JP5614321B2 - Surface plasmon measuring device and surface plasmon measuring method - Google Patents

Description

  The present invention relates to a surface plasmon measuring device and a surface plasmon measuring 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 the measurement by the surface plasmon resonance method (SPR) or the surface plasmon excitation fluorescence method (SPFS), this enhanced electric field is used.

  In measurement by the surface plasmon resonance method (SPR), a substance to be measured is captured on the surface of the metal film. Further, the amount of reflected light, the amount of scattered light, the resonance angle, and all or part of these changes are measured, and the presence / absence of the substance to be measured, the capture amount of the substance to be measured, etc. are specified.

  In measurement by surface plasmon excitation fluorescence spectroscopy (SPFS), a measurement target substance is captured on the surface of a metal film, and a fluorescently labeled substance that is fluorescently labeled is bound to the captured measurement target substance. The enhanced electric field acts on the fluorescent label, and surface plasmon excitation fluorescence is emitted from the fluorescent label. Further, the amount of surface plasmon excitation fluorescence is measured, and the presence / absence of the substance to be measured, the trapped amount of the substance to be measured, etc. are specified.

  The evanescent wave leaking from the interface is a p-wave component of light incident on the interface. Therefore, in order to improve the accuracy and sensitivity of measurement by the surface plasmon resonance method or the surface plasmon excitation fluorescence spectroscopy, it is necessary to stabilize the intensity of the p-wave component incident on the interface.

  On the other hand, an analysis chip including a metal film and a dielectric medium is preferably made disposable for each measurement in consideration of the efficiency of measurement work, safety, and the like. For this reason, the dielectric medium is preferably made of an inexpensive resin. However, since the density distribution is not uniform in the resin molding, the dielectric medium made of the resin generates birefringence, and the degree of the birefringence differs for each dielectric medium. Birefringence also causes rotation of the polarization direction of the excitation light traveling through the dielectric medium. Therefore, when the dielectric medium is made of resin, there is a problem that the p-wave component of the excitation light incident on the reflecting surface is reduced, and the degree of reduction of the p-wave component is different for each analysis chip.

  Patent Document 1 relates to a technique for maximizing the intensity of a p-wave component of excitation light in surface plasmon measurement. Patent Document 1 proposes adjusting the polarization direction of excitation light so that the intensity of the s-wave component of reflected light is minimized.

JP 2009-236709 A

  In Patent Document 1, the intensity of reflected light that is affected by the rotation of the polarization direction is measured not only in the section from the incident surface to the reflecting surface of the dielectric medium but also in the section from the reflecting surface to the emitting surface. For this reason, the rotation of the polarization direction in the section from the incident surface to the reflecting surface of the dielectric medium directly related to the exudation of the evanescent wave is not properly corrected, and the intensity of the p-wave component incident on the reflecting surface is stable. The measurement accuracy and sensitivity were insufficient.

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

The first to seventh aspects of the present invention are directed to a surface plasmon measurement apparatus that performs measurement by surface plasmon excitation fluorescence spectroscopy or surface plasmon resonance.

  (1) In the first aspect of the present invention, a light source, a structure, a polarization direction adjusting mechanism, a reading mechanism, and a first control unit are provided. The structure includes a dielectric medium and a conductor film. The dielectric medium includes an entrance surface, a reflection surface, and an exit surface. The first main surface of the conductor film is in close contact with the reflecting surface, and the second main surface of the conductor film is not in close contact with the reflecting surface. A first index indicating the degree of rotation of the main polarization direction of the excitation light in the section from the incident surface to the reflection surface is added to the structure.

  The light source emits excitation light. The excitation light is incident on the incident surface, the excitation light incident on the incident surface is totally reflected on the reflecting surface, and the excitation light totally reflected on the reflecting surface is emitted from the emitting surface. The polarization direction adjusting mechanism adjusts the polarization direction of the excitation light irradiated to the structure.

The reading mechanism reads the first index. The first control unit acquires the first index from the reading mechanism, and causes the polarization direction adjusting mechanism to set the polarization direction of the excitation light according to the first index. The polarization direction after setting is a polarization direction in which the rotation of the main polarization direction is canceled. The first control unit maintains the polarization direction of the excitation light at the set polarization direction when the light amount of the surface plasmon excitation fluorescence or the light amount of the scattered light by the enhanced electric field of the evanescent wave is measured.

  (2) The second aspect of the present invention adds further matters to the first aspect of the present invention. In the second aspect of the present invention, the first index indicates the rotation angle of the main polarization direction when p-polarized light with respect to the reflecting surface is incident on the incident surface. The polarization direction after setting is a polarization direction obtained by rotating the polarization direction of p-polarized light by an angle obtained by inverting the rotation angle.

(3) In the third aspect of the present invention, a light source, a structure, a polarization direction adjustment mechanism, a reading mechanism, a first control unit, a light amount adjustment mechanism, and a second control unit are provided. The structure includes a dielectric medium and a conductor film. The dielectric medium includes an entrance surface, a reflection surface, and an exit surface. The first main surface of the conductor film is in close contact with the reflecting surface, and the second main surface of the conductor film is not in close contact with the reflecting surface. A first index indicating the degree of rotation of the main polarization direction of the excitation light in the section from the incident surface to the reflection surface is added to the structure. A second index indicating the degree of decrease in intensity of the main polarization direction component of the excitation light in the section is attached to the structure.
The light source emits excitation light. The excitation light is incident on the incident surface, the excitation light incident on the incident surface is totally reflected on the reflecting surface, and the excitation light totally reflected on the reflecting surface is emitted from the emitting surface. The polarization direction adjusting mechanism adjusts the polarization direction of the excitation light irradiated to the structure. The light amount adjusting mechanism adjusts the light amount of the excitation light irradiated to the structure.
The reading mechanism reads the first index and the second index. The first control unit acquires the first index from the reading mechanism, and causes the polarization direction adjusting mechanism to set the polarization direction of the excitation light according to the first index. The polarization direction after setting is a polarization direction in which the rotation of the main polarization direction is canceled. The second control unit acquires the second index from the reading mechanism, and causes the light amount adjustment mechanism to set the light amount of the excitation light according to the second index. The amount of light after setting is the amount of light that cancels the decrease in intensity of the main polarization direction component.
(4) The fourth aspect of the present invention adds further matters to the third aspect of the present invention. In the fourth aspect of the present invention, the first index indicates a rotation angle of the main polarization direction when p-polarized light with respect to the reflection surface is incident on the incident surface. The polarization direction after setting is a polarization direction obtained by rotating the polarization direction of p-polarized light by an angle obtained by inverting the rotation angle.

(5) The fifth aspect of the present invention adds further matters to the third or fourth aspect of the present invention. In the fifth aspect of the present invention, the second index indicates a ratio of the intensity of the main polarization component incident on the reflection surface to the reference intensity when p-polarized light with respect to the reflection surface is incident on the incident surface. The light amount of the excitation light after setting is a light amount obtained by multiplying the reference light amount by the reciprocal of the ratio.

(6) In the sixth aspect of the present invention, a light source, a structure, a light amount adjustment mechanism, a reading mechanism, and a control unit are provided. The structure includes a dielectric medium and a conductor film. The dielectric medium includes an entrance surface, a reflection surface, and an exit surface. The first main surface of the conductor film is in close contact with the reflecting surface, and the second main surface of the conductor film is not in close contact with the reflecting surface. An index indicating the degree of decrease in intensity of the main polarization direction component of the excitation light in the section from the incident surface to the reflection surface is added to the structure.

The light source emits excitation light. The excitation light is incident on the incident surface, the excitation light incident on the incident surface is totally reflected on the reflecting surface, and the excitation light totally reflected on the reflecting surface is emitted from the emitting surface. The light amount adjustment mechanism adjusts the entire light amount of the excitation light irradiated to the structure.

The reading mechanism reads the index. The control unit acquires the index from the reading mechanism, and causes the light amount adjustment mechanism to set the entire light amount of the excitation light according to the index. The amount of light after setting is the amount of light that cancels the decrease in intensity of the main polarization direction component.

(7) In the seventh aspect of the present invention, a light source, a structure, a light amount adjustment mechanism, a reading mechanism, and a control unit are provided. The structure includes a dielectric medium and a conductor film. The dielectric medium includes an entrance surface, a reflection surface, and an exit surface. The first main surface of the conductor film is in close contact with the reflecting surface, and the second main surface of the conductor film is not in close contact with the reflecting surface. An index indicating the degree of decrease in intensity of the main polarization direction component of the excitation light in the section from the incident surface to the reflection surface is added to the structure. The index indicates a ratio of the intensity of the main polarization component incident on the reflection surface to the reference intensity when p-polarized light with respect to the reflection surface is incident on the incident surface.
  The light source emits excitation light. The excitation light is incident on the incident surface, the excitation light incident on the incident surface is totally reflected on the reflecting surface, and the excitation light totally reflected on the reflecting surface is emitted from the emitting surface. The light amount adjusting mechanism adjusts the light amount of the excitation light irradiated to the structure.
  The reading mechanism reads the index. The control unit acquires the index from the reading mechanism, and causes the light amount adjustment mechanism to set the light amount of the excitation light according to the index. The light amount after the setting is a light amount obtained by multiplying the reference light amount by the reciprocal of the ratio, and is a light amount that cancels the decrease in the intensity of the main polarization direction component.

The eighth to tenth aspects of the present invention are directed to a surface plasmon measurement method in which measurement is performed by surface plasmon excitation fluorescence spectroscopy or surface plasmon resonance.

(8) In the eighth aspect of the present invention, a first dielectric medium and a second dielectric medium are prepared. The first dielectric medium includes a first incident surface, a reflection surface, and a first emission surface, and is transparent to excitation light. The second dielectric medium includes a second incident surface and a second emission surface, is transparent to the excitation light, and does not generate birefringence.

  The reflective surface and the second incident surface are stretched to produce a composite of the first dielectric medium and the second dielectric medium. The complex is irradiated with evaluation light. The evaluation light enters the first incident surface, passes through the reflecting surface and the second incident surface, and exits from the second exit surface.

  The degree of rotation of the main polarization direction of the evaluation light in the section from the first entrance surface to the second exit surface is measured.

  After the degree of rotation of the main polarization direction is measured, the composite is separated into a first dielectric medium and a second dielectric medium, and a structure including the first dielectric medium and the conductor film is manufactured. Is done. The first main surface of the conductor film is in close contact with the reflecting surface, and the second main surface of the conductor film is not in close contact with the reflecting surface. A first index indicating the degree of rotation of the main polarization direction is attached to the structure.

  The first index attached to the structure is read, and the polarization direction of the excitation light is set according to the first index. The polarization direction of the excitation light after setting is a polarization direction in which the rotation of the main polarization direction is canceled.

  The structure is irradiated with excitation light, and measurement is performed by surface plasmon resonance or surface plasmon excitation fluorescence spectroscopy. The excitation light enters the first incident surface, is totally reflected by the reflecting surface, and exits from the first emitting surface.

(9) The ninth aspect of the present invention adds further matters to the eighth aspect of the present invention. In the eighth aspect of the present invention, the degree of decrease in intensity of the main polarization direction component of the evaluation light in the section is measured. A second indicator indicating the degree of strength reduction is attached to the structure.

  The second index attached to the structure is read, and the amount of excitation light is set according to the second index. The amount of excitation light after setting is the amount of light that cancels the decrease in intensity of the main polarization direction component.

(10) In the tenth aspect of the present invention, a first dielectric medium and a second dielectric medium are prepared. The first dielectric medium includes a first incident surface, a reflection surface, and a first emission surface, and is transparent to excitation light. The second dielectric medium includes a second incident surface and a second emission surface, is transparent to the excitation light, and does not generate birefringence.

  The reflective surface and the second incident surface are stretched to produce a composite of the first dielectric medium and the second dielectric medium. The complex is irradiated with evaluation light. The evaluation light enters the first incident surface, passes through the reflecting surface and the second incident surface, and exits from the second exit surface.

  The degree of decrease in intensity of the main polarization direction component of the evaluation light in the section from the first incident surface to the second emission surface is measured.

  After the degree of decrease in the intensity of the main polarization direction component is measured, the composite is separated into the first dielectric medium and the second dielectric medium, and the structure includes the first dielectric medium and the conductor film. A body is made. The first main surface of the conductor film is in close contact with the reflecting surface, and the second main surface of the conductor film is not in close contact with the reflecting surface. The structure is accompanied by an index indicating the degree of decrease in the intensity of the main polarization direction component.

  The index attached to the structure is read, and the amount of excitation light is set according to the index. The amount of excitation light after setting is the amount of light that cancels the decrease in intensity of the main polarization direction component.

  The structure is irradiated with excitation light, and measurement is performed by surface plasmon resonance or surface plasmon excitation fluorescence spectroscopy. The excitation light enters the first incident surface, is totally reflected by the reflecting surface, and exits from the first emitting surface.

According to the first and eighth aspects of the present invention, the polarization direction of the excitation light incident on the reflection surface is less affected by the rotation of the main polarization direction by the dielectric medium, and the measurement accuracy and sensitivity are improved.

  According to the second aspect of the present invention, the main polarization direction of the excitation light incident on the reflection surface coincides with the polarization direction of the p-polarized light, and the measurement accuracy and sensitivity are improved.

According to the third, fourth, sixth, ninth, and tenth aspects of the present invention, the intensity of the main polarization direction component of the excitation light incident on the reflecting surface is not easily affected by the rotation of the polarization direction by the dielectric medium. Thus, measurement accuracy and sensitivity are improved.

According to the fifth aspect of the present invention, the main polarization direction of the excitation light incident on the reflecting surface coincides with the polarization direction of the p-polarized light, the intensity of the main polarization direction component is stabilized, and measurement accuracy and sensitivity 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 a figure which shows the polarization direction of excitation light. It is a figure which shows the polarization direction of excitation light. It is a figure which shows the polarization direction of excitation light. It is a figure which shows the polarization direction of excitation light. It is a figure which shows the polarization direction of excitation light. It is a figure which shows the polarization direction of excitation light. It is a figure which shows the polarization direction of excitation light. It is sectional drawing of an analysis chip. It is a disassembled perspective view 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 with the outline of the procedure of the detection of an antigen. It is a schematic diagram which shows the state to which the reflected light amount sensor was added. It is a flowchart which shows the procedure of actual measurement of a 1st parameter | index and a 2nd parameter | index. It is a schematic diagram which shows the measuring apparatus which measures the 1st parameter | index and the 2nd parameter | index.

(Outline of measurement)
This preferred embodiment relates to surface plasmon excited fluorescence measurements that detect antigens by surface plasmon excited fluorescence spectroscopy.

  In this preferred embodiment, excitation light is incident on the interface between the prism and the conductor film, and surface plasmon excitation fluorescence is generated. A first index indicating the degree of rotation of the main polarization direction of the excitation light in the prism and a second index indicating the degree of decrease in intensity of the main polarization direction component of the excitation light in the prism are attached to the analysis chip. The main polarization direction of the excitation light is set according to the first index to a polarization direction in which the rotation of the main polarization direction by the prism is canceled. The amount of excitation light is set according to the second index to the amount of light that cancels the decrease in intensity of the main polarization direction component due to the prism.

(Outline of surface plasmon excitation fluorescence measuring device)
The schematic diagram of FIG. 1 shows an embodiment of a surface plasmon excitation fluorescence measuring 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 attached to and detached from the surface plasmon excitation fluorescence measurement apparatus 1000, and is exchanged for each specimen 9020. However, the analysis chip 1006 may be reused.

  The excitation light 9000 guided to the prism 1024 is incident on the incident surface 1026 of the prism 1024, and the excitation light 9000 incident on the incident surface 1026 is totally reflected by the reflecting surface 1028 of the prism 1024 and reflected by the reflecting surface 1028. Light 9004 exits from the exit surface 1030 of the prism 1024.

  The incident angle θ of the excitation light 9000 on the reflecting surface 1028 is scanned by the incident angle adjusting mechanism 1032, the amount of the measuring light 9002 is measured by the photomultiplier tube 1010, and the resonance angle θr is specified. After 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 1041.

  A barcode label 1034 is attached to the analysis chip 1006. The barcode 1224 formed on the barcode label 1034 includes a first index indicating the degree of rotation of the main polarization direction of the excitation light 9000 in the prism 1024 and a decrease in intensity of the main polarization direction component of the excitation light 9000 in the prism 1024. A second index indicating the degree of is described. The first index and the second index are read by the barcode reader 1036. “Main polarization direction” means the polarization direction in which the intensity is maximum. “Main polarization direction component” means a component that changes to the main polarization direction.

  The polarization direction of the excitation light 9000 applied to the analysis chip 1006 is adjusted by the polarization direction adjustment mechanism 1038, and is set according to the first index in the polarization direction in which the rotation of the main polarization direction of the excitation light 9000 in the prism 1024 is canceled. The

  The light quantity of the excitation light 9000 irradiated to the analysis chip 1006 is adjusted in the laser diode unit 1002 and set to the light quantity that cancels the decrease in intensity of the main polarization direction component of the excitation light 9000 in the prism 1024 according to the second index. The

  The components of the surface plasmon excitation fluorescence measuring apparatus 1000 are controlled by the control calculation unit 1040, and the control calculation unit 1040 performs calculation.

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

  The polarization direction control unit 1100 acquires the first index from the barcode reader 1036 and causes the polarization direction adjustment mechanism 1038 to set the polarization direction of the excitation light 9000 according to the first index. The polarization direction of the excitation light 9000 after setting is a polarization direction in which the rotation of the main polarization direction by the prism 1024 is canceled. Thereby, the main polarization direction of the excitation light 9000 incident on the reflection surface 1028 is less affected by the rotation of the main polarization direction by the prism 1024, the intensity of the p-wave component incident on the reflection surface 1028 is stabilized, and the measurement sensitivity and Accuracy is improved.

  The cancellation of the rotation of the main polarization direction of the excitation light 9000 by the prism 1024 means that the polarization direction of the excitation light 9000 incident on the reflection surface 1028 is close to the polarization direction of the excitation light 9000 incident on the incidence surface 1026. This means that the polarization direction of the excitation light 9000 incident on the reflection surface 1028 is matched with the polarization direction of the excitation light 9000 incident on the incidence surface 1026.

  The light quantity control unit 1102 acquires the second index from the barcode reader 1036 and causes the laser diode unit 1002 to set the light quantity of the excitation light 9000 according to the second index. The light amount of the excitation light 9000 after setting is a light amount that cancels the decrease in intensity of the main polarization direction component by the prism 1024. As a result, the intensity of the main polarization direction component of the excitation light 9000 incident on the reflection surface 1028 is less affected by the rotation of the polarization direction by the prism 1024, the intensity of the p-wave component incident on the reflection surface 1028 is stabilized, and the measurement Sensitivity and accuracy are improved.

  The cancellation of the decrease in the intensity of the main polarization direction component of the excitation light 9000 by the prism 1024 means that the intensity of the main polarization direction component of the excitation light 9000 incident on the reflection surface 1028 approaches the reference intensity. This means that the intensity of the main polarization direction component of the excitation light 9000 incident on the surface 1028 matches the reference intensity.

  It is desirable that both the polarization direction correction by the polarization direction control unit 1100 and the light amount correction by the light amount control unit 1102 are performed, but only one of the polarization direction correction and the light amount correction may be performed.

(Example of correction)
The graphs from FIG. 3 to FIG. 9 show the polarization state of the excitation light 9000. The p-axis and s-axis directions in the graphs of FIGS. 3 to 9 are the polarization directions of p-polarized light and s-polarized light, respectively. The p-polarized light and the s-polarized light are respectively linearly polarized light that becomes a p-wave component and an s-wave component with respect to the reflecting surface 1028 on the assumption that there is no rotation of the polarization direction by the prism 1024.

  In the first example of correction, when linearly polarized light polarized in the p-axis direction as shown in FIG. 3 enters the incident surface 1026, it is polarized in the direction rotated by the rotation angle β from the p-axis direction as shown in FIG. This process is executed when the linearly polarized light is incident on the reflecting surface 1028. In this case, the polarization direction of the excitation light 9000 is corrected by the polarization direction control unit 1100. As a result, as shown in FIG. 5, linearly polarized excitation light 9000 polarized in a direction rotated by an angle −β obtained by reversing the rotation angle β from the p-axis direction is incident on the incident surface 1026, as shown in FIG. Then, linearly polarized excitation light 9000 polarized in the direction of the p-axis enters the reflecting surface 1028. Thereby, the main polarization direction of the excitation light 9000 incident on the reflection surface 1028 matches the polarization direction of the p-polarized light, the intensity of the p-wave component is stabilized, and the measurement accuracy and sensitivity are improved.

  When the first example of correction is executed, the rotation angle β of the main polarization direction of the excitation light 9000 in the section R1 from the incident surface 1026 to the reflecting surface 1028 is the first index. The polarization direction control unit 1100 causes the polarization direction adjustment mechanism 1038 to set the polarization direction of the excitation light 9000 according to the rotation angle β of the main polarization direction.

  In the second example of correction, when linearly polarized light that is polarized in the p-axis direction and the light amount D coincides with the reference light amount Dref as shown in FIG. 3 is incident on the incident surface 1026, as shown in FIG. This is executed when elliptically polarized light that is mainly polarized in the direction rotated by the rotation angle β and whose intensity I of the main polarization direction component is weaker than the reference intensity Iref is incident on the reflecting surface 1028. In this case, the polarization direction control unit 1100 corrects the polarization direction of the excitation light 9000, and the laser diode unit 1002 corrects the light amount D of the excitation light 9000. As a result, as shown in FIG. 8, linearly polarized excitation light 9000 that is polarized in a direction rotated by an angle −β obtained by reversing the rotation angle β from the p-axis direction is incident on the incident surface 1026, as shown in FIG. In addition, elliptically polarized excitation light 9000 mainly polarized in the direction of the p-axis is incident on the reflecting surface 1028. Further, the light amount D of the excitation light 9000 incident on the incident surface 1026 is corrected by a correction coefficient times the reference light amount Dref, and the intensity I of the main polarization direction component of the excitation light 9000 incident on the reflection surface 1028 is corrected to the reference intensity Iref. The As a result, the main polarization direction of the excitation light 9000 incident on the reflecting surface 1028 coincides with the polarization direction of the p-polarized light, the intensity I of the main polarization direction component is stable, the intensity of the p-wave component is stable, and the measurement accuracy and Sensitivity is improved.

  When the second example of correction is executed, the rotation angle β of the main polarization direction of the excitation light 9000 in the section R1 is the first index. Further, the ratio I / Iref of the intensity I of the main polarization direction component of the excitation light 9000 incident on the reflecting surface 1028 to the reference intensity Iref is the second index. The polarization direction control unit 1100 causes the polarization direction adjustment mechanism 1038 to set the polarization direction of the excitation light 9000 according to the rotation angle β of the main polarization direction. The light amount control unit 1102 causes the laser diode unit 1002 to set the light amount of the excitation light 9000 according to the ratio I / Iref. The correction coefficient multiplied by the reference light amount Dref is the reciprocal Iref / I of the ratio I / Iref. The reference light amount Dref is reflected when there is no rotation of the polarization direction of the excitation light 9000 by the prism 1024, that is, when the linearly polarized light incident on the incident surface 1026 enters the reflecting surface 1028 as linearly polarized light and the main polarization direction does not change. This is the amount of light at which the intensity of the main polarization direction component of the excitation light 9000 incident on the surface 1028 becomes the reference intensity Iref.

  The polarization direction control unit 1100 and the light amount control unit 1102 are embedded computers that execute software. One built-in computer may be responsible for the functions of the polarization direction control unit 1100 and the light amount control unit 1102, or two or more built-in computers may be responsible for the functions of the polarization direction control unit 1100 and the light amount control unit 1102. Also good. Hardware without software may be responsible for all or part of the functions of the polarization direction control unit 1100 and the light amount control unit 1102. The hardware may be an electronic circuit such as an operational amplifier or a comparator, or may be a mechanical mechanism such as a link mechanism.

(Analysis chip)
The schematic diagram of FIG. 10 shows a cross section of the analysis chip. The schematic diagram of FIG. 11 is a perspective view showing a state in which the components of the analysis chip are disassembled in the overlapping direction.

  As shown in FIGS. 10 and 11, in the analysis chip 1006, the prism 1024, the conductor film 1202, the flow path forming seal 1204, the antigen capturing body 1206, the flow path forming lid 1208, the sealing seal 1210, and the barcode label. 1034 is overlaid.

  The flow path forming seal 1204 and the sealing seal 1210 are made of double-sided adhesive tape. The joint surface 1214 of the composite 1212 of the prism 1024, the conductor film 1202 and the antigen capturing body 1206 and the joint surface 1216 of the flow path forming lid 1208 are pasted together by a flow path forming seal 1204. A sealing seal 1210 is attached to the sealing surface 1218 of the flow path forming lid 1208.

  The first main surface 1220 of the conductor film 1202 is in close contact with the reflecting surface 1028, and the second main surface 1222 of the conductor film 1202 is not in close contact with the reflecting surface 1028. The antigen capturing body 1206 is fixed on the second main surface 1222.

  When the evanescent wave that exudes from the reflection surface 1028 and the plasmon in the conductor film 1202 resonate when the excitation light 9000 is totally reflected on the reflection surface 1028, the electric field of the evanescent wave is enhanced by the conductor film 1202. The The analysis chip 1006 is desirably a structure in which each piece has a length of several millimeters or several centimeters, but may be replaced with a small structure or a large structure that is not included in the category of “chip”. Good.

(Barcode label)
A barcode 1224 is formed on the barcode label 1034. The bar code 1224 describes a first index and a second index. As a result, the analysis chip 1006 is attached with the first index and the second index. The barcode 1224 is provided at a position where it can be read from the barcode reader 1036 without hindering the incident of the excitation light 9000, the emission of the measurement light 9002, the emission of the reflected light 9004, the injection of the sample 9006 and the fluorescent labeling liquid 9008, and the like.

  Instead of the bar code 1224, a hologram, characters, symbols, a two-dimensional bar code, or the like may be formed on the label, and the first index and the second index may be described on these labels. These may be formed directly on the analysis chip 1008. A notch may be formed in the analysis chip 1006, and the first index and the second index may be described by the notch.

  The first index and the second index may be actually measured or theoretically estimated, but are preferably actually measured by the measurement method described later.

  The first index may be a rotation angle β or the like that directly represents the degree of rotation of the main polarization direction of the excitation light 9000 in the section R1, or the rotation of the main polarization direction of the excitation light 9000 in the section R1. It may be information that is the basis for deriving the degree. For example, when the degree of rotation of the main polarization direction of the excitation light 9000 in the section R1 is actually measured, original data output from a sensor, an encoder, or the like of the measurement apparatus may be used. Similarly, the second index may be a ratio I / Iref or the like that directly represents a decrease in intensity of the main polarization direction component of the excitation light 9000 in the interval R1, or the excitation light 9000 in the interval R1. It may be information serving as a basis for deriving the degree of decrease in intensity of the main polarization direction component. For example, when the degree of intensity of the main polarization direction component of the excitation light 9000 in the section R1 is actually measured, original data output from a sensor, an encoder, or the like of the measurement apparatus may be used.

(prism)
The prism 1024 is a trapezoidal column. One inclined side surface of the trapezoidal column is an incident surface 1026, the larger parallel side surface of the trapezoidal column is a reflecting surface 1028, and the other inclined side surface of the trapezoidal column is an output surface 1030. The prism 1024 is preferably an isosceles trapezoidal column. Thereby, the incident surface 1026 and the exit surface 1030 are symmetric, and the S wave component of the reflected light 9004 is less likely to stay inside the prism 1024. The prism 1024 may be other than the trapezoidal column. As long as the entrance surface 1026, the reflection surface 1028, and the exit surface 1030 are provided, the prism 1024 may be replaced with a shape having a shape not included in the category of the prism.

  The prism 1024 is a dielectric medium made of a material transparent to the excitation light 9000. The prism 1024 is made of glass, resin, or the like. The prism 1024 is preferably made of a resin having a refractive index of 1.4 to 1.6 and a small birefringence. The prism 1024 is made of, for example, a cycloolefin polymer.

  The prism 1024 may be manufactured in any way, for example, by injection molding. In the injection molding, after the molten thermoplastic resin material is introduced into the mold, the mold is cooled to form a molded body.

(Conductor film)
The conductor film 1202 is made of a conductor that generates surface plasmon resonance. The conductor film 1202 is preferably made of gold. However, the conductor film 1202 may be made of a metal such as silver, copper, or aluminum, or an alloy containing these metals.

  The conductor film 1202 is a thin film. The film thickness of the conductor film 1202 is desirably 100 nm or less, and more desirably 40 to 50 nm. However, the film thickness of the conductor film 1202 may be outside this range.

  The conductor film 1202 is formed by sputtering, vapor deposition, plating, or the like. However, the conductor film 1202 may be formed by other methods.

(Antigen trap)
The antigen capturing body 1206 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 1206, the antigen capturing body 1206 does not move.

  Antigen capturing body 1206 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 1206, the antigen to be detected contained in the sample liquid 9006 binds to the antibody contained in the antigen capturing body 1206 and is detected in the sample liquid 9006. The antigen of interest is captured by the antigen capturing body 1206.

  The antigen capturing body 1206 is patterned by, for example, a rubber applicator. The diameter of the antigen capturing body 1206 is typically 2 mm, and the layer thickness of the antigen capturing body 1206 is typically 100 nm or less.

(Flow path forming tape and flow path forming lid)
A flow path 1226 is formed in the flow path forming tape 1204. The flow path 1226 is a hole that penetrates one main surface and the other main surface of the flow path forming tape 1204. The flow path 1226 includes an inlet side flow path 1228, a reaction chamber 1230, and a reservoir side flow path 1232.

  An inlet side channel 1234 and a reservoir side channel 1236 are also formed in the channel forming lid 1208. The inlet side channel 1234 and the liquid reservoir side channel 1236 are holes that penetrate the joint surface 1216 and the sealing surface 1218. The opening on the sealing surface 1218 side of the inlet side channel 1234 becomes an injection port 1238. Near the sealing surface 1218 of the liquid reservoir side channel 1236 becomes a wide liquid reservoir 1240. The inlet side channel 1234 and the reservoir side channel 1236 of the channel forming lid 1208 are connected to the inlet side channel 1228 and the reservoir side channel 1232 of the channel forming tape 1204, respectively.

  The planar position of the reaction chamber 1230 and the planar position of the antigen capturing body 1206 are matched, and the antigen capturing body 1206 is exposed to the reaction chamber 1230.

  The flow path forming tape 1204 and the flow path forming lid 1208 form a flow path structure in which a flow path is formed as a whole. The flow path forming tape 1204 and the flow path forming lid 1208 may be integrated.

  When the sample liquid 9006 or the fluorescent labeling liquid 9008 is injected into the analysis chip 1006, the reaction chamber 1230 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 1206, respectively. To touch.

  The flow path forming tape 1204 and the flow path forming lid 1208 prevent the sample liquid 9006 or the fluorescent labeling liquid 9008 from scattering and make the amount of the sample liquid 9006 or the fluorescent labeling liquid 9008 in contact with the antigen capturing body 1206 constant. Contribute to. However, the flow path forming tape 1204 and the flow path forming lid 1208 may be omitted, and the sample liquid 9006 or the fluorescent labeling liquid 9008 may be dropped directly on the antigen capturing body 1206.

(Laser diode unit)
The schematic diagram of FIG. 12 shows a laser diode unit.

  As shown in FIG. 12, in the laser diode unit 1002, a 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 an irradiation region 1800, which will be described later, close to a circle, the laser diode 1300 has a laser diode holding mechanism in a posture that includes an optical path of the excitation light 9000 and includes an elliptical short axis in a plane perpendicular to the reflection surface 1028. Held by 1306.

  The amount of laser light emitted from the laser diode 1300 is controlled by the power supply control circuit 1312. By adjusting the amount of laser light emitted from the laser diode 1300, the amount of excitation light 9000 applied to the analysis chip 1006 is also adjusted. The light quantity of the excitation light 9000 applied to the analysis chip 1006 may be adjusted by inserting / removing a neutral density filter in the optical path of the excitation light 9000 by an insertion / extraction mechanism.

  When the wavelength and light amount of the laser light emitted from the laser diode 1300 depend on the temperature, preferably, the temperature adjustment circuit 1308 measures the light amount of the sub beam 9014 by the photodiode 1310 and measures the light amount of the sub beam 9014. The power supplied to the laser diode 1300 is regression controlled by the power supply control circuit 1312 so that the result is maintained constant. 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 1042, the polarization direction adjustment mechanism 1038, and the shaping optical system 1044. And the incident angle adjusting mechanism 1032 sequentially. When the excitation light 9000 passes through the first wave shaper 1042, the excitation light 9000 is tuned. When the excitation light 9000 passes through the polarization direction adjusting mechanism 1038, the polarization direction of the excitation light 9000 is adjusted. When the excitation light 9000 passes through the shaping optical system 1044, 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 1032, the incident angle θ of the excitation light 9000 on the reflection surface 1028 is adjusted.

(First wave rectifier)
The schematic diagram of FIG. 13 shows a first wave rectifier.

  As shown in FIG. 13, 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 1042. The first wave shaper 1042 may include an optical filter other than these. The first wave adjuster 1042 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. 14 shows a polarization direction adjusting mechanism.

  As shown in FIG. 14, in the polarization direction adjusting mechanism 1038, 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 a polarization direction in which the oozing of the evanescent wave from the reflecting surface 1028 is maximized and a polarization direction in which the oozing of the evanescent wave from the reflecting surface 1028 is eliminated.

  The polarization direction adjusting mechanism 1038 may be replaced with a mechanism that rotates the laser diode unit 1002 or the laser diode 1300 around the optical axis.

(Incident angle adjustment mechanism)
The schematic diagram of FIG. 15 shows an incident angle adjustment mechanism.

  As shown in FIG. 15, the excitation light 9000 is reflected by the reflecting mirror 1600 when passing through the incident angle adjustment mechanism 1032, and the direction in which the excitation light 9000 travels by the reflection is the second direction from the first direction 9016 toward the analysis chip 1006. The direction 9018 is bent.

  When the incident angle θ is adjusted by the incident angle adjusting mechanism 1032, the reflecting mirror position is adjusted by the reflecting mirror position adjusting mechanism 1604 while the reflecting mirror angle is adjusted by the reflecting mirror angle adjusting mechanism 1602. Thereby, only incident angle (theta) is adjusted and the irradiation position X of the excitation light 9000 in the reflective surface 1028 is maintained.

  When the irradiation position X is adjusted by the incident angle adjusting mechanism 1032, the reflecting mirror angle is fixed, and the reflecting mirror position is adjusted by the reflecting mirror position adjusting mechanism 1604. Thereby, only the irradiation position X is adjusted and the incident angle θ is maintained.

  When the incident angle adjustment mechanism 1032 is in a blocking state where the excitation light 9000 is not guided to the incident surface 1026, 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. A non-reflective light absorbing material is attached to the light absorbing surface. When the incident angle adjustment mechanism 1032 is in a non-blocking state where the excitation light 9000 is guided to the incident surface 1026, the reflection mirror angle is adjusted by the reflection mirror angle adjustment mechanism 1602, and the excitation light 9000 is applied to the light reflection surface of the reflection mirror 1600. Is applied. A multilayer film is formed on the light reflecting surface. This makes it difficult for phase shift, dimming, and the like to occur regardless of the phase angle and reflection angle of the excitation light 9000. 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 light reflecting 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 1032 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 an incident angle adjusting mechanism that adjusts the position and angle of the laser diode unit 1002. Alternatively, the incident angle θ and the irradiation position X may be adjusted by an incident angle adjusting mechanism that adjusts the position and angle of the analysis chip 1006.

(Measurement optical system)
The schematic diagram of FIG. 16 shows a measurement light optical system.

  As shown in FIG. 16, the measurement light 9002 emitted from the conductor film 1204 or the antigen capturing body 1206 toward the second main surface 1222 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 in the optical path of the measurement light 9002, and light having the same wavelength as the excitation light 9000 is measured from the measurement light 9002 by the second bandpass filter 1706. Is selectively attenuated. Thereby, the surface plasmon excitation fluorescence is mainly guided to the photomultiplier tube 1010, and the measurement accuracy and sensitivity 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 in 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 analysis chip 1006 by the liquid feeding mechanism 1014, the analysis chip 1006 and the reagent chip 1046 are arranged in the pretreatment chamber 1020. Further, the sample liquid 9006 is sucked from the dilution container 1050 of the reagent chip 1046 by the liquid feed pump 1048, the liquid feed pump 1048 is transported by the liquid feed pump transport mechanism 1052, and the sample liquid 9006 is transferred to the injection port 1238 by the liquid feed pump 1048. Discharged. When the sample liquid 9006 is discharged, the discharge port of the liquid feed pump 1048 breaks the sealing seal 1210 and is inserted into the injection port 1238.

  Even when the fluorescent labeling solution 9008 is injected into the analysis chip 1006 by the liquid feeding mechanism 1014, the analysis chip 1006 and the reagent chip 1046 are arranged in the pretreatment chamber 1020. Further, the fluorescent labeling liquid 9008 is aspirated from the fluorescent labeling liquid container 1054 of the reagent chip 1046 by the liquid feeding pump 1048, the liquid feeding pump 1048 is conveyed by the liquid feeding pump conveyance mechanism 1052, and the fluorescence is supplied to the injection port 1238 by the liquid feeding pump 1048. The labeling liquid 9008 is discharged.

  When the sample 9020 is diluted, the reagent chip 1046 is placed in the pretreatment chamber 1020. The liquid feeding mechanism 1014 feeds the specimen 9020 from the specimen container 1056 to the dilution container 1050, and sends the dilution liquid 9022 from the dilution liquid container 1058 to the dilution container 1050 to prepare the sample liquid 9006.

  The liquid feed pump 1048 is cleaned with the cleaning liquid 9024 accommodated in the cleaning liquid container 1060 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 includes a fluorescently labeled antibody that is fluorescently labeled and binds to the antigen to be detected.

  The injection injected into the analysis chip 1006 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 1048 from the liquid feeding source to the liquid feeding destination, a liquid feeding mechanism that feeds 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. 17 is a plan view showing a desirable relationship between the irradiation region, the measurement region, and the reaction region 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 in which the amount of emitted measurement light 9002 is measured by the photomultiplier tube 1010. The reaction region 1804 is a region where an antigen capturing body 1206 is provided and an antibody and an antigen contained in the antigen capturing body 1206 are combined. 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. As a result, even if the irradiation position X is shifted due to variations in the prism 1024, 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 flowcharts of FIGS. 18 and 19 show an outline of the procedure for detecting an antigen.

  As shown in FIG. 18, the measurement is prepared (step S101), the antibody and the antigen are reacted (step S102), and the analysis chip 1006 is loaded (step S103).

  After the analysis chip is loaded (after step S103), the polarization direction control unit 1100 causes the barcode reader 1036 to read the first indicator (step S104), and obtains the first indicator from the barcode reader 1036. Then, the polarization direction adjustment mechanism 1038 is caused to set the polarization direction of the excitation light 9000 according to the first index (step S106).

  Further, the light quantity control unit 1102 causes the barcode reader 1036 to read the second index (step S107), acquires the second index from the barcode reader 1036 (step S108), and power according to the second index. The supply control circuit 1312 is caused to set the light amount of the excitation light 9000 (step S109).

  The first index and the second index may be read before the analysis chip 1006 is loaded or in parallel with the analysis chip 1006 being loaded. The order of reading the first index and reading the second index may be switched, and the first index and the second index may be read simultaneously. The order of acquisition of the first index and acquisition of the second index may be switched, and the first index and the second index may be acquired simultaneously. The order of setting the polarization direction of the excitation light 9000 and the setting of the light amount of the excitation light 9000 may be switched, and the polarization direction of the excitation light 9000 and the amount of light of the excitation light 9000 may be set in parallel.

  The excitation light 9000 is irradiated to the prism 1024 of the loaded analysis chip 1006.

  As shown in FIG. 19, the incident angle θ is set to the measurement angle θm in a state where the analysis chip 1006 is irradiated with the excitation light 9000 (step S110), and processing that is desirably performed is performed (steps S111 and S112). The analysis chip 1006 is unloaded (step S113), and the antigen and the fluorescently labeled antibody are reacted (step S114).

  Preferably, the irradiation position X is optimized (step S110) after the incident angle θ is set to the measurement angle θm (after step S110) and before the analysis chip 1006 is unloaded (before step S113). S111), the amount of autofluorescence is measured (step S112). Both or one of the optimization of the irradiation position X (step S111) and the measurement of the amount of autofluorescent light (step S112) may be omitted.

  After the antigen and the fluorescently labeled antibody are reacted (after step S114), the analysis chip 1006 is reloaded (step S115), the amount of surface plasmon excitation fluorescence is measured (step S116), and the measurement result is the sample. The number and the like are stored and displayed (step S117), and the surface plasmon excitation fluorescence measuring apparatus 1000 is initialized (step 118). When the surface plasmon excitation fluorescence measuring apparatus 1000 is initialized, the movable object such as the reflecting mirror 1600 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)
In measurement preparation (step S101), the analysis chip 1006 is prepared, and the analysis chip 1006 is arranged in the pretreatment chamber 1020.

  Separately, a reagent chip 1046 is prepared, the reagent chip 1046 is arranged in the pretreatment chamber 1020, and the reagent chip 1046 is pretreated by the liquid feeding mechanism 1014. 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 of the analysis chip 1006 may be performed after the preparation, arrangement, and pretreatment of the reagent chip 1046, or may be performed in parallel with the preparation, arrangement, and pretreatment of the reagent chip 1046.

(Reaction between antibody and antigen)
In the reaction between the antibody and the antigen (step S102), the liquid feeding mechanism 1014 injects the sample liquid 9006 into the analysis chip 1006.

  When the sample liquid 9006 is injected into the analysis chip 1006, the sample liquid 9006 contacts the antigen capturing body 1206, and the antigen contained in the sample liquid 9006 binds to the antibody contained in the antigen capturing body 1206, and The contained antigen is captured by the antigen capturing body 1206.

(Setting incident angle θ to measurement angle θm)
In setting the incident angle θ to the measurement angle θm (step S110), the insertion / extraction mechanism 1708 extracts the second band-pass filter 1706 from the optical path of the measurement light 9002. Thereby, the scattered light is not shielded, and the amount of the scattered light can be measured by the photomultiplier tube 1010.

  Subsequently, in a state where the second band-pass filter 1706 is removed, the incident angle adjusting mechanism 1032 scans the incident angle θ within an angle range where the total reflection condition is satisfied, and the photomultiplier tube 1010 measures the light amount I. To do. Thereby, the amount of scattered light 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 resonance angle θr, which is the incident angle θ at which the light amount Is is maximized, is specified from the relationship Is (θ), and the measurement angle is the incident angle θ at which the measurement is performed from the resonance angle θr. θm is determined. The deviation between the resonance angle θr at which the amount of scattered light is maximized and the incident angle θmax at which the enhancement of the electric field is maximized is small, and the electric field is sufficiently enhanced even if the determined resonance angle θr is directly used as the measurement angle θm. The However, the resonance angle θr may be corrected to the measurement angle θm. When the resonance angle θr at which the amount of scattered light is maximized is obtained, unlike the case where the resonance angle θr at which the amount of reflected light is minimized is obtained, the light amount sensor that measures the amount of surface plasmon excitation fluorescence is the resonance angle θr. The structure of the surface plasmon excitation fluorescence measurement apparatus 1000 is simplified.

  After the measurement angle θm is determined, the incident angle adjustment mechanism 1032 sets the incident angle θ to the measurement angle θm.

(Scanning range of incident angle θ)
The resonance angle θr is generally determined by the material and shape of the prism 1024, the refractive index of the injection, and the like. However, the resonance angle θr slightly changes depending on the type and amount of molecules captured by the antigen capturing body 1206, variation of the prism 1024, 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 is preferentially scanned.

(Optimization of irradiation position)
In the irradiation position optimization (step S111), the photomultiplier tube 1010 measures the light amount I while the incident angle adjusting mechanism 1032 scans the irradiation position X 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.

  After the relationship Is (X) is obtained, the irradiation positions X1 and X2 at which the light amount Is decreases from the maximum value by the same value ΔIs are identified from the relationship Is (X), and the irradiation between the irradiation positions X1 and X2 is performed. The measurement position Xm, which is the position X, is determined.

  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.

  After the measurement position Xm is determined, the reflector position adjustment mechanism 1063 sets the irradiation position X to the measurement position Xm.

  At an incident angle θ near the resonance angle θr, the amount of reflected light 9004 is small, and the reflected light 9004 hardly generates autofluorescence emitted from the prism 1024. 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 weak 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 weak, the measurement of the amount of surface plasmon excitation fluorescence only affects autofluorescence emitted from the vicinity of the reflecting surface 1028.

  When the irradiation region 1800 is arranged at the center of the measurement region 1802 by optimization of the irradiation position (step S111), 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, measurement accuracy and sensitivity are improved.

(Measurement of the amount of autofluorescence)
In the measurement of the amount of autofluorescence (step S112), the photomultiplier tube 1010 measures the amount of light I. As a result, the amount of autofluorescence Iaf is measured. The measurement result is stored in the control calculation unit 1040 until correction described later is completed.

(Reaction between antigen and fluorescently labeled antibody)
In the reaction between the antigen and the fluorescently labeled antibody (step S114), the liquid feeding mechanism 1014 injects the fluorescently labeled liquid 9008 into the analysis chip 1006. When the fluorescent labeling liquid 9008 is injected into the analysis chip 1006, the fluorescent labeling liquid 9008 comes into contact with the antigen capturing body 1206, 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)
In the measurement of the amount of surface plasmon excitation fluorescence (step S116), the insertion / extraction mechanism 1708 inserts the second bandpass filter 1706 into the optical path of the measurement light 9002. Thereby, the scattered light reaching the photomultiplier tube 1010 is reduced, and the accuracy and sensitivity of measurement are improved.

  After the second bandpass filter 1706 is inserted, the incident angle adjustment mechanism 1032 temporarily blocks the excitation light 9000.

  While the excitation light 9000 is blocked, the photomultiplier tube 1010 measures the light quantity I. Thereby, the dark noise amount In is measured.

  After the dark noise amount In is measured, the incident angle adjustment mechanism 1032 resets the incident angle θ to the measurement angle θm.

  After the incident angle θ is reset to the measurement angle θm, the photomultiplier tube 1010 measures the light quantity I. Thereby, the light amount If of the surface plasmon excitation fluorescence at the measurement angle θm is measured. The amount of surface plasmon excitation fluorescence may be measured while the incident angle θ is shifted by a minute angle.

  After the light amount If of the surface plasmon excitation fluorescence is measured, correction is performed by subtracting the light amount Iaf of the autofluorescence and the dark noise amount In from the light amount If of the surface plasmon excitation fluorescence. This improves the accuracy and sensitivity of measurement.

  Among the above-described 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. .

(Measurement of resonance angle θr by reflected light)
Instead of the resonance angle θr at which the amount of scattered light is maximized, a resonance angle θr at which the amount of reflected light 9004 is minimized may be used. When the latter resonance angle θr is used, as shown in FIG. 20, a reflected light amount sensor 1902 for measuring the light amount of the reflected light 9004 is added to the surface plasmon excitation fluorescence measuring apparatus 1000. Further, the reflected light amount sensor 1902 measures the light amount I while the incident angle adjusting mechanism 1032 scans 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. Also, the resonance angle θr, which is the incident angle θ at which the amount of light Ir is minimized, is specified from the relationship Ir (θ), and the measurement angle θm that is the incident angle θ at which the measurement is performed is determined from the resonance angle θr. Since there is a difference between the resonance angle θr at which the amount of scattered light is maximized and the incident angle θ at which the electric field enhancement is maximized, the resonance angle θr is corrected to increase or decrease a minute angle, and the measurement angle θm is determined. The

(Measurement of the first index and the second index)
The flowchart of FIG. 21 shows a procedure for actually measuring the first index and the second index. The schematic diagram of FIG. 22 shows a measuring apparatus that actually measures the first index and the second index.

  As shown in FIGS. 21 and 22, the prism 1024 is prepared (step S131), and the reference prism 1956 is prepared (step S132). The reference prism 1956 is a dielectric medium made of a material that is transparent to the excitation light 9000 and does not generate birefringence. The reference prism 1956 is made of glass, for example. Desirably, the refractive index of the prism 1024 and the refractive index of the reference prism 1956 are the same. As a result, light refraction and reflection at the interface between the prism 1024 and the reference prism 1956 are suppressed, and measurement of the first index and the second index is facilitated. However, even if the refractive index of the prism 1024 and the refractive index of the reference prism 1956 are different, the first index and the second index can be actually measured.

  After the prism 1024 and the reference prism 1956 are prepared (after steps S131 and S132), the reflecting surface 1028 of the prism 1024 and the incident surface 1962 of the reference prism 1956 are put together (step S133). Thus, a composite 1964 of the prism 1024 and the reference prism 1956 is manufactured. In bonding, desirably, matching oil 1954 is interposed between the reflecting surface 1028 of the prism 1024 and the incident surface 1962 of the reference prism 1956. As a result, the gap between the reflecting surface 1028 of the prism 1024 and the incident surface 1962 of the reference prism 1956 is reduced, and the scattering of the evaluation light 9030 between the reflecting surface 1028 of the prism 1024 and the incident surface 1962 of the reference prism 1956 is suppressed. This makes it easier to actually measure the first index and the second index. However, when the adhesion between the reflecting surface 1028 of the prism 1024 and the incident surface 1962 of the reference prism 1956 is good, the matching oil 1964 may be omitted.

  After the composite 1964 is manufactured, the composite 1964 is attached to the measuring device 1948, and the evaluation light 9030 is irradiated to the composite 1964 (step S134). The evaluation light 9030 enters the incident surface 1026 of the prism 1024, passes through the reflecting surface 1028 of the prism 1024 and the incident surface 1962 of the reference prism 1956, and exits from the exit surface 1966 of the reference prism 1956. Evaluation light 9030 is emitted from laser diode 1950, passes through polarization rotator 1952, and enters incident surface 1026 of prism 1024. Desirably, the wavelength, the light amount, and the incident angle of the evaluation light 9030 coincide with the wavelength of the excitation light 9000, the reference light amount Dref, and the expected resonance angle θr, respectively. As a result, the degree of rotation of the main polarization direction and the degree of decrease in the intensity of the main polarization direction component are measured under the same conditions as when the amount of surface plasmon excitation fluorescence is measured, and the polarization direction and light quantity are corrected appropriately. . The evaluation light 9030 is linearly polarized light, and the polarization direction of the evaluation light 9030 is adjusted to the same polarization direction as the p-polarized light with respect to the reflecting surface 1028 of the prism 1024 by a fixed polarization rotator 1952. The laser diode 1950 is, for example, a He—Ne laser and emits a beam having a cross-sectional diameter of 1 mm.

  While the composite 1964 is irradiated with the evaluation light 9030, the degree of rotation of the evaluation light 9030 in the main polarization direction in the section R2 from the incident surface 1026 of the prism 1024 to the output surface 1966 of the reference prism 1956 is measured (step) In step S135, the degree of decrease in intensity of the main polarization direction component of the evaluation light 9030 in the section R2 is measured (step S136).

  Since the reference prism 1956 does not generate birefringence, the degree of rotation of the main polarization direction of the evaluation light 9030 and the degree of decrease in the intensity of the main polarization direction component of the evaluation light 9030 in the section R2 are determined from the incident surface 1026 of the prism 1024 The degree of rotation of the main polarization direction of the evaluation light 9030 and the degree of decrease in the intensity of the main polarization direction component of the evaluation light 9030 in the section R1 up to the reflection surface 1028 of 1024 are identified.

  In the measuring device 1948, the evaluation light 9030 emitted from the emission surface 1966 of the reference prism 1956 passes through the polarization rotator 1958 and reaches the power meter 1960. The polarization rotator 1958 is rotated in units of 15 °, and the light quantity of the evaluation light 9030 is measured by a power meter 1960. Thereby, the rotation angle of the main polarization direction is measured as the degree of rotation of the main polarization direction component of the evaluation light 9030, and the intensity of the main polarization direction component of the evaluation light 9030 relative to the reference intensity is measured as the degree of decrease in the intensity of the main polarization direction component. The ratio is measured. However, the degree of rotation of the evaluation light 9030 in the main polarization direction and the degree of decrease in the intensity of the main polarization direction component of the evaluation light 9030 may be measured by other measurement methods.

  After the degree of rotation of the main polarization direction of the evaluation light 9030 and the degree of decrease in the intensity of the main polarization direction component of the evaluation light 9030 are measured (after steps S135 and S136), the composite 1964 becomes the prism 1024 and the reference prism. 1956 (step S137).

  The separated prism 1024 is used as a member constituting the analysis chip 1006. That is, the analysis chip 1006 including the separated prism 1024, the conductor film 1202, the flow path forming seal 1204, the antigen capturing body 1206, the flow path forming lid 1208, and the sealing seal 1210 is manufactured (step S138).

  The analysis chip 1006 has a bar code in which a first index indicating the rotation angle of the main polarization direction of the evaluation light 9030 and a second index indicating the ratio of the intensity of the main polarization direction component of the excitation light 9030 to the reference intensity are described. A bar code label 1034 on which 1224 is formed is pasted (step S139). Thereby, the first index and the second index are attached to the analysis chip 1006.

(Application to surface plasmon resonance method)
Instead of the measurement by the surface plasmon excitation fluorescence method (SPFS) for measuring the fluorescence excited by the electric field of the enhanced evanescent wave, the surface plasmon resonance method (SPR) for measuring the scattered light by the electric field of the enhanced evanescent wave is used. Measurement may be performed. When measurement by the surface plasmon resonance method is performed, injection of the fluorescent labeling liquid 9008 is omitted, and the amount of reflected light, the amount of scattered light, the resonance angle θr, and all or part of these changes are measured.

  Although the present invention has been described in detail, the above description is illustrative in all aspects, and the present invention is not limited to the above description. Innumerable modifications not illustrated can be envisaged without departing from the scope of the present invention.

1000 Surface plasmon excitation fluorescence measuring apparatus 1002 Laser diode unit 1006 Analysis chip 1010 Photomultiplier tube 1024 Prism 1026 Incident surface 1028 Reflecting surface 1038 Polarization direction adjustment mechanism 1958 Reference prism 1966 Incident surface 1966 Emission surface

Claims (10)

A surface plasmon measuring device for measuring by surface plasmon excitation fluorescence spectroscopy or surface plasmon resonance method,
A light source that emits excitation light;
A dielectric medium transparent to the excitation light, comprising an incident surface on which the 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 reflected by the reflection surface is emitted; Rotating the main polarization direction of excitation light in a section from the incident surface to the reflective surface, comprising a conductor film having 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 A structure attached with a first index indicating the degree of
A polarization direction adjusting mechanism for adjusting the polarization direction of the excitation light applied to the structure;
A reading mechanism for reading the first index;
The first index is obtained from the reading mechanism, and the polarization direction of the excitation light is set to a polarization direction that cancels the rotation of the main polarization direction according to the first index, and the surface plasmon excitation fluorescence is set. A first control unit that maintains the polarization direction of the excitation light in the set polarization direction when the amount of scattered light or the amount of scattered light due to the enhanced electric field of the evanescent wave is measured ;
A surface plasmon measuring device comprising: In the surface plasmon measuring device according to claim 1,
The first index indicates a rotation angle of a main polarization direction when p-polarized light with respect to the reflecting surface is incident on the incident surface,
The first control unit is a surface plasmon measurement device that sets a polarization direction of excitation light to a polarization direction obtained by rotating the polarization direction of p-polarized light by an angle obtained by inverting the rotation angle.   A surface plasmon measuring device for measuring by surface plasmon excitation fluorescence spectroscopy or surface plasmon resonance method,
  A light source that emits excitation light;
  A dielectric medium transparent to the excitation light, comprising an incident surface on which the 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 reflected by the reflection surface is emitted; Rotating the main polarization direction of excitation light in a section from the incident surface to the reflective surface, comprising a conductor film having 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 A structure that is accompanied by a first index that indicates the degree of the above, and that is accompanied by a second index that indicates the degree of decrease in the intensity of the main polarization direction component of the excitation light in the section;
  A polarization direction adjusting mechanism for adjusting the polarization direction of the excitation light applied to the structure;
  A reading mechanism for reading the first index and the second index;
  First control for acquiring the first index from the reading mechanism and causing the polarization direction adjusting mechanism to set the polarization direction of the excitation light to a polarization direction in which the rotation of the main polarization direction is canceled according to the first index. And
  A light amount adjustment mechanism for adjusting the amount of excitation light irradiated to the structure;
  Second control that acquires the second index from the reading mechanism and causes the light amount adjustment mechanism to set the light amount of the excitation light to a light amount that cancels the decrease in the intensity of the main polarization direction component according to the second index. And
A surface plasmon measuring device comprising:   In the surface plasmon measuring device according to claim 3,
  The first index indicates a rotation angle of a main polarization direction when p-polarized light with respect to the reflecting surface is incident on the incident surface,
  The first control unit sets the polarization direction of the excitation light to a polarization direction obtained by rotating the polarization direction of the p-polarized light by an angle obtained by inverting the rotation angle.
Surface plasmon measuring device.   In the surface plasmon measuring device according to claim 3 or claim 4,
  The second index indicates a ratio of the intensity of the main polarization component incident on the reflecting surface to the reference intensity when p-polarized light with respect to the reflecting surface is incident on the incident surface.
  The second control unit causes the light amount of the excitation light to be set to a light amount obtained by multiplying the reference light amount by the reciprocal of the ratio.
Surface plasmon measuring device.   A surface plasmon measuring device for measuring by surface plasmon excitation fluorescence spectroscopy or surface plasmon resonance method,
  A light source that emits excitation light;
  A dielectric medium transparent to the excitation light, comprising an incident surface on which the 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 reflected by the reflection surface is emitted; A conductive film having a first main surface that is in close contact with the reflecting surface and a second main surface that is not in close contact with the reflecting surface; and a main polarization direction component of excitation light in a section from the incident surface to the reflecting surface A structure with an indicator indicating the degree of strength reduction;
  A light amount adjustment mechanism for adjusting the total amount of excitation light irradiated to the structure,
  A reading mechanism for reading the index;
  A controller that acquires the index from the reading mechanism, and causes the light amount adjustment mechanism to set the total light amount of the excitation light to a light amount that cancels the decrease in the intensity of the main polarization direction component according to the index;
A surface plasmon measuring device comprising:   A surface plasmon measuring device for measuring by surface plasmon excitation fluorescence spectroscopy or surface plasmon resonance method,
  A light source that emits excitation light;
  A dielectric medium transparent to the excitation light, comprising an incident surface on which the 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 reflected by the reflection surface is emitted; A conductive film having a first main surface that is in close contact with the reflecting surface and a second main surface that is not in close contact with the reflecting surface; and a main polarization direction component of excitation light in a section from the incident surface to the reflecting surface An index indicating the degree of intensity reduction is attached, and the index indicates the ratio of the intensity of the main polarization component incident on the reflecting surface to the reference intensity when p-polarized light with respect to the reflecting surface is incident on the incident surface. When,
  A light amount adjustment mechanism for adjusting the amount of excitation light irradiated to the structure;
  A reading mechanism for reading the index;
  The amount of light obtained by acquiring the index from the reading mechanism and multiplying the light amount of the excitation light by the reciprocal of the ratio to the reference light amount according to the index and canceling the decrease in the intensity of the main polarization direction component A control unit to be set to
A surface plasmon measuring device comprising:   A surface plasmon measurement method for measuring by surface plasmon excitation fluorescence spectroscopy or surface plasmon resonance method,
  (a) preparing a first dielectric medium having a first incident surface, a reflective surface and a first emission surface and transparent to the excitation light;
  (b) providing a second dielectric medium having a second incident surface and a second emission surface, which is transparent to excitation light and does not generate birefringence;
  (c) bonding the reflective surface and the second incident surface to produce a composite of the first dielectric medium and the second dielectric medium;
  (d) irradiating the composite with evaluation light that is incident on the first incident surface, passes through the reflective surface and the second incident surface, and is emitted from the second exit surface;
  (e) measuring the degree of rotation of the main polarization direction of the evaluation light in the section from the first incident surface to the second emission surface;
  (f) separating the composite into the first dielectric medium and the second dielectric medium after the step (e);
  (g) After the step (f), a structure including a conductor film including the first dielectric medium, 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 A step of producing
  (h) attaching the first index indicating the degree of rotation to the structure;
  (i) reading the first index attached to the structure;
  (j) setting the polarization direction of the excitation light to a polarization direction in which the rotation of the main polarization direction is canceled according to the first index read in the step (i);
  (k) The structure is irradiated with excitation light that is incident on the first incident surface, is totally reflected by the reflecting surface, and is emitted from the first exit surface, and is obtained by surface plasmon resonance or surface plasmon excitation fluorescence spectroscopy. A process of measuring,
A surface plasmon measuring method comprising:   The surface plasmon measuring method according to claim 8,
  (l) measuring the degree of decrease in intensity of the main polarization direction component of the evaluation light in the section;
  (m) attaching a second index indicating the degree of the decrease to the structure;
  (n) reading the second index attached to the structure;
  (o) setting the light amount of the excitation light to a light amount that cancels the decrease in the intensity of the main polarization direction component according to the second index read in the step (n);
A surface plasmon measurement method further comprising:   A surface plasmon measurement method for measuring by surface plasmon excitation fluorescence spectroscopy or surface plasmon resonance method,
  (a) preparing a first dielectric medium having a first incident surface, a reflective surface and a first emission surface and transparent to the excitation light;
  (b) providing a second dielectric medium having a second incident surface and a second emission surface, which is transparent to excitation light and does not generate birefringence;
  (c) bonding the reflective surface and the second incident surface to produce a composite of the first dielectric medium and the second dielectric medium;
  (d) irradiating the composite with evaluation light that is incident on the first incident surface, passes through the reflective surface and the second incident surface, and is emitted from the second exit surface;
  (e) measuring a degree of decrease in intensity of a main polarization direction component of evaluation light in a section from the first incident surface to the second emission surface;
  (f) separating the composite into the first dielectric medium and the second dielectric medium after the step (e);
  (g) After the step (f), a structure including a conductor film including the first dielectric medium, 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 A step of producing
  (h) attaching a second index indicating the degree of the decrease to the structure;
  (i) reading the second index attached to the structure;
  (j) setting the light amount of the excitation light to a light amount that cancels the decrease in the intensity of the main polarization direction component according to the second index read in the step (i);
  (k) The structure is irradiated with excitation light that is incident on the first incident surface, is totally reflected by the reflecting surface, and is emitted from the first exit surface, and is obtained by surface plasmon resonance or surface plasmon excitation fluorescence spectroscopy. A process of measuring,
A surface plasmon measuring method comprising:

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