method for manufacturing the detection chip 10 is not limited to this.

  The detection chip 10 according to the present embodiment includes, for example, 1) a step of preparing a substrate 20 and a frame 40, 2) a step of forming a metal film 30 and a reaction field on the substrate 20, and 3) a metal film. It can be manufactured by performing a step of fixing the frame 40 and the substrate 20 on which the reaction field 30 is formed and the frame 40 to each other. Hereinafter, each step will be described.

  First, the substrate 20 and the frame 40 are prepared. As the substrate 20, a substrate including two types of regions in which a plurality of grooves having different center-to-center distances are formed is prepared. At this time, the two types of regions each include a plurality of regions. For example, two types of regions may be formed by forming a plurality of grooves having different center-to-center distances on a flat resin substrate. The two types of regions are the first divided region 36 or the second divided region 37, respectively. The method for forming the plurality of grooves is not particularly limited, and may be appropriately selected from known methods. For example, the grooves may be formed by a mold forming method, a lithography method, or the like, or the grooves may be formed by performing press working using an original plate having unevenness.

  Further, as a preparation of the frame body 40, a through hole may be formed in a flat resin substrate. The method for forming the through holes is not particularly limited, and may be appropriately selected from known methods. For example, a through hole may be formed in a flat plate by a die molding method or the like. Moreover, you may purchase the flat plate in which the through-hole was formed.

  Next, a metal film 30 and a reaction field are sequentially formed on the substrate 20. The metal film 30 may be formed on a part of the surface of the substrate 20 where the groove is formed, or may be formed on the entire surface. The method for forming the metal film 30 is not particularly limited, and may be appropriately selected from known methods. Examples of the method of forming the metal film 30 include sputtering, vapor deposition, and plating. Next, the capturing body 50 may be directly or indirectly immobilized on the metal film 30. Thereby, a reaction field can be formed on the substrate 20.

Further, the method of fixing the capturing body 50 is not particularly limited. For example, a self-assembled monolayer (hereinafter, referred to as “SAM”) or a polymer film to which the capturing body 50 is bonded may be formed on the metal film 30. 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 50 may be immobilized on the metal film 30 and the capturing body 50 may be bound to the polymer.

  Note that the order of forming the metal film 30 and forming the diffraction grating 35 is not limited to the above method. For example, after forming the metal film 30 on the flat substrate 20, the metal film 30 may be provided with a lattice shape. According to this method, the first divided region 36 and the second divided region 37 can be arranged on the metal film 30.

  Next, the substrate 20 and the frame body 40 are fixed. The method for fixing the frame body 40 on the substrate 20 is not particularly limited. For example, examples of the method of fixing the substrate 20 and the frame body 40 include adhesion using a double-sided tape or an adhesive, laser welding, and ultrasonic welding.

  Through the above procedure, the detection chip 10 according to the present embodiment can be manufactured. The order in which the step of forming the metal film 30 and the reaction field and the step of fixing the substrate 20 and the frame 40 are not limited to this. For example, after fixing the frame 40 on the substrate 20, the metal film 30 and the reaction field may be formed.

  Next, each component of the SPFS device 100 will be described. As described above, the SPFS device 100 includes the light irradiation unit (light irradiation unit) 110, the light receiving unit (light detection unit) 120, the liquid sending unit 130, the transport unit 140, and the control unit (processing unit) 150.

Light irradiation unit 110 irradiates the excitation light α in the metal film 30 of the detection chip 10 held by the chip holder 142 at a predetermined incident angle theta ₁ (diffraction grating 35). At this time, the light irradiation unit 110 is configured such that a plurality of types (two types in the present embodiment) of the divided regions (the first divided region 36 and the second divided region 37) are simultaneously included in the light irradiation spot 60. The metal film 30 is irradiated with the excitation light α (see FIG. 4). The light irradiation unit 110 irradiates the metal film 30 (diffraction grating 35) with p-polarized light with respect to the surface of the metal film 30 so that the diffraction grating 35 generates diffracted light that can be combined with surface plasmons in the metal film 30. I do. The light amount in the irradiation spot 60 on the metal film 30 is preferably substantially uniform, and specifically, the ratio of the light amount between the point where the light amount is maximum and the point where the light amount is minimum is in the range of 4: 6 to 6: 4. Is preferably within the range. The fluorescent light 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.

  Further, the optical axis of the excitation light α is along the arrangement direction (x direction) of the periodic structure in the diffraction grating 35. Therefore, the optical axis of the excitation light α is parallel to the xz plane (see FIG. 1). Since the excitation light α is p-polarized light with respect to the surface of the metal film 30, 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 30. Parallel.

  The light irradiation unit 110 includes a light source unit 111 that emits the excitation light α, and a light source control unit 112 that controls various devices included in the light source unit 111.

  The light source unit 111 emits the excitation light α. For example, the light source unit 111 has a light source for the excitation light α, a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).

  The type of the light source is not particularly limited. Examples of light source types include laser diodes (LDs), light emitting diodes, mercury lamps, and other laser light sources. For example, the wavelength of the excitation light α emitted from the light source is in the range of 400 nm to 1000 nm.

  When the excitation light α emitted from the light source is not a beam, the excitation light α emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like. When the excitation light α emitted from the light source is not monochromatic light, the excitation light α emitted from the light source is converted into monochromatic light by a diffraction grating or the like. Further, when the excitation light α emitted from the light source is not linearly polarized light, the excitation light α emitted from the light source is converted into linearly polarized light by a polarizer or the like.

  The beam shaping optical system includes, for example, a collimator, a bandpass filter, a linear polarization filter, a half-wave plate, a slit, a zoom unit, and the like. The beam shaping optical system may include all of them, or may include some of them.

  The collimator collimates the excitation light α emitted from the light source.

  The band-pass filter converts the excitation light α emitted from the light source into narrow-band light having only the center wavelength. This is because the excitation light α from the light source has a slight wavelength distribution width.

  The linearly polarized light filter converts the excitation light α emitted from the light source into a completely linearly polarized light. The half-wave plate adjusts the polarization direction of the excitation light α so that the light of the p-polarized component enters the metal film 30. The slit and zoom means adjust the contour shape and beam diameter (for example, φ1 mm) of the excitation light α so that the shape and area of the irradiation spot 60 on the metal film 30 have a predetermined shape and area.

  The shape and area of the irradiation spot 60 are not particularly limited, and can be appropriately set according to the shape and area of the plurality of types of divided regions. Examples of the shape of the irradiation spot 60 include a square, a polygon, a circle, and an ellipse. As described above, in the present embodiment, the shape of the irradiation spot 60 is circular.

  The APC mechanism controls the light source so that the output of the light source becomes constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light α using a photodiode (not shown) or the like. Then, the APC mechanism controls the output of the light source to be constant by controlling the input energy by the regression circuit.

  The temperature adjustment mechanism is, for example, a heater, a Peltier element, or the like. The wavelength and energy of the light emitted from the light source may fluctuate depending on the temperature. Therefore, by keeping the temperature of the light source constant by the temperature adjustment mechanism, the wavelength and energy of the light emitted from the light source are controlled to be constant.

  The light source control unit 112 controls various devices included in the light source unit 111 to adjust the power, irradiation time, and the like of the excitation light α from the light source unit 111. The light source control unit 112 includes, for example, a known computer or a microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

  The light receiving unit 120 is configured to allow the light irradiation unit 110 to generate SPR on the metal film 30 in a state where the detection target substance labeled with a fluorescent substance is directly or indirectly bonded to the metal film 30. When the metal film 30 is irradiated with light, fluorescence β emitted from the fluorescent substance on the metal film 30 is detected. As shown in FIG. 1, the light receiving unit 120 passes through the intersection of the optical axis of the excitation light α and the metal film 30 with respect to the light irradiation unit 110 and sandwiches a normal to the surface of the metal film 30. Are located.

  The light receiving unit 120 has a light receiving sensor 121 and a light receiving sensor control unit 122. The light receiving unit 120 may further include a condenser lens group, an aperture stop, a fluorescent filter, and the like.

  The light receiving sensor 121 detects the fluorescence β emitted from the fluorescent substance existing on the metal film 30 and detects the fluorescent image on the metal film 30. The type of the light receiving sensor 121 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 light receiving sensor control unit 122 detects the output value of the light receiving sensor 121, manages the sensitivity of the light receiving sensor 121 based on the detected output value, and controls the sensitivity of the light receiving sensor 121 to obtain an appropriate output value. The light receiving sensor control unit 122 is configured by, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

  The liquid sending unit 130 supplies liquids such as a specimen, a fluorescent labeling liquid, and a washing liquid into a storage portion of the detection chip 10 held by the chip holder 142, and removes these liquids from the storage portion. The liquid sending unit 130 includes a liquid chip 131, a syringe pump 132, and a liquid sending pump driving mechanism 135.

  The liquid chip 131 is a container that stores liquids such as a specimen, a fluorescent labeling liquid, and a washing liquid. As the liquid chip 131, usually, a plurality of containers are arranged according to the type of liquid, or a chip in which a plurality of containers are integrated is arranged.

  The syringe pump 132 includes a syringe 133 and a plunger 134 that can reciprocate in the syringe 133. By the reciprocating motion of the plunger 134, the liquid is sucked and discharged quantitatively. If the syringe 133 is replaceable, washing of the syringe 133 becomes unnecessary. Therefore, it is preferable from the viewpoint of preventing impurities from being mixed. When the syringe 133 is not configured to be replaceable, it is possible to use the syringe 133 without replacing it by adding a configuration for cleaning the inside of the syringe 133.

  The liquid feeding pump driving mechanism 135 includes a driving device for the plunger 134 and a moving device for the syringe pump 132. The driving device of the syringe pump 132 is a device for reciprocating the plunger 134, and includes, for example, a stepping motor. The driving device including the stepping motor can control the amount and speed of liquid supply of the syringe pump 132, and is therefore preferable from the viewpoint of managing the amount of liquid remaining in the detection chip 10. The moving device of the syringe pump 132, for example, freely moves the syringe pump 132 in two directions: an axial direction (for example, a vertical direction) of the syringe 133 and a direction transverse to the axial direction (for example, a horizontal direction). The moving device of the syringe pump 132 is constituted by, for example, a robot arm, a two-axis stage, or a vertically movable turntable.

  The liquid sending unit 130 sucks various liquids from the liquid chip 131 and supplies the liquid to the storage section of the detection chip 10. When the storage part is a flow path, the liquid reciprocates in the flow path by moving the plunger 134, and the liquid in the flow path is stirred. Thereby, it is possible to make the concentration distribution of the liquid uniform and promote a reaction in the flow channel (for example, a primary reaction and a secondary reaction described later). The liquid in the container is sucked again by the syringe pump 132 and discharged to the liquid chip 131 or the like. By repeating these operations, a reaction with various liquids, washing, and the like are performed, and a substance to be detected or the like, which is labeled with a fluorescent substance, can be arranged in the container.

  The transport unit 140 transports and fixes the detection chip 10 to an installation position, a detection position, or a liquid sending position. Here, the “installation position” is a position for installing the detection chip 10 in the SPFS device 100. The “detection position” is a position where the light irradiation unit 110 irradiates the detection chip 10 with the excitation light α and the light-receiving unit 120 detects the fluorescence β generated thereby. Further, the “liquid sending position” is a position at which the liquid sending unit 130 supplies the liquid into the storage part of the detection chip 10 or removes the liquid from the storage part of the detection chip 10.

  The transfer unit 140 includes a transfer stage 141 and a chip holder 142.

  The chip holder 142 is fixed on the transfer stage 141 and holds the detection chip 10 in a detachable manner. The shape of the chip holder 142 is a shape that can hold the detection chip 10 and does not obstruct the optical path of light such as excitation light α, fluorescence β, and reflected light γ.

  The transfer stage 141 moves the chip holder 142 in one direction and the opposite direction. The transfer stage 141 also has a shape that does not hinder the optical path of light such as excitation light α, fluorescence β, and reflected light γ. The transfer stage 141 is driven by, for example, a stepping motor.

  The control unit 150 controls the light source control unit 112, the light receiving sensor control unit 122, the liquid feed pump driving mechanism 135, and the transport stage 141. Further, the control unit 150 also functions as a processing unit for calculating a signal value indicating the presence or the amount of the substance to be detected based on the detection result of the light receiving sensor 121. The control unit 150 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 100 (a detection method according to the present embodiment) will be described. FIG. 6 is a flowchart illustrating an example of an operation procedure of the SPFS device 100.

  First, preparation for detection is performed (step S10). Specifically, the detection chip 10 is prepared, and the detection chip 10 is installed on the chip holder 142 arranged at the installation position of the SPFS device 100. If a humectant exists on the metal film 30 of the detection chip 10, the humectant is removed by washing the metal film 30 so that the capturing body 50 can appropriately capture the substance to be detected.

  Next, in the state where no fluorescent substance is present on the metal film 30, light containing light having the same wavelength as the fluorescence β is detected, and an optical blank value is measured (step S20). Here, the “optical blank value” means the amount of background light emitted above the detection chip 10.

  Specifically, the control unit 150 controls the transport stage 141 to move the detection chip 10 from the installation position to the detection position. Thereafter, the control unit 150 controls the light source control unit 112 to irradiate the excitation light α from the light source unit 111 of the light irradiation unit 110 to a predetermined position of the metal film 30 (diffraction grating 35). Controls the light receiving sensor control unit 122 to detect light with the light receiving sensor 121 and obtain an optical blank value. The measured optical blank value is transmitted to the control unit (processing unit) 150 and stored. Although details will be described later, at this time, the light irradiation unit 110 irradiates the excitation light α to the metal film 30 so that the first divided region 36 and the second divided region 37 are simultaneously included in the irradiation spot 60.

  Next, the substance to be detected in the sample is bound to the capturing body 50 (primary reaction; step S30). Specifically, the control unit 150 controls the transport stage 141 to move the detection chip 10 from the detection position to the liquid sending position. Thereafter, the control unit 150 controls the liquid sending pump driving mechanism 135 to provide the sample in the liquid chip 131 to the inside of the storage unit. Thus, when the target substance is present in the sample, at least a part of the target substance is captured by the capturing body 50 on the metal film 30. Thereafter, the inside of the storage section is washed with a buffer solution or the like to remove substances not captured by the capturing body 50.

  Next, the target substance bound to the capturing body 50 is labeled with a fluorescent substance (secondary reaction; step S40). Specifically, the control unit 150 controls the liquid feed pump driving mechanism 135 to supply the fluorescent labeling liquid in the liquid chip 131 into the storage unit. This allows the substance to be detected to be labeled with the fluorescent substance. The fluorescent labeling solution is, for example, a buffer containing an antibody (secondary antibody) labeled with a fluorescent substance. Thereafter, the inside of the storage section is washed with a buffer or the like to remove free fluorescent substances and the like.

  Next, the excitation light α is irradiated to the metal film 30 (diffraction grating 35) in a state where the detection target substance labeled with the fluorescent substance is directly or indirectly bonded to the metal film 30 (diffraction grating 35). Then, the fluorescence β emitted from the fluorescent substance labeling the substance to be detected on the metal film 30 is detected, and the fluorescence value is measured (Step S50).

  Specifically, the control unit 150 controls the transport stage 141 to move the detection chip 10 from the liquid sending position to the detection position. Thereafter, the control unit 150 controls the light source control unit 112 to emit the excitation light α from the light source unit 111 of the light irradiation unit 110 toward the metal film 30 (diffraction grating 35). At the same time, the control unit 150 controls the light receiving sensor control unit 122 so that the light receiving sensor 121 detects the fluorescence β. Thereby, the light receiving sensor 121 can measure the fluorescence value. The measured fluorescence value is transmitted to the control unit (processing unit) 150 and recorded. At this time, the light irradiation unit 110 irradiates the excitation light α to the metal film 30 so that the first divided region 36 and the second divided region 37 are simultaneously included in the irradiation spot 60. As a result, the fluorescence β emitted from the fluorescent substance labeling the target substance bound on the first divided region 36 and the fluorescent substance labeling the target substance bound on the second divided region 37 The emitted fluorescence β can be detected.

  Finally, a signal value indicating the presence or the amount of the substance to be detected is calculated (step S60). The fluorescence value mainly includes a fluorescence component (signal value) derived from a fluorescent substance that labels the substance to be detected, and an optical blank value derived from noise. Therefore, the control unit (processing unit) 150 can calculate a signal value correlated with the amount of the target substance by subtracting the optical blank value obtained in step S20 from the fluorescence value obtained in step S50. . The signal value is converted to the amount or concentration of the substance to be detected by a calibration curve created in advance.

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

  Since the SPFS device 100 according to the present embodiment uses the detection chip 10 according to the present embodiment, it detects the substance to be detected with high accuracy without scanning the incident angle of the excitation light α to the metal film 30. be able to. Hereinafter, this point will be described. For comparison, a case where a detection chip including a diffraction grating having one kind of grating pitch (hereinafter, also referred to as a “detection chip for comparison”) will be described. 7A and 7B are graphs showing the relationship between the angle of incidence of the excitation light α on the metal film and the amount of reflected light γ of the excitation light α. FIG. 7A is a graph when the detection chip for comparison is used, and FIG. 7B is a graph when the detection chip 10 according to the present embodiment is used.

  As described above, the optimum incident angle of the excitation light α for generating the SPR depends on the grating pitch of the diffraction grating 35, the wavelength of the excitation light α, and the like. In the SPFS device 100, when the excitation light α is applied to the metal film 30 so as to generate SPR, the fluorescence β is emitted from the metal film 30. At this time, when the amount of the reflected light γ is the smallest, the SPR becomes maximum, and the amount of the emitted fluorescent light β also becomes the maximum. The incident angle at this time is called a resonance angle (or enhancement angle). Then, as the incident angle of the excitation light α deviates from the resonance angle, the amount of reflected light γ increases, and the amount of fluorescence β decreases. For this reason, when the relationship between the incident angle of the excitation light α to the metal film 30 and the amount of the reflected light γ is graphed using a conventional detection chip (a detection chip for comparison), the amount of the reflected light γ is represented by the resonance angle. The curve becomes a downward convex curve which becomes the minimum (see FIG. 7A).

  As shown in FIG. 7A, when the detection chip for comparison is used, when the incident angle of the excitation light α changes from the resonance angle (about 9.1 °) by ± 0.4 °, the amount of the reflected light γ increases. I do. An increase in the amount of the reflected light γ at this time corresponds to a decrease of 4% of the signal value. This means that in the SPFS device using the conventional detection chip (detection chip for comparison), if the incident angle of the excitation light α deviates from the resonance angle, the amount of the detected fluorescence β greatly decreases. I do. Therefore, in order to realize highly accurate detection of the substance to be detected, a step for determining an optimum incident angle (resonance angle) of the excitation light α with respect to the metal film is required. Therefore, the conventional SPFS device using the conventional detection chip requires a scanning mechanism for scanning the incident angle of the excitation light α on the metal film. As a result, the SPFS device is increased in size and cost. Further, since it takes time to determine the resonance angle, the detection time becomes long.

  On the other hand, the metal film 30 of the detection chip 10 according to the present embodiment has the diffraction grating 35 including two types of divided regions (the first divided region 36 and the second divided region 37) having different grating pitches. Then, in SPFS apparatus 100 according to the present embodiment, light irradiation unit 110 irradiates metal film 30 with excitation light α such that first divided region 36 and second divided region 37 are simultaneously included in irradiation spot 60. I do. Therefore, as shown in FIG. 7B, the amount of reflected light γ is the amount of reflected light γ from the first divided region 36 (see one broken line in FIG. 7B) and the amount of reflected light γ from the second divided region 37. (See the other broken line in FIG. 7B). Note that the amount of reflected light γ from the first divided region 36 (or the second divided region 37) indicated by a broken line in FIG. 7B indicates that the first divided region 36 (or the second divided region 37) Is the amount of reflected light γ when it is assumed that Therefore, when the area of the first divided region 36 and the area of the second divided region 37 in the irradiation spot 60 are the same, the amount of reflected light γ from the first divided region 36 (or the second divided region 37) Is half the value read from the dashed line in FIG. 7B. Therefore, the total light amount of the reflected light γ from the irradiation spot 60 shown by the solid line in FIG. 7B is calculated as a value of 和 of the sum of the values read from the two curves shown by the broken lines at each incident angle. . When the grating pitches of the first divided region 36 and the second divided region 37 are appropriately set, the total amount of the reflected light γ from the irradiation spot 60 indicated by the solid line in FIG. Also hardly changes. Specifically, in the SPFS device 100 according to the present embodiment using the detection chip 10 according to the present embodiment, the incident angle is ± 0.2 mm from the resonance angle (the incident angle at which the total amount of the reflected light γ is the smallest). Even if it changes by 4 °, the total amount of reflected light γ hardly increases. An increase in the total amount of reflected light γ at this time corresponds to a decrease of only 1.3% of the signal value. This means that in the SPFS device 100 according to the present embodiment, even if the incident angle of the excitation light α to the metal film 30 deviates from the resonance angle, the amount of the detected fluorescence β hardly changes. Therefore, the SPFS apparatus 100 according to the present embodiment can detect the target substance with high accuracy without scanning the incident angle of the excitation light α on the metal film 30.

  On the other hand, in the SPFS device 100 according to the present embodiment, excitation light α is applied to metal film 30 at an incident angle different from the optimum incident angle (resonance angle) for each of first divided region 36 and second divided region 37. I do. Therefore, the excitation light is applied to the metal film 30 at an incident angle between the optimal incident angle (for example, 8.1 °) for the first divided region 36 and the optimal incident angle (for example, 10.1 °) for the second divided region 37. When α is irradiated, the SPR does not reach the maximum in both the first divided region 36 and the second divided region 37. This can also be understood from the fact that the amount of reflected light γ of the curve shown by the solid line is larger than the amount of reflected light γ of the curve shown by the broken line in the graph of FIG. 7B. As a result, the amount of the detected fluorescence β decreases, but this is not a serious problem. For example, by increasing the power of the excitation light α to increase the amount of the detected fluorescence β, the detection accuracy of the target substance can be maintained.

  Further, as shown by the solid line in the graph of FIG. 7B, in the detection chip 10 according to the present embodiment, it is preferable that the amount of the reflected light γ does not change even if the incident angle of the excitation light α is changed. That is, in the graph of FIG. 7B, it is preferable that the ratio of the components derived from the two divided regions indicated by the broken lines be adjusted so that the slope of the curve indicated by the solid line becomes small. That is, it is preferable that the irradiation spot 60 of the excitation light α includes a plurality of types of divided regions at the same area ratio. For example, in the irradiation spot 60, the ratio of the total area of the types of divided regions having the largest total area to the total area of the types of divided regions having the smallest total area is 4: 6 to 6: 4. Is preferably within the range.

(simulation)
Here, the relationship between the grating pitch of the diffraction grating and the resonance angle was simulated. Specifically, the resonance angle when excitation light α having a wavelength of 635 nm was made incident on a diffraction grating having a grating pitch of 350 nm or 400 nm was obtained by simulation. Table 1 shows the results of the simulation. Table 1 shows that the resonance angle changes by 2 ° when the grating pitch differs by 10 nm.

  As shown by the solid line in the graph of FIG. 7B, in the detection chip 10 according to the present embodiment, the first divided region 36 is formed so that the amount of the reflected light γ does not change even if the incident angle of the excitation light α is changed. It is preferable that the grating pitch of the second divided region 37 is set. That is, in the graph of FIG. 7B, it is preferable to adjust the positions of the two curves shown by the broken lines so that the slope of the curve shown by the solid line becomes small. As can be seen from the above simulation, by adjusting the lattice pitch of the first divided region 36 and the second divided region 37, the resonance angle of the first divided region 36 and the second divided region 37 is adjusted, that is, FIG. 7B Can be moved in the horizontal direction at the positions of the two curves indicated by the broken lines in the graph of FIG. Therefore, it is preferable to set the grating pitch of the first divided region 36 and the second divided region 37 so that the amount of the reflected light γ does not change even if the incident angle of the excitation light α is changed.

(effect)
As described above, by using the detection chip 10 according to the present embodiment, in the detection method according to the present embodiment, without scanning the incident angle of the excitation light α with respect to the metal film 30, The substance to be detected can be detected with high accuracy. In the SPFS device 100 and the detection system 1 according to the present embodiment, a scanning mechanism for the incident angle of the excitation light α is not required, and it is possible to prevent the device from increasing in size and cost.

[Embodiment 2]
(Detection system)
FIG. 1 is a diagram illustrating a configuration of a detection system 2 according to the second embodiment. The detection system 2 according to the present embodiment includes the detection chip 210 and the SPFS device 100. Hereinafter, the detection chip 210 and the SPFS device 100 according to the present embodiment will be described.

(Configuration of detection chip and SPFS device)
FIG. 8 is a plan view of the detection chip 210 according to the second embodiment. In the detection chip 210, only the diffraction grating 235 of the metal film 230 is different from the detection chip 10 according to the first embodiment. Therefore, the same components as those described in the first embodiment are denoted by the same reference numerals, and description thereof is omitted. Further, the detection method and the SPFS device using the detection chip 210 according to the present embodiment are the same as the detection method and the SPFS device 100 according to the first embodiment, and thus description thereof will be omitted. FIG. 9 is a schematic diagram showing the relationship between the diffraction grating 235 and the irradiation spot 260 of the excitation light α in the detection chip 210 according to the second embodiment. In the present embodiment, the shape of the irradiation spot 260 is a square shape.

  The metal film 230 is arranged on the substrate 20 with one surface facing the inside of the housing. The metal film 230 includes a diffraction grating 235. The thickness and material of the metal film 230 are not particularly limited, and are the same as those of the diffraction grating 35 in the detection chip 10 according to the first embodiment.

  The diffraction grating 235 generates an evanescent wave when the metal film 230 is irradiated with the excitation light α. The diffraction grating 235 includes a plurality of types of divided regions having different grating pitches. Further, the plurality of types of divided regions each include a plurality of regions. As shown in FIG. 8, in the present embodiment, diffraction grating 235 includes first divided region 236 and second divided region 237 having different grating pitches. In FIG. 8, the first divided region 236 is shown in black, and the second divided region 237 is shown in white. In the present embodiment, the outer shapes of the first divided area 236 and the second divided area 237 in plan view are quadrangular. The divided regions (the first divided region 236 and the second divided region 237) are arranged such that the sides of the first divided region 236 and the sides of the second divided region 237 face each other. The distance between the centers of the adjacent grooves in each of the divided regions (the first divided region 236 and the second divided region 237), the depth of the groove, and the sectional shape of the groove are not particularly limited as long as an evanescent wave can be generated. This is the same as the detection chip 10 according to the first embodiment.

In addition, even if the position of the irradiation spot 260 of the excitation light α on the metal film 230 is shifted, from the viewpoint of suppressing a change in the area ratio of a plurality of types of divided regions included in the irradiation spot 260, The shape and the shape of the plurality of types of divided regions are preferably the same, and are preferably similar. Further, it is preferable that the areas of the respective regions included in the first divided region 236 and the second divided region 237 are substantially equal to each other. Area of each region included in the first divided area 236 and a second divided region 237 is not particularly limited, for example, when the area of radiation spots 260 is 1 mm ^2, it is 0.4 to 0.6 mm ^2. It is preferable that the irradiation spot 260 includes the same number of each of the regions included in the divided regions. On the other hand, if the area of each region included in the divided region is sufficiently small compared to the area of the irradiation spot 260, the shape of the irradiation spot 260 and the shape of the divided region may be different. At this time, the area ratio of each divided area to the irradiation spot 260 is preferably 0.01 or more and 0.5 or less. For example, when the area of the irradiation spot 260 is 1 mm ² and the area of each area included in the divided area is 0.01 mm ² or more and 0.5 mm ² or less, the area of each area included in the divided area is irradiated. It can be said that the spot 260 is sufficiently small.

  As shown in FIG. 9, in the present embodiment, the shape of irradiation spot 260 and the shape of divided regions (first divided region 236 and second divided region 237) are both quadrangular, and have similar shapes. is there. The irradiation spot 260 includes two first divided regions 236 and two second divided regions 237.

(effect)
Even when the detection chip 210 according to the present embodiment is used, similarly to the first embodiment, the substance to be detected can be detected with high accuracy without scanning the angle of incidence of the excitation light α on the metal film 230. it can.

[Embodiment 3]
(Detection system)
FIG. 1 is a diagram illustrating a configuration of a detection system 3 according to a third embodiment. The detection system 3 according to the present embodiment includes the detection chip 310 and the SPFS device 100. Hereinafter, the detection chip 310 and the SPFS device 100 according to the present embodiment will be described.

(Configuration of detection chip and SPFS device)
FIGS. 10A and 10B are diagrams showing a configuration of the detection chip 310 according to the third embodiment. FIG. 10A is a plan view of the detection chip 310, and FIG. 10B is a cross-sectional view taken along line BB in FIG. 10A. In the detection chip 310, only the diffraction grating 335 of the metal film 330 is different from the detection chip 10 according to the first embodiment. Therefore, the same components as those described in the first embodiment are denoted by the same reference numerals, and description thereof is omitted. Further, the detection method and the SPFS device using the detection chip 310 according to the present embodiment are the same as the detection method and the SPFS device 100 according to the first embodiment, and thus description thereof will be omitted. FIG. 11 is a schematic diagram showing a relationship between the diffraction grating 335 and the irradiation spot 360 of the excitation light α in the detection chip 310 according to the third embodiment. In the present embodiment, the shape of irradiation spot 360 is a square shape.

  The metal film 330 is disposed on the substrate 20 with one surface facing the inside of the housing. The metal film 330 includes a diffraction grating 335. The thickness and material of the metal film 330 are not particularly limited, and are the same as those of the diffraction grating 35 in the detection chip 10 according to the first embodiment.

  The diffraction grating 335 generates an evanescent wave when the metal film 330 is irradiated with the excitation light α. The diffraction grating 335 includes a plurality of types of divided regions having different grating pitches. From the viewpoint of widening the incident angle range of the excitation light α in which the reduction rate of the signal value is suppressed, the number of types of the divided regions is preferably three or more. Further, the plurality of types of divided regions each include a plurality of regions. As shown in FIG. 10A, in the present embodiment, as shown in FIGS. 10A and 10B, the number of types of divided regions is three, and diffraction grating 335 has the first divided regions having different grating pitches. An area 336, a second divided area 337 and a third divided area 338 are included. In FIGS. 10A, 10B, and 11, the first divided region 336 is shown in black, the second divided region 337 is shown in white, and the third divided region 338 is shown by hatching. In the present embodiment, the outer shapes of the first divided region 336, the second divided region 337, and the third divided region 338 are quadrangular in plan view. As illustrated in FIG. 10A, the divided region is not adjacent to a different type of divided region in a first direction (y direction) along a plane direction (x direction and y direction) of the metal film 330, and In a second direction (x direction) along a plane direction (x direction and y direction) of the metal film 330, which is orthogonal to one direction (y direction), the metal film 330 is arranged to be adjacent to different types of divided regions.

  The distance between the centers of the adjacent grooves in each of the divided regions (the first divided region 336, the second divided region 337, and the third divided region 338), the depth of the groove, and the sectional shape of the groove may cause an evanescent wave. The configuration is not particularly limited as long as it can be performed, and is similar to the detection chip 10 according to the first embodiment. In the present embodiment, the distance between the centers of adjacent grooves in the first divided region 336 is 400 nm, the distance between the centers of adjacent grooves in the second divided region 337 is 391.5 nm, and the distance between the centers of the adjacent grooves in the third divided region 338 is The distance between the centers of adjacent grooves is 380 nm. At this time, considering the above-described simulation result of the relationship between the grating pitch of the diffraction grating and the resonance angle, the difference between the resonance angle of the first divided region 336 and the resonance angle of the second divided region 337 is 1 0.7 °, and the difference between the resonance angle of the first divided region 336 and the resonance angle of the third divided region 338 is 4 °.

  The shape and area of the irradiation spot 360 are not particularly limited, and are the same as those of the irradiation spot 60 according to the first embodiment. Examples of the shape of the irradiation spot 360 include a square, a polygon, a circle, and an ellipse. As shown in FIG. 11, in the present embodiment, the shape of irradiation spot 360 is a square shape.

  Since the SPFS device 100 according to the present embodiment uses the detection chip 310 according to the present embodiment, it detects the substance to be detected with high accuracy without scanning the incident angle of the excitation light α to the metal film 330. be able to. Hereinafter, this point will be described. For comparison, a case where a detection chip for comparison is used will also be described. 12A and 12B are graphs showing the relationship between the angle of incidence of the excitation light α on the metal film and the amount of reflected light γ of the excitation light α. FIG. 12A is a graph when the detection chip for comparison is used, and FIG. 12B is a graph when the detection chip 310 according to the present embodiment is used.

  As described above, when the detection chip for comparison is used, when the incident angle of the excitation light α changes from the resonance angle (about 9.1 °), the amount of the reflected light γ increases, which decreases the signal value. (See FIG. 12A). At this time, the size of the incident angle range in which the signal reduction rate is suppressed within 1% is about 0.4 °.

  On the other hand, when the detection chip 310 according to the present embodiment is used, the total light amount of the reflected light γ from the irradiation spot 360 shown by the solid line in FIG. This is １ ／ of the sum of the light amounts of the reflected light γ from the divided area 336, the second divided area 337, and the third divided area 338). Then, as shown by the solid line in FIG. 12B, in the SPFS device 100 using the detection chip 310 according to the present embodiment, the magnitude of the incident angle range in which the rate of decrease in the signal value is suppressed to within 1% is: About 2 °. That is, when the detection chip 310 according to the present embodiment is used, the incident angle range in which the reduction rate of the signal value is suppressed within 1% is five times wider than when the detection chip for comparison is used. can do.

  Further, in the SPFS device 100 according to the present embodiment, the metal film 330 has an incident angle (resonance angle) different from the optimal incident angle (resonance angle) for each of the first divided region 336, the second divided region 337, and the third divided region 338. Is irradiated with excitation light α. Therefore, the SPR does not become maximum in any of the divided regions. This can be understood from the fact that the amount of reflected light γ in the curve shown by the solid line is larger than the amount of reflected light γ in the curve shown by the broken line in the graph of FIG. 12B. As a result, the amount of the detected fluorescence β decreases, but this is not a serious problem. For example, by increasing the power of the excitation light α to increase the amount of the detected fluorescence β, the detection accuracy of the target substance can be maintained.

  As shown by the solid line in the graph of FIG. 12B, in detection chip 310 according to the present embodiment, it is preferable that the amount of reflected light γ does not change even if the incident angle of excitation light α is changed. That is, in the graph of FIG. 12B, it is preferable that the ratio of the components derived from the three divided regions indicated by the broken lines be adjusted so that the slope of the curve indicated by the solid line becomes small. At this time, it is preferable that the divided regions are arranged such that a plurality of types of divided regions are included in the irradiation spot 360 of the excitation light α at substantially the same area ratio. For example, the ratio of the total area of the types of divided areas having the largest total area to the total area of the types of divided areas having the smallest total area in the irradiation spot 360 is 4: 6 to Preferably, they are arranged so as to be within the range of 6: 4.

(effect)
Even when the detection chip 310 according to the present embodiment is used, similarly to the first embodiment, the substance to be detected can be detected with high accuracy without scanning the angle of incidence of the excitation light α on the metal film 330. it can. Further, in the detection method, the detection device, and the detection system according to the present embodiment, as compared with the first embodiment, the incident angle range in which the amount of the reflected light γ does not change even when the incident angle of the excitation light α is changed. Can be further expanded.

  This application claims the priority based on Japanese Patent Application No. 2015-124071 filed on June 19, 2015. The entire contents described in the specification and drawings of the application are incorporated herein by reference.

  ADVANTAGE OF THE INVENTION According to the detection chip, the detection method, the detection apparatus, and the detection system which concern on this invention, since a to-be-detected substance can be detected with high reliability, it is useful for a clinical test etc., for example.

1, 2, 3 Detection system 10, 10'210, 310 Detection chip 20 Substrate (support)
30, 230, 330 Metal film 35, 235, 335 Diffraction grating 36, 236, 336 First division area 37, 237, 337 Second division area 338 Third division area 40 Frame 50 Capture body 60, 260, 360 Irradiation spot 100 Surface Plasmon Resonance Fluorescence Analyzer (SPFS)
110 Light irradiation unit (light irradiation unit)
111 light source unit 112 light source control unit 120 light receiving unit (light detection unit)
121 light receiving sensor 122 light receiving sensor control unit 130 liquid sending unit 131 liquid chip 132 syringe pump 133 syringe 134 plunger 135 liquid sending pump driving mechanism 140 transfer unit 141 transfer stage 142 chip holder 150 control unit (processing unit)
α excitation light β fluorescence γ reflected light

Claims (29)

When the metal film of the detection chip is irradiated with light so as to generate surface plasmon resonance, the presence or amount of the substance to be detected is detected by detecting the fluorescence emitted from the fluorescent substance on the metal film. Detection chip used for
A support,
A metal film including a diffraction grating, disposed on the support,
Has,
The diffraction grating includes a plurality of divided regions each having a different grating pitch,
The divided region, when the light in the metal layer in order to emit fluorescence from the fluorescent material is irradiated, a plurality of types of the divided regions to the irradiation spot of the light is arranged to be included at the same time,
The size of the irradiation spot is smaller than the size of the diffraction grating,
When the irradiation spot is moved along the surface direction of the metal film, the position of the irradiation spot before and after the movement is any position on the diffraction grating, and is included in the irradiation spot. The rate of change of the ratio of the area of the plurality of types of divided regions is 30% or less.
Detection chip. When the metal film of the detection chip is irradiated with light so that surface plasmon resonance is generated, the presence or amount of the substance to be detected is detected by detecting the fluorescence emitted from the fluorescent substance on the metal film. Detection chip used for
A support,
A metal film including a diffraction grating, disposed on the support,
Has,
The diffraction grating includes a plurality of divided regions each having a different grating pitch,
The divided regions are arranged such that when the metal film is irradiated with light to emit fluorescence from a fluorescent substance, a plurality of types of the divided regions are simultaneously included in an irradiation spot of the light,
  The incident angle of the excitation light applied to the metal film is an incident angle that is not a resonance angle with respect to the diffraction grating.
Detection chip.   When the irradiation spot is moved along the surface direction of the metal film, the position of the irradiation spot before and after the movement is any position on the diffraction grating and is included in the irradiation spot. 3. The detection chip according to claim 2, wherein the rate of change of the area ratio of each of the plurality of types of divided regions is 30% or less. The ratio of the total area of the divided areas of the type having the largest total area to the total area of the divided areas of the type having the smallest total area in the irradiation spot is 4: 6. The detection chip according to any one of claims 1 to 3, wherein the detection chip is arranged so as to fall within a range of 6 to 4: 6. The detection chip according to any one of claims 1 to 4, wherein the number of types of the divided regions is three or more. The number of types of split regions is an even number, the detection chip according to any one of claims 1-5. The detection chip according to claim 6 , wherein the number of types of the divided regions is two. Wherein the shape of the divided region, a polygonal shape or a circular shape, the detection chip according to any one of claims 1-7. The divided region includes a first divided region and a second divided region having a different lattice pitch from the first divided region,
The shapes of the first divided region and the second divided region are rectangular,
The plurality of first divided regions and the plurality of second divided regions are arranged such that sides of the first divided region and sides of the second divided region face each other.
A detection chip according to claim 7 . The plurality of types of the divided regions are not adjacent to the different types of the divided regions in a first direction along the surface direction of the metal film, and are orthogonal to the first direction. The detection chip according to any one of claims 1 to 8 , wherein the detection chip is arranged so as to be adjacent to the different types of the divided regions in a second direction along the direction. When the metal film of the detection chip is irradiated with light so that surface plasmon resonance is generated, the presence or amount of the substance to be detected is detected by detecting the fluorescence emitted from the fluorescent substance on the metal film. Detection method for
A detection chip having a support and a metal film including a diffraction grating disposed on the support, wherein a substance to be detected, which is labeled with a fluorescent substance, is directly or indirectly bound to the metal film. Process and
In the state where the detection target substance that is labeled with the fluorescent substance is directly or indirectly bound on the metal film, when the metal film is irradiated with light so that surface plasmon resonance is generated, Detecting fluorescence emitted from the fluorescent material on the metal film,
Including
The diffraction grating includes a plurality of divided regions each having a different grating pitch,
Wherein in the step of detecting the fluorescence, by irradiating the light on the metal film as the divided regions of the plurality of types are simultaneously contained in the irradiation spot of the light,
The size of the irradiation spot is smaller than the size of the diffraction grating,
When the irradiation spot is moved along the surface direction of the metal film, the position of the irradiation spot before and after the movement is any position on the diffraction grating and is included in the irradiation spot. The rate of change of the area ratio of each of the plurality of types of divided regions is 30% or less.
Detection method. When the metal film of the detection chip is irradiated with light so that surface plasmon resonance is generated, the presence or amount of the substance to be detected is detected by detecting the fluorescence emitted from the fluorescent substance on the metal film. Detection method for
A detection chip having a support and a metal film including a diffraction grating disposed on the support, wherein a substance to be detected, which is labeled with a fluorescent substance, is directly or indirectly bound to the metal film. Process and
In the state where the detection target substance that is labeled with the fluorescent substance is directly or indirectly bound on the metal film, when the metal film is irradiated with light so that surface plasmon resonance is generated, Detecting fluorescence emitted from the fluorescent material on the metal film,
Including
The diffraction grating includes a plurality of divided regions each having a different grating pitch,
In the step of detecting the fluorescence, the metal film is irradiated with the light so that the plurality of types of the divided regions are simultaneously included in the irradiation spot of the light,
The incident angle of the excitation light applied to the metal film is an incident angle that is not a resonance angle with respect to the diffraction grating.
Detection method. When the irradiation spot is moved along the surface direction of the metal film, the position of the irradiation spot before and after the movement is any position on the diffraction grating and is included in the irradiation spot. 13. The detection method according to claim 12, wherein the rate of change of the area ratio of each of the plurality of types of divided regions is 30% or less. In the irradiation spot, the ratio of the total area of the divided areas of the type having the largest total area to the total area of the divided areas of the type having the smallest total area is 4: 6 to 6: 4. The detection method according to any one of claims 11 to 13, wherein the detection method is within the range. The detection method according to any one of claims 11 to 14 , wherein the number of types of the divided regions is three or more. The detection method according to any one of claims 11 to 15 , wherein the number of types of the divided regions is an even number. 17. The detection method according to claim 16 , wherein the number of types of the divided regions is two. The detection method according to any one of claims 11 to 17 , wherein a shape of the irradiation spot and a shape of the divided region included in the irradiation spot are similar shapes of the same polygon. 19. The detection method according to claim 18 , wherein the shape of the irradiation spot and the shape of the divided region included in the irradiation spot are quadrangular. The shape of the irradiation spot is circular or elliptical,
The shapes of the divided areas are all the same, and are square, polygonal, or circular,
The area ratio of each region included in each of the plurality of types of divided regions to the irradiation spot is 0.01 or more and 0.5 or less, respectively.
The detection method according to any one of claims 11 to 17 . In the step of detecting the fluorescence, the metal film is irradiated with light such that a ratio of a light amount between a point where the light amount is maximum and a point where the light amount is minimum is 4: 6 to 6: 4 in the irradiation spot. The detection method according to any one of claims 11 to 20 , which performs the detection. When the metal film of the detection chip is irradiated with light so that surface plasmon resonance is generated, the presence or amount of the substance to be detected is detected by detecting the fluorescence emitted from the fluorescent substance on the metal film. A detection device for
Support, and a holder for holding a detection chip having a metal film including a diffraction grating, disposed on the support,
A light irradiation unit that irradiates light to the metal film of the detection chip held by the holder,
In a state in which a substance to be detected, which is labeled with a fluorescent substance, is directly or indirectly bound on the metal film, the light irradiating unit causes the metal film to generate surface plasmon resonance on the metal film. When irradiating light, a light detection unit that detects fluorescence emitted from the fluorescent substance on the metal film,
Has,
The diffraction grating includes a plurality of divided regions each having a different grating pitch,
The light irradiation unit irradiates the light to the metal film so as to include a plurality of types of the divided areas simultaneously to the irradiation spot of the light,
The size of the irradiation spot is smaller than the size of the diffraction grating,
When the irradiation spot is moved along the surface direction of the metal film, the position of the irradiation spot before and after the movement is any position on the diffraction grating and is included in the irradiation spot. The rate of change of the area ratio of each of the plurality of types of divided regions is 30% or less.
Detection device. When the metal film of the detection chip is irradiated with light so that surface plasmon resonance is generated, the presence or amount of the substance to be detected is detected by detecting the fluorescence emitted from the fluorescent substance on the metal film. A detection device for
Support, and a holder for holding a detection chip having a metal film including a diffraction grating, disposed on the support,
A light irradiation unit that irradiates light to the metal film of the detection chip held by the holder,
In a state in which a substance to be detected, which is labeled with a fluorescent substance, is directly or indirectly bound on the metal film, the light irradiating unit causes the metal film to generate surface plasmon resonance on the metal film. When irradiating light, a light detection unit that detects fluorescence emitted from the fluorescent substance on the metal film,
Has,
The diffraction grating includes a plurality of divided regions each having a different grating pitch,
The light irradiation unit irradiates the light to the metal film so as to include a plurality of types of the divided areas simultaneously to the irradiation spot of the light,
The incident angle of the excitation light applied to the metal film is an incident angle that is not a resonance angle with respect to the diffraction grating.
Detection device. 24. The detection device according to claim 22 , wherein a shape of the irradiation spot and a shape of the divided region included in the irradiation spot are similar shapes of the same polygonal shape. 25. The detection device according to claim 24 , wherein the shape of the irradiation spot and the shape of the divided region included in the front irradiation spot are square. The shape of the irradiation spot is circular or elliptical,
The shapes of the divided areas are all the same, and are square, polygonal, or circular,
The area ratio of each region included in each of the plurality of types of divided regions to the irradiation spot is 0.01 or more and 0.5 or less, respectively.
The detection device according to claim 22 or 23 . The light irradiating unit irradiates the metal film with light such that a ratio of a light amount between a point where the light amount is maximum and a point where the light amount is minimum is 4: 6 to 6: 4 in the irradiation spot. The detection device according to any one of claims 22 to 26. When the metal film of the detection chip is irradiated with light so that surface plasmon resonance is generated, the presence or amount of the substance to be detected is detected by detecting the fluorescence emitted from the fluorescent substance on the metal film. Detection system for
Support, and disposed on the support, a metal film including a diffraction grating, wherein the diffraction grating, a detection pitch including a plurality of each of a plurality of types of divided regions each having a different grating pitch,
A holder for holding the detection chip, a light irradiating unit that irradiates the metal film of the detection chip held by the holder with light, and a target substance that is labeled with a fluorescent substance on the metal film. When the light irradiation unit irradiates the metal film with light so that surface plasmon resonance occurs on the metal film in a state where the fluorescent material is directly or indirectly coupled, the fluorescent material on the metal film is A light detection unit that detects fluorescence emitted from
Has,
The light irradiation unit irradiates the light to the metal film so as to include a plurality of types of the divided areas simultaneously to the irradiation spot of the light,
The size of the irradiation spot is smaller than the size of the diffraction grating,
When the irradiation spot is moved along the surface direction of the metal film, the position of the irradiation spot before and after the movement is any position on the diffraction grating and is included in the irradiation spot. The rate of change of the area ratio of each of the plurality of types of divided regions is 30% or less.
Detection system. When the metal film of the detection chip is irradiated with light so that surface plasmon resonance is generated, the presence or amount of the substance to be detected is detected by detecting the fluorescence emitted from the fluorescent substance on the metal film. Detection system for
Support, and disposed on the support, a metal film including a diffraction grating, wherein the diffraction grating, a detection pitch including a plurality of each of a plurality of types of divided regions each having a different grating pitch,
A holder for holding the detection chip, a light irradiating unit that irradiates the metal film of the detection chip held by the holder with light, and a target substance that is labeled with a fluorescent substance on the metal film. When the light irradiation unit irradiates the metal film with light so that surface plasmon resonance occurs on the metal film in a state where the fluorescent material is directly or indirectly coupled, the fluorescent material on the metal film is A light detection unit that detects fluorescence emitted from
Has,
The light irradiation unit irradiates the light to the metal film so as to simultaneously include a plurality of types of the divided regions in the irradiation spot of the light,
The incident angle of the excitation light applied to the metal film is an incident angle that is not a resonance angle to the diffraction grating.
Detection system.

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