JP5640873B2 - Surface plasmon excitation fluorescence measurement apparatus and surface plasmon excitation fluorescence measurement method - Google Patents

Description

  The present invention relates to a surface plasmon excitation fluorescence measurement apparatus and a surface plasmon excitation fluorescence measurement method for performing measurement by surface plasmon excitation fluorescence spectroscopy.

  When the excitation light traveling in the dielectric medium is totally reflected at the interface between the conductor film and the dielectric medium, the evanescent wave leaks from the interface, and the plasmon and the evanescent wave in the conductor film interfere with each other. To do. 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. This enhanced electric field is used in measurements by surface plasmon excitation fluorescence spectroscopy (SPFS).

  In the measurement by SPFS, a measurement object is captured on the surface of the conductor film, and the captured measurement object is fluorescently labeled. The enhanced electric field acts on the fluorescently labeled object to be measured, and surface plasmon excitation fluorescence is emitted from the fluorescently labeled object to be measured. Further, the amount of surface plasmon excitation fluorescence is measured, and the presence / absence of the object to be measured, the capture amount of the object to be measured, etc. are specified.

  The electric field enhancement of the evanescent wave depends on the thickness of the conductor film. For this reason, when there is variation in the film thickness of the conductor film, variation in the electric field enhancement intensity of the evanescent wave occurs, variation occurs in the amount of surface plasmon excitation fluorescence, and the measurement accuracy decreases.

  In Patent Document 1, when the amount of surface plasmon excitation fluorescence is measured, the amount of scattered light is measured. Further, the electric field enhancement is obtained from the amount of scattered light, and the amount of surface plasmon excitation fluorescence is corrected by the electric field enhancement.

JP 2009-216532 A

  However, the amount of scattered light varies depending on the state of the surface of the conductor film. For example, the amount of scattered light varies depending on the surface roughness and swell of the conductor film. For this reason, even if correction of patent documents 1 is performed, the accuracy of measurement is insufficient.

  The present invention is made to solve this problem. An object of the present invention is to provide a surface plasmon excitation fluorescence measurement apparatus and a surface plasmon excitation fluorescence measurement method in which the influence of the film thickness of a conductor film is reduced and measurement accuracy is improved.

  The first to sixth aspects of the present invention are directed to a surface plasmon measuring device.

  In the first aspect of the present invention, a conductor film, a dielectric medium, an irradiation mechanism, a measurement mechanism, and a correction unit are provided.

  A sample is supplied to the first main surface of the conductor film. The second main surface of the conductor film and the contact surface of the dielectric medium are in close contact with each other.

  The irradiation mechanism irradiates the dielectric medium with light, and switches the optical path of the light between the first optical path and the second optical path. In the first optical path, light enters the contact surface at an incident angle greater than the critical angle inside the dielectric medium. In the second optical path, light enters the contact surface at an incident angle smaller than the critical angle inside the dielectric medium.

  The measurement mechanism measures the amount of surface plasmon excitation fluorescence and transmitted light. The surface plasmon excitation fluorescence is emitted from the object to be measured included in the sample while the dielectric medium is irradiated with the light passing through the first optical path. The transmitted light is transmitted through the conductor film while the light passing through the second optical path is irradiated on the dielectric medium.

  The correction unit corrects the measurement value of the amount of surface plasmon excitation fluorescence with the measurement value of the amount of transmitted light.

  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 light source, the optical system, and the drive mechanism are provided in the irradiation mechanism. The optical system guides the optical path of the light emitted to the light source to the dielectric medium. The drive mechanism drives the optical system and switches the optical path of light between the first optical path and the second optical path.

  The third aspect of the present invention adds further matters to the first or second aspect of the present invention. In the third aspect of the present invention, the second optical path is perpendicular to the second main surface.

  The fourth aspect of the present invention adds a further matter to any one of the first to third aspects of the present invention. In the fourth aspect of the present invention, a light quantity sensor is provided in the measurement mechanism. The light amount sensor is on the optical path of surface plasmon excitation fluorescence and on the optical path of transmitted light.

  The fifth aspect of the present invention adds further matters to any of the first to fourth aspects of the present invention. In the fifth aspect of the present invention, a transmittance derivation unit, an electric field enhancement specifying unit, and a normalization unit are provided in the correction unit. The transmittance deriving unit obtains a measured value of the amount of transmitted light from the measurement mechanism, and derives an index of the transmittance of the conductor film from the measured value of the amount of transmitted light. The electric field enhancement specifying unit specifies the electric field enhancement from an index of the transmittance of the conductor film. The normalization unit obtains a measurement value of the amount of surface plasmon excitation fluorescence from the measurement mechanism, and normalizes the measurement value of the amount of surface plasmon excitation fluorescence based on the electric field enhancement.

  The sixth aspect of the present invention adds further matters to the fifth aspect of the present invention. In the sixth aspect of the present invention, the film thickness deriving unit and the electric field enhancement deriving unit are provided in the electric field enhancement specifying unit. The film thickness deriving unit derives the film thickness of the conductor film from the index of the transmittance of the conductor film. The electric field enhancement strength deriving unit derives the electric field enhancement strength from the thickness of the conductor film.

  The seventh aspect of the present invention is directed to a surface plasmon excitation fluorescence measurement method.

In the seventh aspect of the present invention, a conductor film and a dielectric medium are prepared. The second main surface of the conductor film and the contact surface of the dielectric medium are in close contact with each other. After the conductor film and the dielectric medium are prepared, the amount of transmitted light passing through the conductor film is measured while light is incident on the contact surface at an incident angle smaller than the critical angle inside the dielectric medium. When the optical path of light does not pass through the flow path formed on the first main surface of the conductor film, before or after the amount of transmitted light is measured or at the same time as the amount of transmitted light is measured. A sample is supplied to the first main surface. When the optical path of light passes through the flow path formed on the first main surface of the conductor film, the sample is supplied to the first main surface after the amount of transmitted light is measured. After the sample is supplied, the amount of surface plasmon excitation fluorescence emitted from the measurement object contained in the sample is measured while light is incident on the contact surface at an incident angle greater than the critical angle inside the dielectric medium. The measured value of the amount of surface plasmon excitation fluorescence is corrected by the measured value of the amount of transmitted light.

  According to the first and seventh aspects of the present invention, the influence of the film thickness of the conductor film is reduced, and the measurement accuracy is improved.

  According to the second aspect of the present invention, the light passing through the first optical path and the light passing through the second optical path are emitted from the same light source, the number of light sources is reduced, and the surface plasmon excitation fluorescence measuring apparatus is Simplified.

  According to the third aspect of the present invention, when the amount of transmitted light is measured, the influence of light reflection at the interface between the conductor film and the dielectric medium is reduced, and the measurement accuracy is improved.

  According to the fourth aspect of the present invention, the amount of surface plasmon excitation fluorescence and the amount of transmitted light are measured by the same light amount sensor, the number of light amount sensors is reduced, and the surface plasmon excitation fluorescence measurement device is simplified. .

  According to the fifth aspect of the present invention, the influence of the change in the electric field enhancement is eliminated, and the measurement accuracy is 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 of a surface plasmon excitation fluorescence measuring device. It is an exploded view of a sensor chip. It is a schematic diagram of the state by which the sample liquid was inject | poured into the flow path. It is a schematic diagram of the state by which the fluorescent labeling liquid was inject | poured into the flow path. It is a schematic diagram of the optical path of excitation light. It is a schematic diagram of the optical path of film thickness measurement light. It is a block diagram of a controller. It is a graph which shows the relationship between the transmittance | permeability and a film thickness. It is a graph which shows the relationship between a film thickness and an electric field enhancement intensity. It is a flowchart which shows operation | movement of a surface plasmon excitation fluorescence measuring device. It is a graph which shows the relationship between an SPR curve and an incident angle, and electric field enhancement.

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

  As shown in FIG. 1, the surface plasmon excitation fluorescence measurement apparatus 1000 includes a sensor chip 1002, an irradiation mechanism 1004, a measurement mechanism 1006, a supply mechanism 1008, and a controller 1010. The sensor chip 1002 includes a gold film 1012, a prism 1014, an antibody solid layer film 1016, and a flow path forming body 1018. The irradiation mechanism 1004 includes a light source 1020, an optical system 1022, and a driving mechanism 1024. The measurement mechanism 1006 includes a charge coupled device (CCD) sensor 1026, a low pass filter 1028, a neutral density (ND) filter 1030, a filter replacement mechanism 1032, and a photodiode 1034. The optical system 1022 includes a linearly polarizing plate 1036, an optical path switching mirror 1038, and a film thickness measurement light introducing mirror 1040.

  The surface plasmon excitation fluorescence measuring apparatus 1000 performs measurement by surface plasmon excitation fluorescence spectroscopy (SPFS). An object to be measured is captured by the antibody solid layer film 1016. The captured object to be measured is fluorescently labeled.

  Further, the excitation light 1042 is guided to the prism 1014, and the amount of the surface plasmon excitation fluorescence 1044 emitted from the fluorescently labeled measurement object is measured.

  Further, the film thickness measurement light 1050 is guided to the prism 1014, and the amount of transmitted light 1046 transmitted through the gold film 1012 is measured. The measurement value of the light amount of the surface plasmon excitation fluorescence 1044 is corrected by the measurement value of the light amount of the transmitted light 1046.

  The amount of transmitted light 1046 depends on the thickness of the gold film 1012. For this reason, this correction reduces the influence of the film thickness of the gold film 1012 on the measurement value of the light amount of the surface plasmon excitation fluorescence 1044, and improves the measurement accuracy. The amount of transmitted light 1046 is less affected by the surface roughness and swell of the gold film 1012 when compared to the amount of scattered light.

  When the light amount of the surface plasmon excitation fluorescence 1044 is measured, the excitation light 1042 is irradiated to the prism 1014. The excitation light 1042 is totally reflected by the contact surface 1048 inside the prism 1014. The incident angle θ1 of the excitation light 1042 to the contact surface 1048 is set to the resonance angle θr. While the excitation light 1042 is applied to the prism 1014, an evanescent wave leaks from the contact surface 1048 to the gold film 1012, and the evanescent wave and the plasmon in the gold film 1012 resonate to enhance the electric field of the evanescent wave. Is done. This enhanced electric field acts on the fluorescently labeled object to be measured, and surface plasmon excitation fluorescence 1044 is emitted from the fluorescently labeled object to be measured. The amount of light of the surface plasmon excitation fluorescence 1044 is measured by the CCD sensor 1026.

  When the light amount of the transmitted light 1046 is measured, the film thickness measurement light 1050 is irradiated on the prism 1014. The incident angle θ2 of the film thickness measurement light 1050 on the contact surface 1048 is set to an angle smaller than the critical angle θc. While the film thickness measurement light 1050 is applied to the prism 1014, the film thickness measurement light 1050 passes through the gold film 1012, and the amount of transmitted light 1046 is measured by the CCD sensor 1026.

{Sensor chip}
The schematic diagram of FIG. 2 is an exploded view of the sensor chip.

  As shown in FIGS. 1 and 2, in the sensor chip 1002, the antibody solid layer film 1016 is fixed on the first main surface 1052 of the gold film 1012, and a flow path is formed on the first main surface 1052 of the gold film 1012. The body 1018 is bonded, and the second main surface 1053 of the gold film 1012 and the contact surface 1048 of the prism 1014 are in close contact with each other. A flow path 1054 is formed in the flow path forming body 1018. The antibody solid layer film 1016 is inside the flow path 1054.

  The sensor chip 1002 is desirably a structure in which the length of each piece is in the range of several millimeters to several centimeters, but a smaller structure or a larger structure not included in the category of “chip”. It may be replaced with a thing.

{Gold film}
The gold film 1012 is a thin film. The film thickness of the gold film 1012 is desirably 30 to 70 nm. However, the film thickness of the gold film 1012 may be outside this range.

  The gold film 1012 is formed by sputtering, vapor deposition, plating, or the like. However, the gold film 1012 may be formed by other methods.

  The gold film 1012 may be replaced with a film made of another type of conductor that generates surface plasmon resonance. For example, the gold film 1012 may be replaced with a film made of a metal such as silver, copper, or aluminum or an alloy containing these metals.

{prism}
The prism 1014 is a trapezoidal column. Desirably, the prism 1014 is an isosceles trapezoidal column. One inclined side surface of the prism 1014 becomes an incident surface 1066 of the excitation light 1042. The narrow parallel side surface of the prism 1014 becomes the incident surface 1068 of the film thickness measurement light 1050. The wide parallel side surface of the prism 1014 becomes a reflection surface 1070 for the excitation light 1042 and an emission surface 1072 for the film thickness measurement light 1050. The other inclined surface of the prism 1014 becomes the exit surface 1074 of the excitation light 1042. The contact surface 1048 occupies all or part of the wide parallel side surface of the prism 1014. The prism 1014 has a shape in which the excitation light 1042 can be incident on the contact surface 1048 at an incident angle θ1 that is equal to or greater than the critical angle θc, and the film thickness measurement light 1050 is incident on the contact surface 1048 at an incident angle θ2 that is smaller than the critical angle θc. It is decided to be able to.

  As long as the excitation light 1042 can be incident on the contact surface 1048 at an incident angle θ1 equal to or greater than the critical angle θc, and the film thickness measurement light 1050 can be incident on the contact surface 1048 at an incident angle θ2 smaller than the critical angle θc, the prism can be used. 1014 may be other than the trapezoidal column, and the prism 1014 may be replaced with a shape not included in the category of “prism”.

  The prism 1014 is a dielectric medium made of a material that is transparent with respect to the excitation light 1042 and the transmitted light 1046. The prism 1014 is made of glass, resin, or the like. Desirably, the prism 1014 is made of a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

  When the prism 1014 is made of resin, the prism 1014 is preferably manufactured by injection molding.

{Channel formation body}
A flow path 1054 is formed in the flow path forming body 1018. A reaction chamber 1076 is provided in the middle of the channel 1054. The reaction chamber 1076 is exposed at the joint surface 1078. When the flow path forming body 1018 is bonded to the gold film 1012, the planar position of the reaction chamber 1076 and the planar position of the antibody solid layer film 1016 are matched. The antibody solid film 1016 is exposed to the reaction chamber 1076. The flow path 1054 is exposed to the non-bonding surface 1080 of the flow path forming body 1018, and the inlet 1082 is formed in the non-bonding surface 1080. The flow path forming body 1018 and the gold film 1012 are bonded together by adhesion, laser welding, ultrasonic welding, clamp pressure bonding, or the like. However, the flow path forming body 1018 and the gold film 1012 may be joined by other methods.

  The flow path forming body 1018 is made of a material transparent to the surface plasmon excitation fluorescence 1044 and the film thickness measurement light 1050. The flow path forming body 1018 is made of, for example, resin.

  The flow path forming body 1018 prevents scattering of the sample solution or the fluorescent labeling solution, and contributes to making the amount of the sample solution or the fluorescent labeling solution in contact with the antibody solid layer film 1016 constant. However, the flow path forming body 1018 may be omitted, and the sample solution or the fluorescent labeling solution may be dropped directly on the antibody solid layer film 1016.

{Supply mechanism}
The supply mechanism 1008 injects the sample solution and the fluorescent labeling solution into the channel 1054. When the sample solution or the fluorescent labeling solution is injected into the channel 1054, the reaction chamber 1076 is filled with the sample solution or the fluorescent labeling solution, respectively, and the antibody solid layer film 1016 on the first main surface 1052 is filled with the sample solution or A fluorescent labeling solution is supplied, and the sample solution or the fluorescent labeling solution contacts the antibody solid layer film 1016.

{Injection of sample liquid and fluorescent labeling liquid into flow channel}
The schematic diagram of FIG. 3 shows a state in which the sample liquid is injected into the flow path. The schematic diagram of FIG. 4 shows a state in which the fluorescent labeling solution is injected into the flow path.

  As shown in FIG. 3, when the sample solution 1056 is injected into the flow channel 1054, the antigen 1058 contained in the sample solution 1056 and the antibody 1064 contained in the antibody solid layer film 1016 are combined by biochemical reaction or immune reaction. Then, the antigen 1058 is captured by the antibody solid film 1016. When the fluorescent labeling solution 1060 is injected into the flow channel 1054 in a state where the antigen 1058 is captured by the antibody solid layer film 1016, the antigen 1058 captured by the antibody solid layer film 1016 and the antigen 1058 are captured as shown in FIG. The fluorescently labeled antibody 1062 contained in the fluorescently labeled solution 1060 is bound by biochemical reaction or immune reaction, and the antigen 1058 captured by the antibody solid layer film 1016 is fluorescently labeled on the fluorescently labeled antibody 1062. The immune reaction is also called an antigen-antibody reaction.

{Sample solution and fluorescent labeling solution}
The sample liquid 1056 is typically a human sample such as blood, but may be a sample from a non-human organism or a non-animal sample. Other pretreatments such as dilution, blood cell separation, reagent mixing, etc. may be performed on the harvest.

  The fluorescent labeling liquid 1060 includes a fluorescently labeled antibody 1062 that binds to the antigen 1058 of the object to be measured. The fluorescently labeled antibody 1062 includes a chemical structure portion that becomes a fluorescent label that emits fluorescence.

  Instead of the sample solution 1056 and the fluorescent labeling solution 1060, a sample made of a gas or a fluid solid and a fluorescently labeled antibody-containing material may be injected into the flow channel 1054, respectively.

{Antibody solid film}
The antibody solid layer film 1016 is made of a non-fluid. Therefore, even if the sample liquid 1056 or the fluorescent labeling liquid 1060 comes into contact with the antibody solid layer film 1016, the antibody solid layer film 1016 does not move.

  The antibody 1064 is uniformly distributed. The capturing body for capturing the object to be measured is changed according to the object to be measured, and the supplement of the object to be measured is not limited to the antibody solid film 1016.

  The antibody solid film 1016 is patterned by, for example, a rubber applicator. The diameter of the antibody solid layer film 1016 is typically 2 mm, and the layer thickness of the antibody solid layer film 1016 is typically 100 nm or less.

{light source}
As shown in FIG. 1, the light source 1020 emits light. Preferably, the light source 1020 is a laser diode, and the light emitted from the light source 1020 is a parallel light beam, linearly polarized light, and monochromatic light.

  The light source 1020 may be other than a laser diode. For example, the light source 1020 may be a light emitting diode, a mercury lamp, a laser other than a laser diode, or the like. When the light emitted from the light source 1020 is not a parallel light beam, the light is converted into a parallel light beam by a lens, a mirror, a slit, or the like. When the light emitted from the light source 1020 is not linearly polarized light, the light is converted into linearly polarized light by a polarizer or the like. When the light emitted from the light source 1020 is not monochromatic light, the light is converted into monochromatic light by a diffraction grating or the like.

{Linear polarizing plate}
The linearly polarizing plate 1036 is on the optical path of the excitation light 1042 and converts the excitation light 1042 into linearly polarized light. The polarization direction of the excitation light 1042 is selected so that the excitation light 1042 is p-polarized with respect to the contact surface 1048. Thereby, the leakage of the evanescent wave increases, the surface plasmon excitation fluorescence 1044 increases, and the sensitivity of measurement is improved.

{Optical system}
The schematic diagram of FIG. 5 shows the optical path of excitation light. The schematic diagram of FIG. 6 shows the optical path of the film thickness measurement light.

  As shown in FIG. 5, when the light emitted from the light source 1020 is used as the excitation light 1042, the light emitted from the light source 1020 is reflected by the optical path switching mirror 1038. The light reflected by the optical path switching mirror 1038 is applied to the prism 1014. The light emitted to the prism 1014 enters the incident surface 1066 of the excitation light 1042, is reflected by the contact surface 1048, and exits from the exit surface 1074 of the excitation light 1042. The incident angle θ1 of the excitation light 1042 to the contact surface 1048 satisfies the total reflection condition θc ≦ θ1. Therefore, when the excitation light 1042 is incident on the contact surface 1048, the excitation light 1042 is totally reflected on the contact surface 1048, and the evanescent wave 1082 leaks to the gold film 1012 as shown in FIG. Be enhanced.

  As shown in FIG. 6, when the light emitted from the light source 1020 is used as the film thickness measurement light 1050, the light emitted from the light source 1020 is reflected by the optical path switching mirror 1038. The light reflected by the optical path switching mirror 1038 is further reflected by the film thickness measurement light introduction mirror 1040. The light reflected by the film thickness measurement light introduction mirror 1040 is irradiated onto the prism 1014. The light irradiated to the prism 1014 enters the incident surface 1068 of the film thickness measurement light 1050, exits from the adhesion surface 1048, enters the second main surface 1053 of the gold film 1012, passes through the gold film 1012, and The light is emitted from the first main surface 1052 of the gold film 1012. The incident angle θ2 of the film thickness measurement light 1050 on the contact surface 1048 does not satisfy the total reflection condition θc ≦ θ2 (θ2 <θc), and is preferably 0 °. When the incident angle θ2 of the film thickness measurement light 1050 on the adhesion surface 1048 is 0 °, the optical path of the film thickness measurement light 1050 is perpendicular to the second main surface 1053, and the film thickness measurement light 1050 is the gold film 1012. , The influence of reflection of the film thickness measurement light 1050 at the interface between the gold film 1012 and the prism 1014 is suppressed, and the measurement accuracy is improved.

  In order to switch the optical path of the light emitted from the light source 1020 between the optical path of the excitation light 1042 and the optical path of the film thickness measurement light 1050, the position and posture of the optical path switching mirror 1038 are changed by the drive mechanism 1024 as shown in FIG. Adjusted. The drive mechanism 1024 is also used to adjust the incident angle θ1 of the excitation light 1044 to the contact surface 1048. The driving mechanism 1024 includes a driving force source such as a motor or a piezoelectric actuator. The drive mechanism 1024 rotates the optical path switching mirror 1036 and adjusts the posture of the optical path switching mirror 1036. The drive mechanism 1024 moves the optical path switching mirror 1036 on the linear stage and adjusts the position of the optical path switching mirror 1036.

  The optical path of light emitted from the light source 1020 by another type of optical system may be switched between the optical path of the excitation light 1042 and the optical path of the film thickness measurement light 1050. For example, both or one of the optical path switching mirror 1038 and the film thickness measurement light introducing mirror 1040 may be replaced with another type of bending optical element such as a prism. Optical elements other than the optical path switching mirror 1038 and the film thickness measurement light introducing mirror 1040 may be provided. The position and orientation of optical elements other than the optical path switching mirror 1038 are adjusted instead of the driving mechanism that adjusts the position and orientation of the optical path switching mirror 1038 or in addition to the driving mechanism that adjusts the position and orientation of the optical path switching mirror 1038. And a mechanism for adjusting the position and posture of the light source 1020 may be provided.

  Desirably, the optical path of the light emitted from the same light source 1020 is switched between the optical path of the excitation light 1042 and the optical path of the film thickness measurement light 1050 by the optical system 1022. Thereby, the excitation light 1042 and the film thickness measurement light 1050 are emitted from the same light source 1020, the number of the light sources 1020 is reduced, and the surface plasmon excitation fluorescence measuring apparatus 1000 is simplified. However, although the number of light sources increases, the light source that emits the excitation light 1042 and the light source that emits the film thickness radiation light 1050 may be separate. The wavelength of the excitation light 1042 and the wavelength of the film thickness measurement light 1050 are desirably the same, but may be different.

{CCD sensor}
The CCD sensor 1026 is on the optical path of the surface plasmon excitation fluorescence 1044 and on the optical path of the transmitted light 1046, and measures both the light quantity of the surface plasmon excitation fluorescence 1044 and the light quantity of the transmitted light 1046. Thereby, the light quantity of the surface plasmon excitation fluorescence 1044 and the light quantity of the transmitted light 1046 are measured by the same CCD sensor 1026, the number of CCD sensors is reduced, and the surface plasmon excitation fluorescence measurement apparatus 1000 is simplified. For this purpose, it is necessary that at least a part of the optical path of the surface plasmon excitation fluorescence 1044 and the optical path of the transmitted light 1046 coincide. However, although the number of CCD sensors increases, the optical path of the surface plasmon excitation fluorescence 1044 and the optical path of the transmission light 1046 may be completely separated, and the CCD sensor for measuring the light quantity of the surface plasmon excitation fluorescence 1044 and the transmitted light 1046 The CCD sensor that measures the amount of light may be separate. The CCD sensor 1026 may be replaced with another type of light quantity sensor such as a photomultiplier tube (PMT).

  When the resonance angle θr is determined from the incident angle θ1 at which the amount of scattered light is maximized instead of the incident angle θ1 at which the amount of reflected light 1084 is minimized, the CCD sensor 1026 is desirably on the optical path of the scattered light. Yes, the amount of scattered light is also measured. However, a CCD sensor separate from the CCD sensor 1026 may measure the amount of scattered light.

{Low-pass filter, neutral density filter and filter replacement mechanism}
The filter replacement mechanism 1032 switches the filter inserted in the optical path between the low pass filter 1028 and the neutral density filter 1030. As shown in FIG. 5, when the light quantity of the surface plasmon excitation fluorescence 1044 is measured, a low-pass filter 1028 is inserted in the optical path of the surface plasmon excitation fluorescence 1044. As shown in FIG. 6, when the amount of transmitted light 1046 is measured, a neutral density filter 1030 is inserted in the optical path of the transmitted light 1046.

  The low-pass filter 1028 transmits light having a wavelength longer than the cutoff wavelength and attenuates light having a wavelength shorter than the cutoff wavelength. The cutoff wavelength is selected within a range from the wavelength of the excitation light 1042 to the wavelength of the surface plasmon excitation fluorescence 1044. For example, when the wavelength of the excitation light 1042 is about 640 nm and the wavelength of the surface plasmon excitation fluorescence 1044 is 670 nm, 650 nm is selected as the cutoff wavelength. When the low-pass filter 1028 is inserted in the optical path of the surface plasmon excitation fluorescence 1044, the excitation light 1042 is attenuated by the low-pass filter 1028, and only a part of the excitation light 1042 reaches the CCD sensor 1026. Passes through the low pass filter 1028 and most of the surface plasmon excitation fluorescence 1044 reaches the CCD sensor 1026. Thereby, when the light quantity of the surface plasmon excitation fluorescence 1044 with a comparatively small light quantity is measured, the influence of the scattered light with a relatively large light quantity is suppressed, and the measurement accuracy is improved.

  The neutral density filter 1030 attenuates light. When the neutral density filter 1030 is inserted in the optical path of the transmitted light 1046, the transmitted light 1046 is attenuated by the neutral density filter 1030, and part of the transmitted light 1046 reaches the CCD sensor 1026. Thereby, the light amount of the transmitted light 1046 having a relatively large light amount and the light amount of the surface plasmon excitation fluorescence 1044 having a relatively small light amount are easily measured by the same CCD sensor 1026.

  When the optical path of the surface plasmon excitation fluorescence 1044 and the optical path of the transmitted light 1046 are separated, the filter replacement mechanism 1032 may be omitted. When the light amount sensor that measures the light amount of the surface plasmon excitation fluorescence 1044 and the light amount sensor that measures the light amount of the transmitted light 1046 are separate and the sensitivity of the light amount sensor that measures the light amount of the transmitted light 1046 is low, the neutral density filter 1030. May be omitted.

{Photodiode}
As shown in FIG. 1, the photodiode 1034 is on the optical path of the excitation light 1042 emitted from the emission surface 1074 of the excitation light 1042, that is, the reflected light 1084, and measures the light quantity of the reflected light 1084. The photodiode 1034 may be measured by another type of light amount sensor such as a phototransistor or a photoresistor. When the resonance angle θr is determined from the incident angle θ1 at which the amount of scattered light is maximized, the photodiode 1034 may be omitted.

{controller}
The block diagram of FIG. 7 shows the controller.

  As shown in FIG. 7, the controller 1010 includes an operation control unit 1100 and a correction unit 1102. The correcting unit 1102 includes a transmittance deriving unit 1104, an electric field enhancement specifying unit 1106, a normalizing unit 1108, and a storage unit 1110. The electric field enhancement specifying unit 1106 includes a film thickness deriving unit 1112 and an electric field enhancement deriving unit 1114.

  The operation control unit 1100 controls hardware components of the surface plasmon excitation fluorescence measurement apparatus 1000 and is responsible for the operation of the surface plasmon excitation fluorescence measurement apparatus 1000. The correction unit 1102 performs calculation and is responsible for correcting the measurement value of the light amount of the surface plasmon excitation fluorescence 1044 by the measurement value of the light amount of the transmitted light 1046.

  The transmittance deriving unit 1104 obtains a measurement value of the amount of transmitted light 1046 from the CCD sensor 1026 and derives an index of the transmittance of the gold film 1012 from the measured value of the amount of transmitted light 1046. The transmittance index may be the transmittance itself or a value corresponding to the transmittance on a one-to-one basis. For example, the film thickness measurement light 1050 in the section from the first main surface 1052 of the gold film 1012 to the CCD sensor 1026 has a constant light quantity that is incident on the second main surface 1053 of the gold film 1012. Can be regarded as constant, the measured value of the amount of transmitted light 1046 itself can be an index of transmittance. The index of transmittance is derived by dividing the measured value of the amount of transmitted light 1046 by the attenuation factor of the neutral density filter 1030 and the amount of light of the film thickness measurement light 1050.

  The electric field enhancement specifying unit 1106 specifies the electric field enhancement from the transmittance index of the gold film 1012.

  The film thickness deriving unit 1112 derives the film thickness of the gold film 1012 from the transmittance index of the gold film 1012. When the film thickness of the gold film 1012 is derived, a relationship 1116 between the index of the transmittance of the gold film 1012 stored in the storage unit 1110 and the film thickness of the gold film 1012 is referred to. The film thickness of the gold film 1012 corresponding to the specified index of the transmittance of the gold film 1012 is specified.

  The graph of FIG. 8 shows the transmittance of the gold film 1012 theoretically obtained when the wavelength of the film thickness measurement light 1050 is 635 nm and the optical constants of the gold film 1012 are n = 0.22273 and k = 3.4533. The relationship with the film thickness of the gold film 1012 is shown.

  The electric field enhancement strength deriving unit 1114 derives the electric field enhancement strength from the film thickness of the gold film 1012. When the electric field enhancement is derived, the relationship 1118 between the film thickness of the gold film 1012 and the electric field enhancement stored in the storage unit 1110 is referred to, and the film thickness of the gold film 1012 derived by the film thickness deriving unit 1112 is referred to. The electric field enhancement corresponding to is specified.

  The electric field enhancement depends on the incident angle θ 1, and the incident angle θ 1 at which the electric field enhancement becomes maximum varies depending on the film thickness of the gold film 1012. Therefore, desirably, the incident angle θ1 that maximizes the electric field enhancement is specified for each film thickness of the gold film 1012, and the excitation light 1042 is incident on the contact surface 1048 at the incident angle θ1 that maximizes the electric field enhancement. The relationship between the thickness of the gold film 1012 and the electric field enhancement is used.

  The graph of FIG. 9 shows an example of the relationship between the film thickness of the gold film 1012 and the electric field enhancement intensity. FIG. 9 shows the relationship between the thickness of the gold film 1012 theoretically obtained for two types of resin materials of the prism 1014, “E48R” and “BK7”, and the electric field enhancement intensity.

  Relationships 1116 and 1118 are typically numeric tables. However, the relationships 1116 and 1118 for which the relationships 1116 and 1118 may be given as arithmetic expressions are theoretically obtained, but may be actually measured.

  The relationships 1116 and 1118 may be combined, and the relationship between the transmittance index of the gold film 1012 and the electric field enhancement strength may be stored in the storage unit 1110. In this case, the electric field enhancement specifying unit 1106 directly derives the electric field enhancement from the transmittance index of the gold film 1012 without passing through the film thickness of the gold film 1012.

  The normalization unit 1108 acquires the measurement value of the light amount of the surface plasmon excitation fluorescence 1044 from the CCD sensor 1026 and standardizes the measurement value of the light amount of the surface plasmon excitation fluorescence 1044 by the electric field enhancement specified by the electric field enhancement specification unit 1106. Turn into. Normalization is performed by dividing the measured value of the amount of light of the surface plasmon excitation fluorescence 1044 by the electric field enhancement intensity.

  The controller 1010 is an embedded computer that executes software. One embedded computer may have the function of the block shown in FIG. 7, or two or more embedded computers may share the function of the block shown in FIG. Hardware without software may be responsible for all or part of the blocks shown in FIG. The hardware is, for example, an electronic circuit such as an operational amplifier or a comparator. All or part of the processing by the blocks shown in FIG. 7 may be executed manually by humans or may be executed outside the surface plasmon excitation fluorescence measuring apparatus 1000.

(Operation of surface plasmon excitation fluorescence measuring device)
The flowchart of FIG. 10 shows the operation of the surface plasmon excitation fluorescence measurement apparatus.

  As shown in FIG. 10, a sensor chip 1002 is prepared, and the sensor chip 1002 is attached to the surface plasmon excitation fluorescence measuring apparatus 1000 (step S101).

  After the sensor chip 1002 is attached, the amount of transmitted light 1046 is measured (step S102). When the amount of transmitted light 1046 is measured, the driving mechanism 1024 and the CCD sensor 1026 are controlled by the operation control unit 1100. The position and orientation of the optical path switching mirror 1038 are adjusted by the drive mechanism 1024, and the optical path of the light emitted from the light source 1020 is set as the optical path of the film thickness measurement light 1050. At the same time, the amount of transmitted light 1046 is measured by the CCD sensor 1026. The measured value of the amount of transmitted light 1046 is transferred to the controller 1010.

  After the amount of transmitted light 1046 is measured, the incident angle θ is set to the measurement angle θm (step S103). When the incident angle θ is set to the measurement angle θm, the driving mechanism 1024 and the photodiode 1034 are controlled by the operation control unit 1100. The position and orientation of the optical path switching mirror 1038 are adjusted by the drive mechanism 1024, and the optical path of the light emitted from the light source 1020 is set as the optical path of the excitation light 1042. Further, the incident angle θ1 is scanned in the vicinity of the expected resonance angle θr. In parallel with the scanning of the incident angle θ1, the amount of the reflected light 1084 is measured by the photodiode 1034. The measured value of the amount of reflected light 1084 is transferred to the controller 1010. From the surface plasmon resonance (SPR) curve showing the relationship between the incident angle θ1 and the amount of reflected light 1084, the resonance angle θr, which is the incident angle θ1 at which the amount of reflected light 1084 is minimized, is specified, and the measurement angle θm is determined from the resonance angle θr. Is determined. Since the resonance angle θr and the incident angle θ1 at which the electric field enhancement becomes maximum are slightly different, it is desirable that the measurement angle θm is determined by adding or subtracting the minute angle to the resonance angle θr. The graph of FIG. 11 shows the relationship between the SPR curve and the incident angle and the electric field enhancement intensity.

  After the incident angle θ1 is set to the measurement angle θm, the sample liquid 1056 and the fluorescent labeling liquid 1060 are sequentially injected into the flow path 1054 (step S104). When the sample solution 1056 and the fluorescent labeling solution 1060 are sequentially injected into the flow path 1054, the supply mechanism 1008 is controlled by the operation control unit 1100. As a result, the sample solution 1056 and the fluorescent labeling solution 1060 are sequentially supplied to the antibody solid layer film 1016, and the fluorescently labeled antigen 1058 is captured by the antibody solid layer film 1016. When the optical path of the film thickness measurement light 1050 passes through the flow path 1054, the sample liquid 1056 and the fluorescent labeling liquid 1060 are preferably injected into the flow path 1054 after the light amount of the transmitted light 1046 is measured. However, when the optical path of the film thickness measurement light 1050 does not pass through the flow path 1054, the sample liquid 1056 and the fluorescent labeling liquid 1060 are injected into the flow path 1054 either before or after the light amount of the transmitted light 1046 is measured. Alternatively, the sample liquid 1056 and the fluorescent labeling liquid 1060 may be injected into the channel 1054 at the same time as the amount of the transmitted light 1046 is measured.

  After the sample solution 1056 and the fluorescent labeling solution 1060 are injected into the channel 1054, the light quantity of the surface plasmon excitation fluorescence 1044 is measured (step S105). When the light amount of the surface plasmon excitation fluorescence 1044 is measured, the CCD sensor 1026 is controlled by the operation control unit 1100. The amount of light of the surface plasmon excitation fluorescence 1044 is measured by the CCD sensor 1026. The measurement value of the light amount of the surface plasmon excitation fluorescence 1044 is transferred to the controller 1010.

  A light amount sensor that measures the light amount of the surface plasmon excitation fluorescence 1044 and a light amount sensor that measures the light amount of the transmitted light 1046 are separate, and a light source 1020 that emits the excitation light 1042 and a light source 1020 that emits the film thickness measurement light 1050 are provided. In the case where the film thickness measurement light 1050 does not pass through the flow path 1054, the light quantity of the transmitted light 1046 may be measured simultaneously with the measurement of the light quantity of the surface plasmon excitation fluorescence 1044.

  After the light quantity of the surface plasmon excitation fluorescence 1044 is measured, the correction value of the light quantity of the surface plasmon excitation fluorescence 1044 is corrected by the measurement value of the light quantity of the transmitted light 1046 in the correction unit 1102 (step S106).

  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.

DESCRIPTION OF SYMBOLS 1000 Surface plasmon excitation fluorescence measuring apparatus 1004 Irradiation mechanism 1006 Measurement mechanism 1012 Gold film 1020 Light source 1022 Optical system 1024 Drive mechanism 1042 Excitation light 1044 Surface plasmon excitation fluorescence 1046 Transmission light 1050 Film thickness measurement light

Claims (7)

A surface plasmon excitation fluorescence measuring device,
A conductor film having a first main surface and a second main surface, the sample being supplied on the first main surface;
A dielectric medium having a close contact surface in close contact with the second main surface;
The dielectric medium is irradiated with light, and is smaller than the critical angle inside the dielectric medium and a first optical path where the light enters the contact surface at an incident angle greater than a critical angle inside the dielectric medium. An irradiation mechanism that switches an optical path of the light with a second optical path where the light is incident on the contact surface at an incident angle;
While the light passing through the first optical path is irradiated on the dielectric medium, surface plasmon excitation fluorescence emitted from the measurement object included in the sample and the light passing through the second optical path are A measurement mechanism that measures the amount of transmitted light that passes through the conductor film while being irradiated on the dielectric medium;
A correction unit that corrects the measurement value of the light amount of the surface plasmon excitation fluorescence measured by the measurement mechanism by the measurement value of the light amount of the transmitted light measured by the measurement mechanism;
A surface plasmon excitation fluorescence measuring apparatus comprising: In the surface plasmon excitation fluorescence measuring device according to claim 1,
The irradiation mechanism is:
A light source that emits the light;
An optical system for guiding the light emitted from the light source to the dielectric medium;
A drive mechanism for driving the optical system and switching the optical path of the light between the first optical path and the second optical path;
A surface plasmon excitation fluorescence measuring apparatus comprising: In the surface plasmon excitation fluorescence measuring device according to claim 1 or 2,
The surface plasmon excitation fluorescence measuring apparatus in which the second optical path is perpendicular to the second main surface. In the surface plasmon excitation fluorescence measuring device according to any one of claims 1 to 3,
The measurement mechanism is
A surface plasmon excitation fluorescence measuring apparatus comprising a light amount sensor on the optical path of the surface plasmon excitation fluorescence and on the optical path of the transmitted light. In the surface plasmon excitation fluorescence measuring device according to any one of claims 1 to 4,
The correction unit is
A transmittance derivation unit that obtains a measurement value of the amount of transmitted light from the measurement mechanism, and derives an index of the transmittance of the conductor film from the measurement value of the amount of transmitted light;
An electric field enhancement specifying part for specifying an electric field enhancement from the transmittance index of the conductor film;
Obtaining a measurement value of the light amount of the surface plasmon excitation fluorescence from the measurement mechanism, a normalization unit that normalizes the measurement value of the light amount of the surface plasmon excitation fluorescence by the electric field enhancement intensity,
A surface plasmon excitation fluorescence measuring apparatus comprising: In the surface plasmon excitation fluorescence measuring device according to claim 5,
The electric field enhancement specifying part is:
A film thickness deriving section for deriving the film thickness of the conductor film from the index of transmittance of the conductor film;
An electric field enhancement derivation unit for deriving the electric field enhancement from the film thickness of the conductor film;
A surface plasmon excitation fluorescence measuring apparatus comprising: A surface plasmon excitation fluorescence measurement method,
(a) preparing a dielectric medium having a conductive film having a first main surface and a second main surface and an adhesion surface in close contact with the second main surface;
(b) after the step (a), measuring the amount of transmitted light transmitted through the conductor film while making light incident on the contact surface at an incident angle smaller than a critical angle inside the dielectric medium;
(c) When the optical path of the light in the step (b) does not pass through the flow path formed on the first main surface , before or after the step (b) or simultaneously with the step (b) When the sample is supplied to the first main surface and the light path of the light in the step (b) passes through the flow path formed on the first main surface, the first main surface is followed by the first main surface after the step (b). Supplying a sample to the main surface of
(d) After the step (c), surface plasmon excitation fluorescence emitted from the object to be measured contained in the sample while making light incident on the contact surface at an incident angle greater than the critical angle inside the dielectric medium Measuring the amount of light,
(e) correcting the measured value of the light amount of the surface plasmon excitation fluorescence measured in the step (d) by the measured value of the transmitted light amount measured in the step (b);
A surface plasmon excitation fluorescence measurement method comprising:

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