JPWO2015194513A1 - Measuring chip, measuring method and measuring device - Google Patents

Abstract

By applying a voltage between the two metal films on the two metal films exposed in the housing portion and between the two metal films of the measurement chip, the electrical properties of the specimen The specific characteristic of the blood content is determined using the measured value obtained by measuring the target characteristic. Using the obtained specific value of the blood content, the first signal value indicating the amount of the substance to be measured in the sample is converted into the second signal value indicating the amount of the substance to be measured in the liquid component of the sample. To do.

Description

  The present invention relates to a measurement method and a measurement apparatus that measure the amount of a substance to be measured in a specimen including at least a part of blood using surface plasmon resonance, and a measurement chip that can be used in the measurement method and the measurement apparatus. .

  In a clinical test or the like, if a minute amount of a substance to be measured such as protein or DNA in blood can be measured highly sensitively and quantitatively, it becomes possible to quickly grasp the patient's condition and perform treatment. Therefore, there is a need for a method that can measure a substance to be measured in blood with high sensitivity and quantitative.

  Surface plasmon resonance (SPR) method and surface plasmon-field enhanced fluorescence spectroscopy (hereinafter referred to as “Surface Plasmon-field enhanced Fluorescence Spectroscopy”) (Abbreviated as “SPFS”). These methods utilize the fact that surface plasmon resonance (SPR) occurs when a metal film is irradiated with light under predetermined conditions (see, for example, Patent Document 1).

  For example, in SPFS, a capture body (for example, a primary antibody) that can specifically bind to a substance to be measured is immobilized on a metal film to form a reaction field for specifically capturing the substance to be measured. When a specimen (for example, blood) containing a substance to be measured is provided in the reaction field, the substance to be measured is bound to the reaction field. Next, when a capture body (for example, a secondary antibody) labeled with a fluorescent substance is provided to the reaction field, the substance to be measured bound to the reaction field is labeled with the fluorescent substance. When the metal film is irradiated with excitation light in this state, the fluorescent substance that labels the substance to be measured is excited by the electric field enhanced by SPR and emits fluorescence. Therefore, the presence or amount of the substance to be measured can be detected by detecting the fluorescence. In SPFS, a fluorescent substance is excited by an electric field enhanced by SPR, so that a substance to be measured can be measured with high sensitivity.

  On the other hand, when measuring a substance to be measured in a liquid by various measuring methods, not limited to the SPR method and SPFS, the measured value is usually the mass of the substance to be measured per unit volume of the liquid and the signal amount corresponding thereto. Etc. Therefore, when blood is used as the specimen, the measured value is indicated by the mass of the substance to be measured per unit volume of the liquid component (plasma or serum) in the blood, the signal amount corresponding thereto, and the like. Since the ratio of the liquid component in the blood varies from person to person, the blood measurement value cannot be uniformly converted into the liquid component measurement value. For this reason, when blood is used as a specimen, the hematocrit value (ratio of the volume of blood cells in the blood) of the blood is measured, and the blood measurement value is measured using the hematocrit value as the measurement value of the liquid component (plasma or serum). The conversion is done. As a conventional method for measuring a hematocrit value, there are a microhematocrit method for centrifuging blood, a method for obtaining a hematocrit value from a hemoglobin concentration of hemolyzed blood (for example, see Patent Document 2), and the like.

JP-A-10-307141 JP 2001-272403 A

  In the measuring method and measuring apparatus using SPR such as SPR method and SPFS, when the above conventional method for measuring hematocrit is adopted, a new apparatus for measuring hematocrit value such as a centrifuge or a hemoglobin concentration measuring apparatus is newly provided. Therefore, the manufacturing cost and the measurement cost increase.

  A first object of the present invention is a measurement method and a measurement device using SPR, and corrects a measurement value using a specific value of a blood content such as a hematocrit value without adding a new device. It is to provide a measurement method and a measurement apparatus that can be performed. The second object of the present invention is to provide a measuring chip that can be used in this measuring method and measuring apparatus.

  In order to solve the above problems, a measurement method according to an embodiment of the present invention is a method for measuring the amount of a substance to be measured in a specimen including at least a part of blood using surface plasmon resonance. A dielectric prism having an entrance surface, an exit surface, and a film formation surface; and a storage unit disposed on the film formation surface, for accommodating the specimen, and spaced apart on the film formation surface, The sample is provided to a measurement chip having at least one capture body fixed and a plurality of metal films, at least a part of which is exposed in the housing portion, and the target substance is captured by the capture body. A voltage between the two metal films in a state in which the specimen exists on and between the two metal films exposed in the housing portion among the plurality of metal films. Applying electricity to the specimen Measuring the characteristics, obtaining the specific value of the blood content in the specimen using the obtained measurement value, and the state in which the substance to be measured and the capturing body are combined, and in the container In the absence of the specimen, the back surface of the metal film corresponding to the region where the capturing body is fixed is irradiated through the incident surface, and excitation light is irradiated to emit fluorescence emitted from the excited fluorescent material. Measuring the amount of light or the amount of excitation light reflected from the film formation surface and emitted from the exit surface to obtain a first signal value indicating the amount of the substance to be measured in the sample; and the blood Converting the first signal value into a second signal value indicating the amount of the substance to be measured in the liquid component of the specimen using the specific value of the content of

  In order to solve the above problems, a measurement chip according to an embodiment of the present invention is a chip for measuring the amount of a substance to be measured in a specimen including at least a part of blood using surface plasmon resonance. A dielectric prism having an entrance surface, an exit surface, and a film formation surface; an accommodation portion disposed on the film formation surface for accommodating the specimen; and spaced apart on the film formation surface. And a plurality of metal films having a capturing body fixed to at least one and at least a part of which is exposed in the container.

  In order to solve the above problems, a measuring apparatus according to an embodiment of the present invention is an apparatus for measuring the amount of a substance to be measured in a specimen including at least a part of blood using surface plasmon resonance. A dielectric prism having an entrance surface, an exit surface, and a film formation surface; an accommodation portion disposed on the film formation surface for accommodating the specimen; and spaced apart on the film formation surface. A holder for holding a measuring chip having a plurality of metal films having a capturing body fixed to at least one and at least a part of which is exposed in the housing, and exposed in the housing An electrical property measuring unit for measuring electrical properties of the specimen by applying a voltage between the two metallic films in a state where the specimen is present on and between the two metallic films. Toward the entrance surface The capturing body is fixed via the incident surface in a state where the light irradiation section for irradiating the target substance, the substance to be measured and the capturing body are combined, and the specimen is not present in the housing section. The back surface of the metal film corresponding to the region is irradiated with excitation light, and the amount of fluorescent light emitted from the excited fluorescent material or reflected from the film formation surface of the prism and emitted from the emission surface A light detection unit for detecting the amount of excitation light; a first signal value indicating the amount of the substance to be measured in the sample based on a detection result of the light detection unit; and the electrical characteristic measurement unit The specific value of the blood content is calculated based on the measurement result of the above, and the first signal value is calculated using the specific value of the blood content and the amount of the substance to be measured in the liquid component of the specimen To convert to a second signal value indicating It has a part, a.

  According to the present invention, in a measurement method and a measurement device using SPR, measurement is performed by using a specific value of the contents of blood without adding a new device and causing a delay in measurement time. The amount of substance can be accurately measured.

1 is a schematic diagram showing a configuration of a surface plasmon excitation enhanced fluorescence analyzer (SPFS apparatus) according to Embodiment 1. FIG. 2A is a plan view of the measurement chip according to Embodiment 1, and FIG. 2B is a cross-sectional view taken along line AA shown in FIG. 2A. 3A is a plan view of the prism, FIG. 3B is a plan view of the frame, and FIG. 3C is a plan view of the flow path lid. It is a flowchart which shows an example of the operation | movement procedure of the apparatus shown by FIG. 5A is a plan view of the measurement chip according to Embodiment 2, and FIG. 5B is a cross-sectional view taken along line AA shown in FIG. 5A. FIG. 6 is a plan view of a measurement chip according to a modification of the second embodiment. FIG. 7A is a plan view of a measurement chip according to a modification of the second embodiment installed in the chip holder, and FIG. 7B is a cross-sectional view taken along line AA shown in FIG. 7A.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Here, as a representative example of the measuring apparatus according to the present invention, a surface plasmon excitation enhanced fluorescence analyzer (SPFS apparatus) that measures the amount of a substance to be measured contained in a specimen including at least a part of blood will be described. In the present embodiment, a case will be described in which the hematocrit value of blood is obtained as the specific value of the blood content. An SPFS apparatus that measures the impedance of the specimen as the electrical characteristics of the specimen for obtaining the hematocrit value of blood will be described. Examples of specimens include blood (blood that has not been separated and diluted), plasma, serum, and diluted solutions thereof.

[Embodiment 1]
FIG. 1 is a schematic diagram showing a configuration of a surface plasmon excitation enhanced fluorescence analyzer (SPFS apparatus) 100 according to an embodiment of the present invention. As shown in FIG. 1, the SPFS device 100 includes an electrical characteristic measurement unit 105, an excitation light irradiation unit 110, a reflected light detection unit 120, a fluorescence detection unit 130, a liquid feeding unit 140, a transport unit 150, and a control unit 160. Have. The SPFS device 100 is used in a state where the measurement chip 10 is mounted on the chip holder 152 of the transport unit 150. Therefore, the measurement chip 10 will be described first, and then each component of the SPFS device 100 will be described.

  FIG. 2 is a schematic diagram showing the measurement chip 10 used in the present embodiment. 2A is a plan view of the measurement chip 10, and FIG. 2B is a cross-sectional view taken along line AA shown in FIG. 2A. The measurement chip 10 includes a prism 20 having an incident surface 21, a film formation surface 22, and an emission surface 23, a first metal film 30 and a second metal film 31 that are spaced apart from each other on the film formation surface 22, A frame 35 disposed on the film formation surface 22 and a flow path lid 40 are provided.

  3A is a plan view of the prism 20, FIG. 3B is a plan view of the frame body 35, and FIG. 3C is a plan view of the flow path lid 40.

  The prism 20, the first metal film 30, the second metal film 31, the frame body 35, and the channel cover 40 form a flow channel 41 (accommodating portion) through which liquid flows. Examples of the liquid include a specimen (blood, plasma, serum, or a diluted solution thereof) containing a substance to be measured, a labeling liquid containing a capturing body labeled with a fluorescent substance, a washing liquid, and the like. Further, the flow path 41 includes a measured portion 42 for accommodating the specimen when measuring the impedance of the specimen. Here, the “measured part” refers to a region in the flow path 41 where a sample to be measured for impedance exists. In the present embodiment, the part to be measured 42 is a region including a part of the first metal film 30 and a part of the second metal film 31.

  The prism 20 is made of a dielectric that is transparent to the excitation light α. The prism 20 has an incident surface 21, a film forming surface 22, and an exit surface 23 (see FIG. 1). The incident surface 21 causes the excitation light α from the excitation light irradiation unit 110 to enter the prism 20. A first metal film 30 and a second metal film 31 are spaced apart from each other on the film formation surface 22. In the present embodiment, the excitation light α incident on the inside of the prism 20 is applied to the first metal film 30 where the substance to be measured is captured. The excitation light α is reflected on the back surface of the first metal film 30 and becomes reflected light β. More specifically, the excitation light α is reflected at the interface (deposition surface 22) between the prism 20 and the first metal film 30 to be reflected light β. The emission surface 23 emits the reflected light β 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 excitation light irradiation unit 110. When the light source of the excitation light α is a laser diode (hereinafter also referred to as “LD”), when the excitation light α returns to the LD, the excitation state of the LD is disturbed, and the wavelength and output of the excitation light α 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” refers to the incident angle when the amount of the reflected light β emitted from the emission surface 23 is minimum when the incident angle of the excitation light α with respect to the first metal film 30 is scanned. means. The “enhancement angle” refers to scattered light having the same wavelength as the excitation light α emitted above the measurement chip 10 (hereinafter referred to as “plasmon”) when the incident angle of the excitation light α on the first metal film 30 is scanned. This means the incident angle when the amount of δ) (referred to as “scattered light”) is maximized. In the present embodiment, 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 °.

  The design of the measuring chip 10 largely determines the resonance angle (and the enhancement angle near the pole). The design factors are the refractive index of the prism 20, the refractive index of the first metal film 30, the thickness of the first metal film 30, the extinction coefficient of the first 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 first metal film 30, but the amount is less than a few degrees.

  The prism 20 is made of an insulator (dielectric material) from the viewpoint of preventing conduction through the prism 20 when a voltage is applied between the first metal film 30 and the second metal film 31. On the other hand, the prism 20 has not a few birefringence characteristics. Examples of the material of the prism 20 include insulating resin and glass. The material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

  The first metal film 30 and the second metal film 31 are spaced apart from each other on the film formation surface 22 of the prism 20. At least a part of the first metal film 30 and the second metal film 31 is exposed in the flow path 41 (measured portion 42). In the present embodiment, the first metal film 30 and the second metal film 31 are used as electrodes for measuring the impedance of the specimen. The first metal film 30 causes an interaction (SPR) between the photon of the excitation light α incident on the film formation surface 22 under the total reflection condition and the free electrons in the first metal film 30, Local field light (generally also referred to as “evanescent light” or “near-field light”) can be generated on the surface of the first metal film 30.

  The material of the first metal film 30 and the second metal film 31 is not particularly limited as long as it can be used as an electrode for measuring the impedance of the specimen and can generate SPR. Examples of the material of the first metal film 30 and the second metal film 31 include gold, silver, copper, aluminum, and alloys thereof. In the present embodiment, the first metal film 30 and the second metal film 31 are gold thin films. The formation method of the first metal film 30 and the second metal film 31 is not particularly limited. Examples of the method for forming the first metal film 30 and the second metal film 31 include sputtering, vapor deposition, and plating. Although the thickness of the 1st metal film 30 and the 2nd metal film 31 is not specifically limited, The inside of the range of 30-70 nm is preferable.

  Further, as shown in FIGS. 2B and 3A, a capturing body 50 for capturing a substance to be measured is immobilized on the surface of the first metal film 30. By immobilizing the capturing body 50, the substance to be measured can be selectively measured. In the present embodiment, the capturing body 50 is uniformly fixed in a predetermined region on the first metal film 30. The region where the capturing body 50 is immobilized serves as a reaction field where a primary reaction and a secondary reaction described later occur. The capturing body 50 fixed to the first metal film 30 is exposed in the flow path 41. The type of the capturing body 50 is not particularly limited as long as the substance to be measured can be captured. In the present embodiment, the capturing body 50 is an antibody specific to the substance to be measured or a fragment thereof.

  As shown in FIG. 2B and FIG. 3B, the frame 35 is disposed between the film formation surface 22 of the prism 20, the first metal film 30 and the second metal film 31, and the flow path lid 40. It is a thin plate having a first through hole 36. The frame body 35 may be disposed directly on the film formation surface 22. The first through hole 36 is a side surface of the flow path 41 through which the liquid flows. The shape of the first through hole 36 is not particularly limited as long as the liquid can flow and the specimen can be accommodated, and can be appropriately selected depending on the application.

  The material of the frame 35 is preferably an insulator from the viewpoint of preventing electrical conduction through the frame 35 during impedance measurement. The material of the frame 35 is not particularly limited as long as it is an insulator, and may be the same material as a known microplate. Examples of the material of the frame 35 include resins such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane, polymethylmethacrylate, and cyclic olefin copolymer. The frame body 35 may be a silicon sheet (double-sided seal) having adhesiveness on both sides. By using a double-sided seal as the frame body 35, the prism 20 (or the first metal film 30 and the second metal film 32), the frame body 35, and the flow path lid 40 can be easily fixed. .

  As shown in FIGS. 2B and 3C, the flow path lid 40 is a thin plate having two through holes arranged on the frame body 35. The two through holes serve as an inlet 60 and an outlet 70, respectively. Both ends of the channel 41 are connected to the inlet 60 and the outlet 70, respectively.

  The channel lid 40 is preferably made of a material that is transparent to the fluorescence γ and the plasmon scattered light δ emitted from the first metal film 30. An example of the material of the flow path lid 40 includes a resin. As long as the portion from which the fluorescent γ and the plasmon scattered light δ are extracted is transparent to the fluorescent γ and the plasmon scattered light δ, the other portion of the channel lid 40 may be formed of an opaque material. The flow path lid 40 is joined to the frame 35 by, for example, adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.

  In the measurement chip 10, instead of the frame body 35, a flow path lid 40 having a flow path groove formed on the back surface thereof may be used. In this case, the channel 41 (accommodating portion) is formed only by the prism 20, the first metal film 30 and the channel lid 40. At this time, the channel lid 40 is preferably an insulator from the viewpoint of preventing conduction through the channel lid 40 during impedance measurement.

  As shown in FIG. 1, the excitation light α enters the prism 20 at the incident surface 21. The excitation light α that has entered the prism 20 is applied to the first metal film 30 at a total reflection angle (an angle at which SPR occurs). By irradiating the first metal film 30 with the excitation light α at an angle at which SPR occurs in this way, localized field light can be generated on the first metal film 30. This localized field light excites a fluorescent substance that labels the substance to be measured present on the first metal film 30 and emits fluorescent γ. The SPFS device 100 measures the amount of the substance to be measured by measuring the amount of the fluorescent γ emitted from the fluorescent substance.

  Next, each component of the SPFS device 100 according to the present embodiment will be described. As described above, the SPFS device 100 includes the electrical characteristic measurement unit 105, the excitation light irradiation unit 110, the reflected light detection unit 120, the fluorescence detection unit 130, the liquid feeding unit 140, the transport unit 150, and the control unit 160.

  The electrical characteristic measurement unit 105 applies a voltage between the first metal film 30 and the second metal film 31 of the measurement chip 10 held by the chip holder 152, and within the flow channel 41 (measurement unit 42). The impedance of the specimen existing on and between the first metal film 30 exposed on the second metal film 31 and the second metal film 31 is measured. The electrical characteristic measurement unit 105 flows between a power source for applying a voltage between the first metal film 30 and the second metal film 31 and between the first metal film 30 and the second metal film 31. An ammeter for measuring current. The type of power supply is not particularly limited, but it is preferable to use an AC power supply. By using alternating current, the impedance measurement of the specimen described later can be performed without changing the composition of the specimen. In the present embodiment, two terminals for voltage application connected to the power source are connected to the first metal film 30 and the second metal film 31 through the inlet 60 and the outlet 70, respectively. The voltage application terminal is preferably exchanged for each measurement in order to contact the specimen. The type of ammeter is, for example, a DC ammeter, an AC ammeter, an ammeter ammeter, or the like.

  The excitation light irradiation unit 110 irradiates the excitation light α toward the incident surface 21 of the measurement chip 10 held by the chip holder 152. When measuring the fluorescence γ or the plasmon scattered light δ, the excitation light irradiation unit 110 enters only the P wave with respect to the first metal film 30 so that the incident angle with respect to the first metal film 30 is an angle that causes SPR. The light exits toward the surface 21. Here, the “excitation light” is light that directly or indirectly excites the fluorescent material. For example, when the excitation light α is applied to the first metal film 30 through the incident surface 21 of the prism 20 at an angle at which SPR occurs, the first metal film 30 generates localized field light that excites the fluorescent material. This is the light that is generated on the surface. The excitation light irradiation unit 110 includes a light source unit 111, an angle adjustment mechanism 112, and a light source control unit 113.

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

  The type of the light source is not particularly limited, and is, for example, a laser diode (LD). Other examples of light sources include light emitting diodes, mercury lamps, and other laser light sources. When the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like. When the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like. 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 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 unit. 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. The band-pass filter turns the excitation light α emitted from the light source into narrowband light having only the center wavelength. This is because the excitation light α from the light source has a slight wavelength distribution width. The linear polarization filter turns the excitation light α emitted from the light source into completely linearly polarized light. The half-wave plate adjusts the polarization direction of the excitation light α so that the P-wave component is incident on the first 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 first metal film 30 is a circle of a predetermined size.

  The APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light α with a photodiode (not shown) or the like. The APC mechanism controls the input energy by a regression circuit, thereby controlling the output of the light source 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 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 adjustment mechanism 112 adjusts the incident angle of the excitation light α with respect to the first metal film 30 (the interface between the prism 20 and the first metal film 30 (film formation surface 22)). The angle adjusting mechanism 112 irradiates the excitation light α at a predetermined incident angle toward a predetermined position of the first metal film 30 via the incident surface 21 of the prism 20, and the optical axis of the excitation light α and the tip The holder 152 is rotated relatively.

  For example, the angle adjustment mechanism 112 rotates the light source unit 111 around an axis (axis perpendicular to the paper surface of FIG. 1) orthogonal to the optical axis of the excitation light α. At this time, the position of the rotation axis is set so that the position of the irradiation spot on the first metal film 30 hardly changes even when the incident angle is scanned. By setting the position of the center of rotation near the intersection of the optical axes of the two excitation lights α at both ends of the scanning range of the incident angle (between the irradiation position on the film forming surface 22 and the incident surface 21), the irradiation position Can be minimized.

  As described above, among the incident angles of the excitation light α to the first metal film 30, the angle at which the light quantity of the plasmon scattered light δ is maximum is the enhancement angle. By setting the incident angle of the excitation light α to the enhancement angle or an angle in the vicinity thereof, it becomes possible to measure the high-intensity fluorescence γ. The basic incident condition of the excitation light α is determined by the material and shape of the prism 20 of the measurement chip 10, the thickness of the first metal film 30, the refractive index of the liquid in the flow channel 41, etc. Depending on the type and amount of the fluorescent material, the shape error of the prism 20, and the like, the optimum incident condition varies slightly. For this reason, it is preferable to obtain an optimal enhancement angle for each measurement.

  The light source control unit 113 controls various devices included in the light source unit 111 to control the emission of the excitation light α from the light source unit 111. The light source control unit 113 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 reflected light detection unit 120 measures the amount of reflected light β generated by irradiating the measurement chip 10 with the excitation light α in order to measure the resonance angle. The reflected light detection unit 120 is disposed on the opposite side of the excitation light irradiation unit 110 with a normal to the first metal film 30 passing through the irradiation spot. The reflected light detection unit 120 includes a light receiving sensor 121, an angle adjustment mechanism 122, and a sensor control unit 123. When the fluorescence detection unit 130 (described later) determines the enhancement angle by measuring the amount of plasmon scattered light δ, the reflected light detection unit 120 may be omitted.

  The light receiving sensor 121 is disposed at a position where the reflected light β is incident, and measures the amount of the reflected light β. The type of the light receiving sensor 121 is not particularly limited. For example, the light receiving sensor 121 is a photodiode (PD).

  The angle adjustment mechanism 122 adjusts the position (angle) of the light receiving sensor 121 according to the incident angle of the excitation light α with respect to the first metal film 30. The angle adjusting mechanism 122 relatively rotates the light receiving sensor 121 and the chip holder 152 so that the reflected light β enters the light receiving sensor 121.

  The sensor control unit 123 controls detection of an output value of the light receiving sensor 121, management of sensitivity of the light receiving sensor 121 based on the detected output value, change of sensitivity of the light receiving sensor 121 for obtaining an appropriate output value, and the like. The sensor 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 fluorescence detection unit 130 detects the fluorescence γ generated by the irradiation of the excitation light α to the first metal film 30. Further, as necessary, the fluorescence detection unit 130 also detects the plasmon scattered light δ generated by the irradiation of the excitation light α to the first metal film 30. The fluorescence detection unit 130 includes a light receiving unit 131, a position switching mechanism 132, and a sensor control unit 133.

  The light receiving unit 131 is disposed in the normal direction of the first metal film 30 of the measurement chip 10. The light receiving unit 131 includes a first lens 134, an optical filter 135, a second lens 136, and a light receiving sensor 137.

  The first lens 134 is, for example, a condensing lens, and condenses light emitted from the first metal film 30. The second lens 136 is an imaging lens, for example, and forms an image of the light collected by the first lens 134 on the light receiving surface of the light receiving sensor 137. The optical path between both lenses is substantially parallel.

  The optical filter 135 is disposed between both lenses. The optical filter 135 guides only the fluorescence component to the light receiving sensor 137 and removes the excitation light component (plasmon scattered light δ) in order to detect the fluorescence γ with a high S / N ratio. Examples of the optical filter 135 include an excitation light reflection filter, a short wavelength cut filter, and a band pass filter. The optical filter 135 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 137 detects fluorescence γ and plasmon scattered light δ. The light receiving sensor 137 has high sensitivity capable of detecting weak fluorescence γ from a minute amount of a substance to be measured. The light receiving sensor 137 is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).

  The position switching mechanism 132 switches the position of the optical filter 135 on or off the optical path in the light receiving unit 131. Specifically, when the light receiving sensor 137 detects the fluorescence γ, the optical filter 135 is disposed on the optical path of the light receiving unit 131, and when the light receiving sensor 137 detects the plasmon scattered light δ, the optical filter 135 is placed on the light receiving unit 131. Placed outside the optical path.

  The sensor control unit 133 controls detection of an output value of the light receiving sensor 137, management of sensitivity of the light receiving sensor 137 based on the detected output value, change of sensitivity of the light receiving sensor 137 for obtaining an appropriate output value, and the like. The sensor control unit 133 is configured by, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.

  The liquid feeding unit 140 supplies a specimen, a labeling liquid, a cleaning liquid, and the like into the flow path 41 of the measuring chip 10 held by the chip holder 152. The liquid feeding unit 140 includes a liquid chip 141, a syringe pump 142, and a liquid feeding pump drive mechanism 143.

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

  The syringe pump 142 includes a syringe 144 and a plunger 145 that can reciprocate within the syringe 144. By the reciprocating motion of the plunger 145, the liquid is sucked and discharged quantitatively. If the syringe 144 can be replaced, the syringe 144 need not be cleaned. For this reason, it is preferable from the viewpoint of preventing contamination of impurities. When the syringe 144 is not configured to be replaceable, it is possible to use the syringe 144 without replacing it by adding a configuration for cleaning the inside of the syringe 144.

  The liquid feed pump driving mechanism 143 includes a driving device for the plunger 145 and a moving device for the syringe pump 142. The drive device of the syringe pump 142 is a device for reciprocating the plunger 145, and includes, for example, a stepping motor. A drive device including a stepping motor is preferable from the viewpoint of managing the amount of liquid remaining in the measurement chip 10 because it can manage the amount and speed of the syringe pump 142. For example, the moving device of the syringe pump 142 freely moves the syringe pump 142 in two directions, ie, an axial direction (for example, a vertical direction) of the syringe 144 and a direction crossing the axial direction (for example, a horizontal direction). The moving device of the syringe pump 142 is configured by, for example, a robot arm, a two-axis stage, or a turntable that can move up and down.

  The liquid feeding unit 140 sucks various liquids from the liquid chip 141 and supplies them to the flow path 41 of the measurement chip 10. At this time, by moving the plunger 145, the liquid reciprocates in the flow path 41 in the measurement chip 10, and the liquid in the flow path 41 is stirred. As a result, it is possible to achieve a uniform concentration of the liquid and promotion of a reaction (for example, an antigen-antibody reaction) in the flow channel 41. From the viewpoint of performing such operations, the measurement chip 10 and the syringe are protected so that the injection port 60 of the measurement chip 10 is protected by a multilayer film and the injection port 60 can be sealed when the syringe 144 penetrates the multilayer film. 144 is preferably configured.

  The liquid in the channel 41 is again sucked by the syringe pump 142 and discharged to the liquid chip 141 and the like. By repeating these operations, reaction with various liquids, washing, and the like can be performed, and a substance to be measured labeled with a fluorescent substance can be placed in the reaction field in the flow path 41.

  The conveyance unit 150 conveys and fixes the measurement chip 10 to the measurement position or the liquid feeding position. Here, the “measurement position” means that the excitation light irradiation unit 110 irradiates the measurement chip 10 with the excitation light α, and the reflected light β, fluorescence γ or plasmon scattered light δ generated thereby is reflected light detection unit 120 or fluorescence detection. This is the position detected by the unit 130. Further, the “liquid feeding position” is a position where the liquid feeding unit 140 supplies the liquid into the flow channel 41 of the measurement chip 10 or removes the liquid in the flow channel 41 of the measurement chip 10. The transport unit 150 includes a transport stage 151 and a chip holder 152. The chip holder 152 is fixed to the transfer stage 151, and holds the measurement chip 10 in a detachable manner. The shape of the chip holder 152 is a shape that can hold the measurement chip 10 and does not disturb the optical paths of the excitation light α, the reflected light β, the fluorescence γ, and the plasmon scattered light δ. For example, the chip holder 152 is provided with an opening through which excitation light α, reflected light β, fluorescence γ, and plasmon scattered light δ pass. The transfer stage 151 moves the chip holder 152 in one direction and the opposite direction. The transport stage 151 also has a shape that does not obstruct the optical paths of the excitation light α, reflected light β, fluorescence γ, and plasmon scattered light δ. The transport stage 151 is driven by, for example, a stepping motor.

  The control unit 160 includes an electrical characteristic measurement unit 105, an angle adjustment mechanism 112, a light source control unit 113, an angle adjustment mechanism 122, a sensor control unit 123, a position switching mechanism 132, a sensor control unit 133, a liquid feed pump drive mechanism 143, and a conveyance. The stage 151 is controlled. Further, the control unit 160 determines whether or not the sample is a sample containing blood based on the measurement result of the electrical characteristic measuring unit 105, that is, whether it is blood or a diluted solution of blood, or serum, plasma, or these It also functions as a processing unit that determines whether it is a diluted solution. Furthermore, when the control unit 160 determines that the sample is blood or a blood dilution liquid, the control unit 160 calculates a hematocrit value of blood contained in the sample based on the measurement result of the electrical characteristic measurement unit 105. Also works. The control unit 160 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.

  Next, the detection operation of the SPFS device 100 (measurement method according to the embodiment of the present invention) will be described. FIG. 4 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 100.

  First, preparation for measurement is performed (step S10). Specifically, the measurement chip 10 is installed in the chip holder 152 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 50 can appropriately capture the substance to be measured.

  Next, using the impedance of the specimen as an index, it is determined whether the specimen is a specimen containing blood (for example, blood or a diluted solution of blood) or other specimen (for example, plasma, serum, or a diluted solution thereof). To do. When the sample is a sample containing blood, the hematocrit value of blood contained in the sample is measured. In the channel 41, the substance to be measured is captured on the first metal film 30 by the antigen-antibody reaction (primary reaction; step S20). The specimen type determination and blood hematocrit measurement may be performed during the step of binding the substance to be measured to the capturing body 50 (that is, in parallel with the primary reaction), or the substance to be measured is captured by the capturing body. It may be carried out after the step of binding to 50 (ie after the primary reaction).

  Specifically, the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the liquid feeding position. Thereafter, the control unit 160 operates the liquid feeding unit 140 to introduce the sample in the liquid chip 141 into the flow channel 41. In the channel 41, the substance to be measured is captured on the first metal film 30 by the antigen-antibody reaction (primary reaction). Next, the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the measurement position. Next, two terminals for voltage application are connected to the first metal film 30 and the second metal film 31 through the inlet 60 and the outlet 70, respectively. Thereafter, the control unit 160 operates the electrical characteristic measurement unit 105 to measure the impedance of the specimen. The measurement of the impedance is performed in the state where the specimen exists on the first metal film 30 exposed in the flow channel 41 (measurement unit 42), on the second metal film 31, and between them. This is performed by applying a voltage between the metal film 30 and the second metal film 31. The measured value is recorded in the control unit 160. At this time, the voltage (about 0.1 to 1 V) applied between the first metal film 30 and the second metal film 31 is very small. In addition, the impedance measurement time (several seconds) is much shorter than the time for performing the primary reaction (several minutes). For this reason, the impedance of the specimen can be measured without affecting the primary reaction. Therefore, the determination of the type of specimen and the measurement of the hematocrit value of blood may be performed while the substance to be measured is bound to the capturing body 50. Thereby, the measurement time of the amount of the substance to be measured can be shortened.

  Next, based on the obtained impedance of the specimen, the control unit 160 determines whether the specimen is a specimen containing blood (for example, blood or a diluted solution of blood) or other specimens (for example, plasma, serum, or these) It is determined whether it is a diluted solution. The determination result is recorded in the control unit 160. When the sample is a sample containing blood, the control unit 160 further calculates the hematocrit value of the blood contained in the sample using the impedance of the sample. Specifically, a calibration curve of impedance and Hct value is acquired in advance, and the measured impedance is converted into an Hct value. A means for calculating the hematocrit value from the impedance value is known.

  Next, the optical blank value is measured (step S30). Here, the “optical blank value” means the amount of background light emitted above the measurement chip 10 in the measurement of fluorescence γ (step S50). Specifically, the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the liquid feeding position. Thereafter, the control unit 160 operates the liquid feeding unit 140 to remove the specimen in the flow channel 41 of the measurement chip 10 and cleans the flow channel 41 using the cleaning liquid in the liquid chip 141. Next, the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the measurement position. Thereafter, the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detection unit 130 so that the back surface of the first metal film 30 corresponding to the region where the capturing body 50 is fixed is interposed via the incident surface 21. While irradiating the excitation light α, the output value (optical blank value) of the light receiving sensor 137 is recorded. At this time, the control unit 160 operates the angle adjustment mechanism 112 to set the incident angle of the excitation light α to the enhancement angle. Further, the control unit 160 controls the position switching mechanism 132 to arrange the optical filter 135 in the optical path of the light receiving unit 131. The measured optical blank value is recorded in the control unit 160.

  Next, the substance to be measured captured on the first metal film 30 is labeled with a fluorescent substance (secondary reaction; step S40). Specifically, the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the liquid feeding position. Thereafter, the control unit 160 operates the liquid feeding unit 140 to introduce a liquid (labeled liquid) containing a capturing body labeled with a fluorescent substance into the flow channel 41 of the measurement chip 10. In the channel 41, the substance to be measured captured on the first metal film 30 is labeled with a fluorescent substance by an antigen-antibody reaction. Thereafter, the labeling liquid in the flow path 41 is removed, and the inside of the flow path 41 is cleaned with a cleaning liquid.

  Next, the amount of fluorescent γ emitted from the vicinity of the surface of the first metal film 30 that does not face the prism 20 is measured to obtain a first signal value indicating the amount of the substance to be measured in the specimen (step S50). ). Specifically, the control unit 160 operates the transfer stage 151 to move the measurement chip 10 to the measurement position. Thereafter, the control unit 160 operates the excitation light irradiation unit 110 and the fluorescence detection unit 130 so that the substance to be measured and the capturing body 50 are combined with each other, and the sample is placed in the channel 41 (accommodating unit). In the absence, the back surface of the first metal film 30 corresponding to the region where the capturing body 50 is fixed from the prism 20 side is irradiated with the excitation light α via the incident surface 21, and the output value of the light receiving sensor 137. Record. At this time, the control unit 160 operates the angle adjustment mechanism 112 to set the incident angle of the excitation light α to the enhancement angle. Further, the control unit 160 controls the position switching mechanism 132 to arrange the optical filter 135 in the optical path of the light receiving unit 131. The control unit 160 subtracts the optical blank value from the detection value, and calculates a first signal value indicating the amount of the substance to be measured in the sample. The first signal value is converted into the amount or concentration of the substance to be measured as necessary. The first signal value is recorded in the control unit 160.

  Next, it is determined whether the sample is a sample containing blood (for example, blood or a diluted solution of blood) or another sample (for example, plasma, serum, or a diluted solution thereof) (step S60). In this step, the determination result of step S20 may be used as it is. When the sample is a sample containing blood (for example, blood or a diluted solution of blood), the process proceeds to the step of converting the signal value using the hematocrit value (step S70). On the other hand, when the sample is another sample (for example, plasma, serum, or a diluted solution thereof), the signal value conversion using the hematocrit value is unnecessary, and thus the detection operation is terminated.

［上記式（１）および式（２）において、Ｈｔはヘマトクリット値であり、ｄｆは希釈液の希釈率である。］When the sample is a sample containing blood, the first signal value measured in step S50 is used as the amount of the substance to be measured in the liquid component of the sample, using the blood hematocrit value measured in step S20. It converts into the 2nd signal value shown (process S70). Specifically, when the specimen is undiluted blood, the first signal value is calculated by multiplying the first signal value by the conversion coefficient c ₁ represented by the following equation (1) to obtain the second signal value. Convert to the signal value of. On the other hand, when the specimen is a diluted blood solution, the first signal value is multiplied by the conversion coefficient c ₂ represented by the following equation (2) to obtain the first signal value as the second signal value. Convert. Note that the second signal value is not particularly limited. The second signal value may be a converted value such as the amount or concentration of the substance to be measured, for example.

[In the above formulas (1) and (2), Ht is the hematocrit value and df is the dilution rate of the diluent. ]

  With the above procedure, the amount of the substance to be measured in the liquid component of the specimen can be measured.

  In the SPFS apparatus according to the present embodiment, the impedance of the specimen can be measured using a metal film provided on the measurement chip. Therefore, the SPFS device according to the present embodiment can correct the signal value by calculating the hematocrit value of the specimen without adding a new device such as a centrifuge or a hemoglobin concentration measuring device. In addition, the SPFS apparatus according to the present embodiment can measure the impedance of the specimen simultaneously with the primary reaction. For this reason, it is not necessary to spend additional time for determining the type of specimen and calculating the hematocrit value. Moreover, impedance can be measured with a small amount of specimen. Therefore, in the SPFS apparatus according to the present embodiment, the amount of the substance to be measured can be accurately measured in a short time with a small amount of specimen.

[Embodiment 2]
In the second embodiment, an SPFS apparatus capable of performing measurement without wetting a voltage application terminal used for impedance measurement with a specimen will be described. Also in the present embodiment, a case where the hematocrit value of blood is obtained as the specific value of the blood content will be described.

  5A is a plan view of measurement chip 200 according to Embodiment 2, and FIG. 5B is a cross-sectional view taken along line AA shown in FIG. 5A. In addition, about the component same as the measurement chip | tip 10 which concerns on Embodiment 1 shown by FIG. 2, the same code | symbol is attached | subjected and the description is abbreviate | omitted.

  In the measurement chip 200 according to the second embodiment, in addition to the first metal film 230 in which the capturing body 50 is captured and the second metal film 231 in which the capturing body 50 is not fixed, the capturing body 50 is further fixed. A third metal film 232 that is not formed is included.

  As shown in FIG. 5A, in the measurement chip 200, the flow of liquid through the prism 20, the first metal film 230, the second metal film 231, the third metal film 232, the frame body 235, and the channel lid 240. A path 241 (accommodating portion) is formed. The flow path 241 includes a measured part 242 for accommodating the specimen when measuring the impedance of the specimen. In the present embodiment, the part to be measured 242 is a region including a part of the second metal film 231 and a part of the third metal film 232. Further, the first through hole 236 of the frame body 235 has a convex portion that becomes the measured portion 242. The frame body 235 and the flow path lid 240 further have a second through hole 280 and a third through hole 290. At least a part of the second metal film 231 and the third metal film 232 is exposed in the flow path 241 (measurement target 242). The second through hole 280 and the third through hole 290 are positions that do not affect the optical paths of the fluorescence γ and the plasmon scattered light δ, and at least one of the second metal film 231 and the third metal film 232. It arrange | positions so that a part may each be exposed. Although not particularly illustrated, the two terminals for voltage application can be connected to the second metal film 31 and the third metal film 232 through the second through hole 280 and the third through hole 290, respectively.

  In the present embodiment, the measurement of the impedance of the specimen is performed on the second metal film 231 and the third metal film 232 that are exposed in the flow path 241 (measurement target 242), and between them. In the existing state, the voltage is applied between the second metal film 231 and the third metal film 232.

  FIG. 6 is a plan view of a measurement chip 300 according to a modification of the second embodiment. 7A is a plan view of the measurement chip 300 installed in the chip holder 352, and FIG. 7B is a cross-sectional view taken along line AA in FIG. 7A.

  As shown in FIG. 6, the frame 335 may have a first notch 380 and a second notch 390 instead of the second through hole 280 and the third through hole 290. Good. Further, the chip holder 352 has two terminals 353 for applying voltage. The two terminals 353 for voltage application are connected to the second metal film 231 and the third metal film 232 through the first notch 380 and the second notch 390, respectively. The two terminals 353 are also used for positioning the measurement chip 300. As shown in FIGS. 7A and 7B, the measurement chip 300 is disposed at an appropriate position by inserting the two terminals 353 into the first notch 380 and the second notch 390.

  As described above, in the measurement chips 200 and 300 according to the present embodiment, the two terminals 353 for voltage application can be connected to the second metal film 231 and the third metal film 232 outside the channel 241. it can. Therefore, when the measurement chips 200 and 300 according to the present embodiment are used, the impedance of the specimen can be measured without wetting the two terminals 353 for voltage application with the specimen. Therefore, a plurality of measurements can be performed without replacing the voltage application terminal 353.

  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 the measurement method and the measurement apparatus using SPFS. For example, the measurement method and the measurement device according to the present invention may be a measurement method and a measurement device using the SPR method. In this case, the measuring apparatus 100 measures the substance to be measured by measuring the amount of reflected light β as the first signal value without measuring the amount of fluorescence γ. Therefore, measurement of the optical blank value (step S50) and secondary reaction (step S60) are unnecessary.

  In the above-described embodiment, from the viewpoint of avoiding unnecessary processing, the measurement method and the measurement apparatus that perform the specimen type determination using the measurement value of the impedance of the specimen have been described. Is not limited to a measurement method and a measurement apparatus including determination of the type of specimen. In this case, the processing unit obtains a hematocrit value of blood regardless of the type of the specimen, and converts the first signal value into the second signal value.

  In the above embodiment, the SPFS device using the measurement chip in which two metal films are arranged and the measurement chip in which three metal films are arranged has been described. However, if the number of metal films is two or more, There is no particular limitation. For example, the number of metal films may be four or more. In this case, by applying a voltage between the two metal films in a state where the specimen exists on and between the two metal films exposed in the accommodating portion among the plurality of metal films, Measure electrical characteristics.

  In the above embodiment, the impedance of the specimen is measured as the electrical characteristics of the specimen in order to obtain the hematocrit value. However, in the measurement method and measurement apparatus according to the present invention, the electrical characteristics of the specimen for obtaining the hematocrit value are not limited to the impedance of the specimen. For example, in the measuring method and measuring apparatus according to the present invention, the zeta potential of the specimen may be measured in order to obtain the hematocrit value. In this case as well, the hematocrit value can be obtained using a measuring chip that utilizes SPR.

In the above embodiment, the case where the hematocrit value is obtained as the specific value of the blood content has been described. However, in the measuring method and measuring apparatus according to the present invention, the specific value of the blood content is not limited to the hematocrit value. Other examples of specific values of blood contents include sodium (Na) concentration, potassium (K) concentration, chlor (Cl) concentration, ionized calcium (iCa) concentration, bicarbonate ion (HCO ₃ ⁻ ) concentration, blood Urea nitrogen concentration, glucose concentration, creatinine concentration, lactic acid concentration, pH, carbon dioxide partial pressure (pCO ₂ ), extracellular fluid base excess (BE), Anion Gap, total carbon dioxide (tCO ² ), oxygen partial pressure (pO) ₂ ) and oxygen saturation (sO ₂ ).

  This application claims priority based on Japanese Patent Application No. 2014-125219 filed on June 18, 2014. The contents described in the application specification and the drawings are all incorporated herein.

  The measurement method and measurement apparatus according to the present invention can measure a substance to be measured in blood with high reliability, and thus are useful for, for example, examination of diseases.

10, 200, 300 Measuring chip 20 Prism 21 Incident surface 22 Deposition surface 23 Emission surface 30, 230 First metal film 31, 231 Second metal film 35, 235, 335 Frame 36, 236 First through hole 40, 240 Channel lid 41, 241 Channel 42, 242 Measurement target 50 Capturing body 60 Injection port 70 Discharge port 100 SPFS device 105 Electrical characteristic measurement unit 110 Excitation light irradiation unit 111 Light source unit 112 Angle adjustment mechanism 113 Light source control Unit 120 reflected light detection unit 121 light receiving sensor 122 angle adjustment mechanism 123 sensor control unit 130 fluorescence detection unit 131 light receiving unit 132 position switching mechanism 133 sensor control unit 134 first lens 135 optical filter 136 second lens 137 light receiving sensor 140 liquid sending unit 1 DESCRIPTION OF SYMBOLS 1 Liquid chip 142 Syringe pump 143 Liquid feed pump drive mechanism 144 Syringe 145 Plunger 150 Transfer unit 151 Transfer stage 152,352 Chip holder 160 Control part 232 3rd metal film 280 2nd through-hole 290 3rd through-hole 353 Voltage application terminal 380 First notch 390 Second notch α excitation light β reflected light γ fluorescence δ plasmon scattered light

Claims (18)

A method for measuring the amount of a substance to be measured in a specimen including at least a part of blood using surface plasmon resonance,
A dielectric prism having an entrance surface, an exit surface, and a film formation surface; a storage unit disposed on the film formation surface; and a storage unit for storing the specimen; and spaced apart on the film formation surface; The sample is provided to a measurement chip having a capture body fixed to one and a plurality of metal films, at least a part of which is exposed in the housing portion, and the substance to be measured is applied to the capture body. Combining, and
The sample is applied by applying a voltage between the two metal films in a state where the sample exists on and between the two metal films exposed in the housing portion among the plurality of metal films. Measuring the electrical characteristics of, and using the obtained measurement value to determine a specific value of the blood content in the specimen;
The metal corresponding to a region where the capturing body is fixed via the incident surface in a state where the substance to be measured and the capturing body are combined and the specimen is not present in the housing portion. Irradiating the back surface of the film with excitation light, and measuring the amount of fluorescence emitted from the excited fluorescent substance or the amount of excitation light reflected from the film formation surface and emitted from the emission surface, Obtaining a first signal value indicative of the amount of the substance to be measured therein;
Converting the first signal value into a second signal value indicating the amount of the substance to be measured in the liquid component of the specimen, using the specific value of the blood content;
Including a measuring method.   The measuring method according to claim 1, wherein the step of obtaining the specific value of the blood content is performed in parallel with the step of binding the substance to be measured to the capturing body. The plurality of metal films include a first metal film to which the capturing body is fixed and a second metal film to which the capturing body is not fixed,
The electrical characteristics of the specimen are measured in the state where the specimen exists on the first metal film, the second metal film, and the space between the first metal film exposed in the container. By applying a voltage between the metal film and the second metal film,
The measuring method according to claim 1 or 2. The plurality of metal films include a first metal film to which the capturing body is fixed, a second metal film to which the capturing body is not fixed, and a third metal film to which the capturing body is not fixed. Including
The electrical characteristics of the specimen are measured in the state where the specimen exists on the second metal film, the third metal film, and the space between the second metal film exposed in the container. The measurement method according to claim 1, wherein the measurement is performed by applying a voltage between the metal film and the third metal film. In the step of obtaining a specific value of the content of the blood, the obtained measurement value is used to determine whether the sample is blood or blood dilution, or serum, plasma, or dilutions thereof. , When it is determined that the specimen is blood or a diluted blood solution, the specific value of the blood content is obtained using the obtained measurement value,
In the step of converting the first signal value into the second signal value, when it is determined that the sample is blood or a blood dilution, it is converted into a second signal value indicating the amount of the substance to be measured. To
The measuring method as described in any one of Claims 1-4.   The measurement method according to any one of claims 1 to 5, wherein the specific value of the blood content is a hematocrit value. In the step of converting the first signal value into the second signal value,
When the specimen is undiluted blood, the first signal value is multiplied by the conversion coefficient c ₁ represented by the following equation (1) to obtain the first signal value. To the signal value of
When the sample is a diluted blood solution, the first signal value is multiplied by a conversion coefficient c ₂ represented by the following equation (2) to obtain the first signal value as the second signal. Convert to value,
The measurement method according to claim 6.

[In the above formulas (1) and (2), Ht is the hematocrit value, and df is the dilution ratio of the diluent. ]   The measurement method according to claim 1, wherein the electrical characteristic of the specimen is an impedance of the specimen. A chip for measuring the amount of a substance to be measured in a specimen including at least a part of blood using surface plasmon resonance,
A dielectric prism having an entrance surface, an exit surface, and a deposition surface;
A storage unit disposed on the film formation surface for storing the specimen;
A plurality of metal films that are spaced apart from each other on the film-forming surface, the capturing body is fixed to at least one, and at least a part of the metal film is exposed in the housing portion;
Having a measuring chip.   The measurement chip according to claim 9, wherein the plurality of metal films include a first metal film to which the capture body is fixed and a second metal film to which the capture body is not fixed.   The plurality of metal films include a first metal film to which the capturing body is fixed, a second metal film to which the capturing body is not fixed, and a third metal film to which the capturing body is not fixed. The measurement chip according to claim 9, comprising: An apparatus for measuring the amount of a substance to be measured in a specimen including at least a part of blood using surface plasmon resonance,
A dielectric prism having an entrance surface, an exit surface, and a film formation surface; a storage unit disposed on the film formation surface; and a storage unit for storing the specimen; and spaced apart on the film formation surface; A holder for holding a measurement chip having a plurality of metal films each having a capturing body fixed to one and at least a part of which is exposed in the housing;
Electricity for measuring electrical characteristics of the specimen by applying a voltage between the two metal films on the two metal films exposed in the accommodating portion and between the two metallic films. Characteristic measurement unit,
A light irradiator for irradiating light toward the incident surface;
The metal corresponding to a region where the capturing body is fixed via the incident surface in a state where the substance to be measured and the capturing body are combined and the specimen is not present in the housing portion. Light that irradiates the back surface of the film with excitation light and detects the amount of fluorescence emitted from the excited fluorescent material, or the amount of excitation light reflected from the film formation surface of the prism and emitted from the emission surface A detection unit;
A first signal value indicating the amount of the substance to be measured in the specimen is calculated based on the detection result of the light detection unit, and the blood content of the blood is calculated based on the measurement result of the electrical property measurement unit A processing unit that calculates a specific value and converts the first signal value into a second signal value indicating the amount of the substance to be measured in the liquid component of the specimen by using the specific value of the contents of the blood When,
A measuring device. The plurality of metal films include a first metal film to which the capturing body is fixed and a second metal film to which the capturing body is not fixed,
The electrical property measurement unit is configured to perform the first metal film on the first metal film exposed in the housing unit, on the second metal film, and in a state where the specimen exists between the first metal film and the second metal film. The measurement apparatus according to claim 12, wherein the electrical characteristics of the specimen are measured by applying a voltage between the second metal film and the second metal film. The plurality of metal films include a first metal film to which the capturing body is fixed, a second metal film to which the capturing body is not fixed, and a third metal film to which the capturing body is not fixed. Including
The electrical property measurement unit is configured to provide the second metal film on the second metal film exposed in the storage unit, the third metal film, and the specimen in a state where the sample exists between the second metal film and the third metal film. The measurement apparatus according to claim 12, wherein the electrical characteristics of the specimen are measured by applying a voltage between the third metal film and the third metal film.   The processing unit determines whether the sample is blood or a blood diluted solution, or serum, plasma, or a diluted solution thereof based on the measurement result of the electrical characteristic measuring unit, and the sample is When it is determined that the blood is a blood or a diluted blood solution, the specific value of the blood content is calculated, and the first signal value is converted into the second signal value. The measuring apparatus as described in any one.   The measurement device according to any one of claims 12 to 15, wherein the specific value of the blood content is a hematocrit value. The processor is
When the specimen is undiluted blood, the first signal value is multiplied by the conversion coefficient c ₁ represented by the following equation (1) to obtain the first signal value. To the signal value of
When the sample is a diluted blood solution, the first signal value is multiplied by a conversion coefficient c ₂ represented by the following equation (2) to obtain the first signal value as the second signal. Convert to value,
The measuring device according to claim 16.

[In the above formulas (1) and (2), Ht is the hematocrit value, and df is the dilution ratio of the diluent. ]   The measurement apparatus according to claim 12, wherein the electrical characteristic of the specimen is an impedance of the specimen.

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