JP5472033B2 - Surface plasmon resonance fluorescence analyzer and surface plasmon resonance fluorescence analysis method - Google Patents

Description

  The present invention relates to a surface plasmon resonance fluorescence analysis apparatus and a surface plasmon resonance fluorescence analysis method for detecting an analyte contained in a sample liquid using surface plasmon resonance (SPR).

  Conventionally, surface plasmon resonance fluorescence analysis (surface plasmon excitation enhanced fluorescence spectroscopy: SPFS) method is known as a method for detecting a sample (a substance to be detected) with high sensitivity in bioassay for detecting protein, DNA, and the like.

  In the SPFS method, in a prism in which a metal film made of gold, silver, or the like is formed on a predetermined surface, excitation light is incident on the metal film from the prism side under total reflection conditions, and the prism and the metal film having different refractive indexes are made to enter. Light (evanescent wave) that exudes from the interface when the excitation light is totally reflected at the interface is used. Specifically, in the SPFS method, the specimen contained in the sample liquid flowed on the surface of the metal film by the evanescent wave that leaks out when the excitation light is totally reflected, or the fluorescent substance (labeling substance) attached to the specimen. The presence or amount of the specimen can be detected by analyzing the fluorescence (excitation fluorescence) that is emitted when is excited.

  In such an optical measurement using the SPFS method, only the P wave component of the excitation light contributes to the excitation of the surface plasmon resonance in the metal film, so that the P wave polarized excitation light is incident on the metal film.

  However, when the specimen is detected by exchanging the prism for each examination, the amount of the P wave component of the excitation light incident on the metal film is not constant even when the same P wave polarized excitation light is irradiated onto the prism. Variations in measurement results occur. This is due to the fact that phase rotation is caused by birefringence of the excitation light incident on the prism.

  Specifically, when the S wave component is generated by the phase rotation due to birefringence, the P wave component of the excitation light entering the prism is reduced. In addition, birefringence is caused by a density difference in the prism that is generated when the prism is molded, and therefore the birefringence is different for each prism. Therefore, even when excitation light having the same polarization direction is irradiated onto the prism, the amount of P wave component of the excitation light incident on the metal film varies for each prism. In particular, when the prism is generally formed of a dielectric material such as a resin having a high degree of birefringence, the variation in the degree of birefringence for each prism is large, and the variation in the amount of light of the P wave component incident on the metal film is significant. Become.

  When the amount of the P wave component of the excitation light incident on the metal film varies, the amount of the evanescent wave (enhanced electric field) oozing based on surface plasmon resonance also varies. In this way, if the amount of the evanescent wave oozes out because the amount of the P wave component of the excitation light incident on the metal film is not constant for each prism, even if the specimen is detected for the same sample liquid, Since the amount of excitation fluorescence to be measured varies, the measurement results (analyte analysis results) vary. That is, when the prism is exchanged for each examination in the optical measurement using the SPFS method, the variation in the degree of birefringence for each prism affects the measurement result of the specimen.

  Therefore, when the specimen is detected by exchanging the prism for each examination, the amount of the P wave component of the excitation light incident on the metal film in each prism is made constant, and the measurement result of the variation in the degree of birefringence for each prism is measured. An analysis apparatus that suppresses the influence on the environment has been developed (see Patent Document 1).

  In this analyzer, a polarization state adjusting unit for adjusting the polarization state of the excitation light is provided on the optical path of the excitation light between the excitation light source and the prism.

  The polarization state adjusting unit includes a plurality of polarizing elements such as a half-wave plate and a quarter-wave plate, a plurality of driving devices such as actuators that respectively drive the polarizing elements, and after being reflected by the metal film. A light receiving unit that measures the amount of excitation light (reflected excitation light). The analyzer provided with the polarization state adjustment unit adjusts the polarization state of the excitation light by driving each polarization element by the driving device and adjusting its position and orientation based on the amount of reflected excitation light. . Specifically, in this analyzer, P-wave polarized excitation light is applied to the metal film by adjusting the inclination of the polarization direction of the excitation light before entering the prism, the extent of the polarization state, and the like with a plurality of polarization elements. Is incident. Thereby, the influence on the measurement result of the dispersion | variation in the degree of birefringence for every prism is suppressed.

JP 2009-236709 A

  However, in the above-described analyzer, since the position and posture of each of the polarization elements are adjusted while feeding back the amount of reflected excitation light measured by the light receiving unit, this adjustment time is required, and it takes time to analyze the specimen. It takes.

  In addition, since the analyzer described above requires a plurality of polarizing elements and a plurality of driving devices that drive each polarizing element, it is difficult to reduce the size of the analyzer.

  Therefore, in view of the above problems, the present invention suppresses the influence of the variation in the degree of birefringence of each prism on the measurement result, and shortens the analysis time and reduces the size of the apparatus. It is an object to provide an analysis apparatus and a surface plasmon resonance fluorescence analysis method.

  Accordingly, in order to solve the above-mentioned problems, the present invention is a surface plasmon resonance fluorescence analyzer for measuring fluorescence emitted by excitation of an electric field based on surface plasmon resonance by a specimen or a fluorescent substance attached to the specimen. A chip holding portion that can hold the analysis chip including the prism on which the metal film is formed, and the metal film of the prism included in the analysis chip held by the chip holding portion. A light source unit that makes linearly polarized excitation light enter the prism, and a half-wave plate disposed on the optical path of the excitation light between the light source unit and the prism, and The half-wave plate is rotatable so that the deflection direction of the excitation light that has passed through the half-wave plate changes with respect to the metal film, and the excitation light is reflected by the metal film. A light measuring unit capable of measuring the amount of light generated in the metal film and a region adjacent to the metal film, and analyzing the specimen based on the amount of fluorescence included in the light measured by the light measuring unit A control processing unit. The control processing unit corrects the amount of fluorescent light measured by the light measurement unit based on a deviation angle that is an angle between a P wave direction with respect to the metal film and the optical principal axis of the prism, and the deviation angle Measuring the light generated in the metal film and the adjacent region by the light measuring unit while rotating the half-wave plate of the polarization direction adjusting unit in a state where the light source unit emits excitation light. It is obtained from the maximum light quantity and the minimum light quantity obtained.

  Further, in order to solve the above-mentioned problems, the present invention is a surface plasmon resonance fluorescence analyzer for measuring fluorescence emitted when a specimen or a fluorescent substance attached to the specimen is excited by an electric field based on surface plasmon resonance, A prism having a metal film formed on the surface thereof, a light source unit that makes linearly polarized excitation light incident on the prism so as to be reflected by the metal film, and the light source unit between the prism and the light source unit. It has a half-wave plate arranged on the optical path of the excitation light, and this half-wave plate can be rotated so that the deflection direction of the excitation light that has passed through the half-wave plate changes with respect to the metal film. Measured by a certain polarization direction adjusting unit, a light measuring unit capable of measuring the amount of light generated in the metal film and a region adjacent thereto by reflecting the excitation light on the metal film, and the light measuring unit And a control processing unit that performs analysis of the specimen based on the light intensity of the fluorescence included in the. The control processing unit corrects the amount of fluorescent light measured by the light measurement unit based on a deviation angle that is an angle between a P wave direction with respect to the metal film and the optical principal axis of the prism, and the deviation angle Measuring the light generated in the metal film and the adjacent region by the light measuring unit while rotating the half-wave plate of the polarization direction adjusting unit in a state where the light source unit emits excitation light. It is obtained from the maximum light quantity and the minimum light quantity obtained.

  According to these inventions, the excitation light is incident on the metal film without being birefringent in the prism by correcting the light quantity of the fluorescence measured in a state where the excitation light is birefringent in the prism based on the deviation angle. A fluorescent light amount (fluorescent light amount in which an error based on birefringence is suppressed) measured when reflected by a metal film is obtained. As a result, even if the prism is replaced for each inspection, it is possible to obtain the amount of fluorescence measured when the excitation light is incident on the metal film without causing phase rotation due to birefringence in each prism. Measurement errors due to variations in the degree of birefringence for each can be effectively suppressed.

  In addition, since the error included in the measurement value can be reduced simply by measuring the amount of light generated in the metal film and its adjacent region while rotating the half-wave plate, the reflection excitation light can be reduced as in a conventional analyzer. It is not necessary to adjust the position and orientation of each of the plurality of polarizing elements while measuring the amount of light and feeding back the measurement results, and the analysis time of the specimen can be shortened.

  Furthermore, according to these inventions, it is only necessary to provide one half-wave plate so as to be rotatable, and a conventional analyzer including a plurality of polarizing elements and a driving device for controlling the position and orientation of each polarizing element. Compared to the above, it is possible to simplify and miniaturize the configuration of the analyzer.

  In the surface plasmon resonance fluorescence analyzer according to the present invention, the polarization direction adjusting unit includes a rotation driving unit that rotates the half-wave plate, and the control processing unit includes the light source unit, the rotation driving unit, and the like. While controlling the light measurement unit and rotating the half-wave plate by the rotation driving unit in a state where the light source unit emits excitation light, the light measurement unit and the region adjacent to the metal film The deviation angle deriving unit for obtaining the deviation angle from the maximum light amount and the minimum light amount obtained by measuring the light generated in the above, and the deviation obtained by the deviation angle deriving unit for the light amount of the fluorescence measured by the light measurement unit. It is preferable to include a correction unit that performs correction based on the angle.

  According to such a configuration, the analysis device automatically performs the derivation of the correction value, the detection of the fluorescence light amount in which the error due to the birefringence is suppressed, and the analysis of the specimen.

At this time, the deviation angle deriving unit is configured such that the deviation angle is θ _i , the maximum light amount is DR _max , the minimum light amount is DR _min , the excitation light is reflected by the metal film, and the surface plasmon is reflected on the metal film. When the surface diffused light amount, which is the light amount of light measured by the light measuring unit when resonance is not occurring, is SK, the deviation angle is obtained by the following equation (1).

  In the surface plasmon resonance fluorescence analyzer, the control processing unit includes a storage unit capable of storing the surface diffusion light amount, and the deviation angle deriving unit uses the surface diffusion light amount stored in the storage unit. If it is configured to obtain the deviation angle by using it, the deviation angle can be obtained without measuring the surface diffused light amount for each examination, and the analysis time of the specimen can be shortened.

  In the surface plasmon resonance fluorescence analyzer of the present invention, in the surface plasmon resonance fluorescence analyzer, when the measured fluorescence (fluorescence emitted from the specimen or a fluorescent substance attached thereto) is larger than the autofluorescence generated in the prism. The light measuring unit measures the amount of the fluorescent light contained in the light generated in the metal film and a region adjacent thereto, and the deviation angle deriving unit is in a state where the light source unit emits excitation light. The deviation angle may be obtained from the maximum fluorescence amount and the minimum fluorescence amount of the fluorescence measured by the light measurement unit while rotating the half-wave plate of the polarization direction adjustment unit by the rotation drive unit.

  In this case, the light measurement unit only needs to measure only the fluorescence contained in the light generated in the metal film and the region adjacent thereto, and the configuration can be simplified.

  Specifically, the light generated in the metal film and the region adjacent thereto due to surface plasmon resonance generated in the metal film includes, for example, scattered light such as plasmon scattered light and Raman scattered light, and fluorescent materials such as evanescent waves. There is fluorescence emitted when excited by (enhanced electric field). Since these scattered light and fluorescent light are greatly different in light quantity, in order to measure each light, a plurality of light quantity measuring devices corresponding to each light quantity can be arranged in a switchable manner or when measuring with one light quantity measuring device. It is necessary to make it possible to adjust the amount of light incident on the light receiving unit by allowing a light reducing member such as a neutral density filter to be put in and out of the light receiving unit of the light amount measuring device, and the configuration of the light measuring unit becomes very complicated. The device cannot be downsized. However, when the light measurement unit measures only the amount of fluorescent light, there is no need for a dimming member or the like, so the configuration can be simplified compared to a light measurement unit that measures scattered light and fluorescence. .

As described above, when the light measurement unit measures only fluorescence, the deviation angle deriving unit, when the deviation angle is θ _i , the maximum fluorescence amount is S _max , and the minimum fluorescence amount is S _min , The deviation angle is obtained by the following equation (2).

Here, in the surface plasmon resonance fluorescence analyzer according to the present invention, even when the deviation angle deriving unit obtains the deviation angle θ _i from the maximum light amount and the minimum light amount, the maximum fluorescence amount and the minimum fluorescence amount described above. Even if the displacement angle θ _i is obtained from the above, a correction value for obtaining the light amount of the fluorescence measured by the light measurement unit when the excitation light incident on the prism is not birefringent is derived. A correction value deriving unit that is obtained from a deviation angle obtained by the unit, and the correction value deriving unit obtains the correction value by the following equation (3), where K is the correction value.

  When the prism is formed of a dielectric, the above effects can be obtained more remarkably. That is, a prism formed of a dielectric material such as a resin has a higher degree of birefringence than a glass prism, so the variation in the degree of birefringence for each prism also increases. However, in the surface plasmon resonance fluorescence analyzer, Since the influence of the variation in the degree of birefringence for each prism on the measurement result is effectively suppressed, the specimen can be detected with high accuracy even if the prism is replaced for each examination.

  Further, in order to solve the above problems, the present invention is a surface plasmon resonance fluorescence analysis method for measuring fluorescence emitted when a specimen or a fluorescent substance attached to the specimen is excited by an electric field based on surface plasmon resonance, A prism having a metal film formed on the surface of the substrate, a preparation step of flowing a sample solution containing the specimen on the metal film, and linearly polarized excitation light for causing surface plasmon resonance in the metal film The excitation light is incident on the prism so as to be reflected by the metal film, and the excitation light is reflected by the metal film while changing the polarization direction of the excitation light with respect to the metal film while maintaining the state. A measuring step for measuring the amount of light generated in the metal film and a region adjacent thereto, and the P wave direction relative to the metal film from the maximum light amount and the minimum light amount measured in the measuring step, A deviation angle derivation step for obtaining a deviation angle that is an angle formed by the optical principal axis of the rhythm, and the amount of fluorescence contained in the light generated in the metal film and an area adjacent thereto when the excitation light is reflected by the metal film And an analysis step of detecting the specimen by correcting the amount of light based on the deviation angle obtained in the deviation angle derivation step.

  According to the present invention, even if the prism is replaced for each inspection, the fluorescence generated when the excitation light is incident on the metal film without causing phase rotation due to birefringence regardless of the variation in the degree of birefringence for each prism. Can be detected, and as a result, the influence of the variation in the degree of birefringence of each prism on the measurement result can be effectively suppressed. .

  As described above, according to the present invention, the surface plasmon resonance fluorescence analyzer capable of suppressing the influence on the measurement result of the variation in the degree of birefringence for each prism, reducing the analysis time, and reducing the size of the apparatus, And a surface plasmon resonance fluorescence analysis method can be provided.

It is a block diagram which shows the structure of the state by which the analysis chip was installed in the surface plasmon resonance fluorescence analyzer which concerns on 1st Embodiment. It is an enlarged view showing a configuration of a chip holding unit and an analysis chip held by the chip holding unit in the surface plasmon resonance fluorescence analyzer. It is a figure which shows the polarization state of the excitation light in the said surface plasmon resonance fluorescence analyzer, and the irradiation area | region of the excitation light in a metal film. It is a figure for demonstrating the measurement optical system of the said surface plasmon resonance fluorescence analyzer. It is a functional block diagram of the control processing part in the surface plasmon resonance fluorescence analyzer of 1st Embodiment and 2nd Embodiment. It is a flowchart which shows the basic sequence when analyzing a sample in the surface plasmon resonance fluorescence analyzer of 1st Embodiment and 2nd Embodiment. It is a flowchart which shows the resonance angle scanning sequence in the surface plasmon resonance fluorescence analyzer of 1st Embodiment. It is a flowchart which shows the optimal position scanning sequence in the surface plasmon resonance fluorescence analyzer of 1st Embodiment and 2nd Embodiment. It is a flowchart which shows the birefringence measurement sequence in the surface plasmon resonance fluorescence analyzer of 1st Embodiment and 2nd Embodiment. It is a flowchart which shows the excitation fluorescence measurement sequence in the surface plasmon resonance fluorescence analyzer of 1st Embodiment. It is a figure explaining the 2nd positioning of the reflective member in the said surface plasmon resonance fluorescence analyzer. FIG. 6 is a diagram for explaining a major axis rotation amount (deviation angle) θ _i and a correction value K. It is a functional block diagram which shows the structure of the state by which the analysis chip was installed in the surface plasmon resonance fluorescence analyzer which concerns on 2nd Embodiment. It is a flowchart which shows the resonance angle scanning sequence in the surface plasmon resonance fluorescence analyzer of 2nd Embodiment. It is a flowchart which shows the excitation fluorescence measurement sequence in the surface plasmon resonance fluorescence analyzer of 2nd Embodiment.

  Hereinafter, a first embodiment of the present invention will be described with reference to the accompanying drawings.

  The surface plasmon resonance fluorescence analyzer (hereinafter also simply referred to as “analyzer”) according to the present embodiment uses an evanescent wave (enhanced electric field) that exudes from the reflection interface of excitation light incident on the prism under total reflection conditions. In this apparatus, a fluorescent substance labeled (attached) to a substance to be detected (hereinafter also simply referred to as “specimen”) is excited, and the specimen is detected by detecting the amount of fluorescence generated thereby. .

  As shown in FIG. 1, the analyzer includes a chip holding unit 12 that holds an analysis chip 50, an excitation light emitting unit 20 that emits excitation light to the analysis chip 50 held in the chip holding unit 12, and The light measurement unit 40 that measures the intensity of light generated in the analysis chip 50, and various components of the analyzer 10 such as the chip holding unit 12, the excitation light emitting unit 20, and the light measurement unit 40 are controlled and various A control processing unit 14 (control unit) that performs arithmetic processing and a display unit 16 that displays various information such as arithmetic results are provided. The analyzer 10 also includes a preprocessing unit (not shown) that performs preprocessing of blood from the patient. The pretreatment unit accepts a reagent chip (not shown), performs pretreatment (blood cell separation, dilution, mixing, etc.) of blood injected into the reagent chip to generate a sample liquid, This is a portion to be injected into the analysis chip 50. The reagent chip is provided with a plurality of storage units, and each storage unit is individually sealed with a reagent, a diluent, a cleaning solution, and the like in addition to blood and the like.

  As shown in FIG. 2, the analysis chip 50 includes a prism 51, a metal film 55 formed on the surface of the prism 51, and a sample liquid and a cleaning liquid containing a specimen while contacting the metal film 55 on the metal film 55. And a flow path member 57 that forms a flow path 58 through which and the like flow. The analysis chip 50 of this embodiment is replaced every time a sample is detected (analyzed).

  The prism 51 includes an incident surface 52 on which the excitation light α from the excitation light emitting unit 20 is incident, and a film formation surface (predetermined surface) on which the metal film 55 on which the excitation light α incident on the inside is reflected is formed. ) 53 and an emission surface 54 on which excitation light α reflected by the metal film 55 is emitted to the outside of the prism 51, and is formed of transparent glass or resin. The exit surface 54 is a surface that first strikes after the excitation light α is reflected by the metal film 55 (specifically, the interface between the metal film 55 and the film formation surface 53), and the emission surface 54 reflects the excitation light α reflected by the metal film 55. Similar to the incident surface 52, it is formed on the optical surface so that the light of the S wave component does not stay inside the prism 51. The prism 51 of this embodiment has birefringence characteristics. Specifically, the prism 51 is formed of a transparent resin (dielectric material) having a refractive index of about 1.4 to 1.6. The prism 51 may be made of glass. The prism 51 includes an incident surface 52, a film formation surface 53, and an emission surface 54, and the excitation light α incident on the inside from the incident surface 52 is totally reflected by the metal film 55 on the film formation surface 53. The excitation light α (specifically, the S wave component of the excitation light α) may be shaped so as to be emitted from the emission surface 54 to the outside without being irregularly reflected inside.

  The metal film 55 is a metal thin film formed (formed) on the film formation surface 53 of the prism 51, and is formed of gold in this embodiment. The metal film 55 is a member for amplifying an evanescent wave (enhanced electric field) generated when the excitation light α incident on the prism 51 under total reflection conditions is totally reflected at the interface between the metal film 55 and the film formation surface 53. It is. That is, by providing the metal film 55 on the film formation surface 53 and causing surface plasmon resonance in the metal film 55, the excitation light α is totally reflected on the surface without the metal film 55 (film formation surface 53), and evanescent. Compared with the case of generating a wave, the formed evanescent wave can be amplified (that is, an enhanced electric field can be formed in the vicinity of the surface 55a of the metal film 55).

  The material of the metal film 55 is not limited to gold, and may be any metal that causes surface plasmon resonance, and may be, for example, silver, copper, aluminum, or the like (including an alloy).

  A capturing body 56 for capturing a specific antigen is fixed on the surface (surface opposite to the prism 51) 55a of the metal film 55. The capturing body 56 is fixed to the surface 55a of the metal film 55 by surface treatment.

  The flow path member 57 is provided on the film formation surface 53 of the prism 51, and forms a flow path 58 through which the sample liquid flows together with the film formation surface 53. The flow path member 57 is formed of a transparent resin, and is joined to the prism 51 by an adhesive, laser welding, ultrasonic welding, pressure bonding using a clamp member, or the like. The flow path 58 has a shape such that a region where the metal film 55 and the sample solution are in contact with each other is wider than a measurement region of the light measurement unit 40.

  When the analysis chip 50 configured as described above is installed in the pretreatment unit of the analyzer 10, the sample liquid that has been pretreated in the pretreatment unit is injected (supplied) into the flow path 58. The analysis chip 50 into which the sample liquid has been injected is transported to the chip holding unit 12 when the reaction between the capturing body 56 fixed on the metal film 55 and the specimen (specific antigen) is completed. Are held in a predetermined posture.

  The chip holding unit 12 holds the analysis chip 50 when detecting the specimen. Specifically, in the chip holding unit 12, the excitation light α emitted from the excitation light emitting unit 20 enters the prism 51 from the incident surface 52 under total reflection conditions, and the incident excitation light α is converted into the metal film 55. The analysis chip 50 is detachably held in such a posture as to be reflected by the lens. The chip holding unit 12 of this embodiment holds the analysis chip 50 in a posture in which the prism 51 is positioned below the flow path member 57.

  In the analyzer 10, a light absorber is provided in the vicinity of the emission surface 54 of the analysis chip 50 held by the chip holding unit 12 so that the light measurement unit 40 is not affected by the light emitted from the emission surface 54. (Not shown) is arranged.

  The excitation light emitting unit 20 makes the excitation light α enter the prism 51 so as to be reflected by the metal film 55 of the prism 51 included in the analysis chip 50 held by the chip holding unit 12. Specifically, the excitation light emitting unit 20 includes a light source unit 21 that emits linearly polarized excitation light, and an excitation optical system 30 that guides the excitation light α emitted from the light source unit 21 to the incident surface 52 of the prism 51. Have.

  The light source unit 21 includes a light source unit unit 23 that includes an excitation light source 22 and a first wave shaping unit 24 that waves the excitation light α emitted from the excitation light source 22. The light source unit 21 of the present embodiment emits excitation light α downward, and a laser diode is used as the excitation light source 22.

  The light source unit 23 includes an excitation light source 22 and a temperature adjustment circuit 25 that performs temperature adjustment (temperature adjustment) of the excitation light source 22. This light source unit 23 collimates the excitation light α emitted from the excitation light source 22 and also positions the excitation light source 22 so that the excitation light α enters the metal film 55 of the prism 51 from the short axis side. Adjust and hold This is because the shape of the excitation light α emitted from the excitation light source (laser diode) 22 is flat and the polarization direction is substantially biased in one direction even if it is converted to a commutate, so that the posture of the excitation light source 22 is adjusted and maintained. Thus, when the excitation light α is incident on the film formation surface 53 with a total reflection condition (a shallow angle with respect to the film formation surface 53), the contour shape of the irradiation region on the film formation surface 53 of the excitation light α is This is to make it approximately circular (see FIG. 3).

  The temperature adjustment circuit 25 is a regression circuit for adjusting the temperature of the excitation light source (laser diode) 22. Specifically, since the wavelength of the light emitted from the excitation light source 22 and the emission energy vary depending on the temperature, the temperature adjustment circuit 25 uses a photodiode or the like (not shown) to change the amount of light branched from the light of the excitation light α after collimation. ), And the temperature of the excitation light source 22 is adjusted so that the wavelength and the amount of the emitted excitation light α are constant.

  The first wave rectifier 24 harmonizes the excitation light α emitted from the light source unit 23 by a plurality of filters so as to obtain an excitation wavelength having a unique polarization direction. Specifically, the first wave rectification unit 24 includes a first bandpass filter (hereinafter simply referred to as “first BPF”) 26, a linear polarization filter (hereinafter simply referred to as “LPF”) 27, and a first. 1 ND filter (hereinafter simply referred to as “first NDF”) 28. In the first BPF 26, the light emitted from the excitation light source 22 has a slight wavelength distribution width, so that it is filtered into a narrow band of only the center wavelength. Further, the LPF 27 filters the light emitted from the excitation light source 22 into pure linearly polarized light because it has a slight phase difference component. Further, the first NDF 28 is a so-called neutral density filter, and adjusts the light quantity of the excitation light α emitted from the light source unit 21 by reducing the light emitted from the excitation light source 22. Depending on the intensity of the emitted light emitted from the excitation light source 22, the first NDF 28 may not be provided in the first wave shaping unit 24.

  The excitation optical system 30 guides the excitation light α from the light source unit 21 to the prism 51 of the analysis chip 50 held in the chip holding unit 12. This excitation optical system 30 includes a polarization direction adjusting unit 31 that changes the polarization direction of the excitation light α, a shaping optical system 32 that adjusts the contour shape of the beam of the excitation light α, and the excitation light α into the prism 51. An incident path adjusting unit 35 that changes the incident path to change the reflection position of the excitation light α on the metal film 55 and the incident angle θ of the excitation light α with respect to the metal film 55.

  The polarization direction adjusting unit 31 includes a half-wave plate 33 and a rotation driving unit 34 that rotates the half-wave plate 33.

  The half-wave plate 33 is disposed on the optical path of the excitation optical system 30 and is used as a polarization rotator that continuously rotates the polarization direction of the excitation light α. The rotation driving unit 34 rotates the polarization direction of the excitation light α with respect to the metal film 55 by rotating the half-wave plate 33. The rotation drive unit 34 of this embodiment has a step motor, and rotates the half-wave plate 33 by the step motor based on an instruction signal from the control processing unit 14. When the half-wave plate 33 is rotated in this manner, the polarization direction of the excitation light α linearly polarized in the first wave rectifier 24 is rotated, and thereby the P wave in the excitation light α incident on the metal film 55 is rotated. The amount of the component and the amount of the S wave component change. That is, the rotation drive unit 34 rotates the half-wave plate 33 to maximize the evanescent wave in the metal film 55 (that is, the electric field enhancement of the enhanced electric field formed in the vicinity of the surface 55a of the metal film 55). It is possible to freely change the polarization direction from the condition where the degree is the maximum) to the condition where no exudation occurs (that is, the condition where no enhanced electric field is formed near the surface 55a of the metal film 55).

  The shaping optical system 32 adjusts the beam size of the excitation light α and the contour shape of the beam by a slit, a zoom function or the like so that the contour of the irradiation region of the excitation light α in the metal film 55 becomes a circle having a predetermined diameter. . In addition, the irradiation area | region of the excitation light (alpha) in the metal film 55 of this embodiment is adjusted so that it may become smaller than the measurement area | region in the optical measurement part 40 (refer FIG.10 (B)). Thereby, even if the irradiation area | region of the excitation light (alpha) in the metal film 55 shifts | deviates slightly, the light measurement part 40 can measure the light resulting from a surface plasmon resonance and the enhanced electric field based on this.

  The incident path adjustment unit 35 includes a reflection member 36 that reflects the excitation light α from the light source unit 21, and a reflection member drive unit (drive unit) 37 that drives the reflection member 36.

  The reflecting member 36 has a reflecting surface 36a that reflects the excitation light α. In the present embodiment, a reflecting mirror is used as the reflecting member 36. The reflecting member 36 is a dielectric multilayer film that does not cause a phase shift or dimming between the excitation light α before entering the reflection surface 36a and the excitation light α after being reflected by the reflection surface 36a. A dielectric multilayer film is formed on the reflection surface 36a in which the wavelength dependency of both the P wave component and the S wave component at the excitation light wavelength is eliminated. Thereby, the detection accuracy and sensitivity of the sample in the analyzer 10 are improved.

  Further, a non-reflecting light absorbing material (for example, an absorbing ND film, a flocking cloth, etc.) that absorbs the excitation light α without reflecting it is attached to the back surface 36 b of the reflecting member 36.

  The reflecting member driving unit 37 is provided on the stage 37a, a rotation driving mechanism (not shown) that supports and rotates the reflecting member 36, and a reciprocating driving mechanism (not shown) that reciprocates the stage 37a. And have.

  The rotation driving mechanism changes the direction of the reflecting surface 36a by rotating the reflecting member 36 (see arrow β in FIG. 1). Specifically, the rotation drive mechanism is a reflecting surface in a posture orthogonal to a surface (the paper surface of FIG. 1) including the optical path of the excitation light α incident on the reflecting member 36 and the optical path of the excitation light α after being reflected by the reflecting member 36. The reflecting member 36 is rotated based on the instruction signal from the control processing unit 14 so that the rotation of the rotating member 36a along the plane including the optical path is maintained while maintaining the orthogonal posture. The rotation drive mechanism includes a rotation motor, and the reflection member 36 is rotationally driven directly or indirectly by the rotation motor to change the direction of the reflection surface 36a. Further, the rotation drive mechanism may rotate the reflecting member 36 based on an instruction signal from the control processing unit 14 until the excitation light α from the light source unit 21 enters the back surface 36b of the reflecting member 36. In the present embodiment, the reflection member 36 is attached to the rotation drive mechanism so that the center of gravity passes near the rotation center, and the torque of the rotation motor is sufficiently set. The rotary motor of this embodiment is a high-resolution step motor, and can rotate the reflecting member 36 at a predetermined interval by an instruction signal from the control processing unit 14. The predetermined interval is related to the resolution in adjusting the direction of the reflecting surface 36a, and is appropriately set according to the performance of the apparatus.

  The reciprocating drive mechanism linearly moves the stage 37a, that is, the reflecting member 36 along the optical axis of the excitation light α from the light source unit 21 (see arrow γ in FIG. 1). The reciprocating drive mechanism of the present embodiment drives the stage 37a to reciprocate in the optical axis direction of the excitation light α from the light source unit 21, that is, the vertical direction. Specifically, in the reciprocating drive mechanism, the step motor is controlled by an instruction signal from the control processing unit 14, and the stage 37a on which the rotational drive mechanism and the reflecting member 36 are mounted is moved up and down by a screw feed mechanism driven by the step motor. Move back and forth in the direction. That is, the reciprocating drive mechanism moves the reflecting member 36 from the light source unit 21 while keeping the direction of the reflecting surface 36a with respect to the optical axis of the excitation light α from the light source unit 21 constant according to the instruction signal of the control processing unit 14. It is moved to a predetermined position on the optical axis α with high accuracy.

  The light measurement unit 40 includes a light receiving unit 41, a measurement optical system 42 that guides light from the analysis chip 50 to the light receiving unit 41, a second wave shaping unit 43 that waves the light guided in the measurement optical system 42, And the intensity of light generated in the metal film 55 of the analysis chip 50 and a region adjacent thereto (hereinafter also simply referred to as “light generated in the metal film 55”) is measured.

  The light receiving unit 41 receives light and outputs an intensity signal corresponding to the intensity (light quantity). In the present embodiment, a photomultiplier tube (PMT) having a high sensitivity and a high S / N ratio is used as the light receiving unit 41 in order to detect weak light such as fluorescence generated by exciting a fluorescent substance labeled on the specimen. Is used. The light receiving unit 41 is not limited to the PMT, and may be a cooled CCD image sensor or the like.

  As shown in FIG. 4, the measurement optical system 42 is a conjugate optical system that is hardly affected by stray light, and includes a condenser lens 44 and an imaging lens 45. The measurement optical system 42 of this embodiment is a two-group conjugate optical system in which light traveling between groups, that is, between the condenser lens 44 and the imaging lens 45, becomes parallel light or substantially parallel light.

  The second wave shaping unit 43 removes the excitation light component (for example, plasmon scattered light, Raman scattered light, diffused light, etc.) from the light guided in the measurement optical system 42, and the light guided in the measurement optical system 42. Adjust the light intensity. The second wave rectification unit 43 switches the position of the second band pass filter (first optical filter) 46, the second ND filter (second optical filter) 47, and the positions of the filters 46 and 47. Part 48.

  The second bandpass filter (hereinafter simply referred to as “second BPF”) 46 blocks light having the wavelength of the excitation light α (excitation wavelength). As a result, the second BPF 46 causes the light receiving unit 41 to emit light having a wavelength other than the fluorescence (the light generated when the fluorescent substance labeled on the specimen is excited by the enhanced electric field) (for example, leakage light or plasmon from the excitation light emitting unit 20). Scattered light, diffused light, etc.) can be prevented from entering. That is, the second BPF 46 can remove noise components from the light incident on the light receiving unit 41, thereby improving the detection accuracy and sensitivity of weak fluorescence in the light receiving unit 41.

The second ND filter 47 (hereinafter simply referred to as “second NDF”) is a so-called attenuating filter, which attenuates incident light and emits it. The second NDF 47 attenuates plasmon scattered light or diffused light guided in the measurement optical system 42 to detect weak light (fluorescence in this embodiment) (PMT in this embodiment). 41 enables measurement of plasmon scattered light and the like. That is, since the amount of light to be measured for obtaining the incident angle θ ₁ of the excitation light α that maximizes the enhanced electric field is much larger than the amount of excitation fluorescence measured at the time of detection of the specimen, in the case of using the parts 41, by the enhanced electric field by the 2NDF47 to dim the light to be measured to determine the incident angle theta ₁ of the excitation light α that maximizes, prevents light receiving portion 41 from being damaged it can.

  The second BPF 46 and the second NDF 47 share a common holding frame 49 so as to be aligned along the same plane that is substantially perpendicular to the optical axis (specifically, a plane that is substantially orthogonal to the optical axis of the light traveling through the measurement optical system 42). Is held in.

  The position switching unit 48 switches the positions of the second BPF 46 and the second NDF 47 between the filtering position and the retracted position. The filtering position is a position on the optical path in the measurement optical system 42. Specifically, the filtering position is between the condensing lens 44 and the imaging lens 45, and each filter 46, 47 is orthogonal to the optical axis of the parallel light or the substantially parallel light between the lenses 44, 45 and is in parallel. It is a position that crosses light or substantially parallel light. Thereby, the analyzer 10 can detect the sample with high accuracy. This is because when the second BPF 46 or the second NDF 47 is inclined with respect to the optical axis of parallel light or the like traveling between the lenses 44 and 45, the optical axis of the light that has passed through the second BPF 46 or the second NDF 47 is shifted. The measurement accuracy in the measurement unit 40 decreases. On the other hand, the retracted position is a position deviated from the optical path in the measurement optical system 42.

  The position switching unit 48 is configured so that the second NDF 47 is in the retracted position when the second BPF 46 is in the filtering position (see FIG. 4), and the second NDF 47 is in the filtering position when the second BPF 46 is in the retracted position. Switch each position. The position switching unit 48 of the present embodiment reciprocates the holding frame 49 along the plane in which the second BPF 46 and the second NDF 47 are arranged (see the arrow δ in FIG. 4), so that the position of each filter 46, 47 is changed. Switch. Thereby, the position of the two filters 46 and 47 can be switched simultaneously by one drive source.

  The position switching unit 48 switches the positions of the filters 46 and 47 in accordance with an instruction signal from the control processing unit 14.

  In addition, although the 2nd BPF46 and the 2nd NDF47 are provided in the 2nd wave shaping part 43 of this embodiment, if the intensity | strength of the light measured does not exceed the tolerance of the light-receiving part 41, there is no 2nd NDF47. May be. Also, the second NDF 47 is not provided when the light receiving unit is switched depending on the light to be measured, that is, when the light receiving unit 41 for receiving fluorescence and the light receiving unit for receiving light having a light quantity larger than fluorescence are switched. Also good.

  In this embodiment, the positions of the filters 46 and 47 are switched by moving the holding frame 49 back and forth by the position switching unit 48. However, the present invention is not limited to this. For example, each filter 46, 47 is held by a disc-like holding frame so that the second BPF 46 and the second NDF 47 are aligned on the same plane, and the position switching unit rotates about the intermediate position between the second BPF 46 and the second NDF 47. The positions of the filters 46 and 47 may be switched by rotating the disc-shaped holding frame. Further, the position switching unit may have two driving sources, and the switching of the position of the second BPF 46 and the switching of the position of the second NDF 47 may be performed by separate driving sources.

  The control processing unit 14 controls each component constituting the analyzer 10. For example, when the analysis apparatus 10 analyzes a sample, the control processing unit 14 controls the light source unit 21, the polarization direction adjustment unit 31, the incident path adjustment unit 35, the light measurement unit 40, and the like. In the analyzer 10, a resonance angle scanning process, an optimum position scanning process, a birefringence measurement process, an excitation fluorescence measurement process, and the like are performed.

  For example, as illustrated in FIG. 5, the control processing unit 14 of the present embodiment includes a deviation angle deriving unit 141, a correction value deriving unit 142, a correcting unit 143, and the like.

The deviation angle deriving unit 141 obtains a deviation angle (major axis rotation angle) θ _i that is an angle formed between the P wave direction with respect to the metal film 55 and the optical principal axis of the prism 51 having birefringence characteristics. Specifically, the deviation angle deriving unit 141 is adjacent to the metal film 55 by the light measuring unit 40 while rotating the half-wave plate 33 by the rotation driving unit 34 in a state where the light source unit 21 emits the excitation light α. The deviation angle θ _i is obtained from the maximum light amount and the minimum light amount obtained by measuring the light generated in the region to be processed.

The correction value deriving unit 142 obtains a correction value (correction coefficient) K from the deviation angle θ _i obtained by the deviation angle deriving unit 141. This correction value K is used to correct the amount of light measured by the light measurement unit 40. Specifically, the correction value K is obtained by correcting the amount of fluorescent light measured by the light measuring unit 40 with the correction value K, so that the excitation light α incident on the prism 51 is not birefringent (that is, double-refracted). The amount of fluorescent light measured by the light measuring unit 40 when the light enters the metal film 55 in a state in which no phase rotation due to refraction occurs (the amount of fluorescent light in which an error based on birefringence is suppressed) is obtained.

  The correction unit 143 corrects the amount of fluorescent light measured by the light measurement unit 40 using the correction value K obtained by the correction value deriving unit 142. Specifically, the correction unit 143 performs an operation based on an output signal sent from the light measurement unit 40 (specifically, the light receiving unit 41) when analyzing the sample in the analyzer 10, and this light measurement unit Analysis on fluorescence measured by 40 is performed. For example, the correction unit 143 performs counting of the number of fluorescences per unit area detected by the light measurement unit 40, calculation of an increase amount of fluorescence over time, and the like. Then, the correcting unit 143 corrects the counting result of the number of fluorescence and the calculation result of the increase amount of the fluorescence with the correction value K.

  The calculation result in the control processing unit 14 is output to the display unit 16 connected to the control processing unit 14. Details of specific control and calculation by the control processing unit 14 will be described later.

  The display unit 16 displays the calculation result based on the output signal from the control processing unit 14. The display unit 16 may display a calculation result or the like on a screen like a liquid crystal display, or may print out the calculation result or the like like a printer. A combination of these may also be used.

  The analysis of the sample in the analyzer 10 configured as described above will be described below with reference to FIGS. The details of the control of each component of the analyzer 10 by the control processing unit 14 and the calculation performed by the control processing unit 14 will also be described.

  FIG. 6 is a flowchart showing a basic sequence when analyzing a sample in the analyzer 10, FIG. 7 is a flowchart showing a resonance angle scanning sequence, and FIG. 8 is a flowchart showing an optimum position scanning sequence. FIG. 9 is a flowchart showing the birefringence measurement sequence, and FIG. 10 is a flowchart showing the excitation fluorescence measurement sequence.

<Pretreatment process>
Blood or the like is collected from the patient, and the collected blood or the like is injected into the reagent chip. The reagent chip into which the blood or the like has been injected is set in the pretreatment unit of the analyzer 10. The control processing unit 14 uses the preprocessing unit to perform preprocessing (blood cell separation, dilution, mixing, etc.) of the set reagent chip such as blood to generate a sample solution. In this state, when the analysis chip 50 is installed in the preprocessing unit, the control processing unit 14 injects the pretreated sample solution into the flow channel 58 of the analysis chip 50 by the preprocessing unit, and the metal film. The specimen (specific antigen) is captured by the capturing body 56 fixed to the surface of 55 (that is, the capturing body 56 and the specimen are reacted). In this embodiment, the sample labeled with a fluorescent substance (in this embodiment, a fluorescent dye) is captured by the capture body 56, but the present invention is not limited to this, and the analysis chip is captured after the capture body 56 captures the sample. The fluorescent substance may be injected into the fluorescent substance 50 and labeled with respect to the specimen captured by the capturing body 56.

  The analysis chip 50 subjected to the reaction in this way is transported to the chip holding unit 12 and held by the chip holding unit 12 (step S1).

<Temperature control of excitation light source 22>
On the other hand, the excitation light source (in this embodiment, a laser diode) 22 is constantly temperature-controlled by the temperature adjustment circuit 25 and maintained at a constant temperature in order to output output light having a stable wavelength with little wavelength fluctuation. This is indispensable in an apparatus for quantifying proteins in blood because the amount of surface plasmon resonance and the amount of evanescent wave (enhanced electric field) ooze out when the wavelength is shifted. Since it takes time to reach the maintenance temperature, normally, the temperature of the excitation light source 22 is always maintained by the temperature control circuit 25 from when the analyzer 10 is turned on.

<Resonance angle scanning process>
When the analysis chip 50 is held by the chip holding unit 12, the control processing unit 14 performs scanning (resonance angle scanning) under the optimum surface plasmon resonance condition in the analysis chip 50. Based on the result of this scanning, the control processing unit 14 causes the excitation light α to be incident on the metal film 55 at the excitation incident angle θ ₁ , which is the incident angle at which the electric field strength of the enhanced electric field generated in the metal film 55 is the largest. Next, the reflecting member 36 is positioned (first positioning) (step S2).

Specifically, when the control processing unit 14 drives the reflecting member 36 by the reflecting member driving unit 37, the incident condition (excitation incident angle θ) of the excitation light α to the metal film 55 of the prism 51 included in the analysis chip 50 is determined. ₁ ) Scan is performed. Specifically, the intensity of an enhanced electric field (evanescent wave) based on surface plasmon resonance in the metal film 55 of the prism 51 depending on the material, shape, flow path filling liquid (sample liquid) refractive index, and the like of the prism 51 included in the analysis chip 50. The incident angle θ ₁ of the excitation light at which becomes the largest is determined. However, fluctuations in the excitation light incident condition (excitation incident angle θ ₁ ) occur due to the molecular weight of the sample captured by the capturing body 56, the substance constituting the molecule, the manufacturing error on the prism 51 side, and the like. Therefore, the excitation light α is incident on the metal film 55 so that the incident angle is less than ± 10 ° with the excitation incident angle θ _1a based on the design as the center, and based on the intensity of the light generated in the metal film 55 at this time. Thus, the excitation incident angle θ ₁ in the analysis chip 50 is obtained.

  More specifically, the control processing unit 14 first moves the second BPF 46 to the retracted position and moves the second NDF 47 to the filtering position by the position switching unit 48 of the second wave shaping unit 43 (step S21). At this time, the half-wave plate 33 of the polarization direction adjusting unit 31 has the largest P wave component in the excitation light α incident on the metal film 55 when the excitation light α is emitted from the excitation light emitting unit 20. Thus, it is in a state (initial state) determined by design.

  The control processing unit 14 moves the reflecting member 36 to the maximum separation position by the rotation driving mechanism and the reciprocating driving mechanism of the reflecting member driving unit 37 (step S22). The maximum separation position is a state in which the excitation light α is reflected at a specific position of the metal film 55 (within the measurement region of the light measurement unit 40) when the excitation light α is emitted from the excitation light emitting unit 20. In addition, the position of the reflecting member 36 and the direction of the reflecting surface 36a are the incident angle θ at which the evanescent wave does not ooze into the region near the surface 55a of the metal film 55.

  In this state, the control processing unit 14 measures the intensity of light generated in the metal film 55 by the light measuring unit 40, and acquires the result by an output signal from the light measuring unit 40 (specifically, the light receiving unit 41). . The light measured by the light measurement unit 40 when the reflecting member 36 is at the maximum separation position is surface diffused light (or surface scattered light) SK in the prism 51.

  The control processing unit 14 stores the incident angle θ of the excitation light α with respect to the metal film 55 and the intensity of the light measured by the light measurement unit 40 in association with each other (step S23). At this time, since the second BPF 46 is in the retracted position, the light received by the light receiving unit 41 includes light having the same wavelength as the excitation wavelength of the excitation light α. Specifically, the light having the same wavelength as the excitation wavelength is plasmon scattered light, Raman scattered light, diffused light, or the like generated in the metal film 55. Since the light having the same wavelength as the excitation wavelength is enhanced by surface plasmon resonance generated in the metal film 55, the amount of light is sufficiently larger than the excitation fluorescence emitted by the fluorescent substance labeled on the specimen. Therefore, the control processing unit 14 causes the position switching unit 48 to retract the second BPF 46 from the optical path of the measurement optical system 42, so that the light receiving unit 41 can receive the light having the excitation wavelength. The intensity of the generated light can be accurately measured.

  In the present embodiment, the second BPF 46 is retracted to the retracted position in order to measure surface plasmon scattered light, diffused light, or the like having a light amount much larger than that of the excitation fluorescence by the light receiving unit 41 that measures the excitation light having a small amount of light. At the same time, by moving the second NDF 47 to the filtering position, the intensity of both lights (scattered light and the like and excitation fluorescence) can be measured by the same light receiving unit (PMT in the present embodiment) 41.

  In a state where the excitation light α is emitted from the light source unit 21, the control processing unit 14 rotates the direction of the reflection surface 36 a by the rotation drive mechanism so as not to shift the irradiation position on the metal film 55 by the incident path adjustment unit 35. At the same time (Step S24), the position of the reflecting member 36 is moved by the reciprocating drive mechanism (Step S25). Specifically, the control processing unit 14 reflects the position of the reflecting member 36 and the excitation light α reflected by the reflecting surface 36a at that position and enters the prism 51 and reaches a specific position of the metal film 55. The direction of the surface 36a is stored in advance as a table in association with each other. Then, the control processing unit 14 moves the reflecting member 36 by controlling the rotational drive mechanism and the reciprocating mechanism based on this table. Thus, even if the change of the position of the reflecting member 36 and the adjustment of the direction of the reflecting surface 36a are performed by a reciprocating drive mechanism and a rotational drive mechanism that are not mechanically linked to each other, the irradiation of the excitation light α on the metal film 55 Only the incident angle θ with respect to the metal film 55 can be changed without changing the position.

  In step S24 and step S25, one of the steps may be performed first, and then the other step may be performed. Alternatively, both steps may be performed simultaneously. At this time, the light measurement unit 40 measures the intensity of the light generated in the metal film 55 and outputs the measurement result to the control processing unit 14, and the control processing unit 14 stores the measurement result in association with the incident angle θ. (Step S26).

  This is repeated, and the control processing unit 14 measures the light intensity by the light measurement unit 40 while changing the incident angle θ so that the irradiation position on the metal film 55 is not shifted, and stores the measurement result (step S27). ).

When the control processing unit 14 measures the light intensity by the light measuring unit 40 in a predetermined scanning region (for example, an incident angle θ of less than ± 10 ° with the excitation incident angle θ _1a based on the design as the center), the light source unit The emission of the excitation light α from 21 is stopped. Then, the control processing unit 14 selects the maximum value and the minimum value of the stored light amount and stores them (step S28), and at the same time, the position of the reflecting member 36 when the maximum light amount is obtained and the reflection surface 36a. The reflecting member 36 is driven by the reflecting member driving unit 37 so as to be oriented (step S29).

<Optimum position scanning process>
When the first positioning of the reflecting member 36 is finished, the control processing unit 14 reflects the irradiation position (incident position) of the excitation light α onto the metal film 55 so as to be the center of the measurement region of the light measurement unit 40. The member 36 is positioned (second positioning) (step S3).

  Specifically, first, the control processing unit 14 moves the reflecting member 36 to the upper end position by the reciprocating driving mechanism of the reflecting member driving unit 37 (step S31). This upper end position is a position where the incident position is outside the measurement region of the light measurement unit 40 when the excitation light α is incident on the metal film 55 (see position A in FIG. 11A). The control processing unit 14 causes the light source unit 21 to emit the excitation light α when the reflecting member 36 is at this position, and the light measurement unit 40 measures the intensity of the light generated at the metal film 55 at this time, and the measurement result is obtained. Store (step S32). The control processing unit 14 determines whether or not the reflecting member 36 is at the lower end position (step S33). Details of the lower end position will be described later. When determining that the reflecting member 36 is not at the lower end position, the control processing unit 14 moves the reflecting member 36 downward by a predetermined amount by the reciprocating drive mechanism (step S34). At this time, the control processing unit 14 moves the reflecting member 36 only by the reciprocating driving mechanism without rotating the reflecting member 36 by the rotation driving mechanism. That is, the control processing unit 14 moves only the position of the reflecting member 36 without changing the direction of the reflecting surface 36 a with respect to the excitation light α from the light source unit 21. After the reflecting member 36 moves, the control processing unit 14 emits excitation light from the light source unit 21, measures the intensity of light generated at the metal film 55 at this time by the light measuring unit 40, and stores the measurement result ( Step S32). Then, the control processing unit 14 determines whether or not the reflecting member 36 is at the lower end position (step S33). The control processing unit 14 repeats step S32 to step S34 in order until the reflecting member 36 moves to the lower end position.

  The control processing unit 14 determines whether or not the reflecting member has reached the lower end position based on whether or not the light intensity measured by the light measurement unit 40 has decreased by 50% from the maximum light amount among the stored light amounts. to decide. Specifically, since the irradiation region of the excitation light α is outside the measurement region of the light measurement unit 40 in the metal film 55, the amount of light measured by the light measurement unit 40 is small. Then, when the reflecting member 36 gradually moves downward and the irradiation region of the excitation light α enters the measurement region of the light measurement unit 40 (see position B in FIG. 11A), the light measurement unit 40 When the amount of light to be measured gradually increases and the reflecting member 36 further moves downward, the entire irradiation region of the excitation light α is completely included in the measurement region of the light measurement unit 40 (see FIG. 11 (A) (see position C), the amount of light generated by the metal film 55 measured by the light measurement unit 40 is maximized. When the reflecting member 36 further moves downward, the irradiation region of the excitation light α moves outward from the opposite end of the measurement region of the light measurement unit 40 (see position D in FIG. 11A). The amount of light measured by the light measurement unit 40 decreases (that is, vignetting occurs). The control processing unit 14 stores the intensity of these lights in association with the vertical position of the reflecting member 36 when the intensity is measured, and the measured intensity of the light is 50% lower than the maximum light quantity. Judge whether or not. Then, the control processing unit 14 determines that the reflection member 36 has reached the lower end position when the light intensity measured by the light measurement unit 40 falls by 50% of the maximum intensity.

  When determining that the reflecting member 36 has moved to the lower end position, the control processing unit 14 selects a value that is 50% lower than the maximum light amount from each stored light amount value (step S35). At this time, there are two values to be selected (see position B and position D in FIG. 11A). The control processing unit 14 selects the position of the reflection member 36 when the light amount measured by the light measurement unit 40 falls by 50% of the maximum light amount, and the center position thereof (see position Ce in FIG. 11A). Is calculated and stored (step S36). Then, the control processing unit 14 moves the reflection member 36 by the reciprocating drive mechanism, and moves the irradiation region to the determined center position Ce (see step S37, FIG. 11B).

  Thereby, the amount of autofluorescence for each analysis chip 50 measured by the light measurement unit 40 can be made constant. Specifically, fluorescence is generated inside the prism 51 by the excitation light α traveling in the prism 51 (autofluorescence). This fluorescence is weak compared to scattered light or the like (plasmon scattered light or diffused light) generated in the metal film 55, but a fluorescent substance labeled on the specimen is emitted when the concentration of the specimen in the sample liquid is low. Since it becomes the same level as excitation fluorescence, it becomes noise in measurement of excitation fluorescence. Since the autofluorescence is weak, most of the light in the vicinity of the irradiation region of the excitation light α in the metal film 55 can cause noise in the measurement of the excitation fluorescence. Since excitation light α reflected by the metal film 55 is almost eliminated when surface plasmon resonance occurs, autofluorescence from the incident side (left side in FIG. 11A) of the measurement region of the light measurement unit 40 is generated. It becomes a problem. Since the amount of this autofluorescence is proportional to the optical path length, it is necessary to adjust the irradiation position of the excitation light α so that the incident-side optical path length in the measurement region of the light measurement unit 40 is constant. Therefore, the optical path on the incident side of the excitation light α in the measurement region of the light measurement unit 40 is adjusted as described above so that the irradiation position of the excitation light α is always centered in the measurement region of the light measurement unit 40. The length becomes constant, and as a result, the difference in autofluorescence for each analysis chip 50 (prism 51) can be suppressed, and the analysis accuracy of the specimen can be improved.

<Birefringence measurement process>
Next, since the birefringence occurs when the excitation light α travels through the prism 51, the control processing unit 14 measures this birefringence (step S4), and the excitation fluorescence from the fluorescent substance labeled on the specimen is measured. By taking this into account during measurement, the measurement accuracy of the specimen is improved. Specifically, birefringence occurs when light passes through the medium. Birefringence increases when light passes through a dielectric such as resin. This birefringence is caused by a density difference or the like in the medium, and this density difference occurs when the medium is molded. Therefore, the degree of birefringence differs depending on the individual prism 51. Although the phase rotation occurs in the excitation light α traveling in the prism 51 due to birefringence and only the P wave is desired to be incident on the metal film 55, the S wave component is generated in the excitation light α due to the phase rotation due to the birefringence. . The amount of excitation fluorescence excited by the enhanced electric field decreases in accordance with the amount of S wave component generated by this birefringence. For this reason, the control processing unit 14 (specifically, the correction unit 143) corrects this decrease, thereby improving the specimen detection accuracy and sensitivity in the analyzer 10.

  Specifically, the control processing unit 14 (specifically, the deviation angle deriving unit 141) emits the excitation light α from the light source unit 21, and measures the intensity of light generated in the metal film 55 by the light measurement unit 40. At this time, the half-wave plate 33 is in an initial state (step S41). Then, the control processing unit 14 (deviation angle deriving unit 141) rotates the half-wave plate 33 by the rotation driving unit 34 while rotating the half-wave plate 33 with the intensity of the light measured by the light measurement unit 40. It is stored in association with the position (rotation angle from the initial state) (step S42 and step S43). When the control processing unit 14 (deviation angle deriving unit 141) rotates the half-wave plate 33, the P wave component and the S wave component of the excitation light α incident on the metal film 55 increase and decrease. The intensity of the light measured by the light measurement unit 40 increases or decreases while the half-wave plate 33 is rotated. At this time, if the P wave component increases in the excitation light α, the S wave component decreases accordingly, and if the P wave component decreases, the S wave component increases accordingly. The intensity of light measured by the light measurement unit 40 increases as the P wave component increases in the excitation light α incident on the metal film 55, and the intensity of light measured by the light measurement unit 40 increases as the S wave component increases. Becomes smaller. This is because the P wave component contributes to surface plasmon resonance, but the S wave component does not contribute to surface plasmon resonance.

The control processing unit 14 (deviation angle deriving unit 141) repeats step S42 and step S43 in order until the maximum value DR _max and the minimum value DR _min of the amount of light measured by the light measurement unit 40 are obtained (step S44). ). When the maximum value DR _max and the minimum value DR _min are obtained, the control processing unit 14 (deviation angle deriving unit 141) selects the maximum value DR _max and the minimum value DR _min from the stored light amount (step S45). These values are stored, and the rotational position of the half-wave plate 33 when these values are obtained (specifically, the first rotational position when the maximum value DR _max is obtained, and And the second rotational position when the minimum value DR _min is obtained).

Next, the control processing unit 14 (deviation angle deriving unit 141) calculates the following from the stored maximum value DR _max and minimum value DR _min and the intensity of the surface diffused light (SK) stored in the resonance angle scanning step. The deviation angle (major axis rotation amount) θ _i due to birefringence at the prism 51 is obtained by the equation (4). Then, the control processing unit 14 (correction value deriving unit 142) derives the correction value K from the deviation angle θ _{i according} to the equation (5). The control processing unit 14 stores the deviation angle θ _i and the correction value K obtained in this way (step S46).

Then, the control processing unit 14 rotates the half-wave plate 33 to the rotation position when the maximum value (DR _max ) is obtained by the rotation driving unit 34. Accordingly, the excitation light α is incident on the metal film 55 in a state where the P wave component is the largest (that is, in a state where the S wave component is the smallest).

Here, the deviation angle θ _i and the correction value K will be described.

The prism 51 having birefringence characteristics has an optical main axis (slow axis and fast axis), and the relationship between the optical main axis and the polarization direction of incident light (excitation light α in the present embodiment) to the prism 51. Therefore, the polarization state of the excitation light α is changed. For example, when the angle (deviation angle) between the P wave direction with respect to the metal film 55 and the optical principal axis of the prism 51 is θ _i , if the optical principal axis and the polarization direction of the excitation light α do not match, The polarization state of the excitation light α that travels through and enters the metal film 55 becomes elliptically polarized light by phase rotation (see FIG. 12A). When the optical main axis and the polarization direction of the excitation light α are the same, the polarization direction of the excitation light α traveling through the prism 51 and entering the metal film 55 is the same as the optical main axis. The amount of light to be measured is maximized (see FIG. 12B). On the other hand, when the optical main axis and the polarization direction of the excitation light α are shifted by 90 °, the polarization direction of the excitation light α traveling through the prism 51 and entering the metal film 55 is a direction orthogonal to the optical main axis, The amount of light measured by the light measurement unit 40 is minimized (see FIG. 12C).

このズレ角θ_ｉが求まれば、プリズムにおいて複屈折がない場合に光測定部４０により測定される光の光量が以下の式（７）及び式（８）から得られる。

これより、

が得られる。 Thus, when the deviation angle with respect to the P direction is θ _i , the maximum value (maximum light amount value) DR _max is the original light amount ratio (ratio with the light amount measured without birefringence) cos ^2. θ _i and the minimum value (maximum light amount value) DR _min are sin ² θ _{i in} terms of the original light amount ratio. If the maximum value DR _max and the minimum value DR _min are obtained, θ _i can be obtained by the following equation (6).

If the deviation angle θ _i is obtained, the amount of light measured by the light measurement unit 40 when there is no birefringence in the prism can be obtained from the following equations (7) and (8).

Than this,

Is obtained.

<Excitation fluorescence measurement process>
Next, the control processing unit 14 irradiates the reflection member 36 in the state in which the first and second positioning are performed by the light source unit 21 with the excitation light α. As a result, the excitation light α causes surface plasmon resonance in the metal film 55, and the fluorescent substance labeled on the specimen captured by the capture body 56 of the metal film 55 is excited by the enhanced electric field based on the surface plasmon resonance. ). And the control process part 14 measures excitation fluorescence by the light measurement part 40 (step S5).

  Specifically, the control processing unit 14 causes the position switching unit 48 to move the second BPF 46 to the filtering position and retracts the second NDF 47 to the retracted position (step S51: see FIGS. 1 and 4). Then, the control processing unit 14 uses the rotation driving mechanism of the reflecting member driving unit 37 so that the excitation light α emitted from the light source unit 21 is incident on the non-reflecting light absorbing material provided on the back surface 36b of the reflecting member 36. The reflecting member 36 is rotated (step S52). As a result, the excitation light α is not incident on the prism 51, and the control processing unit 14 performs measurement with the light measurement unit 40 in this state, and the output (dark noise DN) from the light measurement unit 40 at this time. Is stored (step S53). The control processing unit 14 rotates the reflecting member 36 again by the rotation driving mechanism of the reflecting member driving unit 37 so that the excitation light α from the light source unit 21 is incident on the reflecting surface 36a of the reflecting member 36 again (step S54). . The direction of the reflecting surface 36a at this time is the direction set in step S29 of the resonance angle scanning process.

The control processing unit 14 emits the excitation light α from the light source unit 21, measures the amount of excitation fluorescence caused by the enhanced electric field generated in the vicinity of the metal film 55 by the light measurement unit 40, and stores this (step S55). . Thereby, the control processing unit 14 obtains the measurement maximum light quantity (first light quantity value) _Smax . This is because, in the birefringence measurement step, the half-wave plate 33 is rotated to the first rotation position where the intensity of the light generated in the metal film 55 becomes the maximum value DR _max when the excitation light α is incident on the metal film 55. This is because the strength of the enhanced electric field in the vicinity of the metal film 55 is maximized.

Next, the control processing unit 14 causes the rotation driving unit 34 to perform the second rotation in which the intensity of light generated in the metal film 55 becomes the minimum value DR _min when the excitation light α is incident on the metal film 55 in the birefringence process. The half-wave plate 33 is rotated to the position (step S56). And the control process part 14 measures the light quantity of excitation fluorescence by the light measurement part 40, and memorize | stores this (step S57). Thereby, the control processing unit 14 obtains the minimum measurement light quantity (second light quantity value) _Smin .

The control processing unit 14 (specifically, the correction unit 143), from the stored measurement maximum light quantity _Smax , measurement minimum light quantity _Smin , and dark noise DN, the following expressions (9-1) to (13) As shown in -2), the light amount h of autofluorescence in the prism is derived. Here, the amount of excitation fluorescence when S _max is obtained is H ₁ , the amount of autofluorescence is h ₁ , the amount of excitation fluorescence when S _min is obtained is H ₂ , and the amount of autofluorescence is h ₂ .

より、

First,

Than,

より、

Also,

Than,

が得られる。 And by the formula (10) and the formula (12),

Is obtained.

から、励起蛍光の光量Ｈを導出し、これを記憶する（ステップＳ５８）。 Then, the control processing unit 14 (correction unit 143) uses the correction value K obtained in the birefringence measurement step,

From this, the light quantity H of the excitation fluorescence is derived and stored (step S58).

Note that if the prism 51 is formed of birefringence or a fine material (including resin), the control processing unit 14 calculates the amount H of excitation fluorescence by the following approximate expression (15). Ask.

Further, in the case of a measurement system with very little dark noise (DN), the control processing unit 14 obtains the amount H of excitation fluorescence by the following approximate expression (16).

<Memory / display process>
As described above, the control processing unit 14 obtains the excitation fluorescence light amount H from which the influence of birefringence is removed, stores this in association with the specimen number, and erases other memory (step S6). In addition, the control processing unit 14 outputs information based on the excitation fluorescence light quantity H stored in association with the specimen number to the display unit 16, and the display unit 16 displays the information.

  Finally, the control processing unit 14 returns the reflecting member 36 to the initial position (step S7) and ends the series of measurements.

  According to the present embodiment, the amount of excitation fluorescence measured by the light measurement unit 40 is corrected by the correction value K, so that the excitation light α is incident on the metal film 55 without birefringence in the prism 51 (metal film The amount of fluorescent light measured when reflected at 55 is obtained (the amount of fluorescent light with reduced error based on birefringence). As a result, even if the prism 51 is replaced for each inspection, the amount of fluorescence measured when the excitation light α is incident on the metal film 55 can be obtained without causing phase rotation due to birefringence in each prism 51. Therefore, measurement errors due to variations in the degree of birefringence for each prism 51 can be effectively suppressed.

  In addition, since the error included in the measurement value can be reduced only by measuring the amount of light generated in the metal film 55 and its adjacent region while rotating the half-wave plate 33, it is like a conventional analyzer. In addition, it is not necessary to adjust the positions and orientations of the plurality of polarizing elements while measuring the amount of reflected excitation light and feeding back the measurement results, thereby shortening the analysis time of the specimen.

  Furthermore, according to the analyzer 10 of the present embodiment, only one half-wave plate 33 is required for adjusting the polarization direction of the excitation light α, and a plurality of polarizing elements and their positions and postures are respectively set. Compared to a conventional analyzer equipped with a driving device to be controlled, the configuration of the analyzer can be simplified and downsized.

  Further, according to the present embodiment, the control processing unit 14 automatically performs the derivation of the correction value K, the detection of the amount of fluorescent light while suppressing the error based on the birefringence, and the analysis of the specimen.

In the analysis apparatus 10, the control processing unit 14 has a storage unit that can store the surface diffusion light amount SK, and the deviation angle deriving unit 141 uses the surface diffusion light amount SK stored in this storage unit. If configured to obtain θ _i , the deviation angle θ _i can be obtained without measuring the surface diffusion light quantity SK for each examination, and the analysis time of the specimen can be shortened.

In addition, according to the present embodiment, it is possible to detect a sample with high sensitivity and high accuracy. Specifically, by measuring the intensity of light generated in the metal film 55 by surface plasmon resonance, the excitation incident angle to the metal film 55 at which the intensity of the enhanced electric field formed in the vicinity of the surface 55a of the metal film 55 is maximized. θ ₁ can be obtained with high accuracy. At this time, since the light generated in the metal film 55 is light having an excitation wavelength such as plasmon scattered light or surface diffused light, the second BPF 46 that blocks this wavelength component is placed in the retracted position, so that the metal is generated based on the surface plasmon resonance. The increase / decrease in the intensity of light generated in the film 55 can be accurately measured. Moreover, at the time of detection of the specimen, by inserting the second BPF 46 on the optical path of the measurement optical system 42, light components having excitation wavelengths such as plasmon scattered light and surface diffused light from the light measured by the light measurement unit 40 can be obtained. As a result, the excitation fluorescence emitted from the fluorescent substance labeled on the specimen can be accurately measured. Therefore, the signal obtained by measurement has a high S / N ratio, and it is possible to detect a sample with high accuracy and high sensitivity.

  Moreover, according to this embodiment, the influence of the autofluorescence in the prism 51 can be removed from the measurement result, thereby ensuring a wide dynamic range.

  Specifically, the S wave in the excitation light when reflected by the metal film 55 from the increase / decrease in the intensity of the light detected by measuring the light generated by the metal film 55 while changing the polarization direction of the excitation light α with respect to the metal film 55. The rotational position of the half-wave plate 33 when the component (component contributing to surface plasmon resonance) is the largest, and the half-wave plate when the P wave component (component contributing to surface plasmon resonance) is the largest. 33 rotational positions can be obtained respectively. Then, by adjusting the half-wave plate 33 so that the polarization direction of the excitation light α is in a state where the S wave component is maximized when reflected by the metal film 55, the excitation fluorescence is measured by the light measurement unit 40. The amount of autofluorescence in the prism 51 can be obtained. As a result, the half-wave plate 33 is adjusted so that when the polarization direction of the excitation light α is incident on the metal film 55, the P-wave component is maximized, and the excitation fluorescence is measured by the light measurement unit 40 based on the result of the measurement. It becomes possible to remove the influence of fluorescence and extract excitation fluorescence. As a result, according to the analyzer 10, the influence of autofluorescence can be suppressed and excitation fluorescence can be detected with high accuracy, and a wide dynamic range is ensured.

  Further, according to the present embodiment, since the incident angle θ of the excitation light α with respect to the metal film 55 can be changed by driving the reflecting member 36, the reflection of the excitation light α on the metal film 55 by changing the incident angle θ. Position shift can be suppressed. That is, according to the configuration described above, the incident angle θ of the excitation light α with respect to the metal film 55 can be changed only by changing / adjusting the position of the reflecting member 36 and the direction of the reflecting surface 36a. Compared to adjusting the incident angle θ by moving the entire excitation optical system consisting of a light source and multiple lenses, the number of movable parts and the weight of the movable part can be reduced. Can be suppressed. As a result, it is possible to suitably suppress the deviation of the reflection position of the excitation light α on the metal film 55 due to changing the incident angle θ of the excitation light α.

  Moreover, in order to cause one excitation light α to enter the prism 51 and generate an enhanced electric field based on surface plasmon resonance in the vicinity of the metal film 55, a plurality of excitation lights α, α,. It is possible to prevent the amount of autofluorescence from increasing as in the case where the light is incident on the prism 51 at the same time. Thereby, in the signal obtained by measuring the light generated in the vicinity of the metal film 55 by surface plasmon resonance, it is possible to suppress a decrease in the SN ratio due to autofluorescence.

Further, according to the present embodiment, the incident angle θ of the excitation light α with respect to the metal film 55 is set with high accuracy so that the electric field strength of the enhanced electric field is maximized by surface plasmon resonance. Specifically, a shift occurs between the absorption peak in the angle spectrum of the reflected light in the metal film 55 and the peak of the electric field intensity of the enhanced electric field, but light caused by surface plasmon resonance (light generated in the metal film 55). ) Matches the peak of the electric field strength of the enhanced electric field. Therefore, the excitation incident on the metal film 55 is adjusted by adjusting the reflection member 36 so that the position of the reflection member 36 and the direction of the reflection surface 36a when the intensity of light generated in the metal film 55 becomes maximum. The light α becomes the excitation incident angle θ _{1 at} which the electric field strength of the enhanced electric field is the largest.

  Next, a second embodiment of the present invention will be described with reference to FIGS. 13 and 5. The same reference numerals are used for the same components as those in the first embodiment, and detailed descriptions are omitted. Only the details will be described.

  In the analyzer 10A of the present embodiment, the configuration of the light measurement unit 40A and the configuration of the control processing unit 14A are different from the configuration of the light measurement unit 40 and the configuration of the control processing unit 14 of the analysis device 10 of the first embodiment (FIG. 1). And FIG. 5).

  Specifically, the second wave shaping unit 43A of the light measurement unit 40A has a second BPF 46. Unlike the first embodiment, the second BRF 46 is fixed on the optical path of the measurement optical system 42. That is, the second wave shaping unit 43A of the present embodiment does not include the second NDF 47 and the position switching unit 48 in the second wave shaping unit 43 of the first embodiment.

Further, the deviation angle deriving unit 141A of the control processing unit 14A obtains the deviation angle θ _i based on the amount of excitation fluorescence measured by the light measurement unit 40A. Specifically, the deviation angle deriving unit 141A has a light amount of excitation fluorescence measured by the light measurement unit 40A while rotating the half-wave plate 33 by the rotation driving unit 34 in a state where the light source unit 21 emits the excitation light α. The deviation angle θ _i is determined from the maximum value (maximum fluorescence amount value) S _max and the minimum value (minimum fluorescence amount value) S _min .

Specifically, when the maximum value S _max and the minimum value S _min are obtained, the deviation angle deriving unit 141A obtains a deviation angle (major axis rotation amount) θ due to birefringence in the prism 51 according to the following equation (17). _i is determined.

  The analysis of the sample in the analyzer 10A configured as described above will be described below with reference to FIGS. 6, 8, 9, 14, and 15. FIG. FIG. 14 is a flowchart showing a resonance angle scanning sequence in the analyzer 10A of this embodiment, and FIG. 15 is a flowchart showing an excitation fluorescence measurement sequence in the analyzer 10A of this embodiment.

<Pretreatment process>
Similar to the first embodiment, the sample liquid generated in the pretreatment process is injected into the analysis chip 50, and the trapping degree 56 and the specimen react. The analysis chip 50 subjected to the reaction is transported to and held by the chip holding unit 12 (step S1).

<Temperature control of excitation light source 22>
As in the first embodiment, the temperature of the excitation light source 22 is controlled by the temperature adjustment circuit 25 so that output light having a stable wavelength with little wavelength fluctuation is output to the excitation light source 22, and is maintained at a constant temperature.

<Resonance angle scanning process>
When the analysis chip 50 is held by the chip holding unit 12, the control processing unit 14A performs resonance angle scanning. Then, the control processing unit 14A performs the first positioning of the reflecting member 36 (step S2).

Specifically, the control processing unit 14 </ b> A drives the reflecting member 36 by the reflecting member driving unit 37, thereby scanning the incident condition (excitation incident angle θ ₁ ) of the excitation light α onto the metal film 55.

  Specifically, the half-wave plate 33 of the polarization direction adjusting unit 31 is in an initial state. Then, the control processing unit 14A causes the reflection surface 36a to be oriented by the rotation drive mechanism so that the irradiation position on the metal film 55 is not shifted by the incident path adjustment unit 35 in a state where the excitation light α is emitted from the light source unit 21. The position of the reflecting member 36 is moved by the reciprocating drive mechanism (step S25) while rotating (step S24). At this time, unlike the analyzer 10 of the first embodiment, the second BPF 46 is fixed on the optical path of the measurement optical system 42, so that the light having the excitation wavelength is blocked by the second BPF 46 and only the excitation fluorescence is present in the light receiving unit 41. Reach. Thereby, the light measurement unit 40A measures the intensity of the excitation fluorescence and outputs the measurement result to the control processing unit 14A, and the control processing unit 14A stores the measurement result in association with the incident angle θ (step S26A).

  This is repeated, and the control processing unit 14A measures the fluorescence intensity by the light measurement unit 40A while changing the incident angle θ so that the irradiation position on the metal film 55 is not shifted, and stores the measurement result (step S27). ).

The control processing unit 14A stops emission of the excitation light α from the light source unit 21 when measuring the intensity of fluorescence by the light measurement unit 40A in a predetermined scanning region. Then, the control processing unit 14A selects the maximum value S _max and the minimum value S _{min of} the stored fluorescence amount and stores them (step S28A), and the reflecting member 36 when the maximum value S _max is obtained. The reflecting member 36 is driven by the reflecting member driving unit 37 so that the position of the reflecting member 36 and the direction of the reflecting surface 36a are aligned (step S29).

<Optimum position scanning process>
When the positioning of the reflecting member 36 is finished, the control processing unit 14A determines that the irradiation position (incident position) of the excitation light α onto the metal film 55 is the center of the measurement region of the light measurement unit 40A, as in the first embodiment. Then, the second positioning of the reflecting member 36 is performed (Step S3 and Steps S31 to S37).

<Birefringence measurement process>
Next, similarly to the first embodiment, the control processing unit 14A measures the birefringence in the prism 51 (step S4), and takes this into consideration when measuring the excitation fluorescence, thereby improving the measurement accuracy of the specimen. .

  Specifically, the control processing unit 14A (specifically, the deviation angle deriving unit 141A) emits the excitation light α from the light source unit 21, and measures the intensity of the excitation fluorescence by the light measurement unit 40A. At this time, the half-wave plate 33 is in an initial state (step S41). Then, the control processing unit 14A (deviation angle deriving unit 141A) rotates the half-wave plate 33 by the rotation driving unit 34 while changing the intensity of the excitation fluorescence measured by the light measurement unit 40A to the half-wave plate 33. The information is stored in association with the rotational position (steps S42 and S43).

Control processing unit 14A (deviation angle deriving section 141A), up to a maximum value of the light amount of the excitation fluorescence measured by the optical measuring unit 40A _{S max} and a minimum value _{S min} is obtained, repeats step S42 and step S43 in this order (step S44). Control processing unit 14A (deviation angle deriving section 141A), when the maximum value _{S max} and a minimum value _{S min} is obtained, elect and these maximum value _{S max} and a minimum value _{S min} from the stored light intensity (step S45) Each of these values is stored.

Next, the control processing unit 14A (deviation angle deriving unit 141A) calculates the deviation angle (long) due to birefringence at the prism 51 from the stored maximum value S _max and minimum value S _{min according} to the above equation (17). (Axis rotation amount) θ _i is obtained. Then, the control processing unit 14A (correction value deriving unit 142) derives the correction value K from the deviation angle θ _i by the same equation (5) as in the first embodiment. The control processing unit 14A stores the deviation angle θ _i thus obtained and the correction value K (step S46).

<Excitation fluorescence derivation process>
Next, the control processing unit 14A derives the amount of excitation fluorescence necessary for analysis of the specimen (step S5A).

  Specifically, the control processing unit 14A rotates and drives the reflecting member driving unit 37 so that the excitation light α emitted from the light source unit 21 enters the non-reflecting light absorbing material provided on the back surface 36b of the reflecting member 36. The reflecting member 36 is rotated by the mechanism (step S52). Accordingly, the control processing unit 14A detects the dark noise DN based on the output of the light measurement unit 40, and stores this (step S53).

The control processing unit 14 (specifically, the correction unit 143) compares the stored maximum value _Smax with a predetermined reference light amount stored in advance (step S155), and if the maximum value _Smax is small, An output signal is output to the display unit 16 to display words such as “impossible to measure” and “sufficiently healthy” (step S156). This reference light amount is a threshold value set to determine whether or not the person who collected the sample is healthy based on the amount of the sample in the sample solution. Specifically, if the maximum value S _max is smaller than the reference light amount, the amount of the sample is small, so the person who collected the sample is judged to be sufficiently healthy, and if it is large, the amount of the sample is large, so the sample was collected. The person is determined to be ill and a detailed analysis of the specimen is performed.

If the maximum value _Smax is equal to or greater than the reference light amount, the control processing unit 14A uses the stored maximum value _Smax , minimum value _Smin, and dark noise DN obtained above as in the first embodiment. The autofluorescence light quantity h in the prism 51 is derived from the above formulas (9-1) to (13-2).

  Then, the control processing unit 14A (correction unit 143) uses the correction value K obtained in the birefringence measurement step to derive the excitation fluorescence light amount H from the above equation (14), and stores this (step S157). ).

<Memory / display process>
As described above, the control processing unit 14A obtains the excitation fluorescence light quantity H from which the influence of birefringence and the like are removed, stores this in association with the specimen number, and erases the other storage (step S6). In addition, the control processing unit 14A outputs information based on the excitation fluorescence light quantity H stored in association with the specimen number to the display unit 16, and the display unit 16 displays the information.

  Finally, the control processing unit 14A returns the reflecting member 36 to the initial position (step S7) and ends a series of measurements.

  According to the analyzer 10A of the present embodiment, the light amount of excitation fluorescence measured by the light measurement unit 40 is corrected with the correction value K, whereby the light amount of fluorescence with reduced error due to birefringence can be obtained. As a result, even if the prism 51 is replaced for each inspection, the amount of fluorescence measured when the excitation light α is incident on the metal film 55 can be obtained without causing phase rotation due to birefringence in each prism 51. Therefore, measurement errors due to variations in the degree of birefringence for each prism 51 can be effectively suppressed. In the analysis apparatus 10A, the above-described effect can be suitably obtained when the excitation fluorescence measured by the light measurement unit 40 is sufficiently larger than the autofluorescence generated in the prism and the deviation angle is sufficiently large.

  In addition, the light measurement unit 40 of the present embodiment only needs to measure only the fluorescence contained in the light generated in the metal film 55 and the region adjacent thereto, and the configuration can be simplified. Specifically, the light generated in the metal film 55 and a region adjacent to the metal film 55 due to surface plasmon resonance generated in the metal film 55 includes, for example, scattered light such as plasmon scattered light and Raman scattered light, a fluorescent material, and the like. There is fluorescence emitted by being excited by an evanescent wave (enhanced electric field). Since these scattered light and fluorescent light are greatly different in light quantity, in order to measure each light, a plurality of light quantity measuring devices corresponding to each light quantity can be arranged in a switchable manner or when measuring with one light quantity measuring device. As in the first embodiment, the second NDF 47 and the like must be able to be taken in and out before the light receiving unit 41 so that the amount of light incident on the light receiving unit 41 can be adjusted, and the configuration of the light measuring unit becomes very complicated. The device cannot be downsized. However, when the light measurement unit 40 measures only the amount of excitation fluorescence, the configuration of the light measurement unit is simpler than the light measurement unit 40 of the first embodiment capable of measuring both scattered light and fluorescence. Can be

  The surface plasmon resonance fluorescence analyzer and the surface plasmon resonance fluorescence analysis method of the present invention are not limited to the first embodiment and the second embodiment described above, and various modifications can be made without departing from the scope of the present invention. Of course, can be added.

  In the analyzers 10 and 10A of the first embodiment and the second embodiment, the analysis chip 50 is replaced every time the sample is detected, but the present invention is not limited to this. That is, the analyzer 10 may be an analyzer 10 in which an analysis chip is incorporated in a part of the analyzers 10 and 10A, and a sample is detected by repeatedly using one analysis chip.

In the first embodiment and the second embodiment, in the resonance angle scanning step, the control processing unit 14 continuously determines the incident angle θ of the excitation light α to the metal film 55 when determining the excitation incident angle θ _1. Although the intensity of light generated in the metal film 55 is measured while changing, it is not limited to this.

For example, the control processing unit 14 measures the intensity of light generated in the metal film 55 at each incident angle θ while intermittently changing the incident angle θ within the first range (for example, by changing the incident angle by 1 °). The incident angle θ is intermittently smaller than the first scan within the second range based on the first scan and the result of the first scan (for example, the incident angle is incremented by 0.1 °) or You may make it perform the 2nd scan which measures the intensity | strength of the light which arises in the metal film 55, changing continuously. That is, the range of the second scan is narrowed by the first scan in discrete steps, and the inside of the narrowed range is finely scanned to obtain the excitation incident angle θ _{1 at} which the light generated in the metal film 55 becomes the maximum light amount. Also good. Note that the second range is included in the first range.

Specifically, for example, the control processing unit 14 changes the incident angle θ in increments of 1 ° within the range of the incident angle θ less than ± 10 ° (first range) around the excitation incident angle θ _1a based on the design. However, the intensity of light generated in the metal film 55 at each incident angle θ is measured (first scanning), and the result is stored in association with the corresponding incident angle θ. Then, in the range of the incident angle θ within ± 1 ° (second range) around the incident angle θ at which the maximum intensity is obtained in the first scanning, the incident angle θ is incremented by 0.1 ° or continuously. , The intensity of light generated in the metal film 55 is measured (second scanning), and the result is stored in association with the corresponding incident angle θ. The control processing unit 14 sets the incident angle θ when the maximum intensity is obtained in the second scanning as the excitation incident angle θ ₁ . By this way, compared to the case of finely scanning the entire first range, enhanced electric field becomes possible to obtain better becomes largest excitation incident angle theta ₁ efficiency.

  Note that in the analyzer 10 that performs the first scan and the second scan in the resonance angle scanning process, and sequentially holds the plurality of analysis chips 50 on the chip holding unit 12 and analyzes the samples one after another. The first scan may be performed only for the first analysis chip 50, and only the second scan may be performed for the second and subsequent analysis chips 50.

Specifically, the control processing unit 14 applies the first analysis chip 50 to the first analysis chip 50 in the same manner as described above, for example, in the range of the incident angle θ less than ± 10 ° around the excitation incident angle θ _1a based on the design (first ), The intensity of light generated at the metal film 55 at each incident angle θ is measured (first scanning), and the result is associated with the corresponding incident angle θ. And remember. Then, in the range of the incident angle θ within ± 3 ° (second range) around the incident angle θ at which the maximum intensity is obtained in the first scanning, the incident angle θ is incremented by 0.1 ° or continuously. , The intensity of light generated in the metal film 55 is measured (second scanning), and the result is stored in association with the corresponding incident angle θ. Then, the control processing unit 14 sets the incident angle θ when the maximum intensity is obtained in the second scanning as the excitation incident angle θ ₁ in the first analysis chip 50.

Next, with respect to the second analysis chip 50, the control processing unit 14 does not perform the first scan, but within the second scan range obtained by the first scan to the first analysis chip 50. The intensity of light generated in the metal film 55 is measured while changing the incident angle θ in increments of 0.1 ° or continuously (second scanning), and the result is stored in association with the corresponding incident angle θ. Then, the control processing unit 14 sets the incident angle θ when the maximum intensity is obtained in the second scanning as the excitation incident angle θ ₁ in the second analysis chip 50. In this way, for the second and subsequent analysis chips 50, the analysis time can be shortened by performing only the scan (second scan) within the second range obtained by the first analysis chip 50. Can be achieved.

DESCRIPTION OF SYMBOLS 10,10A Surface plasmon resonance fluorescence analyzer 12 Chip | tip holding | maintenance part 14,14A Control processing part 20 Excitation light emission part 21 Light source part 30 Excitation optical system 33 1/2 wavelength plate 34 Rotation drive part 35 Incident path adjustment part 36 Reflective member 36a Reflecting surface 37 Reflecting member driving unit 40, 40A Light measuring unit 41 Light receiving unit 42 Measuring optical system 44 Condensing lens 45 Imaging lens 46 Second BPF (first optical filter)
47 Second NDF (second optical filter)
48 Position switching unit 50 Analysis chip 51 Prism 53 Film formation surface (predetermined surface)
55 Metal Films 141 and 141A Deviation Angle Deriving Unit 142 Correction Value Deriving Unit 143 Correction Unit DN Dark Noise K Correction Value (Correction Coefficient)
S _max measurement maximum light quantity, maximum value S _min measurement minimum light quantity, minimum value α excitation light θ incident angle θ ₁ excitation incident angle θ _i displacement angle (major axis rotation amount)

Claims (10)

A surface plasmon resonance fluorescence analyzer that measures fluorescence emitted by excitation of an electric field based on surface plasmon resonance by a fluorescent substance attached to the sample or the sample,
A chip holding unit capable of holding an analysis chip including a prism on which a metal film is formed on a predetermined surface so as to be detachable;
A light source unit that makes linearly polarized excitation light enter the prism so as to be reflected by the metal film of the prism included in the analysis chip held by the chip holding unit;
The metal of the excitation light having a half-wave plate disposed on the optical path of the excitation light between the light source unit and the prism, and the half-wave plate having passed through the half-wave plate A polarization direction adjuster that is rotatable so that the deflection direction with respect to the film changes;
A light measuring unit capable of measuring the amount of light generated in the metal film and a region adjacent thereto by reflecting the excitation light on the metal film;
A control processing unit that analyzes the specimen based on the amount of the fluorescence included in the light measured by the light measurement unit, and
The control processing unit corrects the amount of fluorescent light measured by the light measurement unit based on a deviation angle that is an angle formed between a P wave direction with respect to the metal film and the optical principal axis of the prism;
The misalignment angle is measured by the light measuring unit while rotating the half-wave plate of the polarization direction adjusting unit in a state where the light source unit emits excitation light, and the light measuring unit measures light generated in the metal film and a region adjacent thereto. A surface plasmon resonance fluorescence analyzer characterized in that the surface plasmon resonance fluorescence analyzer is obtained from a maximum light amount and a minimum light amount obtained by performing the above. A surface plasmon resonance fluorescence analyzer that measures fluorescence emitted by excitation of an electric field based on surface plasmon resonance by a fluorescent substance attached to the sample or the sample,
A prism having a metal film formed on a predetermined surface;
A light source unit that makes linearly polarized excitation light enter the prism so as to be reflected by the metal film;
The metal of the excitation light having a half-wave plate disposed on the optical path of the excitation light between the light source unit and the prism, and the half-wave plate having passed through the half-wave plate A polarization direction adjuster that is rotatable so that the deflection direction with respect to the film changes;
A light measuring unit capable of measuring the amount of light generated in the metal film and a region adjacent thereto by reflecting the excitation light on the metal film;
A control processing unit that analyzes the specimen based on the amount of the fluorescence included in the light measured by the light measurement unit, and
The control processing unit corrects the amount of fluorescent light measured by the light measurement unit based on a deviation angle that is an angle formed between a P wave direction with respect to the metal film and the optical principal axis of the prism;
The misalignment angle is measured by the light measuring unit while rotating the half-wave plate of the polarization direction adjusting unit in a state where the light source unit emits excitation light, and the light measuring unit measures light generated in the metal film and a region adjacent thereto. A surface plasmon resonance fluorescence analyzer characterized in that the surface plasmon resonance fluorescence analyzer is obtained from a maximum light amount and a minimum light amount obtained by performing the above. The polarization direction adjustment unit has a rotation drive unit that rotates the half-wave plate,
The control processing unit controls the light source unit, the rotation driving unit, and the light measurement unit,
It is obtained by measuring light generated in the metal film and an adjacent area by the light measuring unit while rotating the half-wave plate by the rotation driving unit in a state where the light source unit emits excitation light. A deviation angle deriving unit for obtaining the deviation angle from the maximum light quantity and the minimum light quantity;
The surface plasmon resonance according to claim 1, further comprising: a correction unit that corrects the light amount of the fluorescence measured by the light measurement unit based on a deviation angle obtained by the deviation angle deriving unit. Fluorescence analyzer. The deviation angle deriving unit generates the surface plasmon resonance in the metal film in a state where the deviation angle is θ _i , the maximum light amount is DR _max , the minimum light amount is DR _min , and the excitation light is reflected by the metal film. 4. The deviation angle is obtained by the following expression (1), where SK is a surface diffusion light amount that is a light amount of light measured by the light measurement unit when the light measurement unit is not in a closed state. The surface plasmon resonance fluorescence analyzer described in 1.

The control processing unit has a storage unit capable of storing the surface diffusion light amount,
5. The surface plasmon resonance fluorescence analyzer according to claim 4, wherein the deviation angle deriving unit obtains the deviation angle using the surface diffusion light amount stored in the storage unit. The light measurement unit measures the amount of the fluorescence contained in the light generated in the metal film and a region adjacent thereto,
The deviation angle deriving unit measures the maximum fluorescence of the fluorescence measured by the light measuring unit while rotating the half-wave plate of the polarization direction adjusting unit by the rotation driving unit in a state where the light source unit emits excitation light. The surface plasmon resonance fluorescence analyzer according to claim 3, wherein the deviation angle is obtained from a quantity and a minimum fluorescence quantity. The deviation angle deriving unit obtains the deviation angle by the following equation (2), where θ _i is the deviation angle, S _{max is} the maximum fluorescence amount, and S _{min is the} minimum fluorescence amount. The surface plasmon resonance fluorescence analyzer according to claim 6.

A correction value deriving unit for obtaining a correction value for obtaining the amount of fluorescence measured by the light measurement unit when the excitation light incident on the prism is not birefringent from the deviation angle obtained by the deviation angle deriving unit; Prepared,
8. The surface according to claim 4, wherein the correction value deriving unit obtains the correction value by the following expression (3), where K is the correction value. Plasmon resonance fluorescence analyzer.

  The surface plasmon resonance fluorescence analyzer according to any one of claims 1 to 8, wherein the prism is formed of a dielectric. A surface plasmon resonance fluorescence analysis method for measuring fluorescence emitted when a fluorescent substance attached to a specimen or a specimen is excited by an electric field based on surface plasmon resonance,
Preparing a prism in which a metal film is formed on a predetermined surface, and a step of flowing a sample solution containing the specimen on the metal film;
The excitation light is incident on the prism so that the linearly polarized excitation light for causing surface plasmon resonance in the metal film is reflected by the metal film, and the excitation light for the metal film is maintained while maintaining the state. A measuring step of measuring the amount of light generated in the metal film and a region adjacent thereto by reflecting the excitation light on the metal film while changing the polarization direction;
A deviation angle deriving step for obtaining a deviation angle, which is an angle formed between the P wave direction with respect to the metal film and the optical principal axis of the prism, from the maximum light amount and the minimum light amount measured in the measurement step;
The excitation light is reflected by the metal film to measure the amount of fluorescent light contained in the metal film and the light adjacent to the metal film, and this light quantity is based on the deviation angle obtained in the deviation angle derivation step. A surface plasmon resonance fluorescence analysis method, comprising: an analysis step of detecting the specimen by correcting.

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