JP6717201B2 - Detection method and detection device - Google Patents

Description

The present invention relates to a detection method and a detection device for detecting the presence or the amount of a substance to be detected contained in a sample by using surface plasmon resonance.

If a trace amount of a substance to be detected such as protein or DNA can be detected with high sensitivity and quantitatively in a clinical test or the like, it is possible to quickly grasp the patient's condition and perform treatment. Therefore, there is a demand for a method capable of detecting a trace amount of a substance to be detected with high sensitivity and quantitatively.

Surface Plasmon-field enhanced Fluorescence Spectroscopy (hereinafter abbreviated as "SPFS") is known as a method capable of detecting a substance to be detected with high sensitivity (for example, refer to Patent Document 1). ..

The surface plasmon excitation enhanced fluorescence analysis method described in Patent Document 1 irradiates excitation light while changing the incident angle to the metal thin film so that the prism on which the metal thin film is formed is totally reflected by the metal thin film. The step of measuring the plasmon scattered light generated on the surface of the metal thin film due to the total reflection of the excitation light by the thin film, and the incident angle (enhancing angle) that maximizes the intensity of the measured plasmon scattered light The angle of incidence with respect to the target substance, and the second capture labeled with the substance to be detected and the fluorescent substance on the metal thin film on which the first capture body (eg, primary antibody) capable of specifically binding to the substance to be detected is immobilized. And a detection step of measuring the fluorescence intensity of fluorescence emitted from the fluorescent substance labeled with the substance to be detected on the metal thin film, by irradiating excitation light at a determined enhancement angle And, including.

In the surface plasmon excitation enhanced fluorescence analysis method described in Patent Document 1, when the substance to be detected is provided on the metal thin film, the substance to be detected is bound to the first capturing body. Next, when the second capturing body and the fluorescent substance are provided on the metal thin film to which the substance to be detected is bound, the substance to be detected is labeled with the fluorescent substance. When the metal thin film is irradiated with excitation light in this state, the fluorescent substance labeling the substance to be detected is excited by an electric field enhanced by Surface Plasmon Resonance (hereinafter abbreviated as “SPR”) and emits fluorescence. To do. Therefore, the presence or amount of the substance to be detected can be detected by detecting the fluorescence emitted from the fluorescent substance.

International Publication No. 2011/152064

However, it is known that the enhancement angle used in the surface plasmon excitation enhanced fluorescence analysis method described in Patent Document 1 varies depending on the temperature. Specifically, the enhancement angle changes due to the increase or decrease in the refractive index of the metal thin film due to the temperature change of the metal thin film, the expansion or contraction of the prism due to the temperature change, and the like. In particular, in an analysis method in which the temperature is not adjusted, the temperature at the time of determining the enhancement angle may differ from the temperature at the time of detecting the substance to be detected. There is a problem in that detection cannot be performed and the detection accuracy decreases.

An object of the present invention is to provide a detection method and a detection apparatus that utilize surface plasmon resonance, which can detect a substance to be detected with higher sensitivity.

In order to solve the above problems, a detection method according to an embodiment of the present invention is a detection method for detecting the presence or the amount of a substance to be detected contained in a sample by using surface plasmon resonance. A first step of providing the analyte on the metal film of a chip having a metal film and a capturing body fixed on the metal film to bind the capturing body and the substance to be detected, After the first step, the temperature of the metal film is directly or indirectly measured, and the incident light is incident when the intensity of the plasmon scattered light generated from the metal film due to the temperature of the metal film and the incident light is maximum. Based on the information on the relationship between the enhancement angle which is the angle or the resonance angle which is the incident angle of the incident light when the amount of reflected light of the incident light irradiated on the metal film is the minimum, the incident angle of the incident light is A second step of determining the enhancement angle or the resonance angle, and after the second step, irradiating the metal film with incident light at the enhancement angle or the resonance angle and detecting a signal generated by the incident light. And a third step of detecting the substance to be detected.

Further, in order to solve the above problems, the detection device according to one embodiment of the present invention is a detection device for detecting the presence or the amount of a substance to be detected contained in a sample by using surface plasmon resonance. Then, so that surface plasmon resonance occurs, a light irradiation unit that irradiates the metal film of the chip having a metal film and a capturing body fixed on the metal film with incident light, and irradiates the metal film. A photodetector for detecting a signal generated by incident light, a temperature measuring unit for directly or indirectly measuring the temperature of the metal film, the temperature of the metal film, and a plasmon generated from the metal film by the incident light. A resonance angle, which is the incident angle of the incident light when the intensity of the scattered light is the maximum, which is the incident angle of the incident light, or the amount of the reflected light of the incident light that is applied to the metal film is the minimum, Based on the temperature of the metal film measured by the temperature measuring unit and the information stored in the storage unit, the incident angle of the incident light is the enhancement angle. Or a processing unit for determining the resonance angle, wherein the light irradiation unit irradiates the metal film with incident light at the enhancement angle or the resonance angle determined by the processing unit, The detection unit detects a signal generated by the incident light with which the metal film is irradiated at the enhancement angle or the resonance angle determined by the processing unit.

According to the present invention, it is possible to detect a substance to be detected with higher sensitivity in a detection method and a detection apparatus that utilize surface plasmon resonance.

FIG. 1 is a schematic diagram showing the configuration of the detection device according to the first embodiment. 2A to 2C are diagrams showing the configuration of the chip. FIG. 3 is a flowchart showing the operation of the detection device according to the first embodiment. FIG. 4 is a flowchart showing a method of adjusting the incident angle. FIG. 5 is a graph showing the relationship between the temperature difference of the metal film and the incident angle correction angle. FIG. 6 is a schematic diagram showing the configuration of the detection device according to the second embodiment. 7A and 7B are perspective views of the diffraction grating. 8A and 8B are diagrams schematically showing a second aspect of the chip. FIG. 9 is a flowchart showing the operation of the detection device according to the second embodiment. FIG. 10 is a flowchart showing another operation of the detection device according to the first and second embodiments. FIG. 11 is a flowchart showing a method for correcting fluorescence intensity. FIG. 12 is a graph showing the relationship between the temperature difference of the metal film and the correction coefficient of the fluorescence intensity. FIG. 13 is a schematic diagram showing the configuration of the detection device according to the third embodiment. FIG. 14 is a flowchart showing the operation of the detection device according to the third embodiment.

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

[Embodiment 1]
FIG. 1 is a schematic diagram showing the configuration of a detection device 100 according to the first embodiment of the present invention.

As shown in FIG. 1, the detection device 100 includes a chip holder 70, a light irradiation unit 110, a light detection unit 120, a temperature measurement unit 130, and a control unit 140 including a storage unit 141 and a processing unit 142. Have. The detection device 100 is used with the chip 10 having the prism 20 mounted on the chip holder 70. The detection device 100 according to the first embodiment is a prism coupling (PC)-SPFS device that uses the prism 20. Therefore, the chip 10 having the prism 20 will be described first, and then the detection device 100 will be described.

(Chip configuration)
FIG. 2 is a schematic diagram showing the configuration of the chip. 2A is a perspective view of the chip 10, FIG. 2B is a cross-sectional view of the chip 10, and FIG. 2C is a cross-sectional view of another form of the chip 10′.

As shown in FIGS. 2A and 2B, the chip 10 has a prism 20, a metal film 30, and a channel cover 40. Usually, the chip 10 is replaced after each detection.

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 and an exit surface 23. The incident surface 21 causes the excitation light α from the 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 incident on the inside of the prism 20 is reflected on the back surface of the metal film 30. More specifically, the excitation light α is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30. The emission surface 23 emits the reflected light β of the excitation light α reflected on the back surface of the metal film 30 to the outside of the prism 20.

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 one leg is the entrance surface 21, and the surface corresponding to the other leg is the exit surface 23.

The incident surface 21 is formed so that the excitation light α does not return to the 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 predetermined scanning range. In the present embodiment, the angle formed by the incident surface 21 and the film forming surface 22 and the angle formed by the film forming surface 22 and the emitting surface 23 are both about 80°.

It should be noted that the design of the chip 10 generally determines the enhancement angle. 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 enhancement angle shifts depending on the substance to be detected immobilized on the metal film 30, but the amount thereof is less than several degrees. Here, the “enhancement angle” is the incident angle of the excitation light α that maximizes the amount of plasmon scattered light generated in the metal film 30 by irradiating the metal film 30 with the excitation light α.

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 local interaction on the surface of the metal film 30 occurs. In-situ light can be generated.

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 in the range of 30 to 70 nm.

A capture body for capturing the substance to be detected is fixed to the metal film 30 (reaction field). The capture body specifically binds to the substance to be detected. In the present embodiment, the capturing body is substantially uniformly immobilized on the surface of the metal film 30. The type of capturing body is not particularly limited as long as it can capture the substance to be detected. For example, the capturing body is an antibody (primary antibody) specific to the substance to be detected or a fragment thereof, an enzyme capable of specifically binding to the substance to be detected, and the like.

The method for immobilizing the capturing body is not particularly limited. For example, a self-assembled monolayer (hereinafter referred to as “SAM”) or a polymer film to which a capturing body is bound may be formed on the metal film 30. Examples of SAM include HOOC- _{(CH 2)} 11 film formed by substituted aliphatic thiols such as -SH. Examples of materials that make up the polymeric membrane include polyethylene glycol and MPC polymers. Alternatively, a polymer having a reactive group (or a functional group that can be converted into a reactive group) capable of binding to the capturing body may be immobilized on the metal film 30, and the capturing body may be bound to this polymer.

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 formation surface 22 of the prism 20, the flow path lid 40 may be arranged on the film formation surface 22. A channel groove is formed on the back surface of the channel lid 40, and the channel lid 40 forms a channel 43 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. Both ends of the channel 43 are connected to an inlet 41 and an outlet 42 formed on the upper surface of the channel lid 40, respectively. The contact-type temperature sensor 131 described later is inserted from the inlet 41 or the outlet 42 and measures the temperature of the metal film 30 (see FIG. 2B).

The flow path lid 40 is preferably made of a material transparent to the fluorescence γ and the plasmon scattered light δ 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 and the plasmon scattered light δ to the outside is transparent to the fluorescence γ and the plasmon scattered light δ, the other portion of the flow path lid 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, adhesion with a double-sided tape or an adhesive, laser welding, ultrasonic welding, pressure bonding using a clamp member, or the like.

Note that, as shown in FIG. 2C, the chip 10 ′ may have a well 44 instead of the flow channel 43. In this chip 10 ′, the sample is provided from the opening of the well 44 (the through hole formed in the flow path lid 40 ′), and the temperature of the metal film 30 is measured by the temperature sensor 131.

As shown in FIG. 1, the excitation light α enters the prism 20 from 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). By irradiating the metal film 30 with the excitation light α at such an angle that surface plasmon resonance occurs, localized field light (generally called “evanescent light” or “near-field light”) is radiated 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 the fluorescence γ is emitted. The detection apparatus 100 detects the presence or amount of the substance to be detected by detecting the amount of fluorescence γ emitted from the fluorescent substance.

(Structure of SPFS device)
Next, each component of the detection device 100 will be described. As described above, the detection device 100 includes the chip holder 70, the light irradiation unit 110, the light detection unit 120, the temperature measurement unit 130, and the control unit 140 including the storage unit 141 and the processing unit 142.

The chip holder 70 holds the chip 10. The shape of the chip holder 70 is not particularly limited as long as it can hold the chip 10 and does not interfere with the optical path of the excitation light α. In the present embodiment, the chip holder 70 is configured to hold the chip 10 by gripping the channel cover 40 from the side.

The light irradiation unit 110 emits the excitation light α to the chip 10 (the back surface of the metal film 30) held by the chip holder 70. At the time of measuring the fluorescence γ, the light irradiation unit 110 emits the excitation light α 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.

The light irradiation unit 110 includes a light source unit 111, a first angle adjustment mechanism 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 of the excitation light α, a beam shaping optical system, an APC mechanism, and a temperature adjusting mechanism (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 may include, for example, a collimator, a bandpass filter, a linear polarization filter, a half-wave plate, a slit, a zoom means, or the like. 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 zoom 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 mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light α with a photodiode (not shown) or the like. Then, the APC mechanism controls the output of the light source to be constant by controlling the input energy by the regression circuit. The temperature adjusting mechanism is, for example, a heater or a Peltier element. The wavelength and energy of the light emitted from the light source may change depending on the temperature. Therefore, the temperature and the energy of the light emitted from the light source are controlled to be constant by keeping the temperature of the light source constant by the temperature adjusting mechanism.

The first angle adjusting mechanism 112 adjusts the angle of incidence of the excitation light α on the back surface of the metal film 30 (the interface between the prism 20 and the metal film 30 (deposition surface 22)). The first angle adjusting mechanism 112 irradiates the excitation light α at a predetermined incident angle toward a predetermined position on the back surface of the metal film 30 via the prism 20, so that the optical axis of the excitation light α and the chip 10 (metal film). 30) and are rotated relative to each other. In the present embodiment, the first angle adjusting mechanism 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 incident angle is changed. 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 incident angle (between the irradiation position on the film formation surface 22 and the incident surface 21), the irradiation position The deviation can be minimized.

As described above, among the angles of incidence of the excitation light α on the back surface of the metal film 30, the angle at which the maximum amount of plasmon scattered light δ can be obtained is the enhancement angle. By setting the incident angle of the excitation light α to the angle of the enhancement angle, 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 chip 10, the film thickness of the metal film 30, the refractive index of the liquid in the flow channel 43, 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. In the present embodiment, the preferable emission angle of the excitation light α with respect to the normal line N of the metal film 30 is about 70°.

The light source control unit 113 controls various devices included in the light source unit 111 to control emission of the emission light (excitation light α) of the light source unit 111. The light source control unit 113 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 light detection unit 120 irradiates the fluorescence γ generated by irradiating the back surface of the metal film 30 with the excitation light α at the time of detecting the substance to be detected, and irradiates the back surface of the metal film 30 with the excitation light α at the time of measuring the enhancement angle. And the plasmon scattered light δ generated by The light detection unit 120 includes, for example, a light receiving unit 121, a position switching mechanism 122, and a light receiving sensor control unit 123.

The light receiving unit 121 is arranged in the direction Z normal to the surface of the metal film 30 of the chip 10. The light receiving unit 121 includes a first lens 124, an optical filter 125, a second lens 126 and a light receiving sensor 127.

The first lens 124 is, for example, a condenser lens, and condenses the light emitted from the metal film 30. The second lens 126 is, for example, an image forming lens, and forms an image of the light condensed by the first lens 124 on the light receiving surface of the light receiving sensor 127. The optical path between both lenses is a substantially parallel optical path. The optical filter 125 is arranged between both lenses.

The optical filter 125 guides only the fluorescence component to the light receiving sensor 127, and removes the excitation light component (plasmon scattered light δ) in order to detect the fluorescence γ with a high S/N ratio. Examples of the optical filter 125 include an excitation light reflection filter, a short wavelength cut filter, and a bandpass filter. The optical filter 125 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 127 detects fluorescence γ or plasmon scattered light δ. The light receiving sensor 127 has high sensitivity capable of detecting weak fluorescence γ or plasmon scattered light δ from a minute amount of the substance to be detected. The light receiving sensor 127 is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).

The position switching mechanism 122 switches the position of the optical filter 125 to the optical path in the light receiving unit 121 or to the outside of the optical path. Specifically, the position switching mechanism 122 arranges the optical filter 125 on the optical path of the light receiving unit 121 when the light receiving sensor 127 detects the fluorescence γ, and when the light receiving sensor 127 detects the plasmon scattered light δ, the optical switching is performed. The filter 125 is arranged outside the optical path of the light receiving unit 121. The position switching mechanism 122 is composed of, for example, a rotation drive unit and a known mechanism (turntable, rack and pinion, etc.) that moves the optical filter 125 in the horizontal direction by utilizing the rotational movement.

The light receiving sensor control unit 123 controls detection of the output value of the light receiving sensor 127, management of the sensitivity of the light receiving sensor 127 based on the detected output value, change of the sensitivity of the light receiving sensor 127 to obtain an appropriate output value, and the like. The light receiving sensor control unit 123 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 temperature measuring unit 130 directly or indirectly measures the temperature of the metal film 30. The temperature measurement unit 130 has a temperature sensor 131 and a temperature sensor adjustment mechanism 132. The type of the temperature sensor 131 is not particularly limited as long as it can measure the temperature of the metal film 30, and may be a contact type or a non-contact type. Examples of the contact type temperature sensor 131 include a thermistor thermometer, a resistance temperature detector thermometer, a thermocouple thermometer, and the like. Further, examples of the non-contact type temperature sensor 131 include a radiation thermometer, a two-color thermometer, a thermography, and the like. In the present embodiment, the temperature sensor 131 is a contact type thermometer. The temperature sensor adjustment mechanism 132 sends the position of the temperature sensor 131 and the detected output value to the control unit 140 (storage unit 141).

The control unit 140 is configured by, for example, a known computer or a microcomputer including a storage unit 141, a processing unit 142, a computing device, a control device, an input device, and an output device, and the first angle adjustment mechanism 112 and a light source control unit. 113, the position switching mechanism 122, the temperature sensor adjusting mechanism 132, and the light receiving sensor control unit 123 are controlled. The storage unit 141 stores information on the relationship between the temperature of the metal film 30 and the optimum incident angle of incident light. Specifically, in the present embodiment, the storage unit 141 has a relationship between the temperature of the metal film 30 and the enhancement angle at which the intensity of the plasmon scattered light δ generated from the metal film 30 by the irradiated incident light is maximum. It stores information. This information may be created before the detection of the substance to be detected or may be created externally. The processing unit 142 determines the incident angle of the incident light to be the optimum incident angle (enhancing angle) based on the temperature of the metal film 30 measured by the temperature measuring unit 130 and the information stored in the storage unit 141.

(Detection operation of the detection device)
Next, the detection operation of the detection device 100 (the detection method according to the first embodiment) will be described. FIG. 3 is a flowchart showing an example of an operation procedure of the detection device 100. In this example, a primary antibody is immobilized on the metal film 30 as a capturing body. In addition, a secondary antibody labeled with a fluorescent substance is used as a capturing body used for fluorescent labeling.

First, the measurement is prepared (step S110). Specifically, the chip 10 is prepared, and the chip 10 is placed in the chip holder 70. When the moisturizer is present on the metal film 30 of the chip 10, the moisturizer is removed by washing the metal film 30 so that the primary antibody can properly capture the substance to be detected.

Then, the substance to be detected in the sample is reacted with the primary antibody (primary reaction, step S120). Specifically, a sample is provided on the metal film 30 to bring the sample into contact with the primary antibody. When the substance to be detected is present in the sample, at least part of the substance to be detected binds to the primary antibody. After that, the metal film 30 is washed with a buffer solution or the like to remove the substance not bound to the primary antibody. The types of the sample and the substance to be detected are not particularly limited. Examples of specimens include body fluids such as blood, serum, plasma, urine, nostril, saliva and semen, and diluted solutions thereof. In addition, examples of the substance to be detected include nucleic acids (such as DNA and RNA), proteins (such as polypeptides and oligopeptides), amino acids, sugars, lipids and modified molecules thereof.

Next, the substance to be detected bound to the primary antibody is labeled with a fluorescent substance (secondary reaction, step S130). Specifically, a fluorescent labeling liquid containing a secondary antibody labeled with a fluorescent substance is provided on the metal film 30 to bring the substance to be detected bound to the primary antibody into contact with the fluorescent labeling liquid. The fluorescent labeling solution is, for example, a buffer solution containing a secondary antibody labeled with a fluorescent substance. When the substance to be detected is bound to the primary antibody, at least a part of the substance to be detected is labeled with a fluorescent substance. After labeling with the fluorescent substance, it is preferable to wash the metal film 30 with a buffer solution or the like to remove free secondary antibodies and the like.

The order of the primary reaction (step S120) and the secondary reaction (step S130) is not limited to this. For example, a liquid containing these complexes may be provided on the metal film 30 after the substance to be detected is bound to the secondary antibody. Further, the sample and the fluorescent labeling liquid may be provided on the metal film 30 at the same time.

Next, the incident angle is adjusted (step S140). Specifically, the control unit 140 causes the temperature of the metal film 30 measured by the temperature measurement unit 130 and plasmon scattering generated from the metal film 30 by the excitation light α irradiated on the back surface of the metal film 30 via the prism 20. The incident angle of the excitation light α is determined as the enhancement angle in the metal film 30 based on the information on the relationship with the enhancement angle at which the light intensity is maximum.

Here, a method of obtaining the relationship between the temperature of the metal film 30 and the correction angle of the incident angle will be described in detail with reference to FIGS. 4 and 5. FIG. 4 is a flowchart for explaining a method of adjusting the incident angle. FIG. 5 is a graph showing the relationship between the temperature difference of the metal film and the correction angle of the enhancement angle. The horizontal axis of FIG. 5 is the temperature difference (° C.) of the metal film, and the vertical axis is the correction angle (°) of the incident angle.

It is known that the enhancement angle changes slightly with temperature. Therefore, in the present embodiment, the incident angle is set to the enhancement angle at that temperature according to the temperature of the metal film 30, the excitation light α is irradiated at the incident angle, and the fluorescence emitted from the excited fluorescent substance is emitted. The detection accuracy of the substance to be detected is improved by detecting γ. Specifically, the temperature difference (° C.) between the reference temperature (for example, 20°) of the metal film 30 and the temperature of the arbitrary metal film 30 is previously stored in the storage unit 141, and the correction angle (°) of the incident angle at the temperature difference (°). ) And the information about the relationship with. Here, the incident angle correction angle (°) refers to the temperature of the metal film at the time of detection with respect to the difference value (temperature difference) between the reference temperature of the metal film 30 and the temperature of the metal film 30 at the time of detection. It is the difference value of the enhancement angle from the enhancement angle at the reference temperature for adjusting to the enhancement angle. As shown in FIGS. 4 and 5, in order to obtain the relationship between the temperature difference of the metal film 30 and the correction angle of the incident angle, first, the temperature of any of the plurality of metal films 30 and the metal film 30 concerned. The enhancement angle at the temperature is measured (step S141). When the temperature of the metal film 30 is directly measured, the temperature sensor 131 is inserted into the inlet 41 or the outlet 42 of the channel lid 40, and the temperature sensor 131 is brought into contact with the metal film 30 for measurement. Further, when the temperature of the metal film 30 is jointly measured, the temperature of the flow path lid 40 or the sample is measured with the temperature sensor 131 moved onto the flow path lid 40. At this time, the temperature of the metal film 30 is close to the temperature of the flow path lid 40 or the sample. Next, the difference value between the reference temperature and the temperature of the arbitrary metal film 30 is obtained (step S142). Then, a difference value between the enhancement angle at the reference temperature and the enhancement angle at the temperature of the arbitrary metal film 30 is obtained (step S143). In order to obtain the enhancement angle, the light receiving sensor 127 detects the plasmon scattered light δ while the first angle adjusting mechanism 112 scans the angle of the optical axis of the excitation light α. Then, the incident angle of the excitation light α that maximizes the amount of the plasmon scattered light δ is set as the enhancement angle.

Next, the control unit 140 drives the temperature measuring unit 130 to measure the temperature of the metal film 30 (step S144). Next, the control unit 140 obtains a difference value (temperature difference) between the temperature of the metal film 30 and the reference temperature. Then, the control unit 140 obtains the correction angle of the incident angle corresponding to the difference value, and then determines the enhancement angle corresponding to the temperature of the metal film 30 at the time of detection. Finally, the first angle adjusting mechanism 112 is driven to adjust the incident angle of the excitation light α to be the enhancement angle (step S145).

Next, the substance to be detected is detected (step S150). Specifically, the control unit 140 drives the light source control unit 113 to drive the light receiving sensor control unit 123 while irradiating the excitation light α to the predetermined position of the metal film 30 at the incident angle determined in step S140. The light receiving sensor 127 is controlled so as to detect the intensity of the fluorescence γ emitted from the metal film 30 (the surface of the metal film 30 and its vicinity). At this time, the control unit 140 operates the position switching mechanism 122 to arrange the optical filter 125 on the optical path of the light receiving unit 121. At this time, since the optical filter 125 does not transmit the plasmon scattered light δ, only the fluorescence γ is detected by the light receiving sensor 127. The fluorescence intensity is converted into the amount or concentration of the substance to be detected, if necessary.

The detection device 100 may measure the blank value before the secondary reaction (step S130). In this case, in the step of detecting the substance to be detected (step S150), the blank value is subtracted from the detected value of the fluorescence γ to calculate the amount of fluorescence γ indicating the amount of the substance to be detected in the sample. In the blank value measurement, the control unit 140 irradiates the metal film 30 with the excitation light α while scanning the incident angle of the incident light. Then, the control unit 140 obtains the maximum value of the intensity of the plasmon scattered light generated from the metal film 30 by the irradiation of the excitation light α as the blank value.

As described above, the detection apparatus 100 according to the present embodiment can detect a substance to be detected with an enhancement angle regardless of the temperature of the metal film 30, and thus can detect the substance to be detected with higher sensitivity. You can

[Second Embodiment]
The detection device 200 according to the second embodiment differs from the detection device 100 according to the first embodiment in that it is a grating coupling (GC)-SPFS device that uses the diffraction grating 50. Therefore, the same components as those of the detection device 100 according to the first embodiment are designated by the same reference numerals, and the description thereof will be omitted.

FIG. 6 is a diagram showing the configuration of the detection device 200 according to the second embodiment.

As shown in FIG. 6, the detection device 200 includes a chip holder 80, a light irradiation unit 110, a light detection unit 220, a temperature measurement unit 130, and a control unit 140 including a storage unit 141 and a processing unit 142. Have. The detection device 200 is used with the chip 90 having the diffraction grating 50 mounted on the chip holder 80.

(Chip configuration)
7A and 7B and FIGS. 8A and 8B are schematic diagrams showing the configuration of the chip 90. FIG. 7A is a perspective view of the one-dimensional diffraction grating, and FIG. 7B is a perspective view of the two-dimensional diffraction grating. FIG. 8A is a diagram schematically showing a first aspect of the chip 90, and FIG. 8B is a diagram schematically showing a second aspect of the chip 90′.

As shown in FIGS. 6 to 8, the chip 90 has a substrate 60, a metal film 30, and a channel lid 40. A diffraction grating 50 is formed on the metal film 30. A capturing body (for example, a primary antibody) is immobilized on the diffraction grating 50, and the surface of the diffraction grating 50 also functions as a reaction field for binding the capturing body and the substance to be detected. In addition, in FIG. 6, the capturing body and the substance to be detected are omitted.

The substrate 60 is a support member for the metal film 30. The material of the substrate 60 is not particularly limited as long as it has mechanical strength capable of supporting the metal film 30. Examples of the material of the substrate 60 include inorganic materials such as glass, quartz and silicon, and resins such as polymethylmethacrylate, polycarbonate, polystyrene and polyolefin.

The metal film 30 is arranged on the substrate 60. As described above, the diffraction grating 50 is formed on the metal film 30. When the metal film 30 is irradiated with light, the surface plasmons generated in the metal film 30 are combined with the evanescent waves generated by the diffraction grating 50 to generate surface plasmon resonance. The material, the forming method, and the thickness of metal film 30 in the present embodiment are the same as those of metal film 30 in the first embodiment, the same forming method, and the same thickness.

The diffraction grating 50 produces an evanescent wave when the metal film 30 is irradiated with light. The shape of the diffraction grating 50 is not particularly limited as long as it can generate an evanescent wave. For example, the diffraction grating 50 may be a one-dimensional diffraction grating as shown in FIG. 7A or a two-dimensional diffraction grating as shown in FIG. 7B. In the one-dimensional diffraction grating shown in FIG. 7A, a plurality of parallel ridges are formed on the surface of the metal film 30 at predetermined intervals. In the two-dimensional diffraction grating shown in FIG. 7B, convex portions having a predetermined shape are periodically arranged on the surface of the metal film 30. Examples of the array of convex portions include a square lattice and a triangular (hexagonal) lattice. Examples of the sectional shape of the diffraction grating 50 include a rectangular wave shape, a sine wave shape, and a sawtooth shape.

The method for forming the diffraction grating 50 is not particularly limited. For example, after forming the metal film 30 on the flat plate-shaped substrate 60, the metal film 30 may be provided with an uneven shape. Further, the metal film 30 may be formed on the substrate 60 to which the uneven shape has been previously given. With either method, the metal film 30 including the diffraction grating 50 can be formed.

As shown in FIG. 6, the excitation light α is applied to the metal film 30 (diffraction grating 50) at a predetermined incident angle. In the irradiation region, the surface plasmon generated in the metal film 30 and the evanescent wave generated in the diffraction grating 50 are combined to generate SPR. When a fluorescent substance is present in the irradiation region, the fluorescent substance is excited by the enhanced electric field formed by SPR, and fluorescence γ is emitted. In GC-SPFS, the fluorescence γ is emitted with directivity in a specific direction. The reflected light of the excitation light α hardly occurs.

The chip 90 may have a well 44 instead of the flow channel 43, as shown in FIG. 8B. In this case, the chip 90 ′ provides the sample from the opening of the well 44 (the through hole formed in the flow path cover 40 ′) and measures the temperature of the metal film 30 by the temperature sensor 131.

(Structure of detector)
Next, each component of the detection device 200 will be described. As described above, the detection device 200 includes the diffraction grating 50 including the chip holder 80, the light irradiation unit 110, the light detection unit 220, the temperature measurement unit 130, and the control unit 140 including the storage unit 141 and the processing unit 142. It is a Grating Coupling (GC)-SPFS device utilized.

The chip holder 80 is formed in a box shape whose upper surface is open. The chip holder 80 accommodates the chip 90 therein.

The light irradiation unit 110 irradiates the metal film 30 (diffraction grating 50) of the chip 90 with the excitation light α having a constant wavelength and a constant light amount. At this time, the light irradiation unit 110 irradiates the metal film 30 (diffraction grating 50) with the excitation light α so that the diffraction grating 50 produces diffracted light that can be combined with the surface plasmons in the metal film 30. The optical axis of the excitation light α is along the array direction of the periodic structure in the diffraction grating 50 (x-axis direction in FIGS. 7A and 7B).

The light irradiation unit 110 includes a light source unit 111, a first angle adjustment mechanism 112, and a light source control unit 113.

The first angle adjusting mechanism 112 adjusts the incident angle of the excitation light α on the diffraction grating 50. The first angle adjusting mechanism 112 passes the intersection of the optical axis of the excitation light α and the metal film 30 and uses the straight line on the surface of the metal film 30 as the rotation axis to rotate the optical axis of the excitation light α and the chip 10 (the metal film 30). ) And rotate relative to. In the present embodiment, the first angle adjustment mechanism 112 rotates the light irradiation unit 110 with the straight line on the surface of the metal film 30 as the rotation axis, passing through the intersection of the optical axis of the excitation light α and the metal film 30. .. The incident angle of the excitation light α is appropriately selected according to the pitch of the diffraction grating 50, the wavelength of the excitation light α, the type of metal forming the metal film 30, and the like.

The light detection unit 220 is arranged so as to pass through the intersection of the optical axis of the excitation light α and the metal film 30 with respect to the light irradiation unit 110 and sandwich the normal line N to the surface of the metal film 30. The light detection unit 220 detects the plasmon scattered light δ generated on the metal film 30 or the fluorescence γ emitted from the fluorescent substance on the diffraction grating 50 (reaction field).

The light detection unit 220 includes, for example, a light receiving sensor 127, a second angle adjusting mechanism 221, and a light receiving sensor control unit 123. The light detection unit 220 may further include a condenser lens group, an aperture stop, a fluorescent filter, and the like. The light receiving sensor 127 detects plasmon scattered light δ or fluorescence γ.

The second angle adjusting mechanism 221 adjusts the optical axis of the light receiving sensor 127 so that the light receiving sensor 127 can detect the fluorescence γ. The second angle adjusting mechanism 221 rotates the light receiving sensor 127 with the axis that passes through the intersection of the optical axis of the fluorescence γ and the metal film 30 and is orthogonal to the optical axis of the fluorescence γ as the rotation axis.

The light receiving sensor 127 detects the fluorescence γ and detects the fluorescence image on the metal film 30. For example, the light receiving sensor 127 is a photomultiplier tube having high sensitivity and high S/N ratio. The light receiving sensor 127 may be an avalanche photodiode (APD), a photodiode (PD), a CCD image sensor, or the like.

The condenser lens group (not shown) is arranged between the chip 90 and the light receiving sensor 127 and constitutes a conjugate optical system that is not easily affected by stray light. The condenser lens group forms a fluorescent image on the metal film 30 on the light receiving surface of the light receiving sensor 127.

The fluorescent filter (not shown) is arranged between the chip 90 and the light receiving sensor 127. The fluorescence filter includes, for example, a cut filter and a darkening (ND) filter, removes noise components other than fluorescence γ (for example, excitation light α, external light, etc.) from the light reaching the light receiving sensor 127, and receives light. It adjusts the amount of light reaching 127.

The control unit 140 is configured by, for example, a known computer or a microcomputer including a storage unit 141, a processing unit 142, a control device, an input device and an output device, and the like, the first angle adjustment mechanism 112, the light source control unit 113, and the temperature. The sensor adjustment mechanism 132, the light receiving sensor control unit 123, and the second angle adjustment mechanism 221 are controlled. The storage unit 141 stores information on the relationship between the temperature of the metal film 30 and the optimum incident angle of incident light. Specifically, in the present embodiment, the storage unit 141 has a relationship between the temperature of the metal film 30 and the enhancement angle at which the intensity of the plasmon scattered light δ generated from the metal film 30 by the irradiated incident light is maximum. It stores information. This information may be created before the detection of the substance to be detected or may be created externally. The processing unit 142 determines the incident angle of the incident light to be the optimum incident angle (enhancing angle) based on the temperature of the metal film 30 measured by the temperature measuring unit 130 and the information stored in the storage unit 141.

(Detection operation of the detection device)
Next, the detection operation (detection method) of the detection device 200 according to the second embodiment will be described. The same steps as those of the detection operation of the detection apparatus 100 according to the first embodiment are designated by the same reference numerals, and the description thereof will be omitted.

FIG. 9 is a flowchart showing an example of the operation procedure of the detection device 200. In this example, a primary antibody is immobilized on the metal film 30 (diffraction grating 50) as a capturing body. In addition, a secondary antibody labeled with a fluorescent substance is used as a capturing body used for fluorescent labeling.

First, the measurement is prepared (step S110).

Then, the substance to be detected in the sample is reacted with the primary antibody (primary reaction, step S120).

Next, the substance to be detected bound to the primary antibody is labeled with a fluorescent substance (secondary reaction, step S130).

Then, the incident angle is adjusted (step S240). Specifically, the control unit 140 causes the plasmon scattering generated from the metal film 30 by the temperature of the metal film 30 measured by the temperature measuring unit 130 and the excitation light α irradiated on the surface (diffraction grating 50) of the metal film 30. The incident angle of the excitation light α is determined as the enhancement angle based on the information on the relationship with the enhancement angle at which the intensity of the light δ is maximized. Note that the method of obtaining the relationship between the temperature of the metal film 30 and the enhancement angle is the same as step S140 in the first embodiment except that the surface of the metal film 30 (diffraction grating 50) is irradiated with the excitation light α. Therefore, the description thereof will be omitted.

Next, the substance to be detected is detected (step S150). Specifically, the control unit 140 causes the light source control unit 113 to irradiate the predetermined position on the metal film 30 with the excitation light α at the enhancement angle determined in step S240, while controlling the metal film 30 (the surface of the metal film 30 and its vicinity). ) The light receiving sensor control unit 123 is controlled so as to detect the intensity of the fluorescence γ emitted from above. The fluorescence intensity is converted into the amount or concentration of the substance to be detected, if necessary.

In the detection device 200, the blank value may be measured before the secondary reaction (step S130). In this case, in the step of detecting the substance to be detected (step S150), the blank value is subtracted from the detected value of the fluorescence γ to calculate the amount of fluorescence γ indicating the amount of the substance to be detected in the sample.

As described above, the detection device 200 according to the present embodiment has the same effects as the detection device 100 according to the first embodiment.

(Modification)
Even with the same amount of fluorescent substance, the intensity of fluorescence γ emitted from the fluorescent substance may differ depending on the temperature. Therefore, in the detection method according to the modification of the first embodiment, the intensity of the fluorescence γ is corrected. Other detection operations of the detection devices 100 and 200 according to the first and second embodiments may further include a step S160 of correcting the intensity of the detected fluorescence γ. The same steps as those of the detection operation of the detection devices 100 and 200 according to the first and second embodiments are designated by the same reference numerals and the description thereof will be omitted.

FIG. 10 is a flowchart showing another example of the operation procedure of the detection devices 100 and 200 according to the first and second embodiments.

First, the measurement is prepared (step S110).

Then, the substance to be detected in the sample is reacted with the primary antibody (primary reaction, step S120).

Next, the substance to be detected bound to the primary antibody is labeled with a fluorescent substance (secondary reaction, step S130).

Next, the enhancement angle is adjusted (step S140, step S240).

Next, the substance to be detected is detected (step S150). Specifically, the control unit 140 causes the light source control unit 113 to irradiate the metal film 30 (the metal film 30) with the excitation light α at a predetermined position of the metal film 30 at the enhancement angle determined in the step S140 or the step S240. The light receiving sensor control unit 123 is controlled so as to detect the intensity of the fluorescence γ emitted from the surface and the vicinity thereof.

Next, the detected fluorescence intensity is corrected (step S160). Specifically, the control unit 140 adjusts the fluorescence intensity detected by the light receiving sensor 127 based on the information on the relationship between the temperature of the metal film 30 and the intensity of the fluorescence γ generated by the applied excitation light.

Here, a method of obtaining the relationship between the temperature of the metal film 30 and the correction coefficient of the fluorescence intensity will be described in detail with reference to FIGS. 11 and 12. FIG. 11 is a flowchart for explaining a method of correcting fluorescence intensity. FIG. 12 is a graph showing the relationship between the temperature difference of the metal film and the correction coefficient of the fluorescence intensity. The horizontal axis of FIG. 12 is the temperature difference (° C.) of the metal film, and the vertical axis is the fluorescence. It is a correction coefficient of strength.

As shown in FIGS. 11 and 12, in the storage unit 141, the temperature difference (° C.) between the reference temperature (for example, 20°) of the metal film 30 and the temperature of the arbitrary metal film 30 and the temperature difference thereof. Information on the relationship with the correction coefficient of the fluorescence intensity is stored. Here, the correction coefficient of the fluorescence intensity is an appropriate fluorescence at the temperature of the metal film at the time of detection with respect to the difference value (temperature difference) between the reference temperature of the metal film 30 and the temperature of the metal film 30 at the time of detection. It is the ratio of the fluorescence intensity at an arbitrary temperature to the fluorescence intensity at the reference temperature for adjusting to the intensity. Regarding the relationship between the temperature difference of the metal film 30 and the correction coefficient of the fluorescence intensity, first, the temperatures of the arbitrary plurality of metal films 30 and the fluorescence intensity at the temperature of the metal film 30 are measured (step S161). At this time, the incident angle of the excitation light α is preferably the enhancement angle. Next, the ratio of the fluorescence intensity at an arbitrary temperature to the fluorescence intensity at the reference temperature is obtained (step S162).

Next, the control unit 140 obtains a difference value (temperature difference) between the temperature of the metal film 30 and the reference temperature when the substance to be detected is detected (step S150) (step S163). Then, the control unit 140 obtains the correction coefficient of the fluorescence intensity corresponding to the difference value, and then determines the appropriate fluorescence intensity by using the correction coefficient for the detected fluorescence intensity (step S164). The fluorescence intensity is converted into the amount or concentration of the substance to be detected, if necessary.

In the detection devices 100 and 200 according to the first and second embodiments, the enhancement angle is adjusted based on the information stored in the storage unit 141 in advance, but the information is obtained in the detection operation of the detection devices 100 and 200. You may do it. In this case, the acquisition of the information is performed before the primary reaction (step S120).

Specifically, before the primary reaction, the metal film 30 is heated or cooled, and at the temperature of the metal film 30 and the temperature of the metal film 30, the excitation light α is applied to a predetermined position of the metal film 30. The first angle adjusting mechanism 112 scans the incident angle of the excitation light α with respect to the metal film 30 while irradiating the light. Further, the control unit 140 controls the light detection units 120 and 220 to detect the plasmon scattered light δ from the metal film 30. The reflected light β of the excitation light α reflected by the metal film 30 reaches the light receiving sensor 127. Thereby, the control unit 140 obtains data including the relationship between the incident angle of the excitation light α and the light amount of the plasmon scattered light δ. Then, the control unit 140 analyzes the data and obtains the enhancement angle. Then, information on the relationship between the temperature difference (° C.) between the reference temperature of the metal film 30 and the temperature of the arbitrary metal film 30 and the correction angle (°) of the enhancement angle at the temperature difference may be obtained. Then, the obtained information may be used in steps S140 and S240.

[Third Embodiment]
The detection device 300 according to the third embodiment is different from the detection device 100 according to the first embodiment in that it is an SPR device. Therefore, the same components as those of the detection device 100 according to the first embodiment are designated by the same reference numerals, and the description thereof will be omitted.

FIG. 13 is a schematic diagram showing the configuration of the detection device 300 according to the third embodiment.

As shown in FIG. 13, the detection device 300 includes a chip holder 70, a light irradiation unit 110, a light detection unit 320, a temperature measurement unit 130, and a control unit 140 including a storage unit 141 and a processing unit 142. .. The detection device 300 is used with the chip 10 mounted on the chip holder 70. The same chip holder 70 and chip 10 can be used. The chip holder 70 in the present embodiment does not interfere with the optical path of the excitation light α and the optical path of the reflected light β.

The light detection unit 320 is arranged with respect to the light irradiation unit 110 so as to pass through the intersection of the optical axis of the excitation light α and the metal film 30 and sandwich the normal line N to the surface of the metal film 30. The light detection unit 320 detects the reflected light β of the excitation light α that is incident from the incident surface 21, is reflected by the back surface of the metal film 30, and is emitted from the emitting surface 23.

The light detection unit 320 includes, for example, a light receiving sensor 127, a second angle adjusting mechanism 321, and a light receiving sensor control unit 123. The light detection unit 320 may further include a condenser lens group, an aperture stop, a fluorescence filter, and the like.

The second angle adjusting mechanism 321 adjusts the optical axis of the light receiving sensor 127 so that the light receiving sensor 127 can detect the reflected light β. The second angle adjusting mechanism 321 rotates the light receiving sensor 127 with the axis passing through the intersection of the optical axis of the reflected light β and the back surface of the metal film 30 and orthogonal to the optical axis of the reflected light β as the rotation axis. The light receiving sensor 127 detects the reflected light β. For example, the light receiving sensor 127 is a photomultiplier tube having high sensitivity and high SN ratio.

The control unit 140 is configured by, for example, a known computer or a microcomputer including a storage unit 141, a processing unit 142, a computing device, a control device, an input device, and an output device, and the first angle adjustment mechanism 112 and a light source control unit. 113, the second angle adjustment mechanism 321, the light receiving sensor control unit 123, and the temperature sensor adjustment mechanism 132 are controlled. The storage unit 141 stores information on the relationship between the temperature of the metal film 30 and the optimum incident angle of incident light. Specifically, in the present embodiment, in the storage unit 141, the temperature of the metal film 30 and the amount of the reflected light β reflected by the metal film 30 due to the irradiated incident light and emitted from the emission surface 23 are the minimum. The information on the relationship with the resonance angle is stored. This information may be created before the detection of the substance to be detected or may be created externally. The processing unit 142 determines the incident angle of the incident light to be the optimum resonance angle based on the temperature of the metal film 30 measured by the temperature measuring unit 130 and the information stored in the storage unit 141.

(Detection operation of the detection device)
Next, the detection operation (detection method) of the detection device 300 according to the third embodiment will be described. The same steps as those of the detection operation of the detection apparatus 100 according to the first embodiment are designated by the same reference numerals, and the description thereof will be omitted.

FIG. 14 is a flowchart showing another example of the operation procedure of the detection device 300 according to the third embodiment.

First, the measurement is prepared (step S110).

Then, the substance to be detected in the sample is reacted with the primary antibody (primary reaction, step S120).

Next, the substance to be detected bound to the primary antibody is labeled with a fluorescent substance (secondary reaction, step S130).

Next, the incident angle is adjusted (step S340). Specifically, the control unit 140 measures the temperature of the metal film 30, and measures the temperature of the metal film 30 and the amount of the reflected light β of the excitation light α irradiated on the back surface of the metal film 30 via the prism 20. The incident angle of the excitation light α is determined as the resonance angle based on the information on the relationship with the resonance angle that minimizes. The method of determining the relationship between the temperature of metal film 30 and the enhancement angle is the same as step S140 in the first embodiment except that the resonance angle is determined instead of the enhancement angle. In order to obtain the resonance angle, the light receiving sensor 127 detects the reflected light β while the first angle adjusting mechanism 112 scans the angle of the optical axis of the excitation light α. Then, the incident angle of the excitation light α at which the light amount of the reflected light β is minimized is defined as the resonance angle.

Next, the substance to be detected is detected (step S150). Specifically, the control unit 140 detects the reflected light β of the excitation light α while irradiating the predetermined position of the metal film 30 with the excitation light α by the light source control unit 113 at the resonance angle determined in step S340. Then, the light receiving sensor control unit 123 is controlled. The amount of light is converted into the amount or concentration of the substance to be detected, if necessary.

In the detection device 300, the blank value may be measured before the secondary reaction (step S130). In this case, in the step of detecting the substance to be detected (step S150), the blank value is subtracted from the detected value of the fluorescence γ to calculate the amount of fluorescence γ indicating the amount of the substance to be detected in the sample. In the measurement of the blank value, the control unit 140 irradiates the metal film 30 with the excitation light α via the prism 20 while scanning the incident angle of the incident light. Then, the control unit 140 obtains the minimum value of the reflected light β of the excitation light α reflected by the metal film 30 as the blank value.

As described above, the detection device 300 according to the present embodiment has the same effects as the detection device 100 according to the first embodiment.

This application claims the priority based on Japanese Patent Application No. 2014-253407 filed on Dec. 15, 2014. The contents described in the application specification and drawings are incorporated herein by reference.

In addition, the detection method and the detection device according to the present invention can measure the substance to be detected with high reliability. Therefore, it is expected to contribute to the development, spread and development of a very simple quantitative immunoassay system.

10, 10', 90, 90' Chip 20 Prism 21 Incident surface 22 Film-forming surface 23 Outgoing surface 30 Metal film 40, 40' Channel lid 41 Injection port 42 Discharge port 43 Channel 50 Diffraction grating 60 Substrate 70, 80 Chip Holder 100, 200, 300 Detection device 110 Light irradiation unit 111 Light source unit 112 First angle adjustment mechanism 113 Light source control unit 120, 220, 320 Light detection unit 121 Light receiving unit 122 Position switching mechanism 123 Light receiving sensor control unit 124 First lens 125 Optical filter 126 Second lens 127 Light receiving sensor 130 Temperature measuring unit 131 Temperature sensor 132 Temperature sensor adjusting mechanism 140 Control unit 141 Storage unit 142 Processing unit 221, 321 Second angle adjusting mechanism α Excitation light β Reflected light γ Fluorescence δ Plasmon scattered light

Claims (7)

A detection method for detecting the presence or amount of a substance to be detected contained in a sample by using surface plasmon resonance,
A first step of providing the analyte on the metal film of a chip having a metal film and a capturing body fixed on the metal film, and binding the capturing body and the substance to be detected;
After the first step, the temperature of the metal film is measured directly or indirectly to measure the temperature of the metal film and the incident light when the intensity of plasmon scattered light generated from the metal film due to the incident light is maximum. A second step of determining the incident angle of the incident light to the enhancement angle based on information on the relationship with the enhancement angle which is the incident angle;
After the second step, a third step of detecting the substance to be detected by irradiating the metal film with incident light at the enhancement angle and detecting a signal generated by the incident light.
Detection method. In the first step, on the metal film of a chip having a prism having an incident surface and a film formation surface, a metal film arranged on the film formation surface, and a trapping body fixed to the surface of the metal film. To provide a fluorescent substance for labeling the analyte and the substance to be detected, to bind the capture body and the substance to be detected, and to label the substance to be detected with the fluorescent substance,
In the second step, the relationship between the temperature of the metal film and the enhancement angle at which the intensity of the plasmon scattered light generated from the metal film due to the excitation light applied to the back surface of the metal film through the prism is maximum. Based on the information of, to determine the incident angle of the excitation light to the enhancement angle,
In the third step, the target substance is detected by irradiating the back surface of the metal film with excitation light through the prism and detecting the fluorescence emitted from the fluorescent substance.
The detection method according to claim 1. In the first step, the sample is provided on the diffraction grating of a chip having a metal film having a diffraction grating formed thereon and a capturing body fixed to the diffraction grating and capturing a substance to be detected labeled with a fluorescent substance. And providing a fluorescent substance for labeling the substance to be detected, binding the capture body and the substance to be detected, and labeling the substance to be detected with the fluorescent substance,
In the second step, the excitation is performed based on information on the relationship between the temperature of the metal film and the enhancement angle at which the intensity of plasmon scattered light generated from the metal film by the excitation light with which the diffraction grating is irradiated is maximized. Determining the incident angle of light to be the enhancement angle,
In the third step, the substance to be detected is detected by irradiating the diffraction grating, on which the capturing body is fixed at the enhancement angle, with excitation light, and detecting fluorescence emitted from the fluorescent substance.
The detection method according to claim 1. The method further comprises a fourth step of correcting the intensity of the fluorescence detected in the third step, based on information on the relationship between the temperature of the metal film and the intensity of the fluorescence generated by the applied excitation light. Alternatively, the detection method according to claim 3. A detection device for detecting the presence or amount of a substance to be detected contained in a specimen by using surface plasmon resonance,
A light irradiation unit that irradiates the metal film of a chip having a metal film and a trapping material fixed on the metal film with incident light so that surface plasmon resonance occurs,
A photodetector for detecting a signal generated by incident light applied to the metal film,
A temperature measurement unit for directly or indirectly measuring the temperature of the metal film,
A storage unit that stores information on the relationship between the temperature of the metal film and the enhancement angle that is the incident angle of the incident light when the intensity of the plasmon scattered light generated from the metal film by the incident light is maximum,
A temperature of the metal film measured by the temperature measuring unit, based on the information stored in the storage unit, a processing unit that determines the incident angle of the incident light to the enhancement angle ,
The light irradiation unit irradiates the metal film with incident light at the enhancement angle determined by the processing unit,
The light detection unit detects a signal generated by incident light with which the metal film is irradiated at the enhancement angle determined by the processing unit,
Detection device. The chip has a prism having an incident surface and a film-forming surface, a metal film disposed on the film-forming surface, and a metal substance fixed on the metal film for capturing a substance to be detected labeled with a fluorescent substance. And a capturing body,
The light irradiation unit irradiates the metal film with excitation light through the prism,
The photodetector detects the fluorescence emitted from the fluorescent substance labeled with the substance to be detected by the excitation light applied to the metal film,
The storage unit stores information on the relationship between the temperature of the metal film and the enhancement angle at which the intensity of plasmon scattered light generated from the metal film by the incident light that is irradiated is maximum.
The processing unit determines the incident angle of the excitation light to be the enhancement angle, based on the temperature of the metal film measured by the temperature measuring unit and the information stored in the storage unit,
The detection device according to claim 5 . The chip has a metal film on which a diffraction grating is formed, and a capture body that is fixed to the diffraction grating and captures a substance to be detected that is labeled with a fluorescent substance,
The light irradiation unit irradiates the diffraction grating with excitation light,
The photodetector detects fluorescence emitted from the fluorescent substance labeled with the substance to be detected by the excitation light applied to the diffraction grating,
The storage unit stores information on the relationship between the temperature of the metal film and the enhancement angle at which the intensity of the plasmon scattered light generated in the metal film by the incident light applied is maximum.
The processing unit, based on the temperature of the metal film measured by the temperature measuring unit, and the information stored in the storage unit, determines the incident angle of the excitation light to the enhancement angle,
The detection device according to claim 5 .

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