JP2021056184A - Detection method and detector - Google Patents

Abstract

To provide a detection method with which, while preventing an increase in manufacturing costs, it is possible to align the position of a detection tip with high accuracy.SOLUTION: A detection method includes the steps of: setting the irradiation angle of light at which a first face of a dielectric member is irradiated from a light irradiation unit; acquiring the position accuracy of a detection tip on the basis of the detection result of reflected light on the first face of the first light emitted from the light irradiation unit and detected by a reflected light detection unit; obtaining a shape correction value that is a difference of the shape value of the dielectric member to be formed from the shape value of the dielectric member having been formed; moving the detection tip to a detection position on the basis of the position information and the shape correction value; and detecting the presence or amount of a detection substance having been captured by the detection tip at the detection position.SELECTED DRAWING: Figure 3

Description

The present invention relates to a detection method and a detection device.

In the measurement for detecting a biological substance such as protein or DNA, if a trace amount of the substance to be detected can be detected with high sensitivity and quantitatively, the patient's condition can be immediately grasped and treated. Therefore, there is a demand for an analytical method and an analyzer that can detect weak light caused by a trace amount of a substance to be detected with high sensitivity and quantitatively. As one method for detecting a substance to be detected with high sensitivity, a surface plasmon resonance fluorescence spectroscopy (Surface Plasmon-field enhanced Fluorescence Spectroscopy: SPFS) is known.

SPFS uses a dielectric member in which a metal film is arranged on a predetermined surface. Then, when the metal film is irradiated 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. Since the fluorescent substance that labels the substance to be detected captured on the metal film is excited by this localized field light, the presence or amount of the substance to be detected can be determined by detecting the fluorescence emitted from the fluorescent substance. 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 the excitation light with respect to the metal film with high accuracy. The incident angle of the excitation light with respect to the light cannot be adjusted with high accuracy. Further, in order to detect the substance to be detected with high sensitivity, it is preferable that the shape and position of the irradiation spot of the excitation light and the shape and position of the reaction field on the metal film match. However, if the position of the detection chip is deviated, the shape and position of the irradiation spot of the excitation light cannot be adjusted with high accuracy. On the other hand, it is not preferable from the viewpoint of usability to arrange the detection chip at a predetermined position with high accuracy by the user.

In SPFS, a method of aligning the detection chip has been proposed (see, for example, 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 excitation light toward the trapezoidal column-shaped dielectric member. At this time, the reflected light of the excitation light reflected by the dielectric member is detected by the light receiving sensor. Then, the 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. Generally, the dielectric member is molded by injection molding or the like.

International Publication No. 2015/067044

However, in the dielectric member formed by injection molding, the end portion where the incident surface on which the excitation light is incident and the surface on which the metal film is formed may not have an ideal shape. In the detection chip having the dielectric member with the sagging at the end as described above, the reflected light reflected by the sagging does not enter the light receiving sensor, so that the position information of the detection chip cannot be accurately obtained. In this case, it is conceivable to mold the mold so that the size of the sagging is small, but it is necessary to perform high-precision processing of the mold and control the molding conditions, and to suppress dimensional changes when the mold is replaced due to deterioration. , The manufacturing cost becomes high due to the reduction of individual dimensional differences when molding a large number of pieces at the same time.

Further, if the position accuracy of the capture region formed on the metal film is low, a part of the irradiation spot of the excitation light may deviate from the capture region. In this case as well, since it is necessary to form the capture region with high accuracy, the manufacturing cost increases.

As described above, the dielectric member that is mass-produced has elements that require high accuracy, and it is difficult to secure the accuracy in the mass-produced process, and the manufacturing cost is high.

The present invention provides a detection method and a detection device capable of aligning the positions of the detection chips with high accuracy while preventing an increase in manufacturing cost of the detection chip and the detection device.

The detection method as one means for solving the above-mentioned problems includes a first surface and a second surface on which a metal film is formed, has a dielectric member transparent to light, and contains a substance to be detected. A chip holder for holding the detection chip captured in the reaction field arranged on the surface side of the metal film, a moving stage for moving the chip holder, and the dielectric of the detection chip held in the chip holder. A light irradiation unit that irradiates light toward the first surface of the body member, a reflected light detection unit that detects reflected light that is irradiated from the light irradiation unit and reflected on the first surface, and the substance to be detected. It has a sample light detection unit that detects 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 at a detection position for detection. In a detection method using a detection device, among the light emitted from the light irradiation unit, the reflected light reflected by the first surface is detected on the first surface so that the reflected light detection unit detects the reflected light. On the other hand, the step of setting the irradiation angle of the light emitted from the light irradiation unit and the irradiation spot of the light emitted from the light irradiation unit are the boundary between the first surface and the first surface and the second surface. While moving the detection chip held in the chip holder by the moving stage so as to pass through, the light irradiation unit irradiates light at the irradiation angle set in the step of setting the light irradiation angle. Then, the reflected light is detected by the reflected light detection unit, and the position information of the detection chip held in the chip holder is acquired based on the detection result of the reflected light by the reflected light detection unit. Based on the step of obtaining the shape correction value which is the difference between the shape value of the dielectric member to be formed and the shape value of the formed dielectric member, the obtained position information, and the shape correction value. The detection is performed by moving the detection chip to the detection position and irradiating light from the light irradiation unit with the detection chip in the detection position and detecting the light by the sample light detection unit. The step includes detecting the presence or amount of the substance to be detected captured by the detection chip at the position.

The detection device as one means for solving the above-mentioned problems includes a first surface and a second surface on which a metal film is formed, has a dielectric member transparent to light, and contains a substance to be detected. A chip holder for holding the detection chip captured in the reaction field arranged on the surface side of the metal film, a moving stage for moving the chip holder, and the dielectric of the detection chip held in the chip holder. A light irradiation unit that irradiates light toward the first surface of the body member, a reflected light detection unit that detects reflected light that is irradiated from the light irradiation unit and reflected on the first surface, and the substance to be detected. A sample light detection unit that detects 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 at a detection position for detection, and the chip. It has a halter, the moving stage, the light irradiation unit, the reflected light detection unit, and a control unit that controls the sample light detection unit, and the control unit is among the lights emitted from the light irradiation unit. The irradiation angle of the light emitted from the light irradiation unit is set with respect to the first surface so that the reflected light reflected by the first surface is detected by the reflected light detection unit, and the light irradiation unit emits light. While moving the detection chip held in the chip holder by the moving stage so that the irradiation spot of the light to be irradiated passes through the first surface and the boundary between the first surface and the second surface. , The light irradiation unit irradiates light at the irradiation angle set in the step of setting the light irradiation angle, the reflected light detection unit detects the reflected light, and the reflected light detection unit detects the reflected light. Based on the above, the position information of the detection chip held in the chip holder is acquired, and the shape correction which is the difference between the shape value of the dielectric member to be formed and the shape value of the formed dielectric member. A value is obtained, and the detection chip is moved to the detection position by the movement stage based on the obtained position information and the shape correction value.

According to the present invention, it is possible to provide a detection method and a detection device capable of aligning the positions of the detection chips with high accuracy while preventing an increase in manufacturing cost of the detection chip and the detection device.

FIG. 1 is a diagram schematically showing a configuration of a detection device according to a first embodiment of the present invention. FIG. 2 is a diagram for explaining the first shape correction value. FIG. 3 is a flowchart showing the operation procedure of the detection device. FIG. 4 is a flowchart showing an operation procedure for acquiring position information. 5A to 5H are schematic views for explaining a process of acquiring the position information of the detection chip. FIG. 6 is a diagram showing the relationship between the moving distance of the detection chip and the intensity of the reflected light. 7A and 7B are diagrams for explaining the second shape correction value in the second embodiment of the present invention.

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

[Embodiment 1]
FIG. 1 is a schematic view showing the configuration of a detection device (surface plasmon resonance fluorescence analyzer; SPFS device) 100 according to the first 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 feed unit 140, a transfer unit 150, and a control unit 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 includes a prism (dielectric member) 20 having an incident surface (first surface) 21, a film forming surface (second surface) 22, an emitting surface 23, and a bottom surface (third surface) 24, and a film forming surface 22. It has a metal film 30 formed on the surface, a flow path lid 40 arranged on the film forming surface 22 or the metal film 30, and a storage unit 50 in which the shape correction value of the detection chip 10 is stored. Usually, the detection chip 10 is replaced every analysis. The detection chip 10 preferably has a structure in which each piece has a length of several mm to several cm.

The prism 20 is made of a dielectric material that is transparent to the excitation light α. The prism 20 has an incident surface 21, a film forming surface 22, an emitting 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-forming surface 22. The excitation light α incident on the inside of 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 exit 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 forming surface 22 and connects the incident surface 21 and the exit surface 23. The shapes of the incident surface 21, the film forming surface 22, the emitting surface 23, and the bottom surface 24 are not particularly limited. The shape of the incident surface 21, the film forming surface 22, the emitting surface 23, and the bottom surface 24 may be a flat surface, a curved surface, or any surface may be a flat surface and the other surface may be a curved surface. In the present embodiment, the incident surface 21, the film forming surface 22, the emitting 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 body having a trapezoidal bottom surface. The surface corresponding to one base of the trapezoid is the film-forming surface 22, the surface corresponding to the other bottom of the trapezoid is the bottom surface 24, the surface corresponding to one leg is the incident surface 21, and the other leg 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 excited state of the LD is disturbed and the wavelength and output of the excitation light α fluctuate. .. Therefore, the angle of the incident surface 21 is set so that the excitation light α is not incident perpendicularly to the incident surface 21 in the scanning range centered on the ideal augmentation angle. In the present embodiment, the dihedral angle θ1 of the film forming surface 22 and the incident surface 21 is 80 °, and the dihedral angle θ2 of the film forming surface 22 and the exit surface 23 is 82.5 °. Here, the "dihedral angle" will be described. First, a virtual 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 exit surface 23 in the present embodiment) is assumed. When the intersection of the first plane and the virtual plane is the first virtual intersection and the intersection of the second plane and the virtual plane is the second virtual intersection, the first virtual intersection and the second virtual intersection are The smaller of the two angles is called the "two-sided angle".

The resonance angle (and the augmentation angle in the immediate vicinity thereof) is roughly determined by the design of the detection chip 10. The design elements include 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 fixed to the metal film 30, but the amount is less than a few degrees.

Examples of materials for 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 prism 20 is manufactured, for example, by injection molding.

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

The material of the metal film 30 is not particularly limited as long as it is a metal capable of causing 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 methods for 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 in the range of 30 to 70 nm.

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

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

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

The storage unit 50 stores the shape correction value (first shape correction value) of the prism 20. The form of the storage unit 50 is not particularly limited as long as the shape of the prism 20 can be stored. Examples of the storage unit 50 include IC chips of barcodes, QR codes (registered trademarks), and RFIDs (Radio Frequency Identifiers). In the present embodiment, the storage unit 50 is a barcode.

As described above, the detection chip 10 (prism 20) is formed by, for example, injection molding. Therefore, sagging may occur at the corners of the prism 20. In the present embodiment, the first shape correction value of the detection chip 10 in which the boundary between the incident surface 21 and the film forming surface 22 is sagging will be described. Here, the "shape correction value" refers to a shape error between the shape of the prism 20 to be formed (ideal design shape) and the shape of the prism 20 actually formed (actual actual measurement shape). FIG. 2 is a diagram for explaining the first shape correction value. The thick solid line in FIG. 2 indicates the detection chip 10 in which sagging has occurred, and the broken line indicates the detection chip 10 formed as designed.

The first shape correction value preferably satisfies the following equation (1).
First shape correction value Δx1 = h (tanθa + tanθb) ... Equation (1)
Equation (1) is the distance between the corner portion of the detection chip 10 formed as designed and the end portion of the flat portion of the film-forming surface 22 of the detection chip 10 in which sagging has occurred. In the formula (1), h is the distance between the film-forming surface 22 and the film-forming surface 22 surface side end of the incident surface 21 in the normal direction ND of the metal film 30. θa is an angle formed by the optical axis of the excitation light α emitted from the light source unit 111 and the virtual straight line VL along the normal line. θb is an angle formed by the incident surface 21 and the virtual straight line VL.

As shown in FIG. 1, when the substance to be detected is detected, the excitation light α is incident on the prism 20 from the incident surface 21. The excitation light α incident on the prism 20 is incident on 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 excitation light α at an angle at which surface plasmon resonance occurs, localized field light (generally also referred to as “evanescent light” or “near-field light”) is applied 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 fluorescent γ. The detection device 100 detects the presence or amount of the substance to be detected by detecting the amount of fluorescent γ emitted from the fluorescent substance.

(Detector)
Next, each component of the detection device 100 will be described. As described above, 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 control unit 160.

The excitation light irradiation unit 110 irradiates the detection chip 10 held in 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 angle of incidence with respect to the metal film 30 is an angle that causes surface plasmon resonance. .. Here, the "excitation light" is light that directly or indirectly excites a fluorescent substance. For example, the excitation light α is light that generates localized field light that excites a 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 positioning the detection chip 10, the excitation light irradiation unit 110 irradiates the excitation light α so as to have a predetermined irradiation angle with respect to the normal line of the film forming surface 22.

The excitation light irradiation unit 110 has a configuration for irradiating 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 (relative to the normal line of the film forming surface 22). (Structure for adjusting the irradiation angle of the excitation light α) and. In the present embodiment, the excitation light irradiation unit 110 includes a light source unit 111, an angle adjusting unit 112, and a light source control unit 113.

The light source unit 111 emits excitation light α that is collimated and has a constant wavelength and light amount so that the shape of the irradiation spot on the back surface of the metal film 30 is substantially circular. The light source unit 111 includes, for example, a light source for 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 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 polarizing filter, a half wave plate, a slit, a zoom means, and the like. The beam shaping optical system may include all of these, or may include some of them. 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 narrow-band light having only a 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 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 zoom means adjust the beam diameter and 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 becomes 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 output of the light source to be constant by controlling the input energy with the regression circuit.

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 fluctuate with temperature. Therefore, by keeping the temperature of the light source constant in the temperature adjusting unit, the wavelength and energy of the excitation light α of the light source are controlled to be constant.

When detecting the substance to be detected, the angle adjusting unit 112 determines the emission angle of the excitation light α after being incident on 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 adjusting and acquiring the position information of the detection chip 10, the emission angle (irradiation angle) of the excitation light α (light) before being incident on the prism 20 with respect to the normal line of the film forming 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 around an axis orthogonal to the optical axis of the excitation light α (an axis perpendicular to the paper surface of FIG. 1) as a 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 center of rotation 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 forming surface 22 and the incident surface 21), the irradiation position The deviation can be minimized.

Of 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. High-intensity fluorescence γ can be measured by setting the incident angle of the excitation light α with respect to the metal film 30 at or near the augmentation angle. The basic incident conditions of the excitation light α are 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 flow path, and the like. The optimum incident conditions vary slightly depending on the type and amount of the substance, the shape error of the prism 20, and the like. Therefore, it is preferable to obtain the optimum enhancement angle for each measurement. In the present embodiment, the preferred irradiation angle of the excitation light α with respect to the normal line of the metal film 30 (the 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 irradiate the film-forming 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 the emission of the emitted light (for example, the excitation light α) of the light source unit 111. The light source control unit 113 includes, for example, a known computer including an arithmetic unit, a control device, a storage device, an input device, and an output device, and a microcomputer.

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 operations on the detection chip 10 (for example, injection of a measuring liquid), optical measurement (for example, detection of an augmented angle, measurement of an optical blank value, detection of fluorescence γ), and the like. For the positioning of 10, the reflected light β generated by irradiating the detection chip 10 with the excitation light α is detected. It is preferable that the reflected light detection unit 120 detects the reflected light β for positioning the detection chip 10 before performing the operation on the first detection chip 10.

The light receiving sensor 121 detects the reflected light β 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 reflected light β. 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 reflected light β can be incident. In the present embodiment, the scanning range of the irradiation angle of the excitation light α before being incident on the prism 20 with respect to the normal of the film forming surface 22 (the straight line in the z-axis direction in FIG. 1) is within the range of about 66 to 72 °. Is. The irradiation angle of the excitation light α is set so that the reflected light β from the incident surface 21 travels in the direction of the light receiving sensor 121 on the traveling direction (x-axis direction in FIG. 1) of the transport stage (moving stage) 152. In this way, the light receiving sensor 121 is arranged at a position where the reflected light β is incident (see FIG. 1). Further, a lens for condensing the reflected light β 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 amplification degree of the output of the light receiving sensor 121 in order to obtain an appropriate output value, and the like. To do. The sensor control unit 122 is composed of, for example, a known computer or microcomputer including an arithmetic unit, 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. If necessary, the fluorescence detection unit 130 also detects the 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, an objective lens, and takes in the light emitted from the metal film 30 into the light receiving unit 131. The second lens 136 is, for example, a condensing lens, and condenses the light captured by the first lens 134 on the light receiving surface of the light receiving sensor 137. The optical path between the two lenses is a substantially parallel optical path. The optical filter 135 is arranged between the two 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 at 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 a predetermined light component.

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

The position switching unit 132 switches the position of the optical filter 135 on or out of the optical path of the light receiving unit 131. 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 on the light receiving unit 131. Place it outside the optical path. The position switching unit 132 is composed of, for example, a rotation drive unit and a known mechanism (such as a turntable or a rack and pinion) that moves the optical filter 135 in the horizontal direction by using a rotational motion.

The sensor control unit 133 controls detection of the output value of the light receiving sensor 137, management of the sensitivity of the light receiving sensor 137 based on the detected output value, change of the amplification degree of the output of the light receiving sensor 137 in order to obtain an appropriate output value, and the like. To do. The sensor control unit 133 includes, for example, a known computer including an arithmetic unit, a control device, a storage device, an input device, and an output device, and a microcomputer.

The liquid feeding unit 140 supplies the sample liquid, the labeling liquid, the cleaning liquid, and the like into the flow path 41 of the detection chip 10 held in the chip holder 154. The liquid feed unit 140 includes a chemical liquid tip 141, a syringe pump 142, and a liquid feed pump drive unit 143.

The chemical solution chip 141 is a container for containing a liquid such as a sample solution, a labeling solution, or a cleaning solution. As the chemical solution chip 141, a plurality of containers are usually arranged according to the type of liquid, or a chip in which a plurality of containers are integrated is arranged.

The syringe pump 142 is composed of a syringe 144 and a plunger 145 capable of reciprocating in the syringe 144. The reciprocating motion of the plunger 145 quantitatively sucks and drains the liquid. If the syringe 144 is replaceable, cleaning of the syringe 144 becomes unnecessary. Therefore, the syringe 144 is preferably replaceable from the viewpoint of preventing impurities from being mixed in. If the syringe 144 is not configured to be replaceable, the syringe 144 can be used without replacement by further adding a configuration for cleaning the inside of the syringe 144.

The liquid feed pump drive unit 143 includes a drive device for the plunger 145 and a moving device for the syringe pump 142. The driving device of the syringe pump 142 is a device for reciprocating the plunger 145, and includes, for example, a stepping motor. A drive device including a stepping motor is preferable from the viewpoint of controlling the residual liquid amount of the detection tip 10 because the liquid feeding amount and the liquid feeding speed of the syringe pump 142 can be controlled. The moving device of the syringe pump 142, for example, freely moves the syringe pump 142 in two directions, an axial direction (for example, a vertical direction) of the syringe 144 and a direction crossing the axial direction (for example, a horizontal direction). The moving device of the syringe pump 142 is composed of, for example, a robot arm, a two-axis stage, or a vertically movable turntable.

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

The liquid in the flow path 41 is sucked again by the syringe pump 142 and discharged to the chemical solution chip 141 or the like. By repeating these operations, reactions with various liquids, washing, and the like can be carried out, and a substance to be detected labeled with a fluorescent substance can be placed in the reaction field in the flow path 41.

The transport unit 150 transports the detection chip 10 to the detection position or the liquid feed position and fixes it. Here, the “detection 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 accordingly. The "liquid feeding position" is a position where the liquid feeding unit 140 supplies the liquid into the flow path 41 of the detection chip 10 or removes the liquid in the flow path 41 of the detection chip 10. The transport unit 150 includes a transport stage 152 and a tip holder 154. The chip holder 154 is fixed to the transport stage 152 and holds the detection chip 10 detachably. The shape of the chip holder 154 is a shape that can hold the detection chip 10 and does not obstruct the optical paths of the excitation light α, the reflected light β, and the fluorescence γ. For example, the chip holder 154 is provided with an opening through which the excitation light α, the reflected light β, and the fluorescence γ pass. The transport stage 152 moves the tip holder 154 in one direction (x-axis direction in FIG. 1) and vice versa. The transport 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 transfer stage 152. Further, the control unit 160 acquires the first shape correction value, and based on the detection result of the reflected light detection unit 120, acquires the position information of the detection chip 10 held in the chip holder 154, and also by the transfer stage 152. It also functions as a position adjusting unit for moving the chip holder 154 to move the detection chip 10 to an appropriate detection position or liquid feeding position. The control unit 160 includes, for example, a known computer including an arithmetic unit, a control device, a storage device, an input device, and an output device, and a microcomputer.

(Detection method)
Next, the detection operation of the detection device 100 (the detection method according to the first embodiment of the present invention) will be described. FIG. 3 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 control unit 160 acquires the first shape correction value from the storage unit 50 of the detection chip 10 (step S110). In the present embodiment, since the storage unit 50 is a bar code, the bar code is read and the first shape correction value is acquired.

Next, the control unit 160 operates the transfer stage 152 to move the detection chip 10 to the vicinity of the detection position (step S120).

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 S130). In this step, 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 β reflected by the excitation light α on the incident surface 21 is detected by the reflected light detection unit 120. To do. In the process of setting the light irradiation angle, the emission angle of the reflected light β with respect to the incident surface 21 depends on the irradiation position of the excitation light α, the irradiation angle, the angle of the surface of the prism 20 through which the excitation light passes, the refractive index of the prism 20, and the like. decide.

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

Next, the control unit 160 operates the excitation light irradiation unit 110, the reflected light detection unit 120, and the transfer stage 152 to acquire the position information of the detection chip 10 (step S140). In this step, the detection chip 10 held in the chip holder 154 is conveyed to the transport stage so that the irradiation spot of the excitation light α emitted from the excitation light irradiation unit 110 passes over the boundary between the incident surface 21 and the film forming surface 22. While moving by 152, the excitation light is irradiated from the excitation light irradiation unit 110 at the irradiation angle set in step S130, the reflected light β is detected by the reflected light detection unit 120, and the reflected light β is detected by the reflected light detection unit 120. Based on the detection result of the reflected light β, the position information of the detection chip 10 held in the chip holder 154 is acquired. As a result, the relative positional deviation between the position of the detection chip 10 and the detection position or the liquid feeding position can be specified.

FIG. 4 is a flowchart for explaining a step (step S140) of acquiring the position information of the detection chip 10. 5A to 5H are schematic views for explaining a step (step S140) of acquiring the position information of the detection chip 10. FIG. 6 is a graph showing the relationship between the moving distance of the detection chip 10 and the intensity of the reflected light β. Here, a case where an ideal detection chip 10A (prism 20A) to be formed and an actual detection chip 10B (prism 20B) formed are used will be described. Here, the detection chip 10A means a detection chip 10 formed (should be formed) as designed, with no sagging at the corners and no burrs. Further, in the present embodiment, the detection chip 10B means the detection chip 10 in which the corner portion is sagging. The horizontal axis of FIG. 6 shows the amount of movement of the detection chips 10A and 10B, and the vertical axis shows the intensity of the reflected light β incident on the light receiving sensor 121. 5A to 5H in FIG. 6 correspond to FIGS. 5A to 5H, respectively. The solid line in FIG. 6 shows the result of the detection chip 10A, and the dotted line shows the result of the detection chip 10B.

In the case of the ideal detection chip 10A, as shown in FIG. 4, in the step of acquiring the position information of the detection chip 10A (step S140), first, the reflected light β is emitted by the light receiving sensor 121 while moving the detection chip 10A. Detect (step S141). Specifically, as shown in FIGS. 5A and 6, when the detection chip 10A is located at a position away from the light source unit 111, when the light source unit 111 emits the excitation light α, the excitation light α is the detection chip 10A. The excitation light α is not incident on the light source, and the reflected light β is not generated. Therefore, the reflected light β from the detection chip 10A does not enter the light receiving sensor 121 of the reflected light detection unit 120.

When the detection chip 10A 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 the boundary between the incident surface 21 and the film forming surface 22 (hereinafter, also referred to as “edge portion”). Say.) Is reached. In this case, as shown in FIGS. 5B and 6, some of the excitation lights α do not enter the prism 20. Further, of the excitation light α, some other excitation light α is reflected by the incident surface 21 and is incident on the light receiving sensor 121 as reflected light β.

Further, when the detection chip 10A is 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 FIGS. 5C and 5D and FIG. 6, the reflected light β reflected by the detection chip 10A is 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 the edge portion, and the irradiation angle of the excitation light α with respect to the incident surface 21 is set so that the reflected light β is incident.

On the other hand, in the case of the actual detection chip 10B, as shown in FIG. 4, in the step of acquiring the position information of the detection chip 10B (step S140), first, the reflected light β by the light receiving sensor 121 while moving the detection chip 10B. Is detected (step S141). Specifically, as shown in FIGS. 5E and 6, when the detection chip 10B is located at a position away from the light source unit 111, when the light source unit 111 emits the excitation light α, the excitation light α is the detection chip 10B. The excitation light α is not incident on the light source, and the reflected light β is not generated. Therefore, the reflected light β from the detection chip 10B does not enter the light receiving sensor 121 of the reflected light detection unit 120.

As shown in FIGS. 5F and 6, even if the actual detection chip 10B is brought closer to the light source unit 111 side from this state to the position where the reflected light is incident on the light receiving sensor 121 in the ideal detection chip 10A, the excitation light α Does not reach the incident surface 21. This is because the actual detection chip 10B is sagging.

As shown in FIGS. 5G and 6, when the detection chip 10B is further 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 reaches the sagging portion. However, since the excitation light α that has reached the sagging portion is reflected in a direction different from that of the light receiving sensor 111, it does not enter the light receiving sensor 121.

As shown in FIGS. 5H and 6, when the detection chip 10B is further brought closer to the light source unit 111 side from this state, the irradiation spot of the excitation light α reaches the incident surface 21. In this case, some of the excitation lights α do not enter the detection chip 10A. Further, of the excitation light α, some other excitation light α is reflected by the incident surface 21 and is incident on the light receiving sensor 121 as reflected light β.

Although not particularly shown, when the detection chip 10B 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. ..

At this time, the light receiving sensor 121 monitors the amount of reflected light β, and when the amount of received light reaches a predetermined set value, acquires the position information of the detection chip 10 (step S142; YES, step S143). On the other hand, when the amount of received light received by the light receiving sensor 121 does not reach a predetermined set value, it is determined that there is a problem in the shape of the detection chip 10 or the installation in the chip holder 154, and the process proceeds to abnormal processing (step S142). ; NO). In the abnormality processing, for example, the measurement may be stopped and a warning may be displayed on the operation screen, or the movement start position of the detection chip 10 may be changed in step S141 to execute steps S141 and S142 (step S144). ..

As described above, in the ideal detection chip 10A, when the excitation light α reaches the edge portion, the reflected light β is incident on the light receiving sensor 121. On the other hand, in the actual detection chip 10B, even if the excitation light α reaches the sagging portion, the reflected light β does not enter the light receiving sensor 121, and only when the excitation light α reaches the flat portion of the incident surface 21. The reflected light β is incident on the light receiving sensor 121. Therefore, in receiving the reflected light β by the light receiving sensor 121, the edge portion of the detection chip 10A and the end portion of the incident surface 21 of the detection chip 10B on the film forming surface 22 side perform the same function. Further, as shown in FIG. 6, it can be seen that the position information of the detection chip 10B is displaced by a distance A from the position information of the detection chip 10A.

The position of the detection chips 10A and 10B may be specified by using the minimum value of the light amount of the reflected light β, the maximum value of the light amount of the reflected light β, or the middle of the light amount of the reflected light β. Values may be used (see Figure 6). The minimum value of the light amount of the reflected light β and the maximum value of the light amount of the reflected light β are the points where the edge of the irradiation spot of the excitation light α reaches the edge portion, or the irradiation spot of the excitation light α is the sagging portion of the incident surface 21. It indicates that it has reached the side end. Therefore, if the irradiation spot diameter of the excitation light α is taken into consideration, the position of the edge portion or the side end portion of the incident surface 21 on the sagging portion side can be specified, and as a result, the position of the detection chip 10 can be specified. On the other hand, the intermediate value of the amount of the reflected light β indicates whether the center of the irradiation spot of the excitation light α reaches the edge portion or the end portion on the side of the sagging portion of the incident surface 21. When the intermediate value of the light amount of the reflected light β 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 positions of the detection chips 10A and 10B 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 positions of the detection chips 10A and 10B by using the intermediate value of the amount of the reflected light β of the excitation light α.

Next, the first shape correction value is added to the acquired position information of the detection chip 10 to obtain the movement amount for moving the detection chip 10 (step S145). The first shape correction value is actually formed because it is a shape error between the shape of the prism 20 to be formed (ideal design shape) and the shape of the prism 20 actually formed (actual measured shape). The correct amount of movement can be obtained by adding the first shape correction value to the position information based on the prism 20.

Next, the control unit 160 operates the transfer stage 152 based on the movement amount acquired in the step S140 to move the detection chip 10 to the liquid feeding position (step S150).

Next, the control unit 160 operates the liquid feeding unit 140 to clean the inside of the flow path 41 with the cleaning liquid and inject the sample liquid (step S160). If a moisturizer is present in the flow path 41 of the detection chip 10, the inside of the flow path 41 is washed before introducing the sample solution so that the trapping body can properly capture the substance to be detected. To remove.

Next, the control unit 160 moves the chip holder 154 by the transfer stage 152 based on the position information of the detection chip 10 acquired in the step S140, and moves the detection chip 10 to an appropriate detection position (step S170). At this time, since the detection chip 10 is moved based on the acquired position information and the first shape correction value, 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 detection position with the excitation light α, and plasmons having the same wavelength as the excitation light α. The scattered light is detected to detect the augmentation angle (step S180). Specifically, the control unit 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 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 amount of plasmon scattered light is maximum as the enhancement angle.

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 detection position with the excitation light α, and the output value (optical) of the light receiving sensor 137. The blank value) is recorded (step S190). At this time, the control unit 160 operates the angle adjustment unit 112 to set the angle of incidence of the excitation light α on the metal film 30 as 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 transfer stage 152 based on the position information of the detection chip 10 acquired in the step S140 to move the detection chip 10 to the liquid feeding position (step S200).

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 path 41 of the detection chip 10 (step S210). In the flow path 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 flow path 41 is removed, and the inside of the flow path is washed with the cleaning liquid.

Next, after washing, the control unit 160 operates the liquid feeding unit 140 to introduce a liquid (labeled liquid) containing a secondary antibody labeled with a fluorescent substance into the flow path 41 of the detection chip 10 (step S220). ). In the flow path 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 that, the labeling liquid in the flow path 41 is removed, and the inside of the flow path is washed with a cleaning liquid.

Next, the control unit 160 moves the detection chip 10 to a detection position where the substance to be detected is detected by the transport stage 152 based on the position information of the detection chip 10 acquired in the step S140 (step S230). 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. At this time, it is preferable to add the design value (correction value) to the position information of the detection chip 10 acquired in step S140, and then add the first shape correction. Here, the predetermined value may be included in the code of the control software, for example, or may be stored in the EEPROM and called up when necessary.

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 detection position with the excitation light α, and the subject captured by the capture body. Fluorescent γ emitted from the fluorescent substance that labels the detection substance is detected (step S240). At this time, the control unit 160 operates the angle adjustment 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 irradiates the excitation light α at an angle different from the irradiation angle of the excitation light α acquired in the step (step S140) of acquiring the position information of the detection chip 10. The control unit 160 subtracts the optical blank value from the detected value to calculate the fluorescence intensity that correlates with the amount of the substance to be detected. The detected fluorescence intensity is converted into the amount and 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 solution can be detected.

The step of acquiring the first shape correction value (step S110) may be acquired at any timing as long as it is before the detection chip 10 is moved to the detection position.

Further, when the irradiation angle of the excitation light α when detecting the substance to be detected is predetermined, the detection of the augmentation angle (step S180) may be omitted. Further, in the above description, after the step of reacting the substance to be detected with the trapped substance (primary reaction, step S210), a step of labeling the substance to be detected with a fluorescent substance (secondary reaction, step S220) was performed. (Two-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, a labeling solution may be added to the sample solution to label the substance to be detected with a fluorescent substance in advance. Further, the sample solution and the labeling solution may be injected into the flow path of the detection chip 10 at the same time. 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 trap. 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 trap. In either case, both the primary reaction and the secondary reaction can be completed by introducing the sample solution into the flow path of the detection chip 10 (one-step method). When the one-step method is adopted in this way, the enhancement angle is detected (step S180) before the antigen-antibody reaction, and before that, the step of setting the irradiation angle of the excitation light α (step S130) and The step (step S140) of acquiring the position information of the detection chip 10 is carried out.

(effect)
As described above, according to the detection device 100 and the detection method according to the present embodiment, the detection chip is based on the first shape correction value which is the shape error between the prism 20 to be formed and the created prism 20. Since the 10 is moved to the detection position, it is possible to suppress a decrease in the alignment accuracy of the detection chip 10 due to the excitation light α incident on the prism 20.

[Embodiment 2]
The detection method and detection device according to the present invention according to the second embodiment differ from the detection method and the detection device according to the first embodiment only in the shape correction value. Therefore, in the present embodiment, only the shape correction value (second shape correction value) will be described.

FIG. 7 is a diagram for explaining the second shape correction value. In FIG. 7, the flow path lid 40 is omitted.

The second shape correction value is obtained based on the difference ΔKx between the distance between the center and the boundary of the reaction field in the prism 20 to be formed and the distance between the center and the boundary of the reaction field in the formed prism 20. The second shape correction value Δx preferably satisfies the following equation (3).
Second shape correction value Δx = ΔKs / (1 + αsp) ... Equation (3)
In the formula (3), ΔKs is the positional deviation of the center of the reaction field before being corrected by αsp. Further, αsp in the formula (3) is a coefficient when the position of the irradiation spot on the metal film 30 changes with respect to the amount of movement of the prism 20. In other words, αsp is a coefficient obtained from the incident angle of the excitation light α at the time of detection, the inclination angle of the incident surface 21 of the prism 20 with respect to the normal of the metal film 30, and the refractive index of the prism 20.

The second shape correction value can be used in the same manner as the above-mentioned first shape correction value. That is, after the amount of received light received by the light receiving sensor 121 reaches a predetermined set value (step S142; YES) and the position information of the detection chip 10 is acquired (step S143), the second shape correction value is added to the position information. Therefore, the correct amount of movement can be obtained.

(effect)
As described above, the inspection device and the detection device according to the present embodiment have the same effects as the detection method and the detection device 100 according to the first embodiment.

The first shape correction value and the second shape correction value are incident on the plane including the straight line perpendicular to the film forming surface 22 and the optical axis of the excitation light α, and the light emitted from the excitation light irradiation unit 110 is incident. It may be a value obtained based on the shape of the end portion of the incident surface 21. Here, "vertical" is a concept that includes variations that occur in the mechanical accuracy (parts and assembly) of the device, and does not have to be completely vertical. Since the end portion is surrounded by the incident surface 21 and the film forming surface 22, it has high shape accuracy. On the other hand, the shape accuracy may not be high in the peripheral portion of the end portion. In addition, it is costly to mold the entire prism 20 with high accuracy.

[Embodiment 3]
The detection method and detection device according to the present invention according to the third embodiment differ from the detection method and detection device according to the first embodiment only in the shape correction value. Therefore, in the present embodiment, only the shape correction value (third shape correction value) will be described.

As described above, the prism 20 has an incident surface (first surface) 21, a film forming surface (second surface) 22, an exit surface 23, and a bottom surface (third surface) 24. The third shape correction value is a value obtained based on the dihedral angle between the film-forming surface 22 and the bottom surface 24. When the detection chip 10 is installed on the chip holder 154 with reference to the bottom surface 24 of the prism 20, if there is an error in the dihedral angle between the film-forming surface 22 and the bottom surface 24, the incident surface 21 and the film-forming surface 22 are tilted. Although an error occurs in the first shape correction value, the detection chip 10 can be moved to the correct position by correcting with the third shape correction value.

The third shape correction value can be used in the same manner as the first shape correction value and the second shape correction value described above. That is, after the amount of received light received by the light receiving sensor 121 reaches a predetermined set value (step S142; YES) and the position information of the detection chip 10 is acquired (step S143), the third shape correction value is added to the position information. Therefore, the correct amount of movement can be obtained.

(effect)
As described above, the detection method and the inspection device according to the present embodiment have the same effects as the detection method and the detection device 100 according to the first embodiment.

In the above embodiment, the light emitted in both the step of acquiring the position information and the step of detecting the substance to be detected is the excitation light α, but the light emitted in both steps is not the same. May be 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 position information of the detection chip 10 does not have to be the excitation light α. Further, the amount of light emitted in the step of acquiring the position information and the step of detecting the substance to be detected does not have to 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 thereto. For example, the present invention can also be applied to a detection method and a detection device using the SPR method. In this case, the fluorescence detection unit 130 reflects the light corresponding to the amount of the substance to be detected captured by the detection chip 10 on the film forming surface 22 of the prism 20 instead of the fluorescence γ, and emits the light on the emitting surface 23. Is detected. 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 does not have to have the metal film 30.

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

10 Detection chip 20 Prism 21 Incident surface 22 Formation surface 23 Exit surface 24 Bottom surface 30 Metal film 40 Flow path lid 41 Flow path 50 Storage unit 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 1st lens 135 Optical filter 136 2nd lens 137 Light receiving sensor 140 Liquid feeding unit 141 Chemical solution chip 142 Syringe pump 143 Liquid transfer pump drive unit 144 Syringe 145 Plunger 150 Transfer unit 152 Transfer stage 154 Chip holder 160 Control unit α Excitation light β Reflected light γ Fluorescence

Claims (8)

It has a first surface and a second surface on which a metal film is formed, has a dielectric member transparent to light, and the substance to be detected is captured in a reaction field arranged on the surface side of the metal film. A chip holder for holding the detection chip and
A moving stage for moving the tip holder and
A light irradiation unit that irradiates light toward the first surface of the dielectric member of the detection chip held by the chip holder.
A reflected light detection unit that detects reflected light that is emitted from the light irradiation unit and reflected on the first surface, and
Specimen light detection that detects the light generated according to the amount of the substance to be detected captured by the detection chip by irradiating the light from the light irradiation unit at the detection position for detecting the substance to be detected. Department and
It is a detection method using a detection device having
Of the light emitted from the light irradiation unit, the light emitted from the light irradiation unit to the first surface so that the reflected light reflected by the first surface is detected by the reflected light detection unit. The process of setting the irradiation angle and
The detection chip held in the chip holder so that the irradiation spot of the light emitted from the light irradiation unit passes through the first surface and the boundary between the first surface and the second surface. While moving by the moving stage, light is irradiated from the light irradiation unit at the irradiation angle set in the step of setting the light irradiation angle, the reflected light is detected by the reflected light detection unit, and the reflected light detection unit is used. The step of acquiring the position information of the detection chip held in the chip holder based on the detection result of the reflected light by
A step of obtaining a shape correction value which is a difference between the shape value of the dielectric member to be formed and the shape value of the formed dielectric member.
A step of moving the detection chip to the detection position based on the obtained position information and the shape correction value.
The substance to be detected captured by the detection chip at the detection position by irradiating light from the light irradiation unit and detecting the light by the sample light detection unit while the detection chip is in the detection position. And the process of detecting the presence or amount of
Detection methods, including. The detection chip further has a storage unit for storing the shape correction value, and has a storage unit.
The shape correction value is stored in the storage unit.
The detection method according to claim 1. The detection method according to claim 1 or 2, wherein the shape correction value satisfies the following formula (1).
Shape correction value Δx = h (tanθa + tanθb) ... Equation (1)
In the formula (1), the distance between the second surface and the second surface side end of the first surface in the normal direction of the metal film is h, and the light emitted from the light irradiation unit. Let θa be the angle formed by the optical axis of No. 1 and the virtual straight line along the normal line, and let θb be the angle formed by the first surface and the virtual straight line. The shape correction value is obtained based on the distance between the center and the boundary of the reaction field in the dielectric member to be formed and the distance between the center of the reaction field and the boundary in the formed dielectric member. The detection method according to claim 1 or 2, which is a value. The detection method according to claim 3 or 4, wherein the shape correction value is a value obtained based on the shape of the end portion of the first surface to which the light emitted from the light irradiation unit is incident. The dielectric member further has a third surface that is disposed so that it faces the second surface.
The shape correction value is a value obtained based on the dihedral angle of the second surface and the third surface.
The detection method according to claim 1. In the step of moving the detection chip to the detection position, the detection chip is moved to the detection position based on the shape correction value after the detection chip is moved based on the position information. The detection method according to any one of 5. It has a first surface and a second surface on which a metal film is formed, has a dielectric member transparent to light, and the substance to be detected is captured in a reaction field arranged on the surface side of the metal film. A chip holder for holding the detection chip and
A moving stage for moving the tip holder and
A light irradiation unit that irradiates light toward the first surface of the dielectric member of the detection chip held by the chip holder.
A reflected light detection unit that detects reflected light that is emitted from the light irradiation unit and reflected on the first surface, and
Specimen light detection that detects the light generated according to the amount of the substance to be detected captured by the detection chip by irradiating the light from the light irradiation unit at the detection position for detecting the substance to be detected. Department and
A control unit that controls the chip holder, the moving stage, the light irradiation unit, the reflected light detection unit, and the sample light detection unit.
Have,
The control unit
Of the light emitted from the light irradiation unit, the light emitted from the light irradiation unit to the first surface so that the reflected light reflected by the first surface is detected by the reflected light detection unit. Set the irradiation angle and
The detection chip held in the chip holder so that the irradiation spot of the light emitted from the light irradiation unit passes through the first surface and the boundary between the first surface and the second surface. While moving by the moving stage, light is irradiated from the light irradiation unit at the irradiation angle set in the step of setting the light irradiation angle, the reflected light is detected by the reflected light detection unit, and the reflected light detection unit is used. Based on the detection result of the reflected light by, the position information of the detection chip held in the chip holder is acquired, and the position information is acquired.
A shape correction value, which is the difference between the shape value of the dielectric member to be formed and the shape value of the formed dielectric member, is obtained.
Based on the obtained position information and the shape correction value, the detection chip is moved to the detection position by the movement stage.
Detection device.

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