WO2014171139A1 - Measurement abnormality detection method and surface plasmon-field enhanced fluorescence measurement device - Google Patents

Abstract

The present invention pertains to a method for detecting measurement abnormalities in measurements made using surface plasmon-field enhanced fluorescence spectroscopy. A sensor chip is provided that has a prism comprising a dielectric, a conductor film disposed on a surface of the prism, and a capturing body fixed onto the conductor film. Light is irradiated from the prism side to the conductor film so as to be totally reflected at the interface of the prism and the conductor film, and optical characteristic values for detecting measurement abnormalities are measured. Measurement abnormalities are detected on the basis of the optical characteristic values.

Description

Measurement abnormality detection method and surface plasmon excitation enhanced fluorescence measurement apparatus

The present invention relates to a method for detecting a measurement abnormality in measurement using surface plasmon excitation enhanced fluorescence spectroscopy, and a surface plasmon excitation enhanced fluorescence measurement apparatus for executing the measurement abnormality detection method.

Conventionally, surface plasmon excitation enhanced fluorescence spectroscopy (Surface Plasmon-field enhanced Fluorescence Spectroscopy: hereinafter abbreviated as “SPFS”) is known as a method for measuring a substance to be measured such as protein and DNA with high sensitivity (for example, “SPFS”). Patent Documents 1 and 2).

Patent Documents 1 and 2 disclose a surface plasmon resonance fluorescence measuring apparatus for measuring a substance to be measured using SPFS. In these devices, a sensor having a prism made of a dielectric, a conductor film (for example, a metal film) formed on one surface of the prism, and a capturing body (for example, an antibody) fixed on the conductor film. A tip is attached. When the sample liquid containing the substance to be measured is provided on the conductor film, the substance to be measured is captured by the capturing body (primary reaction). The captured substance to be detected is further labeled with a fluorescent substance (secondary reaction).

In this state, when the excitation light is applied to the conductor film from the prism side so as to be totally reflected at the interface between the prism and the conductor film, evanescent light oozes from the conductor film when the excitation light is reflected at the interface. Plasmons in the conductive film interfere with evanescent light. When the incident angle of the excitation light to the interface is set to the resonance angle and the plasmon and the evanescent light resonate, the electric field of the evanescent light is remarkably enhanced. In surface plasmon excitation fluorescence spectroscopy (SPFS), this enhanced electric field is used, and this electric field excites a fluorescent substance that labels the substance to be measured. Therefore, when the substance to be measured exists on the conductor film, the fluorescence emitted from the fluorescent substance is observed. The measuring device measures the amount of fluorescence and detects the presence or amount of the substance to be measured.

On the other hand, Patent Document 3 discloses a method of detecting a biomolecule using a white interference method. In this method, a biomolecule is detected by detecting a change in a three-dimensional shape on the surface of a substrate (biosensor) on which a probe biomolecule is fixed using a white interference method. Patent Document 3 also describes that quality control of a biosensor is performed by measuring the three-dimensional shape of the biosensor surface using white interference.

International Publication No. 2012/042805 International Publication No. 2012/108323 JP 2007-10557 A

The measurement of the substance to be measured using SPFS is very sensitive. For this reason, various factors such as changes in the sensor chip or sample solution due to storage and nonspecific adsorption of contaminants in the sample solution (for example, blood cell components when the sample solution is blood) greatly affect the measurement results. Resulting in. Therefore, from the viewpoint of improving the reliability of the measurement result, it is preferable that various abnormalities such as an abnormality of the sensor chip and an abnormality occurring during the measurement can be detected.

An object of the present invention is to provide a measurement abnormality detection method in measurement using SPFS.

In order to solve the above problems, a method for detecting a measurement abnormality according to an embodiment of the present invention is a method for detecting a measurement abnormality in measurement by surface plasmon excitation enhanced fluorescence spectroscopy, comprising: (a-1) from a dielectric And (b-1) preparing a sensor chip having a prism, a conductor film disposed on the surface of the prism, and a capturing body fixed on the conductor film; Irradiating the conductor film with light from the prism side so as to be totally reflected at the interface with the body film, and measuring an optical characteristic value for detecting a measurement abnormality; and (b-2) the optical characteristic. Detecting a measurement abnormality based on the value.

In order to solve the above problems, a surface plasmon excitation enhanced fluorescence measurement device according to an embodiment of the present invention is a surface plasmon excitation enhanced fluorescence measurement that measures a substance to be measured using surface plasmon excitation enhanced fluorescence spectroscopy. The apparatus detects a measurement abnormality in measurement using surface plasmon excitation enhanced fluorescence spectroscopy by the above-described measurement abnormality detection method.

According to the present invention, the reliability of measurement results using SPFS can be improved. Further, according to the present invention, by detecting a measurement abnormality at an early stage of measurement, it is possible to reduce losses such as reagents and measurement time due to the measurement abnormality.

FIG. 1 is a schematic diagram showing a configuration of a surface plasmon excitation enhanced fluorescence measuring apparatus. FIG. 2 is a cross-sectional view of the sensor chip. FIG. 3 is a flowchart showing an example of the measurement procedure. 4A to 4G are flowcharts showing an example of a measurement procedure when a measurement abnormality is inspected in measurement using SPFS. FIGS. 5A to 5C are flowcharts showing an example of a measurement procedure when a measurement abnormality is inspected in measurement using SPFS. FIG. 6 is a photograph of the gold thin film surface after the storage environment acceleration test. FIG. 7A is a graph showing the relationship between the area ratio of circular ridges and the amount of plasmon scattered light. FIG. 7B is a graph showing the relationship between the area ratio of circular ridges and the reflectance at resonance. FIG. 7C is a graph showing the relationship between the area ratio of the circular ridges and the blank light amount. FIG. 8A is a graph showing the relationship between the incident angle of excitation light and the reflectance. FIG. 8B is a graph showing the relationship between the incident angle of excitation light and the amount of scattered light.

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

[Surface plasmon excitation enhanced fluorescence measurement device]
(Configuration of measuring device)
First, a surface plasmon excitation enhanced fluorescence measuring apparatus (hereinafter also referred to as “measuring apparatus”) that measures a substance to be measured using SPFS will be described. FIG. 1 is a schematic diagram showing a configuration of a surface plasmon excitation enhanced fluorescence measuring apparatus 100 according to an embodiment of the present invention.

As shown in FIG. 1, the measurement apparatus 100 includes a pretreatment chamber 110, a measurement chamber 120, a liquid feeding unit 130, a chip transport unit 140, a light source unit 150, an excitation light optical system 160, a chip holder 170, and a measurement light optical system. 180, a measurement light detection unit 190, a reflected light detection unit 200, a control calculation unit 210, and a display unit 220.

In the measuring apparatus 100, the substance to be measured is measured with the sensor chip 300 mounted on the chip holder 170. Here, “measuring the substance to be measured” means performing at least one of detecting the presence of the substance to be measured and detecting the amount of the substance to be measured. As will be described later, the sensor chip 300 includes a prism 310 having an incident surface 312, a reflecting surface 314, and an emitting surface 316, a conductor film 320 formed on the reflecting surface 314, and a trap disposed on the conductor film 320. And a flow path member 340 disposed on the conductor film 320 (see FIG. 2). The sensor chip 300 is disposed in the pretreatment chamber 110 when a liquid such as a sample solution, a fluorescent labeling solution, or a buffer solution is injected into the flow path 342 (indicated by a two-dot chain line in FIG. 1), and is measured in the measurement chamber. 120 (shown by a solid line in FIG. 1).

During measurement, the excitation light α emitted from the light source unit 150 is guided to the sensor chip 300 by the excitation light optical system 160. While the excitation light α is incident on the sensor chip 300, the measurement light β and the reflected light γ are emitted from the sensor chip 300. The measurement light β is guided to the measurement light detection unit 190 by the measurement light optical system 180. The reflected light γ is guided to the reflected light detection unit 200.

Hereinafter, each component of the surface plasmon excitation enhanced fluorescence measuring apparatus 100 will be described.

In the pretreatment chamber 110, the liquid feeding unit 130 injects a liquid such as a sample liquid, a fluorescent labeling liquid, or a cleaning liquid into the flow path 342 of the sensor chip 300, or causes the liquid in the flow path 342 to flow outside the flow path 342. To discharge. The liquid feeding unit 130 includes, for example, a liquid feeding pump 132 and a liquid feeding pump transport unit 134. In the pretreatment chamber 110, a reagent chip 136 into which a sample solution, a fluorescent labeling solution, a cleaning solution, etc. are dispensed is also arranged. The liquid feed pump 132 sucks the liquid from the reagent chip 136 or the sensor chip 300 and discharges the liquid to the reagent chip 136 or the sensor chip 300. For example, the liquid feed pump 132 sucks liquid from one well of the reagent chip 136 and discharges the liquid into the flow path 342 of the sensor chip 300. In some cases, the liquid feed pump 132 may suck liquid from one well of the reagent chip 136 and discharge the liquid to another well of the reagent chip 136. The liquid feed pump transport unit 134 moves the liquid feed pump 132 onto the reagent chip 136 or the sensor chip 300 according to the operation of the liquid feed pump 132.

The chip transport unit 140 transports the sensor chip 300 into the pretreatment chamber 110 during preprocessing, and transports the sensor chip 300 into the measurement chamber 120 during measurement. The sensor chip 300 conveyed into the measurement chamber 120 is held by the chip holder 170.

The light source unit 150 is collimated and emits excitation light α having a constant wavelength and light amount. The light source unit 150 includes, for example, a laser diode as an excitation light source, a collimator that collimates the excitation light α emitted from the laser diode, and a temperature adjustment circuit for making the wavelength and the light amount of the excitation light α constant. (Both are not shown). The wavelength and light amount of the excitation light α emitted from the laser diode varies with temperature. For this reason, the temperature adjustment circuit monitors the amount of light branched from the collimated excitation light α with a photodiode, etc., and adjusts the temperature of the laser diode so that the wavelength and the amount of the excitation light α are constant. To do. The excitation light α emitted from the laser diode is usually linearly polarized light.

The type of light source included in the light source unit 150 is not particularly limited, and may not be a laser diode. Examples of light sources include light emitting diodes, mercury lamps, and other laser light sources. When the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like. When the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like. Furthermore, 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 excitation light optical system 160 adjusts various parameters of the excitation light α emitted from the light source unit 150 and then totally reflects the excitation light α at the interface between the prism 310 and the conductor film 320 (the reflection surface 314 of the prism 310). As shown, the excitation light α is guided to the sensor chip 300. As illustrated in FIG. 1, the excitation light optical system 160 includes, for example, a first wave adjuster 162, a polarization direction adjustment unit 164, a shaping optical system 166, and an incident angle adjustment unit 168.

The first wave shaper 162 waves the excitation light α. The first wave adjuster 162 includes, for example, a first band pass filter, a linear polarization filter, and a neutral density filter (all not shown). The first bandpass filter turns the excitation light α into narrowband light having only the center wavelength. The linearly polarizing filter turns the excitation light α into completely linearly polarized light. The neutral density filter adjusts the amount of excitation light α.

The polarization direction adjusting unit 164 adjusts the polarization direction of the excitation light α so that the P wave component is mainly incident on the conductor film 320. The polarization direction adjusting unit 164 includes, for example, a half-wave plate and a half-wave plate rotating unit (both not shown). The half-wave plate is rotated by a half-wave plate rotating unit. The polarization direction of the excitation light α that passes through the half-wave plate is adjusted by the rotation angle of the half-wave plate.

The shaping optical system 166 adjusts the beam diameter, contour shape, and the like of the excitation light α so that the shape of the irradiation spot at the interface between the prism 310 and the conductor film 320 (the surface of the conductor film 320) is a predetermined size. . The shaping optical system 166 is, for example, a slit or a zoom optical system.

The incident angle adjusting unit 168 adjusts the incident angle θ (see FIG. 1) of the excitation light α to the interface between the prism 310 and the conductor film 320 (the reflecting surface 314 of the prism 310). The incident angle adjusting unit 168 includes, for example, a reflecting mirror, a reflecting mirror angle adjusting unit, and a reflecting mirror position adjusting unit (all not shown). The reflecting mirror angle adjustment unit adjusts the incident angle θ of the excitation light α by rotating the reflecting mirror. The reflecting mirror position adjustment unit moves the reflecting mirror so as to cancel the movement of the irradiation spot due to the change in the incident angle θ. Thereby, only incident angle (theta) can be adjusted, without changing the position of an irradiation spot.

The chip holder 170 holds the sensor chip 300 in the measurement chamber 120. The sensor chip 300 is irradiated with the excitation light α from the excitation light optical system 160 while being held by the chip holder 170. At this time, measurement light β such as scattered light having the same wavelength as the excitation light α and fluorescence emitted from the fluorescent material is emitted upward from the surface of the conductor film 320 that does not face the prism 310. Further, the excitation light α reflected at the interface between the prism 310 and the conductor film 320 is emitted from the emission surface 316 as reflected light γ (see FIG. 2).

The measurement light optical system 180 guides the measurement light β emitted upward from the sensor chip 300 to the measurement light detection unit 190. The measuring light optical system 180 is disposed so as to face a surface of the conductor film 320 of the sensor chip 300 held by the chip holder 170 that does not face the prism 310. More specifically, the measurement light optical system 180 is arranged on a straight line that passes through the irradiation spot of the excitation light α on the surface of the conductor film 320 and is perpendicular to the surface of the conductor film 320. The measurement light optical system 180 includes, for example, a condenser lens, a second wave rectifier, and an imaging lens (all not shown).

The condensing lens and the imaging lens constitute a conjugate optical system that is not easily affected by stray light. The light traveling between the condenser lens and the imaging lens is substantially parallel light. The condenser lens and the imaging lens form an image of the measurement light β (for example, a fluorescent image) on the conductive film 320 on the light receiving surface of the measurement light detection unit 190.

The second wave shaper is disposed so as to be positioned between the condenser lens and the imaging lens. The second wave shaper blocks light having the wavelength of the excitation light α, thereby allowing light other than the wavelength of the fluorescence (for example, leakage light from the light source unit 150 or light having the same wavelength as the excitation light α from the conductor film 320). (Scattered light or the like) is prevented from reaching the measurement light detector 190. That is, the second wave rectifier removes a noise component from the light reaching the measurement light detection unit 190, and contributes to improvement in fluorescence detection accuracy and sensitivity. The second wave shaper includes, for example, a second band pass filter and an insertion / extraction unit. The position of the second band pass filter is switched by the insertion / extraction unit. When measuring the amount of fluorescent light, the second band pass filter is inserted into the optical path of the measurement light β. On the other hand, when measuring the amount of scattered light having the same wavelength as the excitation light α (plasmon scattered light described later), the second bandpass filter is removed from the optical path of the measurement light β.

The insertion / extraction unit may further insert / remove a neutral density filter between the condenser lens and the imaging lens. When measuring the amount of fluorescent light, the neutral density filter is removed from the optical path of the measurement light β. On the other hand, when measuring the amount of scattered light (plasmon scattered light) having the same wavelength as the excitation light α, the neutral density filter is inserted into the optical path of the measurement light β. As a result, the difference between the light amounts of the fluorescence and the plasmon scattered light guided to the measurement light detection unit 190 becomes small, and the measurement of the light amount in the measurement light detection unit 190 becomes easy.

The measurement light detection unit 190 acquires an image of the measurement light β on the conductor film 320 and measures the amount of the measurement light β. For example, the measurement light detection unit 190 measures the amount of fluorescent light on the conductor film 320 and the amount of plasmon scattered light. The measurement light detection unit 190 is, for example, a photomultiplier tube or a cooled charge coupled device (CCD) camera.

The reflected light detection unit 200 measures the amount of reflected light γ. The reflected light detection unit 200 is, for example, a photomultiplier tube or a cooled charge coupled device (CCD) camera.

The control calculation unit 210 performs control of each driving unit, quantification of the amount of light received by the measurement light detection unit 190 and the reflected light detection unit 200, and the like. The control arithmetic unit 210 is, for example, a computer that executes software.

The display unit 220 is connected to the control calculation unit 210 and displays a measurement status, a measurement result, and the like.

(Configuration of sensor chip)
Next, the sensor chip 300 attached to the chip holder 170 of the measuring apparatus 100 will be described. FIG. 2 is a cross-sectional view of the sensor chip 300.

2, the sensor chip 300 includes a prism 310, a conductor film 320, a capturing body 330, and a flow path member 340. The sensor chip 300 is normally replaced for each measurement of the substance to be measured. The sensor chip 300 is preferably a structure having a length of several mm to several cm for each piece, but is a smaller structure or a larger structure not included in the category of “chip”. Also good.

The prism 310 is made of a dielectric that is transparent to the excitation light α. The prism 310 includes an incident surface 312 that allows the excitation light α from the excitation light optical system 160 to enter the prism 310, a reflection surface 314 that reflects the excitation light α incident on the prism 310, and a reflection surface 314 that reflects the reflection surface 314. And an emission surface 316 for emitting the excited excitation light α to the outside of the prism 310. The shape of the prism 310 is not particularly limited. For example, the prism 310 is a column having a trapezoidal bottom surface. In this case, the surface corresponding to one base of the trapezoid is the reflecting surface 314, the surface corresponding to one leg is the entrance surface 312, and the surface corresponding to the other leg is the exit surface 316. The trapezoid serving as the bottom surface is preferably an isosceles trapezoid. Thereby, the entrance surface 312 and the exit surface 316 are symmetric, and the S wave component of the reflected light γ is less likely to stay in the prism 310. For example, the angle between the incident surface 312 and the reflecting surface 314 and the angle between the reflecting surface 314 and the emitting surface 316 are both about 80 °. Examples of the material of the prism 310 include resin and glass. The material of the prism 310 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

The conductor film 320 is formed on the reflection surface 314 of the prism 310. The conductor film 320 amplifies evanescent light (enhanced electric field) generated when the excitation light α is totally reflected by the reflecting surface 314 of the prism 310. That is, by generating surface plasmon resonance in the conductive film 320 on the reflective surface 314, the excitation light α is totally reflected on the surface without the conductive film 320 (reflective surface 314) to generate evanescent light. , The formed evanescent light can be amplified. The material of the conductor film 320 is not particularly limited as long as surface plasmon resonance can be generated. Examples of the material of the conductor film 320 include metals such as gold, silver, copper, and aluminum, and alloys thereof. The method for forming the conductor film 320 is not particularly limited. Examples of the method for forming the conductor film 320 include sputtering, vapor deposition, and plating. The thickness of the conductor film 320 is not particularly limited, but is preferably in the range of 30 to 70 nm.

The capturing body 330 is fixed on a surface of the conductive film 320 that does not face the prism 310, and captures the substance to be measured. The capturing body 330 is uniformly fixed to a predetermined region on the conductor film 320. The type of the capturing body is not particularly limited as long as the substance to be measured can be specifically captured. For example, the capturing body is an antibody or a fragment thereof that specifically binds to the substance to be measured.

The flow path member 340 is disposed on the surface of the conductor film 320 that does not face the prism 310. When the conductor film 320 is formed only on a part of the reflection surface 314 of the prism 310, the flow path member 340 may be disposed on the reflection surface 314. The flow path member 340 and the conductor film 320 (and the prism 310) form a flow path 342 through which a liquid such as a sample liquid flows. A reaction chamber 344 exists in the middle of the flow path 342. The capturing body 330 is exposed to the reaction chamber 344. Both ends of the channel 342 are connected to an inlet 346 and an outlet 348 formed on the upper surface of the channel member 340, respectively. When liquid such as sample liquid or fluorescent labeling liquid is injected into the flow path 342, these liquids come into contact with the capturing body 330 in the reaction chamber 344. The flow path member 340 is made of a material that is transparent to the measurement light β (for example, fluorescence or plasmon scattered light). An example of the material of the flow path member 340 includes a resin. The flow path member 340 is bonded to the conductor film 320 or the prism 310 by, for example, adhesion using an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.

As shown in FIG. 2, the excitation light α guided to the prism 310 enters the prism 310 from the incident surface 312. The excitation light α incident on the prism 310 is totally reflected by the reflection surface 314 (interface between the prism 310 and the conductor film 320) to become reflected light γ. The reflected light γ is emitted from the emission surface 316 to the outside of the prism 310. On the other hand, measurement light β (for example, fluorescence or plasmon scattered light) is emitted from the conductor film 320 and the capturing body 330 in the direction of the measurement light optical system 180. When the excitation light α is incident on the reflecting surface 314 while satisfying the total reflection condition, and the evanescent light oozing out from the reflecting surface 314 and the plasmon in the conductor film 320 resonate, the electric field of the evanescent light is caused by the conductor film 320. Is increased, and the amount of fluorescence and plasmon scattered light is increased.

(Measurement procedure)
Next, the measurement operation of the measurement apparatus 100 will be described (see also Patent Documents 1 and 2). FIG. 3 is a flowchart illustrating an example of a measurement procedure of the measurement apparatus 100.

First, preparation for measurement is performed (step S10). Specifically, the sensor chip 300 and the reagent chip 136 are prepared and installed at predetermined locations in the pretreatment chamber 110, respectively. The control calculation unit 210 controls the liquid feeding unit 130 to prepare a sample solution, a fluorescent labeling solution, and the like as necessary, and dispense them into the reagent chip 136.

Next, the substance to be measured in the sample solution and the capturing body 330 are reacted (primary reaction, step S20). Specifically, the control calculation unit 210 causes the liquid feeding unit 130 to inject the sample liquid into the flow path 342 of the sensor chip 300. When the sample liquid is injected into the flow path 342, the sample liquid comes into contact with the capturing body 330. When the substance to be measured exists in the sample liquid, at least a part of the substance to be measured is captured by the capturing body 330. Thereafter, the inside of the flow path 342 is washed with a buffer solution or the like, and the substances not captured by the capturing body 330 are removed. The kind of sample liquid is not specifically limited. Examples of the sample liquid 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 330 is labeled with a fluorescent material (secondary reaction, step S30). Specifically, the control calculation unit 210 causes the liquid feeding unit 130 to inject the fluorescent labeling solution into the flow path 342 of the sensor chip 300. 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 injected into the flow path 342, the fluorescent labeling liquid comes into contact with the capturing body 330. When the substance to be measured is captured by the capturing body 330, at least a part of the substance to be measured is labeled with a fluorescent substance. Thereafter, the inside of the flow path 342 is washed with a buffer solution or the like to remove free fluorescent substances.

Next, the sensor chip 300 is transported into the measurement chamber 120 (step S40). Specifically, the control calculation unit 210 causes the chip transfer unit 140 to transfer the sensor chip 300 into the measurement chamber 120. The sensor chip 300 is attached to the chip holder 170.

Next, the sensor chip 300 is irradiated with the excitation light α so as to have a predetermined incident angle θ, and the amount of fluorescence emitted from the fluorescent material is measured (step S50). Specifically, the control calculation unit 210 includes the light source unit 150 and the excitation light optical system 160 so that the excitation light α is totally reflected on the reflection surface 314 (the interface between the prism 310 and the conductor film 320) of the sensor chip 300. To emit excitation light α. Thereby, the fluorescence (measurement light β) emitted from the fluorescent material excited by the evanescent light is emitted above the sensor chip 300. At the same time, the control calculation unit 210 causes the measurement light detection unit 190 to measure the amount of fluorescence (measurement light β). In addition, the control calculation unit 210 stores the detected value and displays it on the display unit 220 (step S60). The amount of fluorescent light is converted into the amount and concentration of the substance to be measured as necessary.

Finally, the control calculation unit 210 initializes the entire measuring apparatus 100 and ends a series of measurements (step S70). At this time, the movable object such as the incident angle adjustment unit 168 is returned to the initial position.

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

Although not specifically described this time, the optimum incident angle θ (excitation light α) (before the primary reaction (step S20) or between the primary reaction (step S20) and the secondary reaction (step S30) is used. It is preferable to determine (hereinafter also referred to as “measurement angle θm”) (see Patent Documents 1 and 2). Specifically, while measuring the incident angle θ of the excitation light α with respect to the conductor film 320, the measurement light β or the reflected light γ having the same wavelength as the excitation light α is measured to determine the enhancement angle or the resonance angle (enhancement). The definition of the angle and the resonance angle will be described later). This enhancement angle or resonance angle may be directly used as the measurement angle θm, or a value obtained by correcting the enhancement angle or resonance angle may be used as the measurement angle θm.

Further, before the primary reaction (step S20) or between the primary reaction (step S20) and the secondary reaction (step S30), the position of the irradiation spot of the excitation light α is optimized, and the excitation light α It is also preferable to optimize the polarization direction and measure the amount of autofluorescence (see Patent Documents 1 and 2).

In the above description, after the step of reacting the substance to be measured and the capturing body 330 (primary reaction, step S20), the step of labeling the substance to be measured with a fluorescent substance (secondary reaction, step S30) is performed. (2-step method). However, the timing for labeling the substance to be measured with the fluorescent substance is not particularly limited. For example, before injecting the sample liquid into the flow path 342 of the sensor chip 300, a fluorescent substance may be added to the sample liquid and the substance to be measured may be labeled with the fluorescent substance in advance. Further, the sample solution and the fluorescent material may be simultaneously injected into the flow path 342 of the sensor chip 300. In the former case, the sample substance is injected into the flow path 342 of the sensor chip 300, whereby the measurement target substance labeled with the fluorescent substance is captured by the capturing body 330. In the latter case, the substance to be measured is labeled with a fluorescent substance, and the substance to be measured is captured by the capturing body 330. In any case, both the primary reaction and the secondary reaction can be completed by injecting the sample liquid into the flow path 342 of the sensor chip 300 (one-step method).

[Measurement anomaly detection method]
The measurement abnormality detection method according to the present invention can detect measurement abnormality in measurement using SPFS. Here, “measurement abnormality” means an event that reduces the reliability of measurement using SPFS. Examples of measurement abnormalities include defects in the conductor film in the sensor chip, abnormalities on the surface of the sensor region, abnormal reactions in the sensor region, and the like. First, measurement abnormality will be described with reference to the sensor chip 300 (FIG. 2).

The defect of the conductor film is, for example, a circular bulge of the conductor film 320 (see FIG. 6) or peeling of the conductor film 320. These defects occur, for example, due to storage of the sensor chip 300 or liquid feeding into the flow path 342. When defects occur in the conductor film 320 in this way, variations in the electric field at the time of measurement, an increase in background light, a decrease in the capture rate of the substance to be measured, and the like occur, and the reliability of the measurement results decreases.

Abnormalities on the surface of the sensor region include, for example, corrosion of a layer made of the capturing body 330 or an intermediate layer provided on the conductive film 320 to support this layer, or sensor region (the conductive film 320 and the capturing body 330). For example, adhesion of foreign matter to the surface. These abnormalities are caused by, for example, generation of mold or adhesion of foreign matters during storage of the sensor chip 300, lack of cleaning in the flow path 342, or the like. When abnormality occurs on the surface of the sensor region in this way, variations in the electric field at the time of measurement, a decrease in the capture rate of the substance to be measured, and the like occur, and the reliability of the measurement result decreases.

The abnormality in the reaction in the sensor region is, for example, adhesion of foreign matters derived from the sample liquid to the surface of the sensor region. These abnormalities occur, for example, due to non-specific adsorption of contaminants accompanying the injection of the sample liquid into the flow path 342, lack of cleaning in the flow path 342, and the like. Here, the “contaminant” means a protein or glycolipid other than the substance to be measured contained in the sample solution. For example, when the sample solution is blood or a diluted solution thereof, the blood cell component is contained in the contaminants. When a reaction abnormality occurs in the sensor region in this way, an electric field loss occurs during measurement, and the reliability of the measurement result decreases.

Next, a measurement abnormality detection method in measurement using SPFS will be described.

The measurement abnormality detection method according to the present embodiment includes (a-1) a step of preparing the sensor chip 300, and (b-1) a step of irradiating the sensor chip 300 with light to measure a predetermined optical characteristic value. And (b-2) detecting a measurement abnormality based on the optical characteristic value measured in step (b-1). In addition, the measurement abnormality detection method according to the present embodiment includes (b-3) a step of interrupting measurement when a measurement abnormality is detected in step (b-2), or (b-3 ′) the step ( It is preferable to further include a step of notifying the measurement abnormality when the measurement abnormality is detected in b-2) and continuing the measurement and displaying the measurement result.

In step (a-1), the aforementioned sensor chip 300 is prepared. For example, the sensor chip 300 may be manufactured, or the sensor chip 300 may be purchased.

In step (b-1), light is applied to the conductor film 320 from the prism 310 side so as to be totally reflected at the interface between the prism 310 and the conductor film 320, and an optical characteristic value for detecting a measurement abnormality is obtained. taking measurement. The type of optical characteristic value measured in this step is not particularly limited as long as a measurement abnormality can be detected. Examples of the optical characteristic value include a plasmon scattered light amount, an enhancement angle, a resonance angle, a reflected light amount, and a blank light amount. Only one type of these optical characteristic values may be measured, or two or more types may be measured in combination. The type of optical characteristic value to be measured can be appropriately selected according to the type of measurement abnormality to be detected (see Table 1).

In the specification of the present application, the “plasmon scattered light amount” measured to detect a measurement abnormality is a sensor when excitation light α is incident on the conductive film 320 from the prism 310 side at a specific incident angle θ. This means the amount of scattered light having the same wavelength as the excitation light α emitted above the chip 300. The plasmon scattered light amount is measured by the measurement light detection unit 190.

Further, the “enhancement angle” measured to detect measurement abnormality is the scattered light having the same wavelength as the excitation light α emitted above the sensor chip 300 when the incident angle θ of the excitation light α is scanned. This means the incident angle when the amount of light reaches the maximum. The incident angle θ of the excitation light α is adjusted by the incident angle adjusting unit 168.

Further, the “resonance angle” measured to detect measurement abnormality is the minimum amount of reflected light γ emitted from the emission surface 316 of the sensor chip 300 when the incident angle θ of the excitation light α is scanned. Means the incident angle. The incident angle θ of the excitation light α is adjusted by the incident angle adjusting unit 168.

In addition, the “reflected light amount” measured to detect a measurement abnormality is the minimum amount of reflected light γ emitted from the emission surface 316 of the sensor chip 300 when the incident angle θ of the excitation light α is scanned. Mean value. The amount of reflected light is measured by the reflected light detection unit 200. When the resonance angle is the measurement angle θm, the amount of reflected light is the same as the amount of reflected light γ during fluorescence measurement.

Also, the “blank light amount” measured to detect a measurement abnormality means the amount of background light emitted above the sensor chip 300 during fluorescence measurement. The blank light quantity is measured by the measurement light detection unit 190 under the same optical conditions as those during fluorescence measurement before performing the secondary reaction.

In step (b-2), a measurement abnormality is detected based on the optical characteristic value measured in step (b-1). For example, when the plasmon scattered light amount, reflected light amount, or blank light amount exceeds ± 50% from the reference value, it is determined that a measurement abnormality has occurred. If the enhancement angle or resonance angle exceeds ± 0.1 ° from the reference value, it is determined that a measurement abnormality has occurred. The reference value can be appropriately set according to the equipment used, the type of sample, the required measurement accuracy, and the like.

In step (b-3), if a measurement abnormality is detected in step (b-2), the subsequent steps are canceled and the measurement is interrupted. At this time, the fact that the measurement is interrupted may be displayed on the display unit 220 together with the reason. Further, step (b-3 ') may be performed instead of step (b-3). In step (b-3 '), if a measurement abnormality is detected in step (b-2), the measurement is continued thereafter, but a notification that a measurement abnormality has occurred is displayed when the measurement result is displayed. For example, you may display on the display part 220 that a measurement abnormality was detected during a measurement with a measurement result.

The measurement abnormality detection method according to the present embodiment (hereinafter also referred to as “measurement abnormality inspection”) may be implemented in combination with measurement of a substance to be measured. For example, when the measurement of the substance to be measured is the “two-step method” including the primary reaction and the secondary reaction described above, the measurement of the substance to be measured includes (a-1) a step of preparing the sensor chip 300, a-2) a step of binding the substance to be measured in the sample solution and the capturing body 330 (primary reaction), and (a-3) a step of labeling the substance to be measured bound to the capturing body 330 with a fluorescent substance ( Secondary reaction) and (a-4) a step of measuring the amount of fluorescence emitted from the fluorescent substance that labels the substance to be measured (fluorescence measurement). In this case, the measurement abnormality inspection including the steps (b-1) and (b-2) may be performed between the sensor chip preparation (a-1) and the primary reaction (a-2). It may be performed between the primary reaction (a-2) and the secondary reaction (a-3), or between the secondary reaction (a-3) and the fluorescence measurement (a-4). May be done.

When the measurement of the substance to be measured is the above-described “one-step method”, the measurement of the substance to be measured includes (a-1) a step of preparing the sensor chip 300 and (a-2 ′) a label with a fluorescent substance. A step of binding the substance to be measured and the trap 330 (hereinafter also simply referred to as “reaction”), and (a-4) a step of measuring the amount of fluorescence emitted from the fluorescent substance that labels the substance to be measured. (Fluorescence measurement). In step (a-2 ′), a sample containing a substance to be measured that has been previously labeled with a fluorescent substance is brought into contact with the capturing body 330 to bind the substance to be measured that has been labeled with the fluorescent substance and the capturing body 330. Alternatively, the sample and the fluorescent substance are brought into contact with the capturing body 330 to label the target substance with the fluorescent substance, and the target substance and the capturing body 330 are combined. In this case, the measurement abnormality inspection may be performed between the preparation of the sensor chip (a-1) and the reaction (a-2 ′), or the reaction (a-2 ′) and the fluorescence measurement (a− 4).

In any case, since the step (b-1) is performed in the measurement chamber 120, the sensor chip 300 is removed from the pretreatment chamber 110 by the chip transfer unit 140 before the step (b-1). It is transferred into the measurement chamber 120. As described above, when an abnormality is detected in the inspection, the fact may be displayed on the display unit 220 or the subsequent process may be canceled.

Also, the measurement abnormality inspection may be performed a plurality of times. In this case, the measurement abnormality may be inspected using the same optical characteristic value each time, or the measurement abnormality may be inspected using different optical characteristic values each time.

FIG. 4 is a flowchart showing an example of a measurement procedure when a measurement abnormality is inspected in a two-step measurement using SPFS. FIG. 4A shows an example in which the measurement abnormality test (b-1) is performed once before the primary reaction (a-2). FIG. 4B shows an example in which a measurement abnormality test (b-1) is performed once between the primary reaction (a-2) and the secondary reaction (a-3). FIG. 4C shows an example in which a measurement abnormality test (b-1) is performed once between the secondary reaction (a-3) and the fluorescence measurement (a-4). FIG. 4D shows a measurement abnormality test (b-1) before the primary reaction (a-2) and between the primary reaction (a-2) and the secondary reaction (a-3). An example of performing this time is shown. FIG. 4E shows that the measurement abnormality test (b-1) is performed twice before the primary reaction (a-2) and between the secondary reaction (a-3) and the fluorescence measurement (a-4). An example is shown. FIG. 4F shows the measurement abnormality between the primary reaction (a-2) and the secondary reaction (a-3), and between the secondary reaction (a-3) and the fluorescence measurement (a-4). An example of performing the inspection (b-1) twice will be shown. FIG. 4G shows before primary reaction (a-2), between primary reaction (a-2) and secondary reaction (a-3), secondary reaction (a-3) and fluorescence measurement ( An example is shown in which the measurement abnormality inspection (b-1) is performed three times with respect to a-4). In these figures, among the steps shown in FIG. 3, the primary reaction (step S20 shown in FIG. 3), the secondary reaction (step S20 shown in FIG. 3), and the fluorescence measurement (shown in FIG. 3). Steps other than step S50) are omitted for convenience of explanation.

FIG. 5 is a flowchart showing an example of a measurement procedure when a measurement abnormality is inspected in a one-step measurement using SPFS. FIG. 5A shows an example in which the measurement abnormality test (b-1) is performed once before the reaction (a-2 '). FIG. 5B shows an example in which the measurement abnormality test (b-1) is performed once between the reaction (a-2 ') and the fluorescence measurement (a-4). FIG. 5C shows an example in which the measurement abnormality test (b-1) is performed twice before the reaction (a-2 ′) and between the reaction (a-2 ′) and the fluorescence measurement (a-4). Indicates. Also in these drawings, the steps other than the reaction (injection of the sample liquid containing the measurement target substance labeled with the fluorescent substance or the sample liquid and the fluorescent substance into the flow path 342 of the sensor chip 300) and the measurement of the fluorescence are as follows. It is omitted for convenience of explanation.

For example, the circular bulge of the conductor film 320 can be detected with high sensitivity by measuring the plasmon scattered light amount, the reflected light amount, or the blank light amount as an optical characteristic value for detecting a measurement abnormality. In this case, the circular bulge of the conductor film 320 can be detected by performing the inspection at any timing shown in FIGS. 4A to 4G and FIGS. 5A to 5C. As shown in FIGS. 4A and 5A, the loss of the sample solution, the reagent, and the like can be reduced by performing the inspection before the (primary) reaction. On the other hand, as shown in FIGS. 4B, 4C, and 5B, by performing an inspection after the (primary or secondary) reaction, it is possible to detect a circular ridge generated by liquid feeding.

Also, foreign matter adhesion to the surface of the sensor region can be detected with high sensitivity by measuring the enhancement angle or the resonance angle as an optical characteristic value for detecting a measurement abnormality. In this case, as shown in FIG. 4A and FIG. 5A, it is preferable to perform the inspection before the (primary) reaction.

Further, the adhesion of foreign matter derived from the sample liquid to the surface of the sensor region can be detected with high sensitivity by measuring the enhancement angle or the resonance angle as an optical characteristic value for detecting measurement abnormality. In this case, as shown in FIG. 4D and FIG. 5C, it is preferable to perform an inspection by measuring optical property values before (primary) reaction and after (primary) reaction.

Table 1 shows the types of measurement abnormalities that can be detected and their detection sensitivities for each optical characteristic value. In Table 1, “Δ” indicates that it can be detected, and “◯” indicates that it can be detected with high sensitivity.

Note that the sensor chip 300 prepared in the step (a-1) may have information on the optical characteristic value in a normal state. For example, the sensor chip 300 may be provided with a barcode, character information, or the like including information related to the optical characteristic value in a normal state. In this case, in step (b-2), the optical property value in the normal state is compared with the measured optical property value to detect the presence or absence of measurement abnormality.

As described above, the measurement abnormality detection method according to the present embodiment can detect a measurement abnormality before or during measurement in measurement using SPFS, thereby improving the reliability of the measurement result. Can be made. In addition, since the measurement abnormality detection method according to the present embodiment can detect a measurement abnormality at an early stage of measurement, the measurement time loss due to the measurement abnormality can be shortened.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[Example 1]
In Example 1, an example in which a defect in a conductor film of a sensor chip is detected will be described.

(1) Production of sensor chip A prism made of transparent resin was prepared. The shape of the prism is a column having an isosceles trapezoid as a bottom surface. A thin gold film having a thickness of 45 nm was formed on the largest plane of the prism by sputtering.

The starting point of the organic reaction was formed on the surface of the gold thin film by forming a self-assembled monolayer made of thiol having an amino group. Next, a hydrophilic polymer was bonded by an amide coupling reaction to form a support layer on the surface of the gold thin film. Finally, the antibody was immobilized on the support layer using chemical bonding to produce a sensor chip.

Some sensor chips were immersed in water at 80 ° C. to cause defects (circular bulging) in the gold thin film (storage environment acceleration test). FIG. 6 is an optical microscope image of the gold thin film surface after the storage environment acceleration test. It can be seen that a circular ridge is generated on the surface of the gold thin film.

(2) Measurement of optical characteristic value A sensor chip not formed with a circular ridge or a sensor chip formed with a circular ridge was mounted on a chip holder of a surface plasmon excitation enhanced fluorescence measuring apparatus (see FIG. 1).

A laser beam (excitation light) with a wavelength of 635 nm was irradiated onto the gold thin film from the prism side. The incident angle on the gold thin film was scanned, and the relationship between the incident angle and the amount of scattered light having the same wavelength above the gold thin film was examined. For each sensor chip, the incident angle at which the amount of scattered light was maximum was defined as “enhancement angle”, and the amount of scattered light at the enhancement angle was defined as “plasmon scattered light amount”.

Also, the incident angle with respect to the gold thin film was scanned, and the relationship between the incident angle, the amount of light reflected by the gold thin film, and the amount of scattered light having a wavelength of 650 nm or more above the gold thin film was examined. For each sensor chip, the incident angle at which the amount of reflected light is the minimum was defined as “resonance angle”, and the ratio of the reflected light amount to the excitation light amount when the incident angle was the resonance angle was defined as “resonance reflectance”. Further, the amount of scattered light having a wavelength of 650 nm or more when the incident angle is the resonance angle was defined as “blank light amount”.

(3) Measurement result The measurement result of the optical characteristic value of each sensor chip is shown in FIG. FIG. 7A is a graph showing the relationship between the area ratio of circular ridges and the amount of plasmon scattered light (arbitrary unit). FIG. 7B is a graph showing the relationship between the area ratio of circular ridges and the reflectance at resonance. FIG. 7C is a graph showing the relationship between the area ratio of the circular ridges and the blank light quantity (arbitrary unit). In these graphs, a sensor chip having a circular ridge area ratio of 0% is a normal sensor chip that has not been subjected to a storage environment acceleration test. On the other hand, a sensor chip having a circular ridge area ratio of 0.5 to 10% is a sensor chip having a defect in a gold thin film, which was subjected to a storage environment acceleration test.

As shown in FIG. 7A, the plasmon scattered light amount of the normal sensor chip was 20000 or less, whereas the plasmon scattered light amount of the sensor chip having a defect in the gold thin film exceeded 120,000. Further, as shown in FIG. 7B, the resonance reflectance of the normal sensor chip was 1.8% or less, whereas the resonance reflectance of the sensor chip having a defect in the gold thin film was 1. It was over 8%. Further, as shown in FIG. 7C, the blank light amount of a normal sensor chip was 7000 or less, whereas the blank light amount of a sensor chip having a defect in a gold thin film exceeded 8000. From these results, it is understood that the presence or absence of abnormality in the gold thin film of the sensor chip can be detected by examining the plasmon scattered light amount, the resonance reflectance, or the blank light amount.

[Example 2]
In Example 2, the result of simulating whether or not adhesion of foreign matter on the surface of the conductor film can be detected will be described. When a foreign substance adheres to the surface of the conductor film, a foreign substance layer having a different refractive index is formed on the conductor film. Therefore, in this simulation, the adhesion of foreign matters was simulated by arranging layers having different refractive indexes on the conductor film.

(1) Simulation conditions The wavelength of the excitation light was 633 nm. In the following description, “refractive index” means a refractive index for light having a wavelength of 633 nm.

As a sensor chip, a prism made of a material transparent to excitation light (refractive index 1.72), a gold thin film (conductor film; refractive index 0.1726 + 3.4218i) having a thickness of 50.0 nm, and a thickness 1.00 nm A layered body of protein layers (capture layer; refractive index 1.45) was assumed. It was assumed that a layer having a refractive index of 1.235 to 1.435 was disposed as a foreign substance layer on the protein layer. The refractive index of water is 1.335.

Under the above conditions, the gold thin film was irradiated with excitation light from the prism side. Scanning the angle of incidence on the gold thin film, incident angle, reflectance (ratio of the amount of light reflected by the gold thin film to the amount of excitation light) and scattered light (the amount of scattered light of the same wavelength above the gold thin film) The relationship was calculated.

(2) Simulation results The simulation results are shown in FIG. FIG. 8A is a graph showing the relationship between the incident angle of excitation light and the reflectance. FIG. 8B is a graph showing the relationship between the incident angle of excitation light and the amount of scattered light (arbitrary unit). 8A and 8B, the numerical value given to each curve means the refractive index (n) of the foreign material layer. In the graph of FIG. 8A, for each curve, the incident angle at which the reflectance is minimum corresponds to the “resonance angle”. In the graph of FIG. 8B, the incident angle at which the amount of scattered light is maximum corresponds to the “enhancement angle” for each curve. Here, the scattered light amount at this enhancement angle is referred to as “plasmon scattered light amount”.

8A and 8B that the resonance angle and the enhancement angle increase as the refractive index of the foreign material layer increases (the amount of foreign matter attached increases). Therefore, it can be seen that the adhesion of foreign matter to the conductor film of the sensor chip can be detected by examining the resonance angle or the enhancement angle.

This application claims priority based on Japanese Patent Application No. 2013-085580 filed on April 16, 2013. The contents described in the application specification and the drawings are all incorporated herein.

The measurement abnormality detection method and the surface plasmon excitation enhanced fluorescence measurement apparatus according to the present invention can measure a substance to be measured with high reliability, and are useful for clinical examinations, for example.

DESCRIPTION OF SYMBOLS 100 Surface plasmon excitation enhanced fluorescence measuring apparatus 110 Pretreatment chamber 120 Measurement chamber 130 Liquid feeding part 132 Liquid feeding pump 134 Liquid feeding pump conveyance part 136 Reagent chip 140 Chip conveyance part 150 Light source unit 160 Excitation light optical system 162 First wave shaper 164 Polarization direction adjusting unit 166 Shaping optical system 168 Incident angle adjusting unit 170 Chip holder 180 Measuring light optical system 190 Measuring light detecting unit 200 Reflected light detecting unit 210 Control calculating unit 220 Display unit 300 Sensor chip 310 Prism 312 Incident surface 314 Reflecting surface 316 Emission surface 320 Conductor film 330 Capture body 340 Channel member 342 Channel 344 Reaction chamber 346 Inlet 348 Outlet

Claims (12)

1. A method for detecting a measurement abnormality in measurement by surface plasmon excitation enhanced fluorescence spectroscopy,
   (A-1) preparing a sensor chip having a prism made of a dielectric, a conductor film disposed on a surface of the prism, and a capturing body fixed on the conductor film;
   (B-1) A step of irradiating light from the prism side to the conductor film so as to totally reflect at the interface between the prism and the conductor film, and measuring an optical characteristic value for detecting a measurement abnormality When,
   (B-2) detecting a measurement abnormality based on the optical characteristic value;
   A method for detecting a measurement abnormality, including:
2. After the step (b-2),
   (B-3) If a measurement abnormality is detected in the step (b-2), stop the measurement, or (b-3 ′) If a measurement abnormality is detected in the step (b-2), measure the measurement. A process of notifying measurement abnormality when continuing and displaying the measurement result,
   The method for detecting a measurement abnormality according to claim 1, further comprising:
3. The sensor chip has information relating to optical characteristic values in a normal state,
   In the step (b-2), the optical property value in the normal state is compared with the measured optical property value to detect the presence or absence of measurement abnormality.
   The method for detecting a measurement abnormality according to claim 1 or 2.
4. After the step (a-1),
   (A-2) bringing the sample into contact with the capturing body and binding the substance to be measured and the capturing body contained in the sample;
   (A-3) labeling the substance to be measured bound to the capture body with a fluorescent substance;
   (A-4) Excitation light is emitted from the prism side to the conductor film so as to be totally reflected at the interface between the prism and the conductor film, and is emitted from the fluorescent substance that labels the substance to be measured. Measuring the amount of fluorescent light,
   Further including
   The step (b-1) is performed between the step (a-1) and the step (a-2), between the step (a-2) and the step (a-3), or Performed between (a-3) and step (a-4),
   The method for detecting a measurement abnormality according to any one of claims 1 to 3.
5. After the step (a-1),
   (A-2 ′) contacting a sample containing a substance to be measured that has been labeled with a fluorescent substance in advance with the capture body to bind the substance to be measured labeled with the fluorescent substance and the capture body; Or a step of bringing a sample and a fluorescent substance into contact with the capturing body, labeling the target substance with the fluorescent substance, and binding the target substance and the capturing body;
   (A-4) Excitation light is emitted from the prism side to the conductor film so as to be totally reflected at the interface between the prism and the conductor film, and is emitted from the fluorescent substance that labels the substance to be measured. Measuring the amount of fluorescent light,
   Further including
   The step (b-1) is performed between the step (a-1) and the step (a-2 ′) or between the step (a-2 ′) and the step (a-4). Carried out,
   The method for detecting a measurement abnormality according to any one of claims 1 to 3.
6. The method for detecting a measurement abnormality according to any one of claims 1 to 5, wherein the optical characteristic value is a plasmon scattered light amount.
7. 6. The method for detecting a measurement abnormality according to claim 1, wherein the optical characteristic value is an enhancement angle.
8. The method for detecting a measurement abnormality according to any one of claims 1 to 5, wherein the optical characteristic value is a resonance angle.
9. The method for detecting a measurement abnormality according to any one of claims 1 to 5, wherein the optical characteristic value is a reflected light amount.
10. The method for detecting a measurement abnormality according to any one of claims 1 to 5, wherein the optical characteristic value is a blank light amount.
11. 4. The measurement abnormality detection method according to claim 3, wherein the sensor chip is provided with a barcode including the information.
12. A surface plasmon excitation-enhanced fluorescence measuring apparatus that measures a substance to be measured using surface plasmon excitation-enhanced fluorescence spectroscopy,
    A measurement abnormality in measurement using surface plasmon excitation enhanced fluorescence spectroscopy is detected by the measurement abnormality detection method according to any one of claims 1 to 11.
    Surface plasmon excitation enhanced fluorescence measuring device.

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