WO2016152707A1 - Measuring method, measuring device and measuring chip - Google Patents

Abstract

First, light is radiated onto a prism of a measuring chip including said prism, comprising a dielectric resin, and a metal film disposed on one surface of the prism, and the intensity of autofluorescent light emitted from the prism is attenuated. An optical blank value is next measured. Then fluorescence emitted from a fluorescent substance marking a substance being measured is detected, and a fluorescence value is measured. Finally, a signal value is calculated by subtracting the optical blank value from the fluorescence value.

Description

Measuring method, measuring device and measuring chip

The present invention relates to a measuring method and measuring apparatus for measuring a substance to be measured, and a measuring chip that can be used for measuring the substance to be measured.

In clinical examinations and the like, if a very small amount of a substance to be measured such as protein or DNA can be measured with high sensitivity and quantity, it becomes possible to quickly grasp the patient's condition and perform treatment. For this reason, there is a need for a measuring apparatus that can measure a minute amount of a substance to be measured with high sensitivity and quantitatively.

As a measuring device capable of measuring a substance to be measured with high sensitivity, a device using surface plasmon resonance fluorescence analysis (Surface Plasmon-field enhanced Fluorescence Spectroscopy: hereinafter abbreviated as “SPFS”) is known. (For example, refer to Patent Document 1).

In the measurement apparatus described in Patent Document 1, a measurement chip having a prism (transparent support), a metal film formed on the prism, and a capturing body (for example, an antibody) fixed on the metal film is used. . When a specimen containing a substance to be measured is supplied onto the metal film, the substance to be measured is captured by the capturing body (primary reaction). The captured substance to be measured is further labeled with a fluorescent substance (secondary reaction). In this state, when the metal film is irradiated with excitation light through the prism at an angle at which surface plasmon resonance occurs, localized field light can be generated on the surface of the metal film. This localized field light selectively excites the fluorescent substance that labels the substance to be measured captured on the metal film, and the fluorescence emitted from the fluorescent substance is observed. In this measuring apparatus, fluorescence is detected and the presence or amount of a substance to be measured is measured. At this time, in order to eliminate the influence of autofluorescence emitted from the measurement chip, an optical blank measurement is performed before performing the secondary reaction. The substance to be measured can be measured with high accuracy by calculating the signal value by subtracting the optical blank value from the detected value of the amount of fluorescence light (hereinafter also simply referred to as “fluorescence value”).

JP 2013-053902 A

Since the fluorescence from the fluorescent substance that labels a very small amount of the substance to be measured is weak, the measurement device using SPFS uses a highly sensitive light-receiving sensor such as a photomultiplier tube (PMT) or avalanche photodiode (APD). It is common to do. However, these high-sensitivity light-receiving sensors are suitable for detecting weak light, but have a problem that high-precision temperature control is required because the light-receiving sensitivity varies greatly with temperature.

Therefore, the present inventors examined using a light receiving sensor (for example, a photodiode (PD)) whose change in light receiving sensitivity is small even when the temperature changes. By increasing the power of the excitation light source (for example, 1 mW / mm ² or more), the present inventors use SPFS to measure a small amount of light even when using a light-sensitive sensor such as a PD. It has been found that substances can be measured with high accuracy.

However, according to the preliminary experiments by the present inventors, it was found that when the measurement chip is irradiated with high-power excitation light, the amount of autofluorescence emitted from the measurement chip is attenuated. In this way, when the amount of autofluorescence emitted from the measurement chip is attenuated, the amount of autofluorescence when measuring fluorescence is smaller than the amount of autofluorescence when measuring the optical blank value. For this reason, when the optical blank value is subtracted from the fluorescence value, a correct signal value cannot be calculated, and a small value is calculated inaccurately.

As described above, in the measurement method and measurement apparatus using SPFS, when irradiating high-power excitation light, the substance to be measured is measured with high accuracy by the attenuation of the amount of autofluorescence emitted from the measurement chip. You may not be able to.

A first object of the present invention is a measurement method and a measurement apparatus using SPFS, and even when high-power excitation light is irradiated, the influence of attenuation of the amount of autofluorescence emitted from the measurement chip Is to provide a measurement method and a measurement apparatus capable of measuring a substance to be measured with high accuracy. A second object of the present invention is a measurement chip that can be used in a measurement method and a measurement apparatus using SPFS, and is emitted from the measurement chip even when irradiated with high-power excitation light. It is to provide a measuring chip capable of measuring a substance to be measured with high accuracy while suppressing fluctuations in the amount of autofluorescent light.

In order to solve the above problems, a measurement method according to an embodiment of the present invention detects fluorescence emitted from a fluorescent substance that is labeled with a substance to be measured, excited by localized field light based on surface plasmon resonance. A measurement method for measuring a signal value indicating the presence or amount of the substance to be measured, wherein the measurement chip includes a prism made of a resin as a dielectric and a metal film disposed on one surface of the prism. A first step of irradiating the prism with light to attenuate the amount of autofluorescence emitted from the prism; and after the first step, the metal film in a state where the fluorescent material is not present on the metal film. A second step of irradiating the metal film with excitation light through the prism to detect light emitted from the measurement chip and measuring an optical blank value so that surface plasmon resonance occurs at After the second step, in the state where the substance to be measured labeled with the fluorescent material exists on the metal film, surface plasmon resonance is generated in the metal film, and the metal film is passed through the prism. A third step of irradiating excitation light to detect fluorescence emitted from the fluorescent material and measuring a fluorescence value; and after the third step, subtracting the optical blank value from the fluorescence value to obtain the signal value A fourth step of calculating.

In order to solve the above problems, a measuring device according to an embodiment of the present invention is equipped with a measuring chip having a prism made of resin as a dielectric and a metal film disposed on one surface of the prism, By irradiating the metal film with excitation light through the prism, a fluorescent substance that labels the substance to be measured existing on the metal film is excited by localized field light based on surface plasmon resonance, and emitted from the fluorescent substance. A measuring device for measuring a signal value indicating the presence or amount of the substance to be measured by detecting the detected fluorescence, a holder for holding the measuring chip, and autofluorescence emitted from the prism A light irradiating unit for irradiating the measurement chip held by the holder with light for attenuating the amount of light and excitation light for exciting the fluorescent material; A light detection unit that detects light emitted from the measurement chip when the irradiation unit irradiates light to the measurement chip; a processing unit that processes a detection value obtained by the light detection unit; and the light irradiation. And a control unit for controlling the operation of the light detection unit, and the control unit is configured to measure the optical blank value in a state where the fluorescent material is not present on the metal film. The light irradiator is controlled to irradiate the metal film with excitation light through the prism so that surface plasmon resonance occurs, and the light detector is configured to detect light emitted from the measurement chip. In order to measure the fluorescence value, the control unit may cause surface plasmon resonance to occur in the metal film in a state where the target substance labeled with the fluorescent substance exists on the metal film. , The light irradiation unit is controlled to irradiate the metal film with excitation light through a mechanism, and the light detection unit is controlled to detect fluorescence emitted from the fluorescent material. The signal value is calculated by subtracting the optical blank value from the value.

In order to solve the above-described problems, a measurement chip according to an embodiment of the present invention detects fluorescence emitted from a fluorescent substance that is labeled with a substance to be measured, excited by localized field light based on surface plasmon resonance. A measuring chip used in a measuring method for measuring the presence or amount of the substance to be measured, comprising a prism made of a resin as a dielectric, and a metal film disposed on one surface of the prism The decay rate of the amount of autofluorescence emitted from the prism is 1 when the prism is irradiated twice with light having the same irradiation energy as the excitation light that irradiates the metal film when detecting the fluorescence. % Or less.

According to the present invention, a substance to be measured can be measured with high sensitivity and high accuracy.

FIG. 1 is a diagram showing a configuration of a surface plasmon excitation enhanced fluorescence measuring apparatus according to an embodiment of the present invention. FIG. 2 is a flowchart showing an example of the operation procedure of the surface plasmon excitation enhanced fluorescence measurement apparatus. FIG. 3A is a graph showing the relationship between the irradiation energy of the excitation light applied to the prism of the measurement chip and the optical blank value. FIGS. 3B and 3C are diagrams for explaining the effect of aging on the prism of the measurement chip. It is a conceptual diagram. FIG. 4 is a graph showing the relationship between the irradiation energy of the light applied to the resin member of the measurement chip and the attenuation factor of the optical blank value.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the following description, as a representative example of the measurement apparatus according to the present invention, a surface plasmon excitation enhanced fluorescence measurement apparatus (hereinafter also referred to as “SPFS apparatus”) that measures a substance to be measured using surface plasmon resonance (SPR). Will be described.

[Embodiment 1]
In Embodiment 1, a measurement apparatus and a measurement method for measuring a substance to be measured using a measurement chip having a resin member that is not aged will be described. Here, “aging” means that the resin member is irradiated with light to intentionally attenuate the amount of autofluorescence emitted from the resin member.

FIG. 1 is a diagram illustrating a configuration of the SPFS apparatus 100 according to the first embodiment. As shown in FIG. 1, the SPFS device 100 includes a chip holder 110 for detachably holding the measurement chip 10, a light irradiation unit (light irradiation unit) 120 for irradiating the measurement chip 10 with light, A light receiving unit (light detection unit) 130 for detecting light (autofluorescence, plasmon scattered light β or fluorescence γ) emitted from the measurement chip 10, a control unit (processing unit) 140 for controlling these, and a measurement chip 10 and a liquid feeding unit (not shown) for feeding liquid. The SPFS device 100 is used with the measurement chip 10 mounted on the chip holder 110. Therefore, the measurement chip 10 will be described first, and then each component of the SPFS device 100 will be described.

(Configuration of measuring chip)
As shown in FIG. 1, the measuring chip 10 includes a prism 20 having an incident surface 21, a film formation surface 22 and an emission surface 23, a metal film 30 formed on the film formation surface 22, and a film formation surface 22. Alternatively, a flow path lid 40 disposed on the metal film 30 is included. Usually, the measuring chip 10 is replaced for each measurement (analysis).

The prism 20 is transparent to the excitation light α and is made of a resin that is a dielectric. The prism 20 has an incident surface 21, a film forming surface 22, and an exit surface 23.

The incident surface 21 causes the excitation light α from the light irradiation unit 120 to enter the prism 20. A metal film 30 is disposed on the film formation surface 22. The excitation light α incident on the inside of the prism 20 is reflected by the metal film 30. More specifically, the light is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30. The emission surface 23 emits the excitation light α reflected by the metal film 30 to the outside of the prism 20.

The shape of the prism 20 is not particularly limited. In the present embodiment, the shape of the prism 20 is a column having a trapezoidal bottom surface. The surface corresponding to one base of the trapezoid is the film formation surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the emission surface 23. The trapezoid serving as the bottom surface is preferably an isosceles trapezoid. Thereby, the entrance surface 21 and the exit surface 23 are symmetric, and the S wave component of the excitation light α is less likely to stay in the prism 20. The incident surface 21 is formed so that the excitation light α does not return to the light irradiation unit 120. This is because when the excitation light α returns to the laser diode that is the excitation light source, the excitation state of the laser diode is disturbed, and the wavelength and output of the excitation light α change. Therefore, the angle of the incident surface 21 is set so that the excitation light α does not enter the incident surface 21 perpendicularly in a scanning range centered on an ideal resonance angle or enhancement angle.

Here, the “resonance angle” means an incident angle when the amount of reflected light emitted from the emission surface 23 is minimized when the incident angle of the excitation light α with respect to the metal film 30 is scanned. The “enhancement angle” refers to scattered light having the same wavelength as the excitation light α emitted above the measurement chip 10 when the incident angle of the excitation light α with respect to the metal film 30 is scanned (hereinafter referred to as “plasmon scattered light”). This means the angle of incidence when the light quantity of β is maximized. For example, the angle between the incident surface 21 and the film formation surface 22 and the angle between the film formation surface 22 and the emission surface 23 are both about 80 °.

Examples of the resin constituting the prism 20 include polymethyl methacrylate (PMMA), polycarbonate (PC), cycloolefin-based polymer, and the like. The resin constituting the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence. When the prism 20 is formed using the resin (composition) as described above, the prism 20 usually emits autofluorescence when irradiated with light.

The metal film 30 is formed on the film formation surface 22 of the prism 20. By providing the metal film 30, an interaction (surface plasmon resonance; SPR) occurs between the photon of the excitation light α incident on the film formation surface 22 under total reflection conditions and the free electrons in the metal film 30. Local field light can be generated on the surface of the film 30. The material of the metal film 30 is not particularly limited as long as it is a metal that causes 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 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.

Although not particularly shown, a capturing body for capturing the substance to be measured is fixed to the surface of the metal film 30 that does not face the prism 20. By fixing the capturing body, it becomes possible to selectively measure the substance to be measured. Thus, at least a part of the surface of the metal film 30 is set as a reaction field. In the present embodiment, the central portion of the surface of the metal film 30 is set as the reaction field. A trap is uniformly fixed in the reaction field. The type of the capturing body is not particularly limited as long as the substance to be measured can be captured. For example, the capturing body is an antibody or a fragment thereof that can specifically bind to the substance to be measured.

The channel lid 40 is disposed on the surface of the metal film 30 that does not face the prism 20 with the channel 41 interposed therebetween. The channel lid 40 may be disposed on the film formation surface 22 with the channel 41 interposed therebetween. The flow path lid 40 and the metal film 30 (and the prism 20) form a flow path 41 through which a liquid such as a specimen, a fluorescent labeling liquid, and a cleaning liquid flows. The reaction field is exposed in the flow path 41. Both ends of the channel 41 are connected to an inlet and an outlet (both not shown) formed on the upper surface of the channel lid 40, respectively. When liquids are injected into the channel 41, these liquids contact the reaction field capturing body in the channel 41.

The channel lid 40 is a resin member made of a material that is transparent to light (plasmon scattered light β and fluorescence γ) emitted from the reaction field of the metal film 30. The material of the channel lid 40 is not particularly limited as long as it is transparent to these lights. As long as these lights can be guided to the light receiving unit 130, a part of the flow path lid 40 may be formed of an opaque material. The channel lid 40 is bonded to the metal film 30 or the prism 20 by, for example, adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.

Note that the resonance angle (and the enhancement angle near the pole) is generally determined by the design of the measurement chip 10. The design factors are the refractive index of the prism 20, the refractive index of the metal film 30, the film thickness of the metal film 30, the extinction coefficient of the metal film 30, the wavelength of the excitation light α, and the like. The resonance angle and the enhancement angle are shifted by the substance to be measured trapped on the metal film 30, but the amount is less than several degrees.

As shown in FIG. 1, the excitation light α guided to the prism 20 enters the prism 20 through the incident surface 21. The excitation light α incident on the prism 20 is incident on the interface (deposition surface 22) between the prism 20 and the metal film 30 so as to have a total reflection angle (an angle at which surface plasmon resonance occurs). The reflected light from the interface is emitted from the prism 20 on the emission surface 23 (not shown). At this time, when the excitation light α passes through the prism, autofluorescence is emitted from the region that is the optical path of the excitation light α in the prism 20. Part of the autofluorescence passes through the metal film 30 and the flow path lid 40 and travels toward the light receiving unit 130. In addition, when the excitation light α is incident on the above-described interface at an angle at which surface plasmon resonance occurs, plasmon scattered light β and fluorescence γ are emitted from the reaction field toward the light receiving unit 130.

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

The chip holder 110 holds the measurement chip 10 at a predetermined position. The measurement chip 10 is irradiated with the excitation light α from the light irradiation unit 120 while being held by the chip holder 110.

The light irradiation unit 120 emits light for aging the measurement chip 10 (hereinafter referred to as “aging light”) and excitation light α (single mode laser light) toward the measurement chip 10 held by the chip holder 110. Irradiate. More specifically, the light source unit 121 irradiates the back surface of the metal film 30 corresponding to the region where the capturing body is fixed from the prism 20 side of the measuring chip 10 with the excitation light α at a total reflection angle. . The light source unit 121 irradiates the prism 20 with aging light so as to overlap at least the optical path of the excitation light α in the prism 20.

The light irradiation unit 120 includes a light source unit 121 that emits excitation light α, an angle adjustment unit 122 that adjusts the incident angle of the excitation light α with respect to the interface (deposition surface 22) between the prism 20 and the metal film 30, and a light source unit. And a light source control unit 123 that controls various devices included in the device 121.

The light source unit 121 emits aging light and excitation light α. For example, the light source unit 121 includes an aging light source, an excitation light α light source, a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).

The type of the light source for aging light is not particularly limited, but from the viewpoint of efficiently performing aging, it is preferably a high power light source capable of emitting light having the same wavelength as that of the excitation light α. Also, the type of the light source of the excitation light α is not particularly limited, but from the viewpoint of using a light-sensitive sensor such as a photodiode (PD) as the light receiving sensor 135, a high-power light source is preferable. In the present embodiment, the light source of the excitation light α also serves as the light source of aging light, and the central wavelength of the excitation light α and the central wavelength of the aging light are the same. The light source of the excitation light α and aging light is, for example, a laser diode (LD). Other examples of light source types include light emitting diodes, mercury lamps, and other laser light sources. For example, the light emission power of the light source is 1 mW / mm ² or more. The light source of aging light and the light source of excitation light α may be different.

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

The beam shaping optical system includes, for example, a collimator, a band pass filter, a linear polarization filter, a half-wave plate, a slit, and a zoom means. The beam shaping optical system may include all of these or a part thereof.

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

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

The linear polarization filter turns the excitation light α emitted from the light source of the excitation light α into completely linearly polarized light. The half-wave plate adjusts the polarization direction of the excitation light α so that the P-wave component light is incident on the metal film 30. The slit and zoom means adjust the beam diameter, contour shape, and the like of the excitation light α so that the shape of the irradiation spot on the back surface of the metal film 30 is a circle of a predetermined size.

The APC mechanism controls the light source of the excitation light α so that the output of the light source of the excitation light α 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 input energy by the regression circuit, thereby controlling the output of the light source of the excitation light α to be constant.

The temperature adjustment mechanism is, for example, a heater or a Peltier element. The wavelength and energy of the light emitted from the light source of the excitation light α may vary depending on the temperature. For this reason, the wavelength and 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 angle adjusting unit 122 adjusts the incident angles of the excitation light α and the aging light to the metal film 30 (the interface between the prism 20 and the metal film 30 (film formation surface 22)). The angle adjusting unit 122 is configured to irradiate the excitation light α and the aging light at a predetermined incident angle on the metal film 30 (deposition surface 22) with a predetermined incident angle. The tip holder 110 is rotated relatively. In the present embodiment, the angle adjustment unit 122 rotates the light source unit 121 around an axis (axis perpendicular to the paper surface of FIG. 1) orthogonal to the optical axis of the excitation light α on the metal film 30. At this time, the position of the rotation axis is set so that the irradiation position on the metal film 30 (deposition surface 22) hardly moves even when the incident angle is scanned. For example, the position of the rotation center is set 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 forming surface 22 and the incident surface 21 of the prism 20). Thus, the deviation of the irradiation position can be minimized.

The light source control unit 123 controls various devices included in the light source unit 121 to adjust the power and irradiation time of the excitation light α and the aging light from the light source unit 121. The light source control unit 123 includes, 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 receiving unit 130 is disposed so as to face the surface of the metal film 30 of the measurement chip 10 held by the chip holder 110 that does not face the prism 20. The light receiving unit 130 detects autofluorescence emitted from the measurement chip 10 and light (plasmon scattered light β or fluorescence γ) emitted from the metal film 30. The light receiving unit 130 includes a first lens 132, an optical filter 133, a second lens 134, and a light receiving sensor 135 disposed in the light receiving optical system unit 131, a position switching mechanism 136, and an optical sensor control unit 137.

The first lens 132 is, for example, a condensing lens, and condenses light emitted from the metal film 30. The second lens 134 is, for example, an imaging lens, and forms an image of the light collected by the first lens 132 on the light receiving surface of the light receiving sensor 135. Between both lenses, the light is a substantially parallel light beam.

The optical filter 133 is disposed between the first lens 132 and the second lens 134. The optical filter 133 removes the excitation light component (plasmon scattered light β) in order to guide only the fluorescence component to the light receiving sensor 135 and detect the fluorescence γ with a high S / N ratio. Examples of the optical filter 133 include an excitation light reflection filter, a short wavelength cut filter, and a band pass filter. The optical filter 133 is, for example, a filter including a multilayer film that reflects a predetermined light component, or a color glass filter that absorbs a predetermined light component.

The light receiving sensor 135 detects autofluorescence, plasmon scattered light β and fluorescence γ emitted from the measurement chip 10. The type of the light receiving sensor 135 is not particularly limited as long as the above object can be achieved. However, it is preferable that the variation in measured values is small even when the amount of received light is increased, and that the change in characteristics due to a temperature change is small. The light receiving sensor 135 is, for example, a photodiode (PD). As described above, the present inventors increase the power of the light source of the excitation light α (for example, 1 mW / mm ² or more), so that the SPFS can be used even when the light receiving sensor 135 that is not highly sensitive like PD is used. It has been confirmed that it is possible to measure a very small amount of a substance to be measured with high accuracy.

The position switching mechanism 136 switches the position of the optical filter 133 on or off the optical path in the light receiving optical system unit 131. Specifically, when the light receiving sensor 135 measures the optical blank value or the fluorescence value, the optical filter 133 is disposed on the optical path in the light receiving optical system unit 131, and when the light receiving sensor 135 detects the plasmon scattered light β, the optical filter 133 is optical. A filter 133 is disposed outside the optical path.

The light sensor control unit 137 detects the output value of the light receiving sensor 135, manages the sensitivity of the light receiving sensor 135 based on the detected output value, and controls the sensitivity of the light receiving sensor 135 to obtain an appropriate output value. The optical sensor control unit 137 includes, 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 control unit 140 controls the angle adjustment unit 122, the light source control unit 123, the position switching mechanism 136, and the optical sensor control unit 137. The control unit 140 also functions as a processing unit for calculating a signal value indicating the presence or amount of the substance to be measured based on the detection result of the light receiving sensor 135. The control unit 140 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

(Operation of SPFS device)
Next, the operation of the SPFS apparatus 100 (measurement method using the SPFS apparatus 100) will be described. FIG. 2 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus.

First, preparation for measurement is performed (step S10). Specifically, the measurement chip 10 is installed in the chip holder 110 of the SPFS device 100. Further, when a humectant is present in the flow channel 41 of the measurement chip 10, the humectant is removed by washing the flow channel 41 so that the capturing body can appropriately capture the substance to be measured.

Next, the enhancement angle is measured (step S20). Specifically, the irradiation angle of the excitation light α with respect to the metal film 30 (deposition surface 22) is scanned while irradiating the predetermined position of the metal film 30 (deposition surface 22) with the excitation light α, and the optimum Determine the angle of incidence. The control unit 140 controls the light source control unit 123 and the angle adjustment unit 122 to irradiate the excitation light α from the light source unit 121 to a predetermined position of the metal film 30 (deposition surface 22), while forming the metal film 30 (the formation film). The incident angle of the excitation light α with respect to the film surface 22) is scanned. Further, the control unit 140 controls the position switching mechanism 136 to move the optical filter 133 out of the optical path of the light receiving optical system unit 131. At the same time, the control unit 140 controls the optical sensor control unit 137 so that the light receiving sensor 135 detects the plasmon scattered light β. The control unit 140 obtains data including the relationship between the incident angle of the excitation light α and the intensity of the plasmon scattered light β. Then, the control unit 140 analyzes the data and determines an incident angle (enhancement angle) that maximizes the intensity of the plasmon scattered light β. The enhancement angle is determined by the material and shape of the prism 20, the thickness of the metal film 30, the refractive index of the liquid in the flow channel 41, etc. It varies slightly due to various factors such as. For this reason, it is preferable to determine the enhancement angle every time measurement is performed. The enhancement angle is determined on the order of about 0.1 °.

Next, the incident angle of the excitation light α with respect to the metal film 30 (deposition surface 22) is set to the enhancement angle determined in step S20 (step S30). Specifically, the control unit 140 controls the angle adjustment unit 122 to set the incident angle of the excitation light α with respect to the metal film 30 (deposition surface 22) as an enhancement angle. In the present embodiment, the light source of the aging light and the light source of the excitation light α are the same, and the optical paths of the aging light and the excitation light α in the prism 20 are the same. Therefore, the optical path of the aging light in the prism 20 is also set by this process. In the subsequent steps, the incident angle of the excitation light α with respect to the metal film 30 (deposition surface 22) remains the enhancement angle.

　［式（１）において、Ｓ１は後述のシグナル値であり、ＯＢは後述の光学ブランク値であり、Ｂは許容可能な測定誤差の上限値（％）である。Ｂは、以下の式（２）により算出される。］
　［式（２）において、Ｓ１は後述のシグナル値であり、Ｓ２は、後述の真のシグナル値である。］ Next, the measurement chip 10 is aged (step S40). Prior to the measurement of the optical blank value (step S50) and the measurement of the fluorescence value (step S80), the control unit 140 causes the prism 20 to emit light of autofluorescence in order to attenuate the amount of autofluorescence emitted from the prism 20. The light irradiation unit 120 is controlled to emit light for attenuating the light. Specifically, the control unit 140 controls the light source control unit 123 to irradiate the resin member (prism 20) of the measurement chip 10 with aging light from the light source unit 121. Thereby, at least in the region including the optical path of the excitation light α in the prism 20, the amount of autofluorescence emitted from the resin member can be attenuated. From the viewpoint of performing measurement with higher accuracy, the control unit 140 attenuates the amount of autofluorescence to the prism 20 until the attenuation rate A of the amount of autofluorescence emitted from the prism 20 satisfies the following formula (1). It is preferable to control the light irradiation unit 120 so that the light for making it irradiate. That is, it is preferable to age the measurement chip 10 until the attenuation rate A of the amount of autofluorescent light emitted from the resin on the optical path of the excitation light α satisfies the following formula (1).

[In Formula (1), S1 is a signal value to be described later, OB is an optical blank value to be described later, and B is an upper limit value (%) of an allowable measurement error. B is calculated by the following equation (2). ]
[In Formula (2), S1 is a later-described signal value, and S2 is a later-described true signal value. ]

Here, the “attenuation rate of the amount of light of autofluorescence” refers to the autofluorescence emitted during the first light irradiation when the resin member of the measurement chip 10 is irradiated twice with light under the same conditions for a unit time. This means the degree of decrease in the amount of autofluorescence emitted during the second light irradiation with respect to the amount of light. The two light irradiations may be performed continuously or intermittently.

Further, from the viewpoint of attenuating the amount of autofluorescence in a shorter time, from the irradiation energy of the excitation light α irradiated in the subsequent measurement of the optical blank value (step S50) and the measurement of the fluorescence value (step S80). It is preferable to irradiate light with high irradiation energy. When aging light having an irradiation energy higher than the irradiation energy of the excitation light α is irradiated, for example, the output of the light source may be increased or the irradiation time may be lengthened. At this time, the irradiation conditions of the aging light can be appropriately set according to the required measurement accuracy, the resin material of the prism 20, and the like. For example, when a high power LD with a wavelength of 660 nm is used, when the upper limit B of the required measurement accuracy is 1% and both S1 and OB are 1000 (au), the attenuation factor A is 1%. In order to make it less than, irradiation power, irradiation time, etc. should just be set so that total irradiation energy may be 225 mW * second / mm < ² > or more. For example, in the present embodiment, the irradiation of light with an irradiation energy of 2.6 mW · sec / mm ² or more is performed, and the measurement chip 10 is aged until the decay rate A of the amount of autofluorescent light satisfies the above equation (1). I do.

Note that the step of measuring the enhancement angle (step S20) and the step of aging the resin member of the measurement chip 10 (step S40) may be performed simultaneously. For example, the power or time of the excitation light α (aging light) irradiated when measuring the enhancement angle may be adjusted so that the amount of autofluorescence emitted from the measurement chip 10 can be attenuated.

Next, in the state where the fluorescent substance that labels the substance to be measured does not exist on the metal film 30, the excitation light α is irradiated to the metal film 30 (deposition surface 22) through the prism 20, and light having the same wavelength as the fluorescence γ. Is measured (optical blank value) (step S50). Here, the “optical blank value” means the amount of background light measured together with the fluorescence in the measurement of the fluorescence value (step S80). This background light is mainly caused by autofluorescence emitted from the measurement chip 10 (prism 20) when the excitation light α is irradiated.

In order to measure the optical blank value, the control unit 140 transmits the excitation light α to the metal film 30 through the prism 20 so that surface plasmon resonance occurs in the metal film 30 in a state where the fluorescent material is not present on the metal film 30. The light irradiation unit 120 is controlled to irradiate, and the light receiving unit 130 is controlled to detect the light emitted from the measurement chip 10. Specifically, the control unit 140 controls the position switching mechanism 136 to move the optical filter 133 onto the optical path of the light receiving optical system unit 131. Next, the control unit 140 controls the light source control unit 123 to emit the excitation light α from the light source unit 121 toward the metal film 30 (film formation surface 22). At the same time, the control unit 140 controls the light sensor control unit 137 so that the light receiving sensor 135 detects the amount of light having the same wavelength as the fluorescence γ. Thereby, the light receiving sensor 135 can accurately measure the amount of light (optical blank value) that becomes noise. The measured value is transmitted to the control unit 140 and recorded as an optical blank value.

In this step, the control unit 140 controls the light irradiation unit 120 and the light receiving unit 130 so as to measure the optical blank value twice, compares the obtained optical blank value, and compares the obtained optical blank value. It may be confirmed that the aging is sufficiently performed (for example, the attenuation rate A of the amount of autofluorescence light satisfies the above formula (1) (for example, A <1%)). .

Next, the substance to be measured in the sample is reacted with the capturing body (primary reaction; step S60). Specifically, the sample is injected into the channel 41 on the liquid feeding unit side, and the sample and the capturing body are brought into contact with each other. When the substance to be measured exists in the specimen, at least a part of the substance to be measured is captured by the capturing body. Thereafter, the inside of the flow path 41 is washed with a buffer solution or the like to remove substances not captured by the capturing body. The type of specimen is not particularly limited. Examples of the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluted solutions thereof.

Next, the substance to be measured captured by the capturing body is labeled with a fluorescent material (secondary reaction; step S70). Specifically, a fluorescent labeling solution is provided in the channel 41. The fluorescent labeling solution is, for example, a buffer solution containing an antibody (secondary antibody) labeled with a fluorescent substance. When the fluorescent labeling liquid is provided to the flow path 41, the fluorescent labeling liquid comes into contact with the substance to be measured, and the substance to be measured is labeled with the fluorescent substance. Thereafter, the inside of the flow path 41 is washed with a buffer solution or the like to remove free fluorescent substances.

Next, in the state where the measurement target substance labeled with a fluorescent substance is present on the metal film 30, the metal film 30 (deposition surface 22) is irradiated with the excitation light α through the prism 20, so that the measurement target substance in the reaction field is irradiated. The fluorescence value from the fluorescent substance to be labeled is measured (step S80). In order to measure the fluorescence value, the control unit 140 allows the metal film to pass through the prism 20 so that surface plasmon resonance occurs in the metal film 30 in a state where the measurement target substance labeled with the fluorescent substance exists on the metal film 30. The light irradiation unit 120 is controlled so as to irradiate 30 with the excitation light α, and the light receiving unit 130 is controlled so as to detect the fluorescence emitted from the fluorescent material. Specifically, the control unit 140 controls the light source control unit 123 to emit the excitation light α from the light source unit 121. At the same time, the control unit 140 controls the optical sensor control unit 137 to detect the fluorescence γ emitted from the fluorescent substance that labels the substance to be measured by the light receiving sensor 135.

Finally, a signal value indicating the presence or amount of the substance to be measured is calculated (step S90). The fluorescence value mainly includes a fluorescent component (signal value) derived from a fluorescent substance that labels the substance to be measured, and a fluorescent component (optical blank value) derived from the autofluorescence of the measurement chip 10. Therefore, the control unit 140 can calculate a signal value correlated with the amount of the substance to be measured by subtracting the optical blank value obtained in step S50 from the fluorescence value obtained in step S80. The signal value is converted into the amount and concentration of the substance to be measured by a calibration curve prepared in advance.

The signal value indicating the presence or amount of the substance to be measured can be measured by the above procedure. As described above, from the viewpoint of measuring the substance to be measured with high accuracy, the control unit 140 determines that the optical path in the measurement chip 10 of the light irradiated to attenuate the amount of autofluorescence is the optical blank value and the fluorescence value. It is preferable to control the light irradiation unit 120 so as to overlap the optical path in the measurement chip 10 of the excitation light α irradiated for measuring the light. That is, it is preferable that the optical path in the measurement chip 10 for light for aging in step S40 and the optical path in the measurement chip 10 for excitation light α in steps S50 and S80 overlap. Also, from the viewpoint of shortening the aging time, the control unit 140 determines that the power of the light irradiated to attenuate the amount of autofluorescence is excitation light irradiated to measure the optical blank value and the fluorescence value. It is preferable to control the light irradiation unit 120 to be higher than the power of α. That is, the power (mW / mm ² ) of the aging light in step S40 is preferably higher than the power (mW / mm ² ) of the excitation light α in step S50 and step S80.

In the present embodiment, the primary reaction (step S60) and the secondary reaction (step S70) are continuously performed, and the measurement chip 10 is placed between the light irradiation unit 120 and the light receiving unit from the liquid feeding unit side between the two steps. It is not moved to the unit 130 side. For this reason, the total time concerning detection can be shortened by the moving time of the measuring chip 10. In addition, the measurement accuracy can be improved by keeping the primary reaction time, the secondary reaction time, and the interval time between the primary reaction and the secondary reaction constant.

Moreover, the order which performs the process (process S50) which measures an optical blank value, and the process (process S60) which performs a primary reaction is not limited to this, An optical blank value is measured after performing a primary reaction. Also good. In this case, although it is necessary to move the measuring chip 10 between the primary reaction and the secondary reaction, the optical blank value can be measured in a state where the substance to be measured is captured by the capturing body. As a result, since the optical blank value can be measured in a state closer to the step of measuring the fluorescence value (step S80), the optical blank value can be measured more accurately, and the measurement accuracy can be further improved.

(Reference Experiment 1)
Here, the result of having investigated about the effect of the aging with respect to the resin member (prism 20) of the measurement chip | tip 10 is shown. In Reference Experiment 1, the optical blank value was measured by intermittently irradiating the metal film 30 of the measurement chip 10 with the excitation light α from the prism 20 side so that the incident angle becomes an enhancement angle. At this time, the prism 20 of the measuring chip 10 was intermittently irradiated with light having an irradiation energy of 22.6 mW · sec / mm ² . A high power LD was used as the light source, and a photodiode (PD) was used as the light receiving sensor 135. The material of the prism 20 is a cycloolefin polymer.

FIG. 3A is a graph showing the relationship between the irradiation energy of the excitation light α irradiated on the prism 20 of the measurement chip 10 and the optical blank value, and FIGS. 3B and 3C show the effects of aging on the prism 20 of the measurement chip 10. It is a conceptual diagram for demonstrating. 3A, the horizontal axis represents the total irradiation energy (mW · second / mm ² ) of the excitation light α irradiated to the prism 20, and the vertical axis represents the optical blank value (au). 3B and 3C are conceptual diagrams showing the relationship between the optical blank value, the fluorescence value, and the signal value. 3B and C, OB (OB1 and OB2) indicates an optical blank value, F indicates a fluorescence value, and S (S1 and S2) indicates a signal value. OB, F, and S satisfy the following formula (3).
S = F-OB (3)

As shown in FIG. 3A, when the prism 20 of the measurement chip 10 is irradiated with the high power excitation light α, the optical blank value decreases logarithmically (or exponentially) as the excitation light α is irradiated. . From this result, it can be seen that the amount of autofluorescence emitted from the measurement chip 10 decreases as the resin member of the measurement chip 10 is irradiated with the excitation light α. It can also be seen that the autofluorescence decay rate decreases as the excitation light α is irradiated.

Here, for comparison, a case where the measurement of the optical blank value and the subsequent measurement of the fluorescence value are performed without aging the measurement chip 10 will be described. In this case, the optical blank value OB1 and the fluorescence value F are measured with a light amount as shown in FIG. 3B (for convenience of explanation, the light amount of the optical blank value is increased). Since the measurement of the fluorescence value F is performed after the measurement of the optical blank value OB1, considering the graph of FIG. Is attenuated, and the true optical blank value OB2 when the fluorescence value F is measured is also attenuated. Therefore, when the signal value S1 is calculated by subtracting the optical blank value OB1 from the fluorescence value F without considering the attenuation of the amount of autofluorescence, as shown in FIG. 3C, the signal value S1 is greater than the true signal value S2. Is also calculated as a smaller value by the amount of attenuation ΔOB (= OB1−OB2) of the optical blank value.

In contrast, in the measurement method and the measurement apparatus according to the present embodiment, aging is performed by irradiating the measurement chip with light before measuring the optical blank value OB1. As shown in FIG. 3A, the attenuation rate of the amount of autofluorescence decreases as light is irradiated. For this reason, it is measured at the time of measuring the optical blank value by irradiating the prism 20 of the measuring chip 10 with light until the attenuation rate of the light amount of the autofluorescence becomes sufficiently small before measuring the optical blank value OB. The difference (ΔOB) between the optical blank value OB1 and the true optical blank value OB2 when measuring the fluorescence value F can be reduced to a negligible level. As a result, even if the optical blank value OB1 is directly subtracted from the fluorescence value F, a signal value indicating the presence or amount of the substance to be measured can be calculated with high accuracy.

(Reference Experiment 2)
Next, the results of examining the aging conditions required to obtain the desired measurement accuracy are shown. Here, when the optical blank value OB1 and the signal value S1 are both 1000 (au), the true signal value S2, the signal value S1 calculated from the fluorescence value F and the optical blank value OB1, and Based on the above, the aging condition required to realize that the upper limit B of the allowable measurement error calculated from the above-described equation (2) is less than 1% was examined. As is clear from the equation (1), when the optical blank value OB1 and the signal value S1 are both 1000 (au), the attenuation factor A of the amount of autofluorescence is the upper limit of the allowable measurement error. It is necessary that the value is smaller than the value B (A <B). For this reason, in order for the upper limit B of the allowable measurement error to be less than 1%, the attenuation rate A of the autofluorescence light amount (optical blank value) needs to be less than 1%.

In Reference Experiment 2, the metal film 30 of the measurement chip 10 is irradiated with excitation light α from the prism 20 side so that the incident angle becomes an enhancement angle (irradiation spot diameter of the excitation light α on the metal film 30: φ1. 5 mm), the measurement of the optical blank value was continuously performed. At this time, the prism 20 of the measuring chip 10 was intermittently irradiated with light having an irradiation energy of 22.6 mW · sec / mm ² . A high power LD was used as the light source, and a photodiode (PD) was used as the light receiving sensor 135. The material of the prism 20 is a cycloolefin polymer.

FIG. 4 is a graph showing the relationship between the irradiation energy of the light applied to the resin member of the measuring chip 10 and the attenuation rate of the optical blank value (the amount of autofluorescence). In FIG. 4, the horizontal axis indicates the total irradiation energy (mW · second / mm ² ) of the excitation light α, and the vertical axis indicates the attenuation rate (%) of the optical blank value. The relationship between the optical blank value and the irradiation energy satisfied the following formula (4) (see FIG. 3A).
Optical blank value = −88 × ln (irradiation energy) +1275 (4)
Moreover, the attenuation factor of the optical blank value was calculated from the following formula (5) based on the optical blank value.
Attenuation rate = | OB _{n + 1} / OB _n −1 | × 100 (5)
[In formula (5), OB _n is an optical blank value after irradiation n times the excitation light α to the measurement chip 10. OB _{n + 1} is an optical blank value after the measurement chip 10 is irradiated with the excitation light α n + 1 times. ]

As FIG. 4 shows, the attenuation factor of the optical blank value became small, so that the resin member of the measurement chip | tip 10 was irradiated with light. This is considered because the decay of autofluorescence is saturated due to the aging of the measurement chip 10. In addition, as described above, in the above experimental conditions, when both OB1 and S1 are 1000 (au), the upper limit value B of an allowable measurement error due to attenuation of the optical blank value is set to less than 1%. Therefore, it was found that the attenuation factor A of the optical blank value also needs to be less than 1%, and light with an irradiation energy of 225 mW · sec / mm ² or more should be irradiated as aging light.

(effect)
As described above, according to the measurement method and the measurement apparatus according to the present embodiment, by aging the measurement chip 10 before the optical blank measurement, the attenuation of the autofluorescence during the irradiation of the excitation light α is suppressed. The optical blank value can be accurately measured. For this reason, according to the measuring method and measuring device concerning this embodiment, it is possible to measure the substance to be measured in the specimen with high accuracy. In the measurement method and measurement apparatus according to this embodiment, since the PD is used as the light receiving sensor 135, the substance to be measured can be measured with high accuracy even if the amount of received fluorescence is large. Therefore, the measurement method and the measurement apparatus according to the present embodiment can measure a substance to be measured with a wide dynamic range.

In the above embodiment, the measurement method and the measurement apparatus using SPFS have been described. However, the measurement method and the measurement apparatus according to the present invention are not limited to this, and the measurement includes a high-power light source and a resin member. It can also be used for other fluorescence measurements using the chip.

[Embodiment 2]
In Embodiment 2, an aspect in which a measurement target substance is measured using a measurement chip that has been aged in advance will be described. Since the measurement apparatus according to the present embodiment is the same as the measurement apparatus according to the first embodiment except that the aging light is not irradiated, description of each component of the measurement apparatus is omitted.

As shown in FIG. 1, the measurement chip 10 ′ according to the second embodiment includes a prism 20 ′, a metal film 30, and a channel lid 40. The measurement chip 10 ′ according to the present embodiment is the same as the measurement chip 10 according to the first embodiment except that the prism 20 ′ is pre-aged, and thus description of each component is omitted.

An example of a method for manufacturing the measurement chip 10 'according to the present embodiment will be described. The measurement chip 10 ′ according to the present embodiment can be manufactured, for example, by performing a process for producing a measurement chip and a process for aging the chip. Hereinafter, each step will be described.

First, a measurement chip is prepared. Specifically, a prism made of resin as a dielectric and a flow path lid are prepared. Next, the metal film 30 is formed on the prepared prism. For example, the metal film 30 may be formed by vacuum deposition on one surface of a prism formed into a desired shape by injection molding. Next, the capturing body is immobilized on the metal film 30. The method for immobilizing the capturing body is not particularly limited, and can be appropriately selected from known methods. Next, the prism on which the metal film 30 and the reaction field are formed and the flow path lid are fixed. For example, the prism and the channel cover may be bonded with a double-sided tape or an adhesive.

Next, the prepared chip is aged. Specifically, the produced chip is irradiated with aging light. The aging light irradiation conditions can be appropriately set according to the type of resin constituting the prism. At this time, the aging of the chip is performed until the decay rate of the amount of autofluorescent light emitted when the resin member (prism) of the chip is irradiated with light becomes a desired value (for example, 1%) or less. For example, when a high power LD with a wavelength of 660 nm is used, the irradiation power (in this case, irradiation energy of 22.6 mW · second / mm ² ), irradiation time, etc. so that the total irradiation energy is about 225 mW · second / mm ^2. Should be set.

By the above procedure, the fluorescent substance that labels the substance to be measured is detected by the fluorescence γ emitted and excited by the localized field light based on the surface plasmon resonance, and the measurement method is used to measure the presence or amount of the substance to be measured. A measurement chip 10 ′ used, which has a prism 20 ′ and a metal film 30, and continuously emits light with the same irradiation energy as the excitation light α irradiated to the metal film 30 when detecting fluorescence. Thus, the measurement chip 10 ′ in which the attenuation rate of the amount of autofluorescent light emitted from the prism 20 ′ is less than a desired value (for example, 1%) when irradiated twice can be manufactured. The attenuation of the amount of autofluorescence due to the aging of the measurement chip 10 'does not recover over time (see FIGS. 3A and 4). For this reason, if aging is performed in advance when the measurement chip 10 ′ is manufactured, it is not necessary to perform aging when measuring the substance to be measured.

The measurement method for measuring the substance to be measured using the measurement chip 10 ′ according to the present embodiment is the same as the measurement method of the first embodiment except that aging is not performed, and thus the description thereof is omitted. .

(effect)
According to the present embodiment, the same effect as in the first embodiment can be obtained without performing aging during measurement.

In the second embodiment, the case of aging after manufacturing the chip has been described. However, the measurement chip 10 ′ may be manufactured by fixing the aged prism 20 ′ and the channel lid 40. .

This application claims priority based on Japanese Patent Application No. 2015-058016 filed on March 20, 2015. The contents described in the application specification and the drawings are all incorporated herein.

The measuring method and measuring apparatus according to the present invention can measure a substance to be measured with high sensitivity and high accuracy, and are useful for clinical examinations, for example.

10, 10 'Measuring chip 20, 20' Prism 21 Incident surface 22 Deposition surface 23 Emission surface 30 Metal film 40 Channel lid 41 Channel 100 Surface plasmon resonance fluorescence analyzer (SPFS device)
DESCRIPTION OF SYMBOLS 110 Chip holder 120 Light irradiation unit 121 Light source unit 122 Angle adjustment part 123 Light source control part 130 Light reception unit 131 Light reception optical system unit 132 1st lens 133 Optical filter 134 2nd lens 135 Light reception sensor 136 Position switching mechanism 137 Optical sensor control part 140 Control unit α Excitation light β Plasmon scattered light γ Fluorescence

Claims (15)

This is a measurement method in which a fluorescent substance that labels a substance to be measured detects fluorescence emitted by being excited by localized field light based on surface plasmon resonance, and measures a signal value indicating the presence or amount of the substance to be measured. And
Irradiating light to the prism of the measuring chip having a prism made of resin as a dielectric and a metal film disposed on one surface of the prism, attenuates the amount of autofluorescence emitted from the prism. 1 process,
After the first step, the measurement is performed by irradiating the metal film with excitation light through the prism so that surface plasmon resonance occurs in the metal film in a state where the fluorescent material is not present on the metal film. A second step of detecting light emitted from the chip and measuring an optical blank value;
After the second step, the metal film is excited through the prism so that surface plasmon resonance occurs in the metal film in a state where the substance to be measured labeled with the fluorescent material exists on the metal film. A third step of irradiating light, detecting fluorescence emitted from the fluorescent material, and measuring a fluorescence value;
A fourth step of calculating the signal value by subtracting the optical blank value from the fluorescence value after the third step;
Including a measuring method. 2. The measurement method according to claim 1, wherein in the first step, the prism is irradiated with light until an attenuation rate A of the light amount of the autofluorescence emitted from the prism satisfies the following formula (1).

[In Formula (1), S1 is the said signal value, OB is the said optical blank value, B is the upper limit (%) of the allowable measurement error. ] The measurement method according to claim 2, wherein in the second step, the optical blank value is measured twice, and it is confirmed that the attenuation rate A of the light amount of the autofluorescence satisfies the formula (1). The optical path in the measurement chip of the light irradiated in the first step overlaps the optical path in the measurement chip of the excitation light irradiated in the second step and the third step. 4. The measuring method according to any one of 3. The measurement method according to any one of claims 1 to 4, wherein the power of the light irradiated in the first step is higher than the power of the excitation light irradiated in the second step and the third step. The measuring method according to any one of claims 1 to 5, wherein the first step is performed when the measuring chip is manufactured. A measurement chip having a prism made of resin as a dielectric and a metal film disposed on one surface of the prism is mounted, and the metal film is irradiated with excitation light through the prism, thereby allowing the metal film to be irradiated on the metal film. A signal value indicating the presence or amount of the substance to be measured by exciting a fluorescent substance that labels the substance to be measured with localized field light based on surface plasmon resonance and detecting the fluorescence emitted from the fluorescent substance. A measuring device for measuring
A holder for holding the measurement chip;
A light irradiating unit that irradiates the measurement chip held by the holder with light for attenuating the amount of autofluorescence emitted from the prism and excitation light for exciting the fluorescent material;
A light detection unit that detects light emitted from the measurement chip when the light irradiation unit irradiates the measurement chip with light; and
A processing unit for processing a detection value obtained by the light detection unit;
A control unit for controlling operations of the light irradiation unit and the light detection unit;
Have
In order to measure an optical blank value, the control unit emits excitation light to the metal film through the prism so that surface plasmon resonance occurs in the metal film in a state where the fluorescent material is not present on the metal film. Controlling the light irradiation unit to irradiate and controlling the light detection unit to detect light emitted from the measurement chip;
In order to measure the fluorescence value, the control unit passes through the prism so that surface plasmon resonance is generated in the metal film in a state where the substance to be measured labeled with the fluorescent material exists on the metal film. Control the light irradiation unit to irradiate the metal film with excitation light, and control the light detection unit to detect fluorescence emitted from the fluorescent material,
In order to attenuate the light amount of the autofluorescence in the prism, the control unit attenuates the light amount of the autofluorescence emitted from the prism before the measurement of the optical blank value and the measurement of the fluorescence value. Controlling the light irradiation unit to irradiate the light of
The processing unit calculates the signal value by subtracting the optical blank value from the fluorescence value.
measuring device. The light source of the light irradiation unit for irradiating light for attenuating the amount of autofluorescence and the light source of the light irradiation unit for irradiating excitation light for exciting the fluorescent material are the same. Yes,
The power of the light source is 1 mW / mm ² or more.
The measuring apparatus according to claim 7. The control unit irradiates the prism with light for attenuating the light amount of the autofluorescence until the attenuation rate A of the light amount of the autofluorescence emitted from the prism satisfies the following formula (1). The measuring device according to claim 7 or 8, which controls the light irradiation unit.

[In Formula (1), S1 is the said signal value, OB is the said optical blank value, B is the upper limit (%) of the allowable measurement error. ] The control unit controls the light irradiation unit and the light detection unit to perform the measurement of the optical blank value twice,
The processing unit confirms that the attenuation rate A of the light amount of the autofluorescence satisfies the following formula (1) based on the measurement result of the optical blank value by the light detection unit.
The measuring apparatus according to any one of claims 7 to 9.

[In Formula (1), S1 is the said signal value, OB is the said optical blank value, B is the upper limit (%) of the allowable measurement error. ] The control unit has an optical path in the measurement chip of light irradiated to attenuate the amount of the autofluorescence, and the measurement chip of excitation light irradiated to measure the optical blank value and the fluorescence value. The measuring apparatus according to any one of claims 7 to 10, wherein the light irradiation unit is controlled so as to overlap with an optical path inside. The controller controls the light so that the power of light emitted to attenuate the amount of autofluorescence is higher than the power of excitation light irradiated to measure the optical blank value and the fluorescence value. The measuring apparatus according to any one of claims 7 to 11, which controls the irradiation unit. A measuring chip used in a measuring method for detecting the fluorescence emitted by a fluorescent substance that labels a substance to be measured, excited by localized field light based on surface plasmon resonance and measuring the presence or amount of the substance to be measured Because
A prism made of resin as a dielectric,
A metal film disposed on one surface of the prism;
Have
When the prism is irradiated twice with light having the same irradiation energy as the excitation light that irradiates the metal film when detecting the fluorescence, the attenuation rate of the amount of autofluorescence emitted from the prism is 1%. Is
Measuring chip. The irradiation energy of the excitation light and the light is 22.6MW · sec / mm ^2, measuring chip according to claim 13. The measurement chip according to claim 13 or 14, wherein the amount of the autofluorescence is attenuated by being irradiated with light.

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