JP6702046B2 - Detection method and detection device - Google Patents

Description

  The present invention relates to a detection method for detecting the presence or amount of a substance to be detected using the detection chip after aligning the detection chip, and a detection device used in the detection method.

  In a measurement for detecting a biological substance such as protein or DNA, if a small amount of the substance to be detected can be detected with high sensitivity and quantitatively, it becomes possible to immediately grasp the condition of the patient and perform treatment. For this reason, there is a demand for an analysis method and an analysis device that detect highly sensitive and quantitatively weak light due to a trace amount of a substance to be detected. Surface plasmon resonance fluorescence spectrometry (Surface Plasmon-field enhanced Fluorescence Spectroscopy: SPFS) is known as one method for detecting a substance to be detected with high sensitivity.

  In SPFS, a dielectric member having a metal film arranged on a predetermined surface is used. Then, by irradiating the metal film with excitation light at an angle at which surface plasmon resonance occurs via the dielectric member, localized field light (enhanced electric field) can be generated on the surface of the metal film. This localized field light excites a fluorescent substance that labels the substance to be detected captured on the metal film.Therefore, by detecting the fluorescence emitted from the fluorescent substance, the presence or amount of the substance to be detected can be detected. Can be detected.

  In SPFS, in order to perform highly sensitive and highly accurate detection, it is necessary to arrange the detection chip at a predetermined position with high accuracy. In order to accurately detect the amount (concentration) of the substance to be detected, it is necessary to adjust the incident angle of excitation light with respect to the metal film with high accuracy. It is not possible to adjust the incident angle of the excitation light with respect to the high precision. On the other hand, disposing the detection chip at a predetermined position with high accuracy by the user is not preferable from the viewpoint of usability (usability).

  In SPFS, a method of aligning the detection chip has been proposed (for example, refer to Patent Document 1). In the method of aligning the detection chip in SPFS described in Patent Document 1, the detection chip is moved while irradiating the dielectric member with excitation light. At this time, the reflected light of the excitation light reflected by the dielectric member is detected by the light receiving sensor. Then, position information of the detection chip is acquired based on the amount of light received by the light receiving sensor, and the detection chip is aligned based on the acquired position information.

International Publication No. 2015/064704

  However, in the method of aligning the detection chip in the SPFS described in Patent Document 1, a part of the excitation light emitted is incident on the dielectric member at the incident surface of the dielectric member, and The light internally reflected in the body member may reach the light receiving sensor. In addition, due to the shape error of the dielectric member or the installation error of the detection chip, a part of the excitation light emitted reaches the light receiving sensor by being internally reflected in the dielectric member. There is. In this way, if not only the light externally reflected on the surface of the dielectric member, but also the light internally reflected on the dielectric member is detected by the light receiving sensor, these become stray light and are more accurately detected by an unexpected detection result. In some cases, the detection chip cannot be aligned.

  Therefore, an object of the present invention is a detection method and a detection device for detecting a substance to be detected by using the detection chip after aligning the detection chip, which is the detection chip by the light incident on the dielectric member. An object of the present invention is to provide a detection method and a detection device capable of suppressing a decrease in alignment accuracy.

  In order to solve the above problems, a detection method according to an embodiment of the present invention includes a first surface, a second surface, and a third surface, has a dielectric member transparent to light, and includes a substance to be detected. A chip holder for holding the detection chip captured on the surface side of the second surface, a moving stage for moving the chip holder, and the dielectric member of the dielectric member of the detection chip held by the chip holder. A light irradiating section that irradiates light toward the first surface and changes an irradiation angle of the light irradiating the first surface, and a part of the light irradiating from the light irradiating section by the dielectric member. A reflected light detection unit that detects reflected light, and a sample light detection unit that detects sample light generated according to the amount of the substance to be detected captured by the detection chip by irradiating light from the light irradiation unit. And a first reflected light of the light emitted from the light emitting unit that is reflected by the first surface and the first surface that is transmitted through the first surface. The irradiation angle of the light irradiated from the light irradiation unit with respect to the first surface is set so that the second reflected light sequentially reflected by the second surface and the third surface are simultaneously detected by the reflected light detection unit. And the detection held by the chip holder so that the irradiation spot of the light emitted from the light irradiation unit passes over the boundary between the first surface and another surface adjacent to the first surface. While the chip is moved by the moving stage, light is emitted from the light irradiation unit at the irradiation angle set in the step of setting the light irradiation angle, and the reflected light detection unit causes the first reflected light and the first reflected light to be emitted. A step of detecting two reflected lights, and acquiring position information of the detection chip held in the chip holder, based on the detection results of the first reflected light and the second reflected light by the reflected light detection unit, Based on the acquired position information of the detection chip, a step of moving the detection chip by the moving stage to a detection position for detecting the sample light, in a state where the detection chip is in the detection position, Irradiating light from the light irradiation unit, by detecting the sample light by the sample light detection unit, a step of detecting the presence or the amount of the substance to be detected captured in the detection chip at the detection position, including.

  In order to solve the above problems, a detection device according to an embodiment of the present invention includes a first surface, a second surface and a third surface, has a dielectric member transparent to light, A chip holder for holding a detection chip in which a detection substance is captured on the surface side of the second surface, a moving stage for moving the chip holder, and the dielectric member of the detection chip held by the chip holder. Of the light emitted from the light irradiator, and a dielectric of the light emitted from the light irradiator, the light irradiator changing the irradiation angle of the light irradiating the first surface with the light. A reflected light detection unit that detects reflected light from a member, and by irradiating light from the light irradiation unit, sample light that detects sample light generated according to the amount of the substance to be detected captured in the detection chip A detection unit, and a control unit that controls the moving stage, the light irradiation unit, the reflected light detection unit and the sample light detection unit, the control unit, of the light emitted from the light irradiation unit. Among them, the first reflected light reflected by the first surface and the second reflected light that is the light emitted from the light irradiation unit is transmitted through the first surface and sequentially reflected by the second surface and the third surface. So as to be detected by the reflected light detection unit at the same time, the light irradiation unit sets an irradiation angle of light, and in order to acquire position information of the detection chip, irradiation of light emitted from the light irradiation unit is performed. An irradiation angle of light while moving the detection chip held by the chip holder by the moving stage so that a spot passes on a boundary between the first surface and another surface adjacent to the first surface. The light is irradiated from the light irradiation unit at the irradiation angle set in the step of setting.

  According to the present invention, even when the light incident on the dielectric member is detected by the reflected light detection unit, the detection chip is accurately aligned to accurately detect the presence or amount of the substance to be detected. Can be detected.

FIG. 1 is a diagram schematically showing a configuration of a detection device according to an embodiment of the present invention. FIG. 2 is a flowchart showing the operation procedure of the detection device. FIG. 3 is a flowchart for explaining the step (step S130) of acquiring the position information of the detection chip. 4A to 4C are schematic diagrams for explaining the step (step S130) of acquiring the position information of the detection chip. 5A to 5C are views for explaining the step (step S134) of adjusting the irradiation angle of the excitation light. 6A and 6B are diagrams for explaining the relationship between the irradiation angle of the excitation light and the emission angle of the reflected light. FIG. 7 is a graph showing an example of the detection result of the first reflected light and the second reflected light by the light receiving sensor. FIG. 8 is a diagram showing another optical path of the excitation light. FIG. 9 is a flowchart for explaining a process of acquiring position information of another detection chip. FIG. 10 is a flowchart for explaining a process of acquiring the position information of another detection chip.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the following description, a detection method and a detection apparatus for detecting a substance to be detected by SPFS will be described as an embodiment of the detection method and the detection apparatus according to the present invention. Is not limited to this.

  FIG. 1 is a schematic diagram showing a configuration of a detection device (surface plasmon resonance fluorescence analysis device; SPFS device) 100 according to an embodiment of the present invention. As shown in FIG. 1, the detection device 100 includes an excitation light irradiation unit 110, a reflected light detection unit 120, a fluorescence detection unit 130, a liquid feeding unit 140, a transport unit 150, and a controller 160. The detection device 100 is used with the detection chip 10 mounted on the chip holder 154 of the transport unit 150. Therefore, the detection chip 10 will be described first, and then each component of the detection device 100 will be described.

(Detection chip)
The detection chip 10 is formed on the film formation surface 22 and a prism (dielectric member) 20 having an incident surface (first surface) 21, a film formation surface (second surface) 22 and an emission surface (third surface) 23. The metal film 30 and the channel cover 40 disposed on the film formation surface 22 or the metal film 30. Usually, the detection chip 10 is replaced after each analysis. The detection chip 10 is preferably a structure with each piece having a length of several mm to several cm, but may be a smaller structure or a larger structure that is not included in the category of “chip”. .

  The prism 20 is made of a dielectric material that is transparent to the excitation light α. The prism 20 has an entrance surface 21, a film formation surface 22, an exit surface 23, and a bottom surface 24. The incident surface 21 causes the excitation light α from the excitation light irradiation unit 110 to enter the inside of the prism 20. A metal film 30 is arranged on the film formation surface 22. The excitation light α that has entered the prism 20 is reflected by the back surface of the metal film 30. More specifically, the excitation light α is internally reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30. The emission surface 23 emits the excitation light α reflected by the metal film 30 to the outside of the prism 20. The bottom surface 24 is arranged so as to face the film formation surface 22, and connects the entrance surface 21 and the exit surface 23. The shapes of the entrance surface 21, the film formation surface 22, the exit surface 23, and the bottom surface 24 are not particularly limited. The shapes of the incident surface 21, the film formation surface 22, the emission surface 23, and the bottom surface 24 may be flat or curved, respectively, and any one of them may be flat and the other may be curved. May be In the present embodiment, the shapes of the incident surface 21, the film formation surface 22, the emission surface 23, and the bottom surface 24 are all flat.

  The shape of the prism 20 is not particularly limited. In the present embodiment, the shape of the prism 20 is a pillar having a trapezoidal bottom surface. The surface corresponding to one bottom side of the trapezoid is the film forming surface 22, the surface corresponding to the other bottom side of the trapezoid is the bottom surface 24, the surface corresponding to one leg is the incident surface 21, and the other leg is The corresponding surface is the exit surface 23.

  The incident surface 21 is formed so that the excitation light α does not return to the excitation light irradiation unit 110. When the light source of the excitation light α is a laser diode (hereinafter also referred to as “LD”), when the excitation light α returns to the LD, the excitation state of the LD is disturbed, and the wavelength and output of the excitation light α are changed. .. Therefore, the angle of the incident surface 21 is set so that the excitation light α does not vertically enter the incident surface 21 in the scanning range centered on the ideal enhancement angle. In the present embodiment, the dihedral angle θa of the film forming surface 22 and the incident surface 21 is 80°, and the dihedral angle θb of the film forming surface 22 and the emitting surface 23 is 82.5° (see FIG. 6). .. Here, the “dihedral angle” will be described. First, an imaginary plane perpendicular to the first plane (the film forming surface 22 in the present embodiment) and the second plane (the incident surface 21 or the emitting surface 23 in the present embodiment) is assumed. When the intersection line of the first plane and the virtual plane is the first virtual intersection line and the intersection line of the second plane and the virtual plane is the second virtual intersection line, the first virtual intersection line and the second virtual intersection line are The smaller one of the two angles formed is referred to as a “dihedral angle”.

  The design of the detection chip 10 generally determines the resonance angle (and the enhancement angle in the immediate vicinity thereof). The design factors are the refractive index of the prism 20, the refractive index of the metal film 30, the film thickness of the metal film 30, the extinction coefficient of the metal film 30, the wavelength of the excitation light α, and the like. The resonance angle and the enhancement angle are shifted by the substance to be detected immobilized on the metal film 30, but the amount thereof is less than several degrees.

  The prism 20 has some birefringence characteristics. Examples of the material of the prism 20 include resin and glass. The material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

  The metal film 30 is arranged on the film formation surface 22 of the prism 20. As a result, an interaction (surface plasmon resonance) occurs between the photon of the excitation light α incident on the film formation surface 22 under the condition of total reflection and the free electron in the metal film 30, and the interaction occurs on the surface of the metal film 30. Localized field light can be generated. The metal film 30 may be disposed on at least a part of the film formation surface 22. That is, the metal film 30 may be arranged on the entire film formation surface 22 or may be arranged on a part of the film formation surface 22. Further, in the present embodiment, since the film formation surface 22 is a flat surface, the back surface of the metal film 30 is also a flat surface.

  The material of the metal film 30 is not particularly limited as long as it is a metal that can generate surface plasmon resonance. Examples of the material of the metal film 30 include gold, silver, copper, aluminum and alloys thereof. In the present embodiment, the metal film 30 is a gold thin film. The method for forming the metal film 30 is not particularly limited. Examples of the method of forming the metal film 30 include sputtering, vapor deposition, and plating. The thickness of the metal film 30 is not particularly limited, but is preferably within the range of 30 to 70 nm.

  Further, although not particularly shown, in this embodiment, a capturing body for capturing a substance to be detected is fixed (disposed) on the surface of the metal film 30 that does not face the prism 20 (the surface of the metal film 30). ing. By immobilizing the capturing body, the substance to be detected can be selectively detected. In the present embodiment, the capturing body is uniformly immobilized in a predetermined region (reaction field) on the metal film 30. The type of capturing body is not particularly limited as long as it can capture the substance to be detected. In the present embodiment, the capturing body is an antibody specific to the substance to be detected or a fragment thereof.

  The flow path lid 40 is arranged on the metal film 30. When the metal film 30 is formed only on a part of the film forming surface 22 of the prism 20, the flow path lid 40 may be arranged on the film forming surface 22. A channel groove is formed on the back surface of the channel lid 40, and the channel lid 40 forms a channel 41 through which the liquid flows together with the metal film 30 (and the prism 20 ). Examples of the liquid include a sample liquid containing a substance to be detected, a labeling liquid containing an antibody labeled with a fluorescent substance, a washing liquid, and the like. The capturing body immobilized on the metal film 30 is exposed in the flow channel 41. Both ends of the channel 41 are connected to an inlet and an outlet (not shown) formed on the upper surface of the channel lid 40, respectively. When the liquid is injected into the flow channel 41, the liquid comes into contact with the capturing body.

  The flow path lid 40 is preferably made of a material transparent to the fluorescence γ emitted from the metal film 30. Examples of the material of the flow path lid 40 include resin. If the portion for extracting the fluorescence γ to the outside is transparent to the fluorescence γ, the other portion of the channel cover 40 may be formed of an opaque material. The flow path lid 40 is bonded to the metal film 30 or the prism 20 by, for example, bonding with a double-sided tape or an adhesive, laser welding, ultrasonic welding, pressure bonding using a clamp member, or the like.

  As shown in FIG. 1, when the substance to be detected is detected, the excitation light α enters the prism 20 through the incident surface 21. The excitation light α that has entered the prism 20 enters the metal film 30 at a total reflection angle (an angle at which surface plasmon resonance occurs). In this way, by irradiating the metal film 30 with the excitation light α at an angle at which surface plasmon resonance occurs, a localized field light (generally called “evanescent light” or “near-field light”) is formed on the metal film 30. Can be generated. The localized field light excites the fluorescent substance that labels the substance to be detected existing on the metal film 30, and emits the fluorescence γ. The detection device 100 detects the presence or amount of the substance to be detected by detecting the amount of fluorescence γ emitted from the fluorescent substance.

(Detection device)
Next, each component of the detection device 100 will be described. As described above, the detection device 100 includes the excitation light irradiation unit 110, the reflected light detection unit 120, the fluorescence detection unit 130, the liquid feeding unit 140, the transport unit 150, and the control unit 160.

  The excitation light irradiation unit 110 irradiates the detection chip 10 held by the chip holder 154 with the excitation light α. When detecting the substance to be detected, the excitation light irradiation unit 110 irradiates only the P wave with respect to the metal film 30 toward the incident surface 21 so that the incident angle with respect to the metal film 30 becomes an angle that causes surface plasmon resonance. .. Here, the "excitation light" is light that directly or indirectly excites the fluorescent substance. For example, the excitation light α is light that causes localized field light that excites the fluorescent substance on the surface of the metal film 30 when the metal film 30 is irradiated through the prism 20 at an angle at which surface plasmon resonance occurs. is there. Further, in the detection device 100 according to the present embodiment, the excitation light α is also used for positioning the detection chip 10. Although the details will be described later, when the detection chip 10 is positioned, the excitation light irradiation unit 110 irradiates the excitation light α at a predetermined irradiation angle with respect to the normal line of the film formation surface 22.

  The excitation light irradiation unit 110 has a configuration for emitting the excitation light α toward the prism 20 and a configuration for scanning the incident angle of the excitation light α with respect to the back surface of the metal film 30 (with respect to the normal line of the film formation surface 22). A configuration for adjusting the irradiation angle of the excitation light α). In the present embodiment, the excitation light irradiation unit 110 includes a light source unit 111, an angle adjustment unit 112, and a light source control unit 113.

  The light source unit 111 emits the excitation light α that is collimated and has a constant wavelength and a constant light amount such that the irradiation spot on the back surface of the metal film 30 has a substantially circular shape. The light source unit 111 includes, for example, a light source for the excitation light α, a beam shaping optical system, an APC unit, and a temperature adjusting unit (all not shown).

  The type of light source is not particularly limited, and is, for example, a laser diode (LD). Other examples of light sources include light emitting diodes, mercury vapor lamps, and other laser light sources. When the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like. When the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like. Further, when the light emitted from the light source is not linearly polarized light, the light 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 means, and the like. The beam shaping optical system may include all or some of these. The collimator collimates the excitation light α emitted from the light source. The bandpass filter converts the excitation light α emitted from the light source into narrowband light having only the central 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 completely linearly polarized light. The half-wave plate adjusts the polarization direction of the excitation light α so that the P-wave component is incident on the metal film 30. The slit and the zooming means adjust the beam diameter and the contour shape of the excitation light α so that the shape of the irradiation spot on the back surface of the metal film 30 becomes a circle of a predetermined size.

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

  The temperature adjusting unit is, for example, a heater or a Peltier element. The wavelength and energy of the excitation light α of the light source may change depending on the temperature. Therefore, the wavelength and energy of the excitation light α of the light source are controlled to be constant by keeping the temperature of the light source constant by the temperature adjusting unit.

  When detecting the substance to be detected, the angle adjusting unit 112 sets the emission angle of the excitation light α after entering the prism 20 with respect to the metal film 30 (the interface between the prism 20 and the metal film 30 (deposition surface 22)). When the position information of the detection chip 10 is adjusted, the exit angle (irradiation angle) of the excitation light α (light) before entering the prism 20 with respect to the normal line of the film formation surface 22 is adjusted. In order to adjust the irradiation angle of the excitation light α, the optical axis of the excitation light α and the chip holder 154 may be relatively rotated.

  For example, when detecting a substance to be detected, the angle adjusting unit 112 rotates the light source unit 111 with the axis orthogonal to the optical axis of the excitation light α (the axis perpendicular to the paper surface of FIG. 1) as the rotation axis. .. At this time, the position of the rotation axis is set so that the position of the irradiation spot on the metal film 30 hardly changes even if the emission angle is scanned. By setting the position of the rotation center near the intersection of the optical axes of the two excitation lights α at both ends of the scanning range of the emission angle (between the irradiation position on the film formation surface 22 and the incident surface 21), the irradiation position The deviation can be minimized.

  Among the angles of incidence of the excitation light α on the metal film 30, the angle at which the maximum amount of plasmon scattered light can be obtained is the enhancement angle. By setting the incident angle of the excitation light α with respect to the metal film 30 to the enhancement angle or an angle in the vicinity thereof, it becomes possible to measure the high-intensity fluorescence γ. The basic incident condition of the excitation light α is determined by the material and shape of the prism 20 of the detection chip 10, the film thickness of the metal film 30, the refractive index of the liquid in the channel, and the like. The optimum incident condition slightly varies depending on the type and amount of the substance, the shape error of the prism 20, and the like. Therefore, it is preferable to find the optimum enhancement angle for each measurement. In the present embodiment, the preferable irradiation angle of the excitation light α with respect to the normal line of the metal film 30 (straight line in the z-axis direction in FIG. 1) is about 70°.

  Further, when acquiring the position information of the detection chip 10, the angle adjusting unit 112 may be rotated about the same axis as in the case of detecting the substance to be detected, or may be irradiated onto the film formation surface 22. The position of the rotation axis may be set so that the position of the irradiation spot on the incident surface 21 hardly changes even if the angle is scanned.

  The light source control unit 113 controls various devices included in the light source unit 111 to control emission of emitted light (for example, excitation light α) of the light source unit 111. The light source control unit 113 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 excitation light irradiation unit 110 may adjust the emission angle (irradiation angle) of the excitation light α by moving the optical system in the light source. In this case, the detection device 100 can be simplified and downsized.

  The reflected light detection unit 120 is a detection chip for performing an operation (for example, injection of a measurement solution) to the detection chip 10 and an optical measurement (for example, detection of an enhancement angle, measurement of an optical blank value, detection of fluorescence γ). For positioning 10, the first reflected light β1 and the second reflected light β2 generated by irradiating the detection chip 10 with the excitation light α are detected. The reflected light detection unit 120 preferably detects the first reflected light β1 and the second reflected light β2 for positioning the detection chip 10 before the first operation of the detection chip 10. Here, the “first reflected light β1” is the light emitted from the light source (the excitation light α in the present embodiment) reflected by the incident surface 21. The “second reflected light β2” is the light emitted from the light source that is incident on the incident surface 21, is sequentially reflected by the film formation surface 22 and the emission surface 23, and is emitted from the incident surface 21. The optical path of the second reflected light β2 is not particularly limited as long as it is incident on the incident surface 21 and then sequentially reflected by the film formation surface 22 and the emission surface 23. In the present embodiment, after the second reflected light β2 is incident on the incident surface 21, the second reflected light β2 is sequentially reflected on the film formation surface 22 and the emission surface 23 without further reflection on another surface or further transmission on another surface. It is the light emitted from the incident surface 21.

  The light receiving sensor 121 detects the first reflected light β1 and the second reflected light β2 generated by the irradiation of the excitation light α. The type of the light receiving sensor 121 is not particularly limited as long as it can detect the first reflected light β1 and the second reflected light β2. For example, the light receiving sensor 121 is a photodiode (PD), an area sensor, or the like. The size of the light receiving surface of the light receiving sensor 121 is preferably larger than the beam diameter of the excitation light α. For example, when the beam diameter of the excitation light α is about 1.0 to 1.5 mm, the length of one side of the light receiving surface of the light receiving sensor 121 is preferably 3 mm or more. When the light receiving sensor 121 is an area sensor, a plurality of pixels are arranged on the light receiving surface.

  The light receiving sensor 121 is arranged at a position where the first reflected light β1 and the second reflected light β2 can enter. The light receiving sensor 121 is preferably arranged at a position where the first reflected light β1 and the second reflected light β2 of the excitation light α emitted at an angle different from that at the time of detecting the fluorescence γ are incident. In the present embodiment, the scanning range of the irradiation angle of the excitation light α before entering the prism 20 with respect to the normal line of the film formation surface 22 (straight line in the z-axis direction in FIG. 1) is within a range of about 66 to 72°. Is. The excitation light α so that the first reflected light β1 and the second reflected light β2 from the incident surface 21 travel toward the light receiving sensor 121 on the traveling direction (x-axis direction in FIG. 1) side of the transport stage (moving stage) 152. Set the irradiation angle of. In this way, the light receiving sensor 121 is arranged at a position where the first reflected light β1 and the second reflected light β2 are incident (see FIG. 1). Further, a lens for condensing the first reflected light β1 and the second reflected light β2 on the light receiving sensor 121 may be arranged between the light receiving sensor 121 and the incident surface 21.

  The sensor control unit 122 controls detection of the output value of the light receiving sensor 121, management of the sensitivity of the light receiving sensor 121 based on the detected output value, change of the sensitivity of the light receiving sensor 121 to obtain an appropriate output value, and the like. The sensor control unit 122 is composed of, 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 fluorescence detection unit 130 detects the fluorescence γ generated by irradiating the metal film 30 with the excitation light α. Further, if necessary, the fluorescence detection unit 130 also detects plasmon scattered light generated by irradiating the metal film 30 with the excitation light α. The fluorescence detection unit 130 includes, for example, a light receiving unit 131, a position switching unit 132, and a sensor control unit 133.

  The light receiving unit 131 is arranged in the normal direction (z-axis direction in FIG. 1) of the metal film 30 (deposition surface 22) of the detection chip 10. The light receiving unit 131 includes a first lens 134, an optical filter 135, a second lens 136, and a light receiving sensor 137.

  The first lens 134 is, for example, a condenser lens and collects the light emitted from the metal film 30. The second lens 136 is, for example, an image forming lens, and forms an image of the light condensed by the first lens 134 on the light receiving surface of the light receiving sensor 137. The optical path between both lenses is a substantially parallel optical path. The optical filter 135 is arranged between both lenses.

  The optical filter 135 guides only the fluorescence component to the light receiving sensor 137 and removes the excitation light component (plasmon scattered light) in order to detect the fluorescence γ with a high S/N ratio. Examples of the optical filter 135 include an excitation light reflection filter, a short wavelength cut filter, and a bandpass filter. The optical filter 135 is, for example, a filter including a multilayer film that reflects a predetermined light component, but may be a colored glass filter that absorbs the predetermined light component.

  The light receiving sensor 137 detects the fluorescence γ. The light receiving sensor 137 has a high sensitivity that is capable of detecting the weak fluorescence γ from the minute amount of the substance to be detected. The light receiving sensor 137 is, for example, a photomultiplier tube (PMT), an avalanche photodiode (APD), or a highly sensitive photodiode (PD).

  The position switching unit 132 switches the position of the optical filter 135 to the optical path in the light receiving unit 131 or the optical path outside the optical path. Specifically, when the light receiving sensor 137 detects fluorescence γ, the optical filter 135 is arranged on the optical path of the light receiving unit 131, and when the light receiving sensor 137 detects plasmon scattered light, the optical filter 135 is placed in the light receiving unit 131. Place it outside the optical path. The position switching unit 132 is configured by, for example, a rotation driving unit and a known mechanism (turntable, rack and pinion, or the like) that moves the optical filter 135 in the horizontal direction by utilizing the rotational movement.

  The sensor control unit 133 controls the detection of the output value of the light receiving sensor 137, the management of the sensitivity of the light receiving sensor 137 based on the detected output value, and the change of the sensitivity of the light receiving sensor 137 to obtain an appropriate output value. The sensor control unit 133 is composed of, 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 feeding unit 140 supplies the sample liquid, the labeling liquid, the cleaning liquid, etc. into the flow channel 41 of the detection chip 10 held by the chip holder 154. The liquid sending unit 140 includes a drug solution chip 141, a syringe pump 142, and a liquid sending pump drive unit 143.

  The chemical liquid chip 141 is a container that stores liquid such as a sample liquid, a labeling liquid, and a cleaning liquid. As the chemical liquid chip 141, 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 142 includes a syringe 144 and a plunger 145 capable of reciprocating inside the syringe 144. The reciprocating movement of the plunger 145 quantitatively sucks and discharges the liquid. If the syringe 144 is replaceable, it is not necessary to wash the syringe 144. Therefore, it is preferable that the syringe 144 is replaceable from the viewpoint of preventing impurities from entering. When the syringe 144 is not configured to be replaceable, it is possible to use the syringe 144 without replacement by further adding a configuration for cleaning the inside of the syringe 144.

  The liquid feed pump driving unit 143 includes a driving device for the plunger 145 and a moving device for the syringe pump 142. The drive device of the syringe pump 142 is a device for reciprocating the plunger 145, and includes, for example, a stepping motor. A driving device including a stepping motor is preferable from the viewpoint of managing the residual liquid amount of the detection chip 10 because it can control the liquid feeding amount and the liquid feeding speed of the syringe pump 142. For example, the moving device of the syringe pump 142 freely moves the syringe pump 142 in two directions, an axial direction (for example, a vertical direction) of the syringe 144 and a direction transverse to the axial direction (for example, a horizontal direction). The moving device of the syringe pump 142 is constituted by, for example, a robot arm, a biaxial stage, or a vertically movable turntable.

  The liquid feeding unit 140 sucks various liquids from the chemical liquid chip 141 and supplies the liquids into the flow channel 41 of the detection chip 10. At this time, by moving the plunger 145, the liquid reciprocates in the flow channel 41 in the detection chip 10 and the liquid in the flow channel 41 is agitated. As a result, it is possible to realize uniform liquid concentration and promotion of reaction (for example, antigen-antibody reaction) in the flow channel 41. From the viewpoint of performing such an operation, the injection port of the detection chip 10 is protected by a multilayer film, and the detection chip 10 and the syringe 144 are sealed so that the injection port can be sealed when the syringe 144 penetrates the multilayer film. It is preferably configured.

  The liquid in the flow path 41 is again sucked by the syringe pump 142 and discharged to the drug solution chip 141 or the like. By repeating these operations, it is possible to carry out reactions with various liquids, washing, etc., and place the substance to be detected labeled with the fluorescent substance in the reaction field in the flow channel 41.

  The transport unit 150 transports and fixes the detection chip 10 to the measurement position or the liquid transfer position. Here, the “measurement position” is a position where the excitation light irradiation unit 110 irradiates the detection chip 10 with the excitation light α, and the fluorescence detection unit 130 detects the fluorescence γ generated therewith. Further, the “liquid feeding position” is a position where the liquid feeding unit 140 supplies the liquid into the flow channel 41 of the detection chip 10 or removes the liquid from the flow channel 41 of the detection chip 10. The transfer unit 150 includes a transfer stage 152 and a chip holder 154. The chip holder 154 is fixed to the transfer stage 152 and detachably holds the detection chip 10. The shape of the chip holder 154 is such that it can hold the detection chip 10 and does not interfere with the optical paths of the excitation light α, the first reflected light β1, the second reflected light β2, and the fluorescence γ. For example, the chip holder 154 is provided with an opening for passing the excitation light α, the first reflected light β1, the second reflected light β2, and the fluorescence γ. The transfer stage 152 moves the chip holder 154 in one direction (x-axis direction in FIG. 1) and the opposite direction. The transfer stage 152 is driven by, for example, a stepping motor or the like.

  The control unit 160 controls the angle adjustment unit 112, the light source control unit 113, the sensor control unit 122, the position switching unit 132, the sensor control unit 133, the liquid feed pump drive unit 143, and the transport stage 152. In addition, the control unit 160 acquires the position information of the detection chip 10 held by the chip holder 154 based on the detection result of the reflected light detection unit 120, and moves the chip holder 154 by the transfer stage 152 to perform detection. It also functions as a position adjustment unit that moves the chip 10 to an appropriate measurement position or liquid transfer position. The control unit 160 is composed of, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

(Detection method)
Next, the detection operation of the detection device 100 (the detection method according to the embodiment of the present invention) will be described. FIG. 2 is a flowchart showing an example of the operation procedure of the detection device 100.

  First, the detection chip 10 is installed in the chip holder 154 of the detection device 100 (step S100).

  Next, the controller 160 operates the transfer stage 152 to move the detection chip 10 to the vicinity of the measurement position (step S110).

  Next, the control unit 160 operates the excitation light irradiation unit 110 and the reflected light detection unit 120 to set the irradiation angle of the light emitted from the excitation light irradiation unit 110 (step S120). In this step, the first reflected light β1 in which the excitation light α is reflected by the incident surface 21 and the second reflected light β2 in which the excitation light α is transmitted through the incident surface 21 and sequentially reflected by the film formation surface 22 and the emission surface 23 are provided. The irradiation angle of the light emitted from the excitation light irradiation unit 110 with respect to the incident surface 21 is set so that the reflected light is detected by the reflected light detection unit 120 at the same time. In the step of setting the light irradiation angle, the emission angles of the first reflected light β1 and the second reflected light β2 with respect to the incident surface 21 are the irradiation position of the excitation light α, the irradiation angle, and the angle of the surface of the prism 20 through which the excitation light passes. , The refractive index of the prism 20, etc.

  The initial irradiation angle of the excitation light α is set based on, for example, the dihedral angle of the film forming surface 22 and the incident surface 21 and the dihedral angle of the film forming surface 22 and the emitting surface 23. In this way, by setting the initial irradiation angle of the excitation light α as an eigenvalue corresponding to the shape of the prism 20, when detecting the substance to be detected by properly using two types of detection chips 10 having different shapes of the prism 20. , It becomes unnecessary to adjust the irradiation angle of the excitation light α by largely scanning the initial irradiation angle so that the reflected light from the prism 20 enters the optimum position of the reflected light detection unit 120, or the excitation light Since it is not necessary to adjust the irradiation angle of α, the measurement time can be shortened. That is, in the step of S132 of FIG. 3, the determination is less likely to transit to the flow of NO, and the measurement time can be shortened. Here, as a method for the detection device 100 to recognize the shape of the prism 20, for example, a bar code in which the shape information of the prism 20 (type of detection chip or shipping inspection data) is recorded is arranged on the detection chip 10, and detection is performed. There is a method in which the barcode is read by a barcode reader (not shown) in the apparatus 100, and the control unit 160 recognizes the read value. The barcode reader may be arranged outside the device. Alternatively, the user may input the shape information of the prism 20 to the detection device 100 using a barcode reader. Further, the shape information of the prism 20 may be recorded on a medium other than the barcode. Further, the method of reading the information on the shape of the prism 20 does not need to use the bar code reader.

  Further, if the shape information including the shape error generated when the prism 20 is manufactured is recorded in the barcode reader, the shape information is input to the detection device 100 so that the shape generated when the prism 20 is manufactured. It is possible to correct the deviation of the emission angles of the first reflected light β1 and the second reflected light β2 due to an error. Thereby, the position information of the detection chip 10 can be acquired with high accuracy and with a short measurement time.

  The initial irradiation angle of the excitation light α may be set according to the refractive index of the prism 20. In this way, by setting the initial irradiation angle of the excitation light α according to the refractive index of the prism 20, two types of detection chips 10 having the prism 20 formed of different materials are used properly to detect a substance to be detected. In this case, it is not necessary to scan and adjust the irradiation angle of the excitation light α largely with respect to the initial irradiation angle so that the reflected light from the prism 20 enters the optimum position of the reflected light detection unit 110. Therefore, the measurement time can be shortened.

  Further, if the refractive index information including the refractive index error generated when manufacturing the prism 20 is recorded in the barcode reader, when the prism 20 is manufactured by inputting the refractive index information to the detection device 100. It is possible to correct the deviation of the emission angles of the first reflected light β1 and the second reflected light β2 due to the generated refractive index error. Thereby, the position information of the detection chip 10 can be acquired with high accuracy and with a short measurement time.

The initial irradiation angle of the excitation light α may be set based on both the shape of the prism 20 and the refractive index of the prism 20. In this way, by setting the initial irradiation angle of the excitation light α as an eigenvalue corresponding to the shape of the prism 20 and the refractive index of the prism 20, two types of detection chips 10 having different shapes of the prism 20 are selectively used. When detecting a detection substance, the irradiation angle of the excitation light α needs to be largely scanned and adjusted with respect to the initial irradiation angle so that the reflected light from the prism 20 enters the reflected light detection unit 110 at an optimum position. Or there is no need to adjust the irradiation angle from the initial irradiation angle, so the measurement time can be shortened. More specifically, it is desirable that the initial irradiation angle of the excitation light α be set near the irradiation angle θ _i that satisfies the following formula.
sin(θ _a −θ _i )=n ₁ cos θ _b (3)
[In the formula (3), θ _a is the dihedral angle of the film formation surface 22 and the incident surface 21, and θ _i is from the excitation light irradiation unit 110 before incidence on the prism 20 with respect to the normal line of the film formation surface 22. Is the irradiation angle of the excitation light, n ₁ is the refractive index of the prism 20, and θ _b is the dihedral angle of the film formation surface 22 and the emission surface 23.

  Furthermore, if information including a shape error and a refractive index error that occur when the prism 20 is manufactured is recorded in the barcode reader, by inputting the information to the detection device 100, the shape error and the Since the deviation of the emission angles of the first reflected light β1 and the second reflected light β2 due to the refractive index error can be corrected, the position information of the detection chip 10 can be acquired with high accuracy and with a short measurement time.

  As described above, it is preferable that the irradiation angle of the excitation light α from the excitation light irradiation unit 110 at the time of starting the acquisition of the position information of the detection chip 10 is set to a fixed value stored in the detection device 100 in advance.

  Next, the controller 160 operates the excitation light irradiation unit 110, the reflected light detection unit 120, and the transport stage 152 to acquire the position information of the detection chip 10 (step S130). In this step, the irradiation spot of the excitation light α emitted from the excitation light irradiation unit 110 is on the boundary between the incident surface 21 and the other surface adjacent to the incident surface 21 (the back surface of the flow path lid 40 in the present embodiment). While the detection chip 10 held by the chip holder 154 is moved by the transport stage 152 so as to pass through, the excitation light is emitted from the excitation light irradiation unit 110 at the irradiation angle set in step S120 to detect reflected light. The unit 120 detects the first reflected light β1 and the second reflected light β2, and holds them in the chip holder 154 based on the detection results of the first reflected light β1 and the second reflected light β2 detected by the reflected light detection unit 120. The positional information of the detected detection chip 10 is acquired. Thus, the relative positional deviation amount between the position of the detection chip 10 and the measurement position or the liquid transfer position obtained can be specified. As described above, in the present embodiment, the other surface adjacent to the entrance surface 21 is the back surface of the flow path lid 40, but the other surface adjacent to the entrance surface 21 of the prism 20 facing the film formation surface 22. It may be the bottom.

  FIG. 3 is a flowchart for explaining the step (step S130) of acquiring the position information of the detection chip 10. FIG. 4 is a schematic diagram for explaining the step (step S130) of acquiring the position information of the detection chip 10. FIG. 5 is a diagram for explaining the step (step S134) of adjusting the irradiation angle of the excitation light α. FIG. 6 is a graph showing an example of the detection result of the first reflected light β1 and the second reflected light β2 by the light receiving sensor 121.

  As shown in FIG. 3, in the step of obtaining the position information of the detection chip 10 (step S130), first, the light receiving sensor 121 detects the first reflected light β1 and the second reflected light β2 while moving the detection chip 10. (Step S131). Specifically, as shown in FIG. 4A, when the detection chip 10 is located away from the light source unit 111, when the light source unit 111 emits the excitation light α, the excitation light α is reflected by the flow path lid 40. To the lower side (transport stage 152 side). Therefore, the reflected light β from the detection chip 10 does not enter the light receiving sensor 121 of the reflected light detection unit 120. When the detection chip 10 is located away from the light source unit 111, the excitation light α does not enter the detection chip 10 and the first reflected light β1 and the second reflected light β2 are not generated.

  When the detection chip 10 is brought closer to the light source unit 111 side from this state, the irradiation spot of the excitation light α emitted from the light source unit 111 is a boundary portion between the prism 20 and the flow path lid 40 (hereinafter referred to as “edge portion”). If the joint surface between the prism 20 and the flow path cover 40 is sufficiently thin, the edge portion cannot reach the boundary portion between the prism surfaces 21 and 22.). In this case, as shown in FIG. 4B, a part of the excitation light α of the excitation light α is reflected by the flow path cover 40 and does not enter the light receiving sensor 121. A part of the other excitation light α of the excitation light α is reflected by the incident surface 21 and enters the light receiving sensor 121 as the first reflected light β1. Further, in the present embodiment, a part of the other excitation light α out of the excitation light α is transmitted through the incident surface 21, is sequentially reflected by the film formation surface 22 and the emission surface 23, and then from the incident surface 21. The light is emitted and enters the light receiving sensor 121 as the second reflected light β2. Therefore, the first reflected light β1 and the second reflected light β2 reflected by the detection chip 10 enter the light receiving sensor 121.

  When the detection chip 10 is further brought closer to the light source unit 111 side, all the irradiation spots of the excitation light α on the incident surface 21 irradiated from the light source unit 111 reach the incident surface 21 of the prism 20. Therefore, as shown in FIG. 4C, the first reflected light β1 reflected by the detection chip 10 and the second reflected light β2 are incident on the light receiving sensor 121. As described above, in the present embodiment, the irradiation spot is moved between the incident surface 21 and another surface adjacent to the incident surface 21, so that the first reflected light β1 and the second reflected light β2 are incident. , The irradiation angle of the excitation light α with respect to the incident surface 21 is set.

  Here, the light amounts of the first reflected light β1 and the second reflected light β2 will be examined. Here, the prism 20 has a refractive index of 1.5, the incident surface 21 has a reflectance of 4%, the metal film 30 has a reflectance of about 90%, and the exit surface 23 has a reflectance of about 90%. Assume 4%. In this case, when the light amount of the excitation light α is 100%, the light amount of the first reflected light β1 is 4% of the light amount of the excitation light α. Further, when the light amount of the excitation light α is 100%, the light amount of the second reflected light β2 is about 3.5% of the light amount of the excitation light α. That is, when the light amount of the excitation light α is 100%, the reflected light having a maximum light amount of about 7.5% is incident on the light receiving sensor 121.

  In the example described above, the case where the first reflected light β1 and the second reflected light β2 are incident on the light receiving sensor 121 even if the detection chip 10 is moved is shown. However, the initial irradiation angle of the excitation light α and the shape of the prism 20 are shown. Depending on an error or an installation error, the first reflected light β1 and the second reflected light β2 may not be incident on the light receiving sensor 121 at the same time even if the detection chip 10 is moved. That is, the light amount received by the light receiving sensor 121 may not be the maximum light amount described above. In the present embodiment, the irradiation angle of the excitation light α is adjusted so that the first reflected light β1 and the second reflected light β2 are simultaneously incident on the light receiving sensor 121 even if the detection chip 10 is moved.

  Specifically, the irradiation angle of the excitation light α is based on the detection value of the light receiving sensor 121 which changes with the irradiation angle of the excitation light α by the excitation light irradiation unit 110 with the detection chip 10 fixed. To adjust. In the present embodiment, the irradiation angle of the excitation light α before entering the prism 20 with respect to the normal line of the film formation surface 22 is gradually changed. When the irradiation angle of the excitation light α is extremely small, the light receiving sensor 121 does not detect the first reflected light β1 and the second reflected light β2. Then, when the irradiation angle of the excitation light α is gradually increased, only the second reflected light β2 enters the light receiving sensor 121, and subsequently, the first reflected light β1 and the second reflected light β2 enter the light receiving sensor 121. Then, when the irradiation angle of the excitation light α is further increased, the second reflected light β2 does not enter the light receiving sensor 121, and subsequently, the first reflected light β1 also does not enter the light receiving sensor 121. Then, the determination as to whether or not the first reflected light β1 and the second reflected light β2 are incident on the light receiving sensor 121 is made based on whether or not the received light amount (detection value) of the light receiving sensor 121 reaches a set value. In the present embodiment, the set value of the amount of received light by the light receiving sensor 121 is the excitation light emitted from the excitation light irradiation unit 110 with the total light amount of the first reflected light β1 and the second reflected light β2 detected by the light receiving sensor 121. It is 7.5% of the light amount of α. When the received light amount of the light receiving sensor 121 reaches the set value (step S132; YES), the position information of the detection chip 10 is acquired based on the received light amount (step S133).

  Further, regarding the adjustment of the irradiation angle of the excitation light α, the irradiation angle may be minutely changed in any direction from the initial position of the irradiation angle. The change amount of the irradiation angle at this time may be, for example, 0.01 deg or 0.1 deg. Position detection can be performed with high accuracy by reducing the amount of change in the irradiation angle. On the other hand, if the change amount of the irradiation angle is increased, the measurement can be performed quickly.

  FIG. 5 is a diagram for explaining the step (step S134) of adjusting the irradiation angle of the excitation light α. FIG. 5A is an optical path diagram of the excitation light α when the irradiation angle of the excitation light α before entering the prism 20 on the film formation surface 22 is small, and FIG. 5B is before the incidence on the prism 20 on the film formation surface 22. 5C is an optical path diagram of the excitation light α when the irradiation angle of the excitation light α is large, and FIG. 5C is a schematic diagram showing an example of the detection result of the first reflected light β1 and the second reflected light β2 by the light receiving sensor 121. ..

  As shown in FIGS. 5A to 5C, the irradiation angle of the excitation light α changes with the change of the irradiation angle of the excitation light α by the excitation light irradiation unit 110 with the detection chip 10 fixed. Is preferably adjusted based on the detected value of. In the present embodiment, the irradiation angle of the excitation light α before entering the prism 20 with respect to the normal line of the film formation surface 22 is gradually changed. When the irradiation angle of the excitation light α is extremely small, the light receiving sensor 121 does not detect the first reflected light β1 and the second reflected light β2 (see the area A surrounded by a dotted line in FIG. 5C). As shown in FIG. 5A, when the irradiation angle of the excitation light α is gradually increased, only the second reflected light β2 is incident on the light receiving sensor 121 (see the area B surrounded by a dotted line in FIG. 5C). As shown in FIG. 5B, when the irradiation angle of the excitation light α is further increased, the first reflected light β1 and the second reflected light β2 are incident on the light receiving sensor 121 (see a region C surrounded by a dotted line in FIG. 5C). .. In this way, the irradiation angle of the excitation light α before the incidence on the prism 20 with respect to the normal line of the film formation surface 22 so that the first reflected light β1 and the second reflected light β2 are incident on the light receiving sensor 121 at the same time is predetermined. It turns out that it is limited to the range of. When the irradiation angle of the excitation light α is further increased, the second reflected light β2 does not enter the light receiving sensor 121, and only the first reflected light β1 enters the light receiving sensor 121 (see a region D surrounded by a dotted line in FIG. 5C). ). When the irradiation angle of the excitation light α is extremely large, the first reflected light β1 and the second reflected light β2 are not detected by the light receiving sensor 121 (see the area E surrounded by the dotted line in FIG. 5C). Whether or not the first reflected light β1 and the second reflected light β2 are incident on the light receiving sensor 121 is determined by the light receiving sensor 121 when the first reflected light β1 and the second reflected light β2 are incident on the light receiving sensor 121. The light amount is set in advance, and it is determined whether or not the detection result of the light receiving sensor 121 reaches the received light amount.

  At this time, the irradiation angle of the excitation light α before entering the prism 20 with respect to the normal line of the film formation surface 22 for the first reflected light β1 and the second reflected light β2 to enter the light receiving sensor 121 is determined by the positioning of the detection chip 10. Is the irradiation angle of the excitation light α. In addition, the irradiation angle of the excitation light α that is the smallest for the first reflected light β1 and the second reflected light β2 to enter the light receiving sensor 121, and the first reflected light β1 and the second reflected light β2 enter the light receiving sensor 121. It is preferable to set an intermediate angle between the maximum irradiation angle of the excitation light α and the irradiation angle of the excitation light α in the positioning of the detection chip 10. If the shape and the refractive index of the detection chip 100 used in the detection apparatus 100 are one, a step of adjusting the irradiation angle of the excitation light α to a fixed value and adjusting the irradiation angle of the excitation light α to an optimum position (described later) S134) may not be performed.

  Further, the irradiation angle of the excitation light α is substantially parallel to the first reflected light β1 and the second reflected light β2 so that the irradiation spots of the first reflected light β1 and the second reflected light β2 on the light receiving sensor 121 are not largely separated. It is preferable to set the angle so that By doing so, the light receiving sensor 121 can be downsized.

Here, the relationship between the irradiation angle of the excitation light α and the emission angles of the first reflected light β1 and the second reflected light β2 will be described. FIG. 6 is a diagram for explaining the relationship between the irradiation angle of the excitation light α and the emission angles of the first reflected light β1 and the second reflected light β2. FIG. 6A is a diagram for explaining the irradiation angle of the excitation light α, the emission angle of the first reflected light β1 and the emission angle of the second reflected light β2, and FIG. 6B is the irradiation angle of the excitation light α. 3 is a graph showing the relationship between the output angles of the first reflected light β1 and the second reflected light β2. The horizontal axis of FIG. 6A is the irradiation angle of the excitation light α. The vertical axis represents the emission angle of the first reflected light β1 or the second reflected light β2. In the vertical axis of FIG. 6A, the emission angle of the reflected light toward the upper side (flow path lid 40) of the virtual plane including the film formation surface 22 is set to be positive, and the lower side of the virtual plane including the film formation surface 22 (transport stage). The emission angle of the reflected light toward 152) is shown as a minus. The black square symbol in FIG. 6B shows the result of the first reflected light β1, and the black circle symbol shows the result of the second reflected light β2. In the present embodiment, the dihedral angle θ _a of the film forming surface 22 and the incident surface 21 is 78°, the dihedral angle θ _b of the film forming surface 22 and the emitting surface 23 is 84°, and the prism 20 of the prism 20 is formed. The refractive index is 1.527.

As shown in FIG. 6A, the irradiation angle θ _i of the excitation light α is an angle with respect to the normal line of the film formation surface 22. The emission angle θ ₁ of the first reflected light β ₁ is an angle with respect to the virtual plane including the film formation surface 22, and the emission angle θ ₂ of the second reflected light β ₂ is an angle with respect to the virtual plane including the film formation surface 22. is there. As shown in FIG. 6B, it can be seen that the emission angle of the first reflected light β1 decreases as the irradiation angle of the excitation light α increases. Further, it can be seen that the emission angle of the second reflected light β2 increases as the irradiation angle of the excitation light α increases. From this graph, it is understood that the irradiation angle of the excitation light α before entering the prism 20 with respect to the normal line of the film formation surface 22 is preferably in the range of about 66 to 72°.

Further, the irradiation angle of the excitation light α is preferably adjusted so as to satisfy the following formula (1).
sin(θ _a −θ _i −3°)<n ₁ cos θ _b <sin(θ _a −θ _i +3°) (1)
[In the formula (1), θ _i is the irradiation angle of the excitation light from the excitation light irradiation unit 110 before incidence on the prism 20 with respect to the normal line of the film formation surface 22, and n ₁ is the refractive index of the prism 20. , Θ _a is the dihedral angle of the film forming surface 22 and the incident surface 21, and θ _b is the dihedral angle of the film forming surface 22 and the emitting surface 23].

  When the irradiation angle of the excitation light α is adjusted so as to satisfy the above formula (1), the first reflected light β1 and the second reflected light β2 enter the light receiving sensor 121 as two substantially parallel light beams (light fluxes). Here, “substantially parallel” means that the smaller angle of the angles formed by the first reflected light β1 and the second reflected light β2 is less than 3°. Thereby, the interval between the first reflected light β1 and the second reflected light β2 is kept substantially constant (does not spread), so that the light receiving sensor 121 can be downsized. In addition, since the range in which the first reflected light β1 and the second reflected light β2 are incident on the light receiving surface of the light receiving sensor 121 can be reduced, the light receiving sensor 121 is used when a minute formation error occurs in the detection chip 10. However, it can receive light properly.

The irradiation angle of the excitation light α is more preferably adjusted so as to satisfy the following expression (2).
sin(θ _a −θ _i −1.5°)<n ₁ cos θ _b <sin(θ _a −θ _i +1.5°)
(2)
[In the formula (2), θ _i is the irradiation angle of the excitation light from the excitation light irradiation unit 110 before incidence on the prism 20 with respect to the normal line of the film formation surface 22, and n ₁ is the refractive index of the prism 20. , Θ _a is the dihedral angle of the film forming surface 22 and the incident surface 21, and θ _b is the dihedral angle of the film forming surface 22 and the emitting surface 23]
At this time, the expression (2) means that the smaller angle of the angles formed by the first reflected light β1 and the second reflected light β2 is less than 1.5°.

  Then, again while moving the detection chip 10, the light receiving sensor 121 detects the first reflected light β1 and the second reflected light β2 (step S131). When the amount of light received by the light receiving sensor 121 reaches the set value (step S132; YES), the positional information of the detection chip 10 is acquired (step S133).

  FIG. 7 is a graph showing an example of the detection result of the first reflected light β1 and the second reflected light β2 by the light receiving sensor 121. In this example, while the detection chip 10 is moved in one direction (x-axis direction) by the transport stage 152 as in step S131, the light receiving sensor 121 changes the total light amount of the first reflected light β1 and the second reflected light β2. It was measured. FIG. 7 also shows three approximate straight lines.

  As shown in FIG. 7, even when the detection chip 10 is moved in the X-axis direction, the reflected light (the first reflected light β1 or the second reflected light β2) is not measured by the light receiving sensor 121 in the initial stage. This is because the excitation light α is reflected by the flow path lid 40, heads toward the lower side (transport stage 152 side), and does not enter the light receiving sensor 121 (see FIG. 4A). When the detection chip 10 continues to move, the light amounts of the first reflected light β1 and the second reflected light β2 incident on the light receiving sensor 121 gradually increase. This is because a part of the excitation light α is reflected by the incident surface 21 and enters the light receiving sensor 121 as the first reflected light β1. Further, another part of the excitation light α enters the incident surface 21, is sequentially reflected by the film formation surface 22 and the emission surface 23, and is emitted from the incident surface 21 as the second reflected light β2, which is a light receiving sensor. This is because it is incident on 121 (see FIG. 4B). If the movement of the detection chip 10 is further continued, the light amounts of the first reflected light β1 and the second reflected light β2 incident on the light receiving sensor 121 become constant. This is because all of the first reflected light β1 and the second reflected light β2 enter the light receiving sensor 121 (see FIG. 4C).

  In FIG. 7, the first half of the horizontal part, the inclined part, and the second half of the horizontal part are linearly approximated. Point A in the graph is the intersection of the approximate straight line in the first half of the horizontal portion and the approximate straight line in the inclined portion. Point B is an intersection of the approximate straight line of the inclined portion and the approximate straight line of the horizontal portion in the latter half. Point C is the midpoint between points A and B. Point A corresponds to the minimum value of the amount of reflected light β. Point B corresponds to the maximum value of the amount of reflected light β. Point C corresponds to an intermediate value of the amount of reflected light β.

  In the graph of FIG. 7, any of the points A to C may be used to specify the position of the detection chip 10. Points A and B indicate the points where the edge of the irradiation spot of the excitation light α has reached the edge portion. Therefore, when the irradiation spot diameter of the excitation light α is taken into consideration, the position of the edge portion can be specified, and as a result, the position of the detection chip 10 can be specified. On the other hand, the point C indicates the point where the center of the irradiation spot of the excitation light α reaches the edge portion. When the point C is used, the position of the edge portion can be specified without considering the irradiation spot diameter of the excitation light α, and as a result, the position of the detection chip 10 can be specified. Therefore, from the viewpoint of suppressing the influence of the irradiation spot diameter of the excitation light α, it is preferable to specify the position of the detection chip 10 using the intermediate value of the light amount of the reflected light β of the excitation light α.

  Thus, the position of the detection chip 10 can be specified by irradiating the detection chip 10 with the excitation light α and simultaneously detecting the first reflected light β1 and the second reflected light β2 of the excitation light α.

  Next, the control unit 160 operates the transport stage 152 to move the detection chip 10 to the liquid feeding position based on the position information of the detection chip 10 acquired in step S130 (step S140).

  Next, the controller 160 operates the liquid sending unit 140 to wash the inside of the flow path 41 with the washing liquid (step S150). When the moisturizer is present in the flow channel 41 of the detection chip 10, the flow channel 41 is washed before introducing the sample solution so that the capturing body can properly capture the substance to be detected. To remove.

  Next, the control unit 160 moves the chip holder 154 by the transport stage 152 based on the position information of the detection chip 10 acquired in step S130 to move the detection chip 10 to an appropriate measurement position (step S160). At this time, since the detection chip 10 is moved based on the acquired position information, the detection chip 10 can be moved to the detection position with high accuracy.

  Next, the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detection unit 130 to irradiate the detection chip 10 arranged at an appropriate measurement position with the excitation light α, and at the same time, the plasmon having the same wavelength as the excitation light α. The scattered light is detected to detect the enhancement angle (step S170). Specifically, the controller 160 operates the excitation light irradiation unit 110 to scan the incident angle of the excitation light α with respect to the metal film 30, and operates the fluorescence detection unit 130 to detect the plasmon scattered light. At this time, the control unit 160 operates the position switching unit 132 to arrange the optical filter 135 outside the optical path of the light receiving unit 131. Then, the control unit 160 determines the incident angle of the excitation light α with respect to the metal film 30 when the light amount of the plasmon scattered light is maximum as the enhancement angle. The enhancement angle is preferably different from the irradiation angle of the excitation light α acquired in the step of acquiring the positional information of the detection chip 10 (step S130). If the enhancement angle is the same as the irradiation angle of the excitation light α acquired in the step of acquiring the position information of the detection chip 10 (step S130), the first reflected light β1 and the second reflected light β1 reflected from the detection chip 10 The reflected light β2 is reflected by the light receiving surface of the light receiving sensor 137, and stray light may be generated due to the reflected light traveling to the fluorescence detection unit 130 side, which may deteriorate the measurement accuracy. Furthermore, if the procedure of performing the step of injecting the sample liquid (step S150) is performed before the step of obtaining the position detection information (step S130), the vicinity of the metal film 30 is obtained when the position information of the detection chip 10 is obtained. Therefore, plasmons due to the excitation light α are generated, the amount of the excitation light α reflected by the film formation surface 22 changes, the amount of light detected by the detection sensor 121 changes, and the position of the detection chip 10 can be accurately detected. It may disappear.

  Next, the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detection unit 130 to irradiate the detection chip 10 arranged at an appropriate measurement position with the excitation light α, and outputs the output value (optical) of the light receiving sensor 137. The blank value) is recorded (step S180). At this time, the control unit 160 operates the angle adjusting unit 112 to set the incident angle of the excitation light α to the metal film 30 to the enhancement angle. Further, the control unit 160 controls the position switching unit 132 to arrange the optical filter 135 in the optical path of the light receiving unit 131.

  Next, the control unit 160 operates the transport stage 152 to move the detection chip 10 to the liquid feeding position based on the position information of the detection chip 10 acquired in step S120 (step S190).

  Next, the control unit 160 operates the liquid feeding unit 140 to inject the sample liquid in the chemical liquid chip 141 into the flow channel 41 of the detection chip 10 (step S200). In the flow channel 41, the substance to be detected is captured on the metal film 30 by the antigen-antibody reaction (primary reaction). After that, the sample liquid in the channel 41 is removed, and the inside of the channel is washed with the washing liquid.

  Then, the controller 160 operates the liquid sending unit 140 after cleaning to introduce the liquid (label liquid) containing the secondary antibody labeled with a fluorescent substance into the flow channel 41 of the detection chip 10 (step S210). ). In the channel 41, the substance to be detected captured on the metal film 30 is labeled with a fluorescent substance by an antigen-antibody reaction (secondary reaction). After this, the marker liquid in the channel 41 is removed, and the inside of the channel is washed with the washing liquid.

  Next, the control unit 160 moves the detection chip 10 to the detection position where the transport stage 152 detects the substance to be detected based on the position information of the detection chip 10 acquired in step S130 (step S220). At this time, since the detection chip 10 is moved based on the acquired position information, the detection chip 10 can be moved to the measurement position with high accuracy.

  Next, the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detection unit 130 to irradiate the detection chip 10 arranged at an appropriate measurement position with the excitation light α, and at the same time, the target captured by the capturing body. The fluorescence γ emitted from the fluorescent substance labeling the detection substance is detected (step S230). At this time, the control unit 160 operates the angle adjusting unit 112 to set the emission angle of the excitation light α to the enhancement angle. Further, it is preferable that the excitation light irradiation unit 110 emits the excitation light α at an angle different from the irradiation angle of the excitation light α acquired in the step of acquiring the position information of the detection chip 10 (step S130). The control unit 160 subtracts the optical blank value from the detected value, and calculates the fluorescence intensity correlated with the amount of the substance to be detected. The detected fluorescence intensity is converted into the amount or concentration of the substance to be detected, if necessary.

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

  When the irradiation angle of the excitation light α when detecting the substance to be detected is predetermined, the enhancement angle detection (step S170) may be omitted. In the above description, after the step of reacting the substance to be detected with the capturing body (first reaction, step S200), the step of labeling the substance to be detected with a fluorescent substance (secondary reaction, step S210) is performed. (2-step method). However, the timing of labeling the substance to be detected with the fluorescent substance is not particularly limited. For example, before introducing the sample solution into the flow path of the detection chip 10, the labeling liquid may be added to the sample solution to label the substance to be detected with a fluorescent substance in advance. Further, the sample liquid and the labeling liquid may be simultaneously injected into the flow channel of the detection chip 10. In the former case, by injecting the sample solution into the flow path of the detection chip 10, the substance to be detected labeled with the fluorescent substance is captured by the capturing body. In the latter case, the substance to be detected is labeled with a fluorescent substance and the substance to be detected is captured by the capturing body. In either case, both the primary reaction and the secondary reaction can be completed by introducing the sample solution into the channel of the detection chip 10 (one-step method). When the one-step method is adopted as described above, the enhancement angle is detected (step S170) before the antigen-antibody reaction, and further, the step of setting the irradiation angle of the excitation light α (step S120) and The step (step S130) of acquiring the position information of the detection chip 10 is performed.

  Further, the excitation light α may be emitted from the incident surface 21 as the second reflected light β2 via a path different from the path described above. FIG. 8 is a diagram showing another optical path of the excitation light α in the prism 20. As shown in FIG. 8, the second reflected light β2 enters the incident surface 21, is sequentially reflected by the film forming surface 22 and the emitting surface 23, is then reflected by the bottom surface of the prism 20, and then is incident from the incident surface 21. It may be emitted. In this case, it is preferable that the light reflected by the emission surface 23 be totally reflected by the bottom surface 24. For example, the bottom surface 24 may be a mirror surface.

  In addition, in the detection operation of the detection device 100 (the step of acquiring the position information of the detection chip 10), a malfunction of the detection chip 10 or the detection device 100 may be detected. 9 and 10 are flowcharts for explaining a process of acquiring position information of another detection chip 10. In the step of acquiring the position information of the detection chip 10, when the detection value by the light receiving sensor 121 is less than or equal to a predetermined value, the irradiation angle of the excitation light α emitted from the excitation light irradiation unit 110 is adjusted and the light receiving sensor 121 is again detected. Or the step of acquiring the positional information of the above-described detection chip 10 only in that the detection by the light receiving sensor 121 is stopped in the next step of detecting the substance to be detected (step S130). Different from Therefore, the same steps as those in step S130 are designated by the same reference numerals, and the description thereof will be omitted.

  As shown in FIG. 9, in the step of acquiring the position information of the detection chip 10, when the amount of light received by the light receiving sensor 121 when the detection chip 10 is brought close to the light source unit 111 is equal to or less than a predetermined value (step S132; NO). The irradiation angle of the excitation light α is adjusted (step S134), and the light receiving sensor 121 receives the first reflected light β1 and the second reflected light β2 again while bringing the detection chip 10 closer to the light source unit 111. At this time, the step of adjusting the irradiation angle of the excitation light α (step S134) is repeated a plurality of times (step S135; NO), and when the number of repetitions reaches a predetermined number (for example, 3 times) (step S135; YES). ) The process may be terminated without detecting the substance to be detected. As described above, in the process of acquiring the position information of the detection chip 10, it is possible to detect a defect in the detection chip 10 or the detection device 100. Further, as shown in FIG. 2, since a defect can be detected at a relatively early stage of the measurement flow, the measurement error can be notified to the user as soon as possible, thus reducing the burden on the user.

  Further, as shown in FIG. 10, when the light receiving sensor 121 is an area sensor having a plurality of pixels, the first reflected light β1 and the second reflected light β2 are incident in the step of acquiring the position information of the detection chip 10. The position information of the detection chip 10 is acquired based on the total detection value of all the pixels on the light receiving surface of the area sensor (step S132), and the position of the pixel on the light receiving surface at which the first reflected light β and the second reflected light β have reached It may further include a step of determining whether to stop the detection by the light receiving sensor 121 based on the above. When the first reflected light β1 and the second reflected light β2 are detected at the predetermined positions (step S136; YES), the process proceeds to the step of moving the detection chip 10 to the liquid feeding position (step S140). On the other hand, when the first reflected light β1 or the second reflected light β2 is not detected at the predetermined position (step S136; NO), the process ends without detecting the substance to be detected. If the first reflected light β1 or the second reflected light β2 is not detected at a predetermined position, the detection chip 10 may be installed outside the specified range, or the detection chip 10 or the detection device 100 may be defective. Can be detected and erroneous measurement can be prevented.

  As described above, when the amount of light received by the light receiving sensor 121 is extremely small, or when the first reflected light β1 and the second reflected light β2 do not reach a predetermined position, the error in the installation position of the detection chip 10 and the device Since a defect can be detected, erroneous measurement of the substance to be detected can be prevented.

(effect)
As described above, according to the detection device 100 and the detection method according to the present embodiment, the first reflected light β1 and the second reflected light β2 are detected while irradiating the incident surface 21 with the excitation light α. Since the angle of incidence on the prism 20 with respect to the normal line of the film formation surface 22 is set, it is possible to suppress the deterioration of the alignment accuracy of the detection chip 10 due to the excitation light α incident on the prism 20.

  In the above embodiment, the light irradiated in the step of acquiring position information and the step of detecting the substance to be detected is the excitation light α, but the light irradiated in both steps is not the same. Good. That is, if the light emitted in the step of detecting the substance to be detected is the excitation light α, the light emitted in the step of acquiring the positional information of the detection chip 10 may not be the excitation light α. Further, the light amounts of the light irradiated in the step of acquiring the position information and the step of detecting the substance to be detected may not be the same.

  Further, in the above embodiment, the detection method and the detection device 100 using SPFS have been described, but the detection method and the detection device 100 according to the present invention are not limited to this. For example, the present invention can be applied to a detection method and a detection device using the SPR method. In this case, the fluorescence detection unit 130 reflects the sample light according to the amount of the substance to be detected captured by the detection chip 10 not on the fluorescence γ but on the film forming surface 22 of the prism 20 and emits it on the emitting surface 23. Detect light. Further, the present invention can also be applied to a detection method and a detection device using an evanescent fluorescence method in which a fluorescent substance that labels a substance to be detected is excited with evanescent light without using SPR. In this case, the detection chip 10 may not have the metal film 30.

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

10 detection chip 20 prism 21 incident surface 22 film formation surface 23 emission surface 24 bottom surface 30 metal film 40 flow channel lid 41 flow channel 100 detection device 110 excitation light irradiation unit 111 light source unit 112 angle adjustment unit 113 light source control unit 120 reflected light detection Unit 121 Light receiving sensor 122 Sensor control unit 130 Fluorescence detection unit 131 Light receiving unit 132 Position switching unit 133 Sensor control unit 134 First lens 135 Optical filter 136 Second lens 137 Light receiving sensor 140 Liquid sending unit 141 Chemical liquid chip 142 Syringe pump 143 Liquid sending Pump drive unit 144 Syringe 145 Plunger 150 Transport unit 152 Transport stage 154 Chip holder 160 Control unit α Excitation light β1 First reflected light β2 Second reflected light γ Fluorescence

Claims (16)

Chip for holding a detection chip having a dielectric member that includes a first surface, a second surface and a third surface and is transparent to light, and a substance to be detected is trapped on the surface side of the second surface With a holder,
A moving stage for moving the chip holder,
Setting the irradiation angle to irradiate light against the first surface of the dielectric member of the detection chip held by the chip holder, light is irradiated to the first surface at the irradiation angle and the light irradiation section you,
Of the light emitted from the light irradiation unit, a reflected light detection unit that detects reflected light by the dielectric member,
By irradiating light from the light irradiation unit, a sample light detection unit that detects the sample light generated according to the amount of the substance to be detected captured in the detection chip,
A detection method using a detection device having:
Of the light emitted from the light irradiator, the first reflected light reflected by the first surface and the second reflected light transmitted through the first surface and sequentially reflected by the second surface and the third surface And a step of setting an irradiation angle of the light irradiated from the light irradiation unit with respect to the first surface, such that and are simultaneously detected by the reflected light detection unit,
The detection chip held by the chip holder is moved so that the irradiation spot of the light emitted from the light irradiation unit passes on the boundary between the first surface and another surface adjacent to the first surface. While moving by the stage, light is irradiated from the light irradiation unit at the irradiation angle set in the step of setting the light irradiation angle, and the first reflected light and the second reflected light are emitted by the reflected light detection unit. Detecting, based on the detection result of the first reflected light and the second reflected light by the reflected light detection unit, acquiring the position information of the detection chip held in the chip holder,
Based on the acquired position information of the detection chip, a step of moving the detection chip by the moving stage to a detection position for detecting the sample light,
In the state where the detection chip is at the detection position, the detection target captured by the detection chip at the detection position is irradiated with light from the light irradiation unit and the sample light is detected by the sample light detection unit. Detecting the presence or amount of a substance,
The detection method including.   The detection method according to claim 1, wherein each of the first surface, the second surface, and the third surface is a flat surface.   The second reflected light passes through the first surface, is sequentially reflected by the second surface and the third surface, and then is transmitted through the first surface without being further reflected or transmitted by another surface. The detection method according to claim 1, wherein the detection light is light emitted from the dielectric member.   The irradiation angle of the light irradiated from the said light irradiation part is set according to the shape of the said dielectric member in the process of setting the irradiation angle of light, The any one of Claims 1-3. Detection method.   In the step of setting the light irradiation angle, the light irradiation angle of the light emitted from the light irradiation unit is set to a dihedral angle of the second surface and the first surface and a dihedral angle of the second surface and the third surface. The detection method according to claim 1, wherein the detection method is set based on the surface angle.   The irradiation angle of the light irradiated from the said light irradiation part is set according to the refractive index of the said dielectric member in the process of setting the irradiation angle of light. Detection method.   The detection method according to claim 1, wherein in the step of setting the light irradiation angle, the light irradiation angle of the light emitted from the light irradiation unit is set in the detection device in advance.   In the step of setting the light irradiation angle, the light irradiation angle of the light emitted from the light irradiation unit is set based on the detection result of the reflected light detection unit. The detection method described.   In the step of setting the light irradiation angle, the light irradiation angle of the light emitted from the light irradiation unit changes in accordance with the change of the light irradiation angle of the light irradiation unit in the state where the detection chip is fixed. The detection method according to claim 8, wherein the detection method is set based on a detection value of the reflected light detection unit.   In the step of setting the light irradiation angle, the light irradiation angle of the light emitted from the light irradiation unit is set such that the first reflected light and the second reflected light are substantially parallel to each other. 10. The detection method according to any one of 9 to 10. 11. In the step of setting the light irradiation angle, the light irradiation angle of the light irradiated from the light irradiation unit is set so as to satisfy the following formula (1): 11. Detection method.
sin(θ _a −θ _i −3°)<n ₁ cos θ _b <sin(θ _a −θ _i +3°) (1)
[In Formula (1), θ _i is the irradiation angle of the light emitted from the light irradiation unit before the incidence on the dielectric member with respect to the normal to the second surface, and θ _a is the second surface and Is the dihedral angle of the first surface, θ _b is the dihedral angle of the second surface and the third surface, and n ₁ is the refractive index of the dielectric member]   In the step of acquiring the position information of the detection chip, when the detection value in the reflected light detection unit is a predetermined value or less, by adjusting the irradiation angle of light from the light irradiation unit again in the reflected light detection unit The detection method according to any one of claims 1 to 11, wherein the detection by the reflected light detection unit is stopped in the subsequent step of detecting or detecting the substance to be detected. The reflected light detection unit has an area sensor having a plurality of pixels,
In the step of acquiring the position information of the detection chip, the position information of the detection chip is calculated based on a total detection value of all pixels of the light receiving surface of the area sensor on which the first reflected light and the second reflected light are incident. In the next step of detecting the substance to be detected, based on the position of the pixel of the light receiving surface that the first reflected light and the second reflected light have reached, the detection by the sample light detection unit is performed. Judge whether or not,
The detection method according to any one of claims 1 to 12. A metal film is disposed on at least a part of the second surface,
In the step of detecting the substance to be detected, the light irradiation section is held by the chip holder at an angle different from the irradiation angle of the light irradiated toward the first surface in the step of setting the light irradiation angle. Light is emitted toward the metal film of the detection chip, and the sample light detection unit is a surface generated by the light emitted from the light irradiation unit entering the dielectric member and being irradiated on the metal film. By detecting the sample light due to plasmon resonance, to detect the presence or amount of the substance to be detected,
The detection method according to any one of claims 1 to 13. On the surface of the surface of the metal film opposite to the dielectric member, a capturing body for capturing the substance to be detected is arranged,
In the step of detecting the substance to be detected, excitation light is irradiated from the light irradiation unit,
The sample light detection unit irradiates the metal film with the excitation light to detect fluorescence emitted from a fluorescent substance labeled with the substance to be detected,
The detection method according to claim 14. A chip for holding a detection chip which has a dielectric member including a first surface, a second surface and a third surface and is transparent to light, and in which a substance to be detected is trapped on the surface side of the second surface. With a holder,
A moving stage for moving the chip holder,
A light irradiator that irradiates light toward the first surface of the dielectric member of the detection chip held by the chip holder and changes an irradiation angle of light irradiated onto the first surface. ,
Of the light emitted from the light emitting unit, a reflected light detection unit that detects the reflected light by the dielectric member,
By irradiating light from the light irradiation unit, a sample light detection unit that detects the sample light generated according to the amount of the substance to be detected captured in the detection chip,
A control unit that controls the moving stage, the light irradiation unit, the reflected light detection unit, and the sample light detection unit;
Have
The control unit is
Of the light emitted from the light irradiation unit, the first reflected light reflected by the first surface and the light emitted from the light irradiation unit pass through the first surface, and the second surface and the second surface. The irradiation angle of light is set to the light irradiation unit so that the second reflection light sequentially reflected by the three surfaces is simultaneously detected by the reflection light detection unit,
In order to obtain the position information of the detection chip, an irradiation spot of light emitted from the light irradiation unit passes on a boundary between the first surface and another surface adjacent to the first surface, While moving the detection chip held by a chip holder by the moving stage, irradiate light from the light irradiation unit at the irradiation angle set in the step of setting the irradiation angle of light,
Detection device.

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