JPWO2012108323A1 - Surface plasmon excitation fluorescence measurement apparatus and surface plasmon excitation fluorescence measurement method - Google Patents

Abstract

Provided are a surface plasmon excitation fluorescence measurement device and a surface plasmon excitation fluorescence measurement method that improve the sensitivity and accuracy of antigen detection. The antigen is captured by the antigen capturing body of the analysis chip, and the fluorescently labeled antibody is bound to the antigen captured by the antigen capturing body. The amount of surface plasmon excitation fluorescence emitted from the fluorescent label when the excitation light enters the analysis chip is measured. The resonance angle θr is measured after the antigen is captured by the antigen capturing body and before the fluorescently labeled antibody is bound to the antigen. The measurement angle θm is determined from the resonance angle θr. The incident angle θ of the excitation light to the reflection surface is set to the resonance angle θr, and the amount of surface plasmon excitation fluorescence is measured.

Description

  The present invention relates to a surface plasmon excitation fluorescence measurement device and a surface plasmon excitation fluorescence measurement method.

  When the excitation light traveling in the dielectric medium is totally reflected at the interface between the metal film and the dielectric medium, the evanescent wave leaks from the interface, and the plasmon and the evanescent wave in the metal film interfere with each other. When the incident angle of the excitation light to the interface is set to the resonance angle and the plasmon and the evanescent wave resonate, the electric field of the evanescent wave is remarkably enhanced. In surface plasmon excited fluorescence spectroscopy (SPFS), this enhanced electric field is used. That is, the antigen is captured on the surface of the metal film, the captured antigen is fluorescently labeled, and an enhanced electric field is allowed to act on the fluorescent label. When the enhanced electric field acts on the fluorescent label, fluorescence is emitted from the fluorescent label. In surface plasmon excitation fluorescence spectroscopy, the amount of fluorescence emitted from a fluorescent label is measured.

  Patent Document 1 relates to detection of an antigen by surface plasmon excitation fluorescence spectroscopy. In Patent Document 1, the resonance angle is measured after the antigen is fluorescently labeled.

Japanese Patent No. 4370383

  Since the intensity of the electric field is significantly increased when the incident angle of the excitation light is set to the resonance angle, it is important to accurately identify the resonance angle when detecting antigen by surface plasmon excitation fluorescence spectroscopy. .

  However, in the prior art, the resonance angle is measured before the antigen is captured, the change in the resonance angle due to the antigen concentration or the like and the variation in the amount of change are ignored, and the resonance angle is not accurately specified. Or, as in Patent Document 1, the resonance angle was measured after the antigen was fluorescently labeled, causing the fading of the fluorescent dye. Both of these cause a decrease in the amount of surface plasmon excitation fluorescence, and cause a large variation in the amount of surface plasmon excitation fluorescence. Therefore, in the prior art, the sensitivity and accuracy of antigen detection are not sufficient.

  The present invention is made to solve this problem. An object of the present invention is to provide a surface plasmon excitation fluorescence measurement device and a surface plasmon excitation fluorescence measurement method that improve the sensitivity and accuracy of antigen detection.

  The first to seventh aspects of the present invention relate to a surface plasmon excitation fluorescence measuring apparatus.

  (1) In the first aspect, a dielectric medium, a conductor film, and an antigen capturing body are provided. The dielectric medium is transparent to the excitation light. The first main surface of the conductor film is in close contact with the reflective surface of the dielectric medium. The second main surface of the conductor film does not adhere to the reflective surface of the dielectric medium. The antigen capturing body is fixed on the second main surface. The antigen capturer includes an antibody that binds to the antigen.

  Excitation light emitted from the light source is incident on the incident surface of the dielectric medium. The excitation light incident on the incident surface is totally reflected on the reflecting surface. The excitation light totally reflected by the reflecting surface is emitted from the emitting surface.

  While the incident angle of the excitation light to the reflection surface is scanned, the amount of light emitted from the conductor film and the antigen capturing body in the direction toward the second main surface is measured, and the relationship between the incident angle and the amount of light is obtained. From the relationship between the incident angle and the light amount, the resonance angle at which the light amount is maximized is specified, and the measurement angle is determined from the resonance angle. The incident angle is set to the measurement angle, the amount of light emitted from the conductor film and the antigen capturing body in the direction in which the second main surface faces is measured, and the first measurement result is obtained.

  The relationship between the incident angle and the amount of light is obtained after the sample has contacted the antigen capturing body and before the fluorescently labeled antibody-containing material has contacted the antigen capturing body. The fluorescently labeled antibody-containing material includes a fluorescently labeled antibody that is fluorescently labeled and binds to an antigen.

  (2) The difference between the first aspect and the second aspect is in specifying the resonance angle. In the second aspect, the amount of light emitted from the reflecting surface is measured while scanning the incident angle of the excitation light on the reflecting surface, and the resonance angle at which the amount of light is minimized is specified from the relationship between the incident angle and the amount of light. The

  (3) The third aspect adds further matters to the first or second aspect. In the third aspect, the direction in which the second main surface faces from the conductor film and the antigen capturing body before the fluorescently labeled antibody-containing material is brought into contact with the antigen capturing body and after the incident angle is set to the measurement angle The amount of light emitted to is measured and a second measurement result is obtained. Further, the second measurement result is subtracted from the first measurement result.

  (4) The fourth aspect adds further matters to the third aspect. In the fourth aspect, the amount of light emitted from the conductor film and the antigen capturing body toward the second main surface is measured while the irradiation position of the excitation light is scanned, and the relationship between the irradiation position and the amount of light is can get. From the relationship between the irradiation position and the light amount, the first irradiation position and the second irradiation position at which the light amount decreases from the maximum value by the same value are specified, and the middle of the first irradiation position and the second irradiation position is determined as the measurement position. Is done. The irradiation position is set to the measurement position.

  (5) The fifth aspect adds further matters to any of the first to fourth aspects. In the fifth aspect, the amount of light emitted from the conductor film and the antigen capturing body in the direction in which the second main surface faces is measured in a blocking state where excitation light is not guided to the incident surface, and the third measurement result Is acquired. Further, the third measurement result is subtracted from the first measurement result.

  (6) The sixth aspect adds further matters to any of the first to fifth aspects. In the sixth aspect, the amount of light emitted from the conductor film and the antigen capturing body toward the second main surface is measured while the polarization direction of the excitation light is scanned, and the relationship between the polarization direction and the amount of light is determined. can get. From the relationship between the polarization direction and the light amount, the polarization direction in which the light amount is maximized is specified, and the measurement polarization direction in which the measurement is performed is determined. The polarization direction is set in the measurement method.

  (7) The seventh aspect adds further matters to any one of the first to sixth aspects. In the seventh aspect, a band-pass filter that selectively attenuates light having the same wavelength as the excitation light while the light amount is measured to obtain the first measurement result is provided from the conductor film and the antigen capturing body. It is inserted into the optical path of the light emitted in the direction in which the main surface of 2 faces.

  The eighth and ninth aspects of the present invention relate to a surface plasmon excitation fluorescence measurement method.

  (8) In the eighth aspect, a structure including a dielectric medium, a conductor film, and an antigen capturing body is prepared. The dielectric medium is transparent to the excitation light. The first main surface of the conductor film is in close contact with the reflective surface of the dielectric medium. The second main surface of the conductor film does not adhere to the reflective surface of the dielectric medium. The antigen capturer includes an antibody that binds to the antigen. The antigen capturing body adheres to and is fixed on the second main surface.

  The excitation light emitted from the light source enters the incident surface of the dielectric medium, is totally reflected by the reflecting surface, and exits from the emitting surface.

  The amount of scattered light is measured while the incident angle of the excitation light on the reflecting surface is scanned, and the relationship between the incident angle and the amount of light is obtained. From the relationship between the incident angle and the light amount, the resonance angle at which the light amount is maximized is specified, and the measurement angle is determined from the resonance angle. The incident angle is set as the measurement angle, and the amount of surface plasmon excitation fluorescence is measured.

  The relationship between the incident angle and the amount of light is obtained after the sample has contacted the antigen capturing body and before the fluorescently labeled antibody-containing material has contacted the antigen capturing body. The fluorescently labeled antibody-containing material includes a fluorescently labeled antibody that is fluorescently labeled and binds to an antigen.

  (9) The difference between the eighth aspect and the ninth aspect is in specifying the resonance angle. In the ninth aspect, the light amount of the reflected light is measured while scanning the incident angle of the excitation light on the reflecting surface, and the resonance angle at which the light amount is minimized is specified from the relationship between the incident angle and the light amount.

  According to the first, second, eighth, and ninth aspects, the relationship between the incident angle and the amount of light is measured after the sample contacts the antigen capturing body, and the resonance angle is accurate regardless of the antigen concentration in the sample. Specified. In addition, the relationship between the incident angle and the amount of light is measured before the fluorescently labeled antibody-containing material contacts the antigen-capturing body, and the fluorescent dye is less likely to fade due to excitation light. These improve the sensitivity and accuracy of antigen detection.

  According to the first and eighth aspects, the deviation between the resonance angle and the incident angle at which the enhancement of the electric field is maximized is reduced, and the sensitivity and accuracy of antigen detection are improved.

  According to the third aspect of the present invention, the measurement result of the amount of autofluorescence is reduced, the influence of autofluorescence is suppressed, and the sensitivity and accuracy of antigen detection are improved.

  According to the fourth aspect of the present invention, the optical path length of the excitation light in the measurement region is constant, the influence of the amount of autofluorescence is constant, and the sensitivity and accuracy of antigen detection are improved.

  According to the fifth aspect of the present invention, the measurement result of dark noise is reduced, the influence of dark noise is suppressed, and the sensitivity and accuracy of antigen detection are improved.

  According to the sixth aspect of the present invention, the decrease of the P wave component due to the birefringence of the dielectric medium is eliminated, and the sensitivity and accuracy of antigen detection are improved.

  According to the seventh aspect of the present invention, surface plasmon excitation fluorescence is mainly guided to the light quantity sensor, and the sensitivity and accuracy of antigen detection are improved.

  These and other objects, features, aspects and advantages of the invention will become more apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.

It is a schematic diagram which shows embodiment of a surface plasmon excitation fluorescence measuring device. It is a block diagram of a control calculating part. It is sectional drawing of an analysis chip. It is a schematic diagram of a laser diode unit. It is a schematic diagram of a 1st wave shaper. It is a schematic diagram of a polarization direction adjustment mechanism. It is a schematic diagram of an incident angle adjustment mechanism. It is a schematic diagram of a measurement light optical system. It is a top view which shows the relationship between a reaction area | region, a measurement area | region, and an irradiation area | region. It is a flowchart with the outline of the procedure of the detection of an antigen. It is a flowchart of the procedure of a measurement preparation. It is a flowchart of the procedure of the setting to the measurement angle of an incident angle. It is a flowchart of the procedure of optimization of an irradiation position. It is a graph which shows the relationship between an irradiation position and the light quantity of scattered light. It is a flowchart of the procedure of optimization of the polarization direction of excitation light. It is a flowchart which shows the procedure of the measurement of the light quantity of surface plasmon excitation fluorescence. It is a schematic diagram which shows the state to which the reflected light amount sensor was added.

(Outline of measurement)
This desirable embodiment relates to a surface plasmon excitation fluorescence measuring apparatus and a surface plasmon excitation fluorescence measurement method for detecting an antigen by surface plasmon excitation fluorescence spectroscopy.

  In this desirable embodiment, the antigen is captured by the antigen capturing body of the analysis chip, and the fluorescently labeled antibody is bound to the antigen captured by the antigen capturing body. The amount of surface plasmon excitation fluorescence emitted from the fluorescent label when the excitation light enters the analysis chip is measured. The resonance angle θr is measured after the antigen is captured by the antigen capturing body and before the fluorescently labeled antibody is bound to the antigen. Since the resonance angle θr is measured after the antigen is captured by the antigen capturing body, the resonance angle θr is accurately specified regardless of the disturbance factor of the resonance angle θr such as the antigen concentration. Since the resonance angle θr is measured before the fluorescently labeled antibody is bound to the antigen, the fluorescent dye is not easily faded by excitation light. These improve the accuracy and sensitivity of antigen detection.

(Outline of surface plasmon excitation fluorescence measuring device)
The schematic diagram of FIG. 1 shows a preferred embodiment of a surface plasmon excitation fluorescence measurement apparatus.

  In the surface plasmon excitation fluorescence measuring apparatus 1000, the excitation light 9000 emitted from the laser diode unit 1002 is guided to the analysis chip 1006 by the excitation light optical system 1004. While the excitation light 9000 is incident on the analysis chip 1006, the measurement light 9002 and the reflected light 9004 are emitted from the analysis chip 1006. The measurement light 9002 is guided to the photomultiplier tube 1010 by the measurement light optical system 1008. The reflected light 9004 is absorbed by the light absorber 1012.

  A sample liquid 9006 and a fluorescent labeling liquid 9008 are sequentially injected into the analysis chip 1006 by a liquid feeding mechanism 1014. The analysis chip 1006 is arranged in the measurement chamber 1016 and is held in the analysis chip holding mechanism 1018 when measurement is performed. The analysis chip 1006 is placed in the pretreatment chamber 1020 when the sample solution 9006 or the fluorescent labeling solution 9008 is injected. The analysis chip 1006 is loaded from the pretreatment chamber 1020 to the measurement chamber 1016 and unloaded from the measurement chamber 1016 to the pretreatment chamber 1020 by the analysis chip transport mechanism 1022. The analysis chip 1006 is desirably attached to and detached from the surface plasmon excitation fluorescence measurement apparatus 1000 and is replaced for each specimen 9020. However, the analysis chip 1006 may be reused.

  After the sample liquid 9006 is injected into the analysis chip 1006 and before the fluorescent labeling liquid 9008 is injected into the analysis chip 1006, the incident angle adjustment mechanism 1034 scans the incident angle θ of the excitation light 9000, and the measurement light 9002 is scanned. Is measured by the photomultiplier tube 1010, and the resonance angle θr is specified. After the fluorescent labeling liquid 9008 is injected into the analysis chip 1006 and the incident angle θ is set to the measurement angle θm, the light quantity of the measurement light 9002 is measured by the photomultiplier tube 1010. The measurement result is displayed on the display 1024.

  The components of the surface plasmon excitation fluorescence measuring apparatus 1000 are controlled by the control calculation unit 1026, and the control calculation unit 1026 performs calculation.

(Control calculation part)
The block diagram of FIG. 2 shows the control calculation unit 1026.

  The contents of the control and calculation by the control calculation block group such as the preprocessing control unit 1104 shown in FIG. 2 will be mentioned in the description of the antigen detection procedure. These control operation block groups are embedded computers that execute software. One embedded computer may be responsible for the functions of these control arithmetic block groups, or two or more embedded computers may be responsible for the functions of these control arithmetic block groups. Hardware without software may be responsible for all or part of the functions of these control operation block groups. The hardware may be an electronic circuit or a mechanical mechanism such as a link mechanism.

(Analysis chip)
The schematic diagram of FIG. 3 shows a cross section of the analysis chip.

  As shown in FIG. 3, in the analysis chip 1006, the first main surface 1202 of the conductor film 1200 is in close contact with the reflecting surface 1206 of the prism 1204, and the second main surface 1210 of the conductor film 1200 is the reflecting surface 1206. It does not adhere to. The antigen-capturing body 1208 is fixed to the second main surface 1210, and the joint surface 1214 of the flow path forming body 1212 is joined. The excitation light 9000 guided to the prism 1204 is incident on the incident surface 1216, and the excitation light 9000 incident on the incident surface 1216 is totally reflected by the reflection surface 1206 in the irradiation region 1800, and the reflected light 9004 is emitted from the emission surface 1218. Scattered light and fluorescence are emitted from the conductor film 1200 and the antigen capturing body 1208 to the side where the second main surface 1210 faces. When the excitation light 9000 is incident on the reflecting surface 1206 while satisfying the total reflection condition, and the evanescent wave that oozes from the reflecting surface 1206 resonates with the plasmon in the conductor film 1200, the conductor film 1200 causes the evanescent wave to resonate. The electric field is increased, and the amount of scattered light and fluorescence increases. The analysis chip 1006 is preferably a structure in which each piece has a length of several millimeters to several centimeters, but is replaced with a smaller structure or a larger structure not included in the category of “chip”. May be.

(prism)
The prism 1204 is a trapezoidal column. One inclined side surface of the trapezoidal column is an incident surface 1216, the wide parallel side surface of the trapezoidal column is a reflecting surface 1206, and the other inclined side surface of the trapezoidal column is an output surface 1218. The prism 1204 is preferably an isosceles trapezoidal column. Thereby, the incident surface 1216 and the exit surface 1218 are symmetric, and the S wave component of the reflected light 9004 is less likely to stay inside the prism 1204. As long as the entrance surface 1216, the reflection surface 1206, and the exit surface 1218 are provided, the prism 1204 may be other than the trapezoidal column, and the prism 1204 may be replaced with a shape not included in the category of “prism”.

  The prism 1204 is a dielectric medium made of a material that is transparent to the excitation light 9000. The prism 1204 is made of glass, resin, or the like. The prism 1204 is preferably made of a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

(Conductor film)
The conductor film 1200 is made of a conductor that generates surface plasmon resonance. The conductor film 1200 is preferably made of gold. However, the conductor film 1200 may be made of a metal such as silver, copper, or aluminum, or an alloy containing these metals.

  The conductor film 1200 is a thin film. The film thickness of the conductor film 1200 is desirably 30 to 70 nm. However, the film thickness of the conductor film 1200 may be outside this range.

  The conductor film 1200 is formed by sputtering, vapor deposition, plating, or the like. However, the conductor film 1200 may be formed by other methods.

(Antigen trap)
The antigen capturing body 1208 is made of a non-fluid body. Therefore, even when the sample liquid 9006 or the fluorescent labeling liquid 9008 comes into contact with the antigen capturing body 1208, the antigen capturing body 1208 does not move.

  Antigen capturing body 1208 includes an antibody that binds to the antigen to be detected. The antibody is evenly distributed. When the sample liquid 9006 containing the antigen to be detected comes into contact with the antigen capturing body 1208, the antigen to be detected contained in the sample liquid 9006 binds to the antibody contained in the antigen capturing body 1208 and is detected in the sample liquid 9006. The antigen of interest is captured by the antigen capturing body 1208.

(Channel formation body)
A flow path 1220 is formed in the flow path forming body 1212. A reaction chamber 1222 is in the middle of the flow path 1220. The reaction chamber 1222 is exposed at the joint surface 1214. The planar position of the reaction chamber 1222 and the planar position of the antigen capturing body 1208 are matched, and the antigen capturing body 1208 is exposed to the reaction chamber 1222. When the sample liquid 9006 or the fluorescent labeling liquid 9008 is injected into the channel 1220, the reaction chamber 1222 is filled with the sample liquid 9006 or the fluorescent labeling liquid 9008, respectively, and the sample liquid 9006 or the fluorescent labeling liquid 9008 is the antigen capturing body 1208. To touch. The flow path 1220 is exposed to the non-bonding surface 1224 of the flow path forming body 1212, and the inlet 1226 for the sample liquid 9006 and the fluorescent labeling liquid 9008 is formed on the non-bonding surface 1224.

  The joint surface 1214 and the second main surface 1210 are joined by adhesion, laser welding, ultrasonic welding, clamp pressure bonding, or the like. However, the bonding surface 1214 and the second main surface 1210 may be bonded by other methods.

  The flow path forming body 1212 is made of a material transparent to scattered light and surface plasmon excitation fluorescence. The flow path forming body 1212 is made of, for example, resin.

  The flow path forming body 1212 prevents the sample liquid 9006 or the fluorescent labeling liquid 9008 from scattering and contributes to making the amount of the sample liquid 9006 or the fluorescent labeling liquid 9008 in contact with the antigen capturing body 1208 constant. However, the flow path forming body 1212 may be omitted, and the sample liquid 9006 or the fluorescent labeling liquid 9008 may be dropped directly on the antigen capturing body 1208.

(Laser diode unit)
The schematic diagram of FIG. 4 shows a laser diode unit.

  As shown in FIG. 4, in the laser diode unit 1002, the beam 9010 emitted from the laser diode 1300 is split into a main beam 9012 and a sub beam 9014 by a beam splitter 1302. The main beam 9012 is collimated by a collimator 1304 and emitted as excitation light 9000 from the laser diode unit 1002.

  Even if the excitation light 9000 is collimated, the cross-sectional shape of the beam of the excitation light 9000 is flat. In order to make the planar shape of the irradiation region 1800 close to a circle, the laser diode 1300 is moved by the laser diode holding mechanism 1306 in such a posture that an elliptical short axis is included in a plane perpendicular to the reflection surface 1206 including the optical path of the excitation light 9000. Retained.

  When the wavelength and light amount of the laser light emitted from the laser diode 1300 depend on the temperature, the laser diode 1300 is desirably controlled by the temperature adjustment circuit 1308. In the temperature adjustment circuit 1308, the light amount of the sub beam 9014 is measured by the photodiode 1310, and the power supplied to the laser diode 1300 is maintained so that the measurement result of the light amount of the sub beam 9014 is kept constant. Is controlled by regression. Thereby, the temperature of the laser diode 1300 is kept constant, and the wavelength and light amount of the excitation light 9000 are kept constant. The photodiode 1310 may be replaced with another light quantity sensor such as a phototransistor or a photoresistor. Since the wavelength of the excitation light 9000 affects the resonance angle θr and the amount of evanescent wave oozing, the temperature adjustment circuit 1308 contributes to the improvement in the accuracy and sensitivity of antigen detection.

  When it takes a long time for the temperature of the laser diode 1300 to reach a steady temperature, power is constantly supplied to the laser diode 1300 from the start of the operation of the surface plasmon excitation fluorescence measurement apparatus 1000.

  The polarization direction of the excitation light 9000 emitted from the laser diode 1300 is generally unevenly distributed in a specific direction.

  The laser diode 1300 may be replaced with another light source such as a light emitting diode, a mercury lamp, or a laser other than the laser diode. When the light emitted from the light source is not a beam, the light 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 is converted into monochromatic light by a diffraction grating or the like. When the light emitted from the light source is not linearly polarized light, the light is converted into linearly polarized light by a polarizer or the like.

(Excitation light optical system)
As shown in FIG. 1, while the excitation light 9000 is guided from the laser diode unit 1002 to the analysis chip 1006 by the excitation light optical system 1004, the first wave rectifier 1028, the polarization direction adjustment mechanism 1030, and the shaping optical system 1032. And the incident angle adjusting mechanism 1034 sequentially. When the excitation light 9000 passes through the first wave shaper 1028, the excitation light 9000 is tuned. The polarization direction of the excitation light 9000 is adjusted so that the P wave component is mainly incident on the reflection surface 1206 when the excitation light 9000 passes through the polarization direction adjusting mechanism 1030. When the excitation light 9000 passes through the shaping optical system 1032, the cross-sectional shape of the beam of the excitation light 9000 is shaped by a slit, a zoom optical system, or the like. When the excitation light 9000 passes through the incident angle adjustment mechanism 1034, the incident angle θ of the excitation light 9000 on the reflection surface 1206 is adjusted.

(First wave rectifier)
The schematic diagram of FIG. 5 shows a first wave rectifier.

  As shown in FIG. 5, the excitation light 9000 sequentially passes through the first bandpass filter 1400, the linear polarization filter 1402, and the neutral density filter 1404 when passing through the first wave shaper 1028. The first wave adjuster 1028 may include other optical filters. The first wave adjuster 1028 may include an optical element other than the optical filter.

  When the excitation light 9000 passes through the first bandpass filter 1400, the wavelength distribution of the excitation light 9000 is narrowed. Thereby, even if the wavelength distribution of the excitation light 9000 emitted from the laser diode unit 1002 is wide, the excitation light 9000 having a narrow wavelength distribution can be obtained. When the wavelength distribution of the excitation light 9000 emitted from the laser diode unit 1002 is sufficiently narrow, the first bandpass filter 1400 may be omitted.

  When the excitation light 9000 passes through the linear polarization filter 1402, the distribution of the polarization direction of the excitation light 9000 is narrowed. Thereby, even if the distribution of the polarization direction of the excitation light 9000 emitted from the laser diode unit 1002 is wide, the excitation light 9000 having a narrow distribution of the polarization direction can be obtained. When the distribution of the polarization direction of the excitation light 9000 emitted from the laser diode unit 1002 is sufficiently narrow, the linear polarization filter 1402 may be omitted.

  When the excitation light 9000 passes through the neutral density filter 1404, the light amount of the excitation light 9000 is decreased. If the amount of excitation light 9000 emitted from the laser diode unit 1002 is appropriate, the neutral density filter 1404 may be omitted.

(Polarization direction adjustment mechanism)
The schematic diagram of FIG. 6 shows a polarization direction adjusting mechanism.

  As shown in FIG. 6, in the polarization direction adjusting mechanism 1030, the half-wave plate 1500 is rotated by the half-wave plate rotation mechanism 1502 around a rotation axis perpendicular to the half-wave plate 1500. Half-wave plate rotating mechanism 1502 rotates half-wave plate 1500 by a rotary stepping motor or the like. The polarization direction of the excitation light 9000 that passes through the half-wave plate 1500 is adjusted by the rotation angle of the half-wave plate 1500. The polarization direction of the excitation light 9000 can be adjusted between the polarization direction in which the oozing of the evanescent wave from the reflecting surface 1206 is maximized and the polarization direction in which the oozing of the evanescent wave from the reflecting surface 1206 is eliminated. In the initial state, the rotation angle of the half-wave plate 1500 is set to the rotation angle at which the P-wave component is expected to be incident on the reflecting surface 1206.

  The polarization direction adjusting mechanism 1030 may be replaced with a mechanism that rotates the laser diode unit 1002 or the laser diode 1300 around the optical axis. When optimization of the polarization direction described later is omitted, the half-wave plate rotation mechanism 1502 may be omitted and the half-wave plate 1500 may be fixed. When the optimization of the polarization direction described later is omitted and the laser diode unit 1002 or the laser diode 1300 is held in a posture where the P-wave component is mainly incident on the reflection surface 1206, the half-wave plate 1500 and the half-wave plate rotation The mechanism 1502 may be omitted.

(Incident angle adjustment mechanism)
The schematic diagram of FIG. 7 shows an incident angle adjustment mechanism.

  As shown in FIG. 7, the excitation light 9000 is reflected by the reflecting mirror 1600 when passing through the incident angle adjustment mechanism 1034, and the traveling direction of the excitation light 9000 is the second direction from the first direction 9016 toward the analysis chip 1006. Bent in direction 9018.

  When the incident angle θ is adjusted by the incident angle adjusting mechanism 1034, the position of the reflecting mirror is set so as to cancel the movement of the irradiation position X due to the change of the incident angle θ while the reflecting mirror angle is adjusted by the reflecting mirror angle adjusting mechanism 1602. The reflecting mirror position is adjusted by the adjusting mechanism 1604. Thereby, only incident angle (theta) is adjusted and the irradiation position X of the excitation light 9000 in the reflective surface 1206 is maintained.

  When the irradiation position X is adjusted by the incident angle adjusting mechanism 1034, the reflecting mirror angle is fixed, and the reflecting mirror position adjusting mechanism 1604 adjusts the reflecting mirror position. Thereby, only the irradiation position X is adjusted and the incident angle θ is maintained.

  When the incident angle adjustment mechanism 1034 is in a blocking state where the excitation light 9000 is not guided to the incident surface 1216, the reflection mirror angle is adjusted by the reflection mirror angle adjustment mechanism 1602, and the excitation light 9000 is applied to the light absorption surface of the reflection mirror 1600. Is applied. When the incident angle adjustment mechanism 1034 is in a non-blocking state where the excitation light 9000 is guided to the incident surface 1216, the reflection mirror angle is adjusted by the reflection mirror angle adjustment mechanism 1602, and the excitation light 9000 strikes the mirror surface of the reflection mirror 1600. It is done. The blocking state and the non-blocking state may be switched by a mechanism other than the reflector angle adjustment mechanism 1602. For example, a light absorber may be inserted into and removed from the optical path of the excitation light 9000.

  The reflecting mirror angle adjustment mechanism 1602 adjusts the reflecting mirror angle by rotating the reflecting mirror 1600 around a rotation axis parallel to the mirror surface by a rotary stepping motor or the like.

  The reflecting mirror position adjusting mechanism 1604 moves the reflecting mirror 1600 together with the reflecting mirror angle adjusting mechanism 1602 along the first direction 9016 by a linear stepping motor or the like on the linear stage or the like to adjust the reflecting mirror position.

  The incident angle adjusting mechanism 1034 has an advantage that the incident angle θ and the irradiation position X can be adjusted by a simple mechanism. However, the incident angle θ and the irradiation position X may be adjusted by a mechanism that adjusts the position and posture of the laser diode unit 1002. Alternatively, the incident angle θ and the irradiation position X may be adjusted by a mechanism that adjusts the position and posture of the analysis chip 1006.

(Measurement optical system)
The schematic diagram of FIG. 8 shows a measurement light optical system.

  As shown in FIG. 8, the measurement light 9002 emitted to the side on which the second major surface 1210 faces is guided to the photomultiplier tube 1010 by the measurement light optical system 1008. In the measurement light optical system 1008, the measurement light 9002 is collected by the condenser lens 1700, converted into parallel light, passes through the second wave multiplier 1702, and is connected to the photomultiplier tube 1010 by the imaging lens 1704. Imaged.

  The condensing lens 1700 and the imaging lens 1704 constitute a conjugate optical system. Thereby, the influence of stray light is suppressed.

  In the second wave rectifier 1702, the second band pass filter 1706 is inserted into and removed from the optical path of the measurement light 9002 by the insertion / extraction mechanism 1708.

  When the amount of surface plasmon excitation fluorescence is measured, a second bandpass filter 1706 is inserted into the optical path of the measurement light 9002, and light having the same wavelength as the excitation light 9000 is selectively transmitted by the second bandpass filter 1706. Attenuated. Thereby, the scattered light is attenuated, and mainly the surface plasmon excitation fluorescence is guided to the photomultiplier tube 1010, and the sensitivity and accuracy of antigen detection are improved. When the amount of scattered light is measured, the second band pass filter 1706 is removed from the optical path of the measurement light 9002.

  A neutral density filter may be inserted into and removed from the optical path of the measurement light 9002 by an insertion / extraction mechanism. When the amount of surface plasmon excitation fluorescence is measured, the neutral density filter is removed from the optical path of the measurement light 9002. When the amount of scattered light is measured, a neutral density filter is inserted into the optical path of the measurement light 9002. Thereby, the difference between the light amounts of the surface plasmon excitation fluorescence guided to the photomultiplier tube 1010 and the scattered light is reduced, and the light amount can be easily measured. The amount of surface plasmon excitation fluorescence may be measured by a relatively high sensitivity photomultiplier tube 1010, and the amount of scattered light may be measured by a relatively low sensitivity photodiode, phototransistor, photoresistor, or the like.

(Photomultiplier tube)
The amount of measurement light 9002 is measured by a photomultiplier tube 1010. The photomultiplier tube 1010 has the advantage of good sensitivity and good signal to noise ratio. However, the photomultiplier tube 1010 may be replaced with another light quantity sensor such as a cooled charge coupled device (CCD) camera.

(Liquid feeding mechanism)
As shown in FIG. 1, when the sample liquid 9006 is injected into the flow path 1220 by the liquid feeding mechanism 1014, the analysis chip 1006 and the reagent chip 1036 are arranged in the pretreatment chamber 1020. In addition, the sample liquid 9006 is sucked from the dilution container 1042 of the reagent chip 1036 by the liquid feed pump 1040, the liquid feed pump 1040 is transported by the liquid feed pump transport mechanism 1044, and the sample liquid 9006 is transferred to the inlet 1226 by the liquid feed pump 1040. Discharged.

  The analysis chip 1006 and the reagent chip 1036 are also arranged in the pretreatment chamber 1020 when the fluorescent labeling liquid 9008 is injected into the flow path 1220 by the liquid feeding mechanism 1014. Further, the fluorescent labeling liquid 9008 is sucked from the fluorescent labeling liquid container 1046 of the reagent chip 1036 by the liquid feeding pump 1040, the liquid feeding pump 1040 is conveyed by the liquid feeding pump conveying mechanism 1044, and the fluorescence is supplied to the inlet 1226 by the liquid feeding pump 1040. The labeling liquid 9008 is discharged.

  In order to inject the sample solution 9006 or the fluorescent labeling solution 9008 into the analysis chip 1006 after the analysis chip 1006 is retracted to the pretreatment chamber 1020, components that obstruct optical measurement are excluded from the measurement chamber 1016. Contributes to controlling pollution. However, the retreat to the pretreatment chamber 1020 may be omitted.

  When the sample 9020 is diluted, the reagent chip 1036 is placed in the pretreatment chamber 1020. By the liquid feeding mechanism 1014, the specimen 9020 is fed from the specimen container 1048 to the dilution container 1042, and the dilution liquid 9022 is sent from the dilution liquid container 1050 to the dilution container 1042, whereby the sample liquid 9006 is prepared.

  The liquid feed pump 1040 is cleaned with the cleaning liquid 9024 accommodated in the cleaning liquid container 1052 as necessary.

  In some cases, the sample 9020 is not diluted and the sample 9020 becomes the sample liquid 9006 as it is. In addition to or in place of dilution, other pretreatments such as blood cell separation and reagent mixing may be performed.

  The specimen 9020 is typically a collected material from a human such as blood, but may be a collected material from a non-human organism or a non-living material.

  The fluorescent labeling solution 9008 contains a fluorescently labeled antibody that is fluorescently labeled by binding to the antigen to be detected.

  The injection injected into the flow path 1220 is typically a liquid, but may be a gas or a fluid solid.

  Instead of the liquid feeding mechanism 1014 that transports the liquid feeding pump 1040 from the liquid feeding source to the liquid feeding destination, a mechanism for feeding the sample liquid 9006 and the fluorescent labeling liquid 9008 by a tube may be provided.

(Relationship between reaction area, measurement area and irradiation area)
The schematic diagram of FIG. 9 is a plan view showing a desirable relationship among the irradiation area, the measurement area, and the reaction area on the reflecting surface. The irradiation region 1800 is a region where the excitation light 9000 is irradiated. The measurement region 1802 is a region where the light amount of the measurement light 9002 is measured by the photomultiplier tube 1010. The reaction region 1804 is a region where an antigen capturing body 1208 is provided and an antibody contained in the antigen capturing body 1208 is bound to an antigen. The planar shapes of the irradiation region 1800 and the measurement region 1802 are preferably circular.

  The irradiation region 1800 and the measurement region 1802 overlap at least. Desirably, the irradiation region 1800 is included in the measurement region 1802, and more desirably, the irradiation region 1800 is disposed in the center of the measurement region 1802. Accordingly, even if the irradiation position is shifted due to the variation of the prism 1204 or the like, the irradiation region 1800 does not easily deviate from the measurement region 1802, and the light amount of the measurement light 9002 is maintained.

  The irradiation region 1800 and the reaction region 1804 overlap at least, preferably the irradiation region 1800 is included in the reaction region 1804, and more preferably, the irradiation region 1800 is disposed at the center of the reaction region 1804.

  The measurement region 1802 and the reaction region 1804 overlap at least, preferably the measurement region 1802 is included in the reaction region 1804, and more preferably, the measurement region 1802 is disposed at the center of the reaction region 1804.

(Outline of antigen detection procedure)
The flowchart of FIG. 10 shows an outline of the procedure for detecting an antigen.

  As shown in FIG. 10, the measurement is prepared (step S101), the antibody and the antigen are reacted (step S102), and the load control unit 1100 loads the analysis chip 1006 on the analysis chip transport mechanism 1022 (step S103). . The excitation light 9000 is irradiated on the prism 1204 of the loaded analysis chip 1006. In a state in which the excitation light 9000 is irradiated on the analysis chip 1006, the incident angle θ is set to the measurement angle θm (step S104), preferably the processing to be performed is performed (steps S105 to S107), and the unload control unit 1102 The analysis chip transport mechanism 1022 unloads the analysis chip 1006 (step S108), and the antigen and the fluorescently labeled antibody are reacted (step S109).

  Preferably, the irradiation position is optimized (step S105) after the incident angle θ is set to the measurement angle θm (after step S104) and before the analysis chip 1006 is unloaded (before step S108). ), The amount of autofluorescence is measured (step S106), and the polarization direction of the excitation light 9000 is optimized (step S107). All or part of the optimization of the irradiation position (step S105), the measurement of the amount of autofluorescence light (step S106), and the optimization of the polarization direction of the excitation light 9000 (step S107) may be omitted.

  After the antigen and the fluorescently labeled antibody are reacted (after step S109), the reload control unit 1104 reloads the analysis chip 1006 to the analysis chip transport mechanism 1022 (step S110), and the amount of surface plasmon excitation fluorescence light Is measured (step S111), the measurement result is stored and displayed together with the sample number and the like (step S112), and the surface plasmon excitation fluorescence measuring apparatus 1000 is initialized (step S113). When the surface plasmon excitation fluorescence measuring apparatus 1000 is initialized, a movable object such as a reflecting mirror is returned to the initial position. The light quantity of surface plasmon excitation fluorescence is converted into the absolute amount, concentration, etc. of an antigen as needed.

(Preparation for measurement)
The flowchart of FIG. 11 shows the procedure for measurement preparation (step S101).

  As shown in FIG. 11, the analysis chip 1006 is prepared (step S1011), and the analysis chip 1006 is arranged in the pretreatment chamber 1020 (step S1012).

  Separately, a reagent chip 1036 is prepared (step S1013), the reagent chip 1036 is arranged in the pretreatment chamber 1020 (step S1014), and the pretreatment control unit 1104 causes the liquid delivery mechanism 1014 to pretreat the reagent chip 1036 ( Step S1015). Thereby, the sample liquid 9006 is prepared and the preparation of the sample liquid 9006 is completed. In addition, the preparation of the fluorescent labeling liquid 9008 is completed.

  The preparation and arrangement (steps S1011 and S1012) of the analysis chip 1006 may be performed after the preparation, arrangement and pretreatment of the reagent chip 1036 (steps S1013, S1014 and S1015). You may perform in parallel with a process (step S1013, S1014, and S1015).

(Reaction between antibody and antigen)
In the reaction between the antibody and the antigen (step S102), the first reaction control unit 1106 causes the liquid feeding mechanism 1014 to inject the sample liquid 9006 into the flow path 1220.

  When the sample liquid 9006 is injected into the flow path 1220, the sample liquid 9006 comes into contact with the antigen capturing body 1208, the antigen contained in the sample liquid 9006 is bound to the antibody contained in the antigen capturing body 1208, and the sample liquid 9006 The contained antigen is captured by the antigen capturing body 1208.

(Setting incident angle θ to measurement angle θm)
The flowchart of FIG. 12 shows the procedure for setting the incident angle θ to the measurement angle θm (step S104).

  As shown in FIG. 12, the filter removal control unit 1108 causes the insertion / extraction mechanism 1708 to remove the second band-pass filter 1706 from the optical path of the measurement light 9002 (step S1041). Thereby, the scattered light is not shielded, and the amount of the scattered light can be measured by the photomultiplier tube 1010.

  With the second bandpass filter 1706 removed, the incident angle scanning control unit 1110 acquires the measurement result of the light amount I from the photomultiplier tube 1010 while causing the incident angle adjusting mechanism 1034 to scan the incident angle θ ( Step S1042). Thus, the amount of scattered light Is is measured during scanning at the incident angle θ, and the relationship Is (θ) between the incident angle θ and the amount of scattered light Is is obtained.

  After the relationship Is (θ) is obtained, the incident angle determination unit 1112 specifies the resonance angle θr, which is the incident angle θ at which the light amount Is is maximized, from the relationship Is (θ), and the measurement is performed from the resonance angle θr. The measurement angle θm, which is the incident angle θ, is determined (step S1043). Since the deviation between the resonance angle θr at which the amount of scattered light Is is maximal and the incident angle θmax at which the electric field enhancement is maximal is small, the injection of the fluorescent labeling liquid 9008 has little effect on the resonance angle θr. Even if the resonance angle θr determined before the injection of 9008 is set to the measurement angle θm as it is, the electric field is sufficiently enhanced. However, the resonance angle θr may be corrected to the measurement angle θm. When the resonance angle θr that maximizes the amount of scattered light Is is required, a light amount sensor that measures the amount of surface plasmon excitation fluorescence is used, unlike the case where the resonance angle θr that minimizes the amount of reflected light Ir is obtained. It becomes possible, and the structure of the surface plasmon excitation fluorescence measuring apparatus 1000 is simplified.

  After the measurement angle θm is determined, the incident angle setting unit 1114 causes the incident angle adjustment mechanism 1034 to set the incident angle θ to the measurement angle θm (step S1044).

  When the relationship Is (θ) is measured after the sample liquid 9006 is injected into the flow path 1220, the resonance angle θr is accurately specified regardless of the antigen concentration or the like of the sample liquid 9006. Further, when the relationship Is (θ) is obtained before the fluorescent labeling liquid 9008 is injected into the flow path 1220, the fluorescent dye is not easily faded by the excitation light 9000. These improve the sensitivity and accuracy of antigen detection.

  It is necessary that the relationship Is (θ) is obtained before the fluorescent labeling liquid 9008 is injected into the flow path 1220 (step S1042), but the measurement angle θm is required before the fluorescent labeling liquid 9008 is injected into the flow path 1220. Is determined (step S1043) and the incident angle θ is not necessarily set to the measurement angle θm (step S1044). Therefore, the measurement angle θm may be determined after the fluorescent labeling liquid 9008 is injected into the flow path 1220 (step S1043), and the incident angle θ may be set to the measurement angle θm (step S1044).

(Scanning range of incident angle θ)
The resonance angle θr is generally determined by the material and shape of the prism 1204, the refractive index of the injection material into the flow path 1220, and the like. However, the resonance angle θr slightly changes depending on the type and amount of molecules captured by the antigen capturing body 1208, variation of the prism 1204, and the like. For this reason, in the scan of the incident angle θ, a range of approximately 10 ° or less including the expected resonance angle θr, which is a range where the total reflection condition is satisfied, is preferentially scanned.

(Optimization of irradiation position)
The flowchart of FIG. 13 shows the procedure of optimization of an irradiation position (step S105).

  As shown in FIG. 13, the irradiation position scanning unit 1116 acquires the measurement result of the light amount I from the photomultiplier tube 1010 while causing the reflecting mirror position adjusting mechanism 1604 to scan the reflecting mirror position (step S1051). While the reflecting mirror position is scanned, the excitation light 9000 moves in parallel while the incident angle θ is maintained at the measurement angle θm, and the irradiation position 1300 is scanned along a straight line. Thereby, the light amount Is of the scattered light is measured during the scanning of the irradiation position X, and the relationship Is (X) between the irradiation position X and the light amount Is of the scattered light is obtained.

  The graph of FIG. 14 shows the relationship Is (X) between the irradiation position X and the amount of scattered light Is.

  As shown in FIG. 14, the light amount Is is substantially constant in the section R in which the irradiation region 1800 is included in the measurement region 1802. However, outside the section R, the irradiation area 1800 deviates from the measurement area 1802, and the light amount Is decreases as the irradiation position X moves away from the section R.

  After the relationship Is (X) is obtained, the irradiation position determination unit 1118 identifies the irradiation positions X1 and X2 where the light amount Is decreases from the maximum value by the same value ΔIs from the relationship Is (X), and the irradiation positions X1 and X2 The middle is determined as the measurement position Xm, which is the irradiation position X at which the measurement is performed (step S1052).

  Desirably, the relationship Is (X) is measured after the measurement angle θ is set to the measurement angle θm. Thereby, the irradiation position X is optimized at the same incident angle θ as when the amount of surface plasmon excitation fluorescence is measured, and the irradiation position X is appropriately optimized.

  Subsequently, the irradiation position setting unit 1120 causes the reflecting mirror position adjusting mechanism 1604 to set the reflecting mirror position to the reflecting mirror position where the irradiation position X becomes the measurement position Xm (step S1053).

  At an incident angle θ in the vicinity of the resonance angle θr, the amount of reflected light 9004 is small, and the reflected light 9004 hardly generates autofluorescence emitted from the prism 1204. For this reason, the optical path length of the excitation light 9000 in the measurement region 1802 greatly affects the amount of autofluorescence, and when the irradiation position X changes, the amount of autofluorescence also changes. The amount of autofluorescence is small compared to the amount of excitation light 9000, but cannot be ignored compared to the amount of surface plasmon excitation fluorescence. However, since the amount of autofluorescence is small, measurement of the amount of surface plasmon excitation fluorescence only affects autofluorescence emitted from the vicinity of the reflecting surface 1206.

  When the irradiation region 1800 is arranged at the center of the measurement region 1802 by the optimization of the irradiation position X (step S105), the optical path length of the excitation light 9000 in the measurement region 1802 is constant, and the influence of the amount of autofluorescence is constant. Thus, the sensitivity and accuracy of antigen detection are improved. Desirably, the irradiation position X is optimized (step S105) before the measurement of the amount of autofluorescence (before step S106). Thereby, the accuracy of measurement of the amount of autofluorescence is improved.

(Measurement of the amount of autofluorescence)
In the measurement of the amount of autofluorescence (step S106), the autofluorescence measurement unit 1122 acquires the measurement result of the amount of light I from the photomultiplier tube 1010. As a result, the amount of autofluorescence Iaf is measured. The measurement result is stored in the autofluorescence measuring unit 1122 until the autofluorescence correction described later is completed.

(Optimization of excitation light polarization direction)
The flowchart of FIG. 15 shows the procedure for optimizing the polarization direction of the excitation light (step S107).

  As shown in FIG. 15, the polarization direction scanning unit 1124 acquires the measurement result of the light amount I from the photomultiplier tube 1010 while causing the polarization direction adjustment mechanism 1030 to scan the polarization direction α (step S1071). As a result, the amount of scattered light Is is measured during scanning in the polarization direction α, and the relationship Is (α) between the polarization direction α of the excitation light 9000 and the amount of scattered light Is is obtained.

  Desirably, the relationship Is (α) is measured after the measurement angle θ is set to the measurement angle θm. Thereby, the polarization direction α is optimized at the same incident angle θ as when the amount of surface plasmon excitation fluorescence is measured, and the polarization direction α is appropriately optimized. More preferably, the relationship Is (α) is measured after the irradiation position X is optimized (after step S105). Thereby, the polarization direction is optimized at the same irradiation position X as when the amount of surface plasmon excitation fluorescence is measured, and the polarization direction α is appropriately optimized.

  After the relationship Is (α) is obtained, the polarization direction determination unit 1126 specifies the polarization direction α from which the light amount Is is maximized from the relationship Is (α), and sets the measurement polarization direction αm that is the polarization direction α in which the measurement is performed. Determination is made (step S1072).

  After the measurement polarization direction αm is determined, the polarization direction setting unit 1128 causes the polarization direction adjustment mechanism 1030 to set the polarization direction α to the measurement polarization direction αm (step S1073).

  Birefringence that cannot be ignored occurs when light travels through a resin medium. This birefringence is caused by a change in the density of the medium depending on the position. Since the change in the density of the medium depending on the position occurs when the resin is molded, the strength of birefringence differs for each prism 1204. Therefore, depending on the prism 1204, even if only the P wave component contributing to the exudation of the evanescent wave is caused to enter the reflecting surface 1206, the S wave component is generated and the P wave component is reduced, and the sensitivity of detecting the antigen is increased. And the accuracy decreases.

  By optimizing the polarization direction of the excitation light (step S107), the decrease of the P wave component due to the variation of the prism 1204 is eliminated, and the sensitivity and accuracy of antigen detection are improved.

(Reaction between antigen and fluorescently labeled antibody)
In the reaction between the antigen and the fluorescently labeled antibody (step S109), the second reaction control unit 1130 causes the liquid feeding mechanism 1014 to inject the fluorescently labeled liquid 9008 into the flow path 1220. When the fluorescent labeling liquid 9008 is injected into the flow path 1220, the fluorescent labeling liquid 9008 comes into contact with the antigen capturing body 1208, and the fluorescently labeled antibody contained in the fluorescent labeling liquid 9008 binds to the antigen.

(Measurement of light intensity of surface plasmon excitation fluorescence)
The flowchart of FIG. 16 shows the procedure for measuring the amount of surface plasmon excitation fluorescence (step S111).

  As shown in FIG. 16, the filter insertion control unit 1132 causes the insertion / extraction mechanism 1708 to insert the second band-pass filter 1706 into the optical path of the measurement light 9002 (step S1111). Thereby, the scattered light reaching the photomultiplier tube 1010 is reduced, and the sensitivity and accuracy of antigen detection are improved.

  After the second band pass filter 1706 is inserted, the blocking control unit 1134 causes the reflecting mirror angle adjustment mechanism 1602 to temporarily block the excitation light 9000 (step S1112).

  While the excitation light 9000 is blocked, the dark noise measurement unit 1136 acquires the measurement result of the light amount I from the photomultiplier tube 1010 (step S1113). Thereby, the dark noise amount In is measured.

  Desirably, the blocking of the excitation light (step S1112) and the measurement of the amount of dark noise (step S1113) are performed by resetting the incident angle θ to the measurement angle θm (step S1114) and measuring the light amount If of the surface plasmon excitation fluorescence. Although it is performed immediately before (Step S1115), it may be performed at other times as long as it is before dark noise correction (Step S1117).

  After the dark noise amount In is measured (after step S1113), the incident angle resetting unit 1136 causes the reflector angle adjusting mechanism 1602 to reset the incident angle θ to the measurement angle θm (step S1114).

  After the incident angle θ is reset to the measurement angle θm, the fluorescence measurement unit 1138 acquires the measurement result of the light amount I from the photomultiplier tube 1010 (step S1115). Thereby, the light amount If of the surface plasmon excitation fluorescence at the measurement angle θm is measured.

  After the surface plasmon excitation fluorescence light amount If is measured, the autofluorescence correction unit 1104 performs correction to subtract the autofluorescence light amount Iaf from the surface plasmon excitation fluorescence light amount If (step S1116), and the dark noise correction unit 1142 Correction for subtracting the dark noise amount In from the light amount If of the plasmon excitation fluorescence is performed (step S1117). This improves the sensitivity and accuracy of antigen detection. Autofluorescence correction (step S1116) and dark noise correction (step S1117) may be performed simultaneously, and autofluorescence correction (step S1116) may be performed after dark noise correction (step S1117).

  Of the above procedures, the procedure executed by the surface plasmon excitation fluorescence measurement apparatus 1000 may be executed manually, or the procedure executed manually may be executed by the surface plasmon excitation fluorescence measurement apparatus 1000.

  Instead of the resonance angle θr at which the light amount Is of the scattered light is maximized, a resonance angle θr at which the light amount Ir of the reflected light 9004 is minimized may be used. When the latter resonance angle θr is used, a reflected light amount sensor 1902 that measures the amount of reflected light 9004 is added to the surface plasmon excitation fluorescence measuring apparatus 1000 as shown in FIG. In step S <b> 1042, the incident angle scanning control unit 1110 acquires the measurement result of the light amount I from the reflected light amount sensor 1902 while causing the incident angle adjustment mechanism 1034 to scan the incident angle θ. As a result, the amount of light Ir of the reflected light 9004 is measured during scanning at the incident angle θ, and the relationship Ir (θ) between the incident angle θ and the amount of light Ir of the reflected light 9004 is obtained. In step S1043, the incident angle determination unit 1112 specifies the resonance angle θr, which is the incident angle θ at which the light amount Ir is minimized, from the relationship Ir (θ), and determines the measurement angle θm from the resonance angle θr. Since there is a difference between the resonance angle θr at which the reflected light quantity Ir is minimized and the incident angle θmax at which the electric field enhancement is maximized, it is desirable that correction is performed to increase / decrease the minute angle to the resonance angle θr, and the measurement angle θm is determined.

  While the invention has been shown and described in detail, the above description is illustrative in all aspects and not restrictive. Thus, it will be appreciated that numerous modifications and variations can be devised without departing from the scope of the invention.

DESCRIPTION OF SYMBOLS 1000 Surface plasmon excitation fluorescence measuring device 1002 Laser diode unit 1006 Analysis chip 1010 Photomultiplier tube 1014 Liquid feeding mechanism 1030 Polarization direction adjustment mechanism 1034 Incident angle adjustment mechanism 1200 Conductor film 1204 Prism 1206 Reflecting surface 1212 Flow path formation body 1216 Incident surface 1218 Emission surface 1220 Flow path 1706 Second band pass filter 9006 Sample solution 9008 Fluorescent labeling solution

Claims (9)

A surface plasmon excitation fluorescence measuring device,
A light source that emits excitation light;
A dielectric that includes an incident surface on which excitation light is incident, a reflection surface that totally reflects the excitation light incident on the incident surface, and an emission surface from which the excitation light that is totally reflected on the reflection surface is emitted. Medium,
A conductor film comprising a first main surface that is in close contact with the reflective surface and a second main surface that is not in close contact with the reflective surface;
An antigen-capturing body comprising an antibody that binds to an antigen, and anchored on the second main surface;
An adjustment mechanism for adjusting the incident angle of the excitation light to the reflecting surface;
A light amount sensor that measures the amount of light emitted from the conductor film and the antigen capturing body in a direction in which the second main surface faces;
A contact mechanism for contacting a sample and a fluorescently labeled antibody-containing substance containing a fluorescently labeled antibody that binds to an antigen with the antigen capturing body;
A first reaction control unit for bringing the sample into contact with the antigen capturing body in the contact mechanism;
A first scanning control unit that obtains a light amount measurement result from the light amount sensor while causing the adjustment mechanism to scan an incident angle after the sample is brought into contact with the antigen capturing body, and obtains a relationship between the incident angle and the light amount When,
A second reaction control unit for contacting the antigen-captured substance with the fluorescently labeled antibody-containing substance after the relationship between the incident angle and the light amount is obtained;
Identifying the resonance angle at which the light amount is maximized from the relationship between the incident angle and the light amount, and determining the measurement angle at which the measurement is performed from the resonance angle;
An incident angle setting unit that causes the adjustment mechanism to set an incident angle to the measurement angle;
A first measurement control unit for obtaining a light amount measurement result from the light amount sensor after the fluorescently labeled antibody-containing material is brought into contact with the antigen capturing body and an incident angle is set to the measurement angle;
A surface plasmon excitation fluorescence measuring apparatus comprising: A surface plasmon excitation fluorescence measuring device,
A light source that emits excitation light;
A dielectric that includes an incident surface on which excitation light is incident, a reflection surface that totally reflects the excitation light incident on the incident surface, and an emission surface from which the excitation light that is totally reflected on the reflection surface is emitted. Medium,
A conductor film comprising a first main surface that is in close contact with the reflective surface and a second main surface that is not in close contact with the reflective surface;
An antigen-capturing body comprising an antibody that binds to an antigen, and anchored on the second main surface;
An adjustment mechanism for adjusting the incident angle of the excitation light to the reflecting surface;
A reflected light amount sensor that measures the amount of light emitted from the exit surface;
A light amount sensor that measures the amount of light emitted from the conductor film and the antigen capturing body in a direction in which the second main surface faces;
A contact mechanism for contacting the antigen-capturing body with a fluorescently labeled antibody-containing material containing a sample and a fluorescently labeled antibody that binds to the antigen;
A first reaction control unit that causes the contact mechanism to inject the sample into the flow path;
A first scanning control unit that obtains a measurement result of the light amount from the reflected light amount sensor while scanning the incident angle after the sample is brought into contact with the antigen capturing body, and obtains a relationship between the incident angle and the light amount When,
A second reaction control unit for contacting the antigen-captured substance with the fluorescently labeled antibody-containing substance after the relationship between the incident angle and the light amount is obtained;
Identifying a resonance angle at which the light amount is minimized from the relationship between the incident angle and the light amount, and determining the measurement angle at which the measurement is performed from the resonance angle;
An incident angle setting unit that causes the adjustment mechanism to set an incident angle to the measurement angle;
A first measurement control unit for obtaining a light amount measurement result from the light amount sensor after the fluorescently labeled antibody-containing material is brought into contact with the antigen capturing body and an incident angle is set to the measurement angle;
A surface plasmon excitation fluorescence measuring apparatus comprising: In the surface plasmon excitation fluorescence measuring device according to claim 1 or 2,
A second measurement control unit that acquires a measurement result of the light amount from the light amount sensor before the fluorescently labeled antibody-containing material is brought into contact with the antigen capturing body and the incident angle is set to the measurement angle;
A first correction unit that subtracts the measurement result of the light quantity acquired by the second measurement control unit from the measurement result of the light quantity acquired by the first measurement control unit;
A surface plasmon excitation fluorescence measurement apparatus further comprising: In the surface plasmon excitation fluorescence measuring device according to claim 3,
The adjustment mechanism adjusts the irradiation position of the excitation light on the reflection surface,
The surface plasmon excitation fluorescence measuring device is:
A second scanning control unit that obtains a light amount measurement result from the light amount sensor while causing the adjustment mechanism to scan the irradiation position, and obtains a relationship between the irradiation position and the light amount;
The first irradiation position and the second irradiation position where the light amount decreases from the maximum value by the same value from the relationship between the irradiation position and the light amount are specified, and the intermediate between the first irradiation position and the second irradiation position is measured. An irradiation position determination unit that determines a measurement position where
An irradiation position setting unit that causes the adjustment mechanism to set an irradiation position to the measurement position;
A surface plasmon excitation fluorescence measurement apparatus further comprising: In the surface plasmon excitation fluorescence measuring device according to claim 1 or 2,
The adjustment mechanism switches between a non-blocking state where excitation light is guided to the incident surface and a blocking state where it is not guided,
The surface plasmon excitation fluorescence measuring device is:
A shut-off control unit that temporarily places the adjusting mechanism in the shut-off state;
A third measurement control unit for obtaining a light quantity measurement result from the light quantity sensor while the adjustment mechanism is in the shut-off state;
A second correction unit that subtracts the measurement result of the light amount acquired by the third measurement control unit from the measurement result of the light amount acquired by the first measurement control unit;
A surface plasmon excitation fluorescence measurement apparatus further comprising: In the surface plasmon excitation fluorescence measuring device according to claim 1 or 2,
A polarization direction adjusting mechanism for adjusting the polarization direction of the excitation light;
A polarization direction scanning unit that obtains a measurement result of the light amount from the light amount sensor while causing the polarization direction adjustment mechanism to scan the polarization direction, and obtains a relationship between the polarization direction and the light amount;
A polarization direction determining unit that identifies a polarization direction in which the light amount is maximized from the relationship between the polarization direction and the light amount, and determines a measurement polarization direction in which the measurement is performed;
A polarization direction setting unit that causes the polarization direction adjustment mechanism to set the polarization direction to the measurement polarization direction;
A surface plasmon excitation fluorescence measurement apparatus further comprising: In the surface plasmon excitation fluorescence measuring device according to claim 1 or 2,
A bandpass filter that selectively attenuates light of the same wavelength as the excitation light;
An insertion / extraction mechanism for inserting / extracting the band-pass filter into / from an optical path of light emitted from the conductor film and the antigen capturing body in a direction in which the second main surface faces.
An insertion / removal control unit that causes the insertion / removal mechanism to insert the band-pass filter while the first measurement control unit acquires a light amount measurement result;
A surface plasmon excitation fluorescence measurement apparatus further comprising: A surface plasmon excitation fluorescence measurement method,
(a) a conductive medium having an entrance surface, a reflection surface and an exit surface and having a dielectric medium that is transparent to excitation light, a first main surface that is in close contact with the reflection surface, and a second main surface that is not in close contact with the reflection surface Providing a structure comprising a body membrane and an antigen-capturing body that contains an antibody that binds to an antigen and is fixed to the second main surface;
(b) preparing a sample;
(c) preparing a fluorescently labeled antibody-containing material containing a fluorescently labeled antibody that binds to an antigen;
(d) contacting the sample with the antigen capturer;
(e) irradiating the structure with excitation light that is incident on the incident surface, is totally reflected on the reflecting surface, and is emitted from the emitting surface;
(f) After the step (d), the amount of scattered light emitted in the direction toward the second main surface is measured while scanning the incident angle of the excitation light on the reflecting surface, and the relationship between the incident angle and the amount of light. And obtaining
(g) after the step (f), bringing the fluorescently labeled antibody-containing material into contact with the antigen-capturing body;
(h) identifying a resonance angle at which the amount of scattered light is maximized from the relationship between the incident angle and the amount of light, and determining a measurement angle at which measurement is performed from the resonance angle;
(i) setting the incident angle to the measurement angle;
(j) measuring the amount of surface plasmon excitation fluorescence after the step (g) and the step (i);
A surface plasmon excitation fluorescence measurement method comprising: A surface plasmon excitation fluorescence measurement method,
(a) a conductive medium having an entrance surface, a reflection surface and an exit surface and having a dielectric medium that is transparent to excitation light, a first main surface that is in close contact with the reflection surface, and a second main surface that is not in close contact with the reflection surface Providing a structure comprising a body membrane and an antigen-capturing body that contains an antibody that binds to an antigen and is fixed to the second main surface;
(b) preparing a sample;
(c) preparing a fluorescently labeled antibody-containing material containing a fluorescently labeled antibody that binds to an antigen;
(d) contacting the sample with the antigen capturer;
(e) irradiating the structure with excitation light that is incident on the incident surface, is totally reflected on the reflecting surface, and is emitted from the emitting surface;
(f) measuring the amount of reflected light emitted from the exit surface while scanning the incident angle of the excitation light to the reflective surface after the step (d), and obtaining a relationship between the incident angle and the amount of light;
(g) after the step (f), bringing the fluorescently labeled antibody-containing material into contact with the antigen-capturing body;
(h) identifying a resonance angle at which the amount of reflected light is minimized from the relationship between the incident angle and the amount of light, and determining a measurement angle at which measurement is performed from the resonance angle;
(i) setting the incident angle to the measurement angle;
(j) measuring the amount of surface plasmon excitation fluorescence after the step (g) and the step (i);
A surface plasmon excitation fluorescence measurement method comprising:

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