JPWO2012093437A1 - Surface plasmon resonance fluorescence analyzer and analysis chip used in the analyzer - Google Patents

Abstract

The present invention includes an excitation light emitting unit that causes excitation light α to enter the prism unit, and a light measurement unit that measures light generated in a region adjacent to the metal film, and the excitation light emitting unit includes an excitation light source, A diameter changing optical system constructed by a plurality of optical lenses and capable of changing the beam diameter of the excitation light α. The diameter changing optical system has an irradiation area of the excitation light α in the metal film of the light measurement unit in the metal film. The beam diameter of the excitation light α is changed so as to be included in the measurement region.

Description

  The present invention relates to a surface plasmon resonance fluorescence analyzer that analyzes a sample contained in a sample solution using surface plasmon resonance (SPR), and an analysis chip used in the analyzer.

  2. Description of the Related Art Conventionally, in a bioassay for detecting protein, DNA, etc., an analyzer using a surface plasmon resonance fluorescence analysis (surface plasmon excitation enhanced fluorescence spectroscopy: SPFS) method as a device for detecting a sample (a substance to be detected) with high sensitivity. It is known (see Patent Document 1).

  In this SPFS method, excitation light is incident on a prism formed with a metal film made of gold, silver, or the like on a predetermined surface so as to satisfy a total reflection condition with respect to the predetermined surface. An evanescent wave (electric field) that exudes from the metal film when the excitation light is reflected is used.

  Specifically, in the above analyzer, when excitation light enters the prism provided with the metal film, the excitation light enters the metal film so as to have a predetermined incident angle (plasmon resonance angle). Surface plasmon resonance occurs in the metal film, and the evanescent wave (electric field) is enhanced. In this state, when the sample solution is flowed so as to contact the surface of the metal film (surface opposite to the prism), the specimen contained in the sample solution or the fluorescent substance (labeling substance) attached to the specimen is enhanced. When excited by an electric field, it emits fluorescence (excitation fluorescence). The analyzer described above detects the presence or amount of the sample with high sensitivity and high accuracy by measuring the excitation fluorescence.

  In the above-described analyzer, when excitation light enters the prism so as to be reflected by the metal film during analysis of the specimen, autofluorescence caused by the excitation light is generated in the prism. This autofluorescence is based on the material characteristics of the prism, and is generated when excitation light (light) travels in the prism. When autofluorescence generated in such a prism enters a light measurement unit or the like that measures excitation fluorescence during analysis of a specimen, the autofluorescence becomes noise and the SN ratio is reduced.

Japanese Patent No. 4370383

  An object of the present invention is to provide a surface plasmon resonance fluorescence analysis capable of suppressing a decrease in the signal-to-noise ratio of a signal obtained by measuring light caused by surface plasmon resonance generated in a metal film in an analyzer utilizing the SPFS method. An apparatus and an analysis chip used in the analysis apparatus are provided.

  In the surface plasmon resonance fluorescence analyzer according to the present invention and the analysis chip used in the analyzer, the irradiation region of the excitation light in the metal film when the excitation light is reflected by the metal film is in the metal film of the light measurement unit. The beam diameter of the excitation light is changed so as to be included in the light measurement region. For this reason, according to the present invention, a surface plasmon resonance fluorescence analyzer capable of suppressing a decrease in the SN ratio of a signal obtained by measuring light caused by surface plasmon resonance generated in a metal film, and the analyzer An analysis chip used in the above can be provided.

  The above and other objects, features and advantages of the present invention will become apparent from the following detailed description and the accompanying drawings.

FIG. 1 is a functional block diagram showing a configuration in a state where an analysis chip is installed in the surface plasmon resonance fluorescence analyzer according to the present embodiment. FIG. 2 is an enlarged view showing the configuration of the chip holding part of the surface plasmon resonance fluorescence analyzer and the analysis chip held by the chip holding part. FIG. 3 is a diagram showing the polarization state of the excitation light in the surface plasmon resonance fluorescence analyzer and the irradiation region of the excitation light on the metal film. 4A is a plan view for explaining a light source holding part of the surface plasmon resonance fluorescence analyzer, and FIG. 4B is a cross-sectional view taken along the line IVB-IVB in FIG. FIG. 5A and FIG. 5B are bottom views for explaining the rotation operation of the light source holding part. FIG. 6A is a diagram for explaining the magnitude relationship among the region where the physiologically active substance is fixed in the metal film, the measurement region, and the irradiation region, and is from a direction along the surface of the metal film. FIG. FIG. 6B is a diagram for explaining the magnitude relationship among the region where the physiologically active substance is fixed in the metal film, the measurement region, and the irradiation region, from a direction orthogonal to the surface of the metal film. FIG. FIG. 7 is a diagram for explaining a measurement optical system of the surface plasmon resonance fluorescence analyzer. FIG. 8 is a flowchart showing a basic sequence for analyzing a specimen in the surface plasmon resonance fluorescence analyzer. FIG. 9 is a flowchart showing the resonance angle scanning sequence of FIG. FIG. 10 is a flowchart showing the optimum position scanning sequence of FIG. FIG. 11 is a flowchart showing the birefringence measurement sequence of FIG. FIG. 12 is a flowchart showing the excitation fluorescence measurement sequence of FIG. FIG. 13A and FIG. 13B are diagrams illustrating the second positioning of the reflecting member in the surface plasmon resonance fluorescence analyzer. FIG. 14A is a diagram showing an autofluorescence region within the detection field of the light measurement unit in the prism, and excitation light having a beam diameter that makes the irradiation region larger than the measurement region is incident on the prism. FIG. 14B is a diagram showing the autofluorescence region in the detection field of the light measurement unit in the prism, and the beam diameter is such that the irradiation region is smaller than the measurement region. It is a figure which shows the state which entered the excitation light in the prism. FIG. 15 is a central longitudinal sectional view of an analysis chip according to another embodiment.

  Hereinafter, an 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 allows excitation light incident on the prism to be reflected at the reflection interface so that the total reflection condition is satisfied with respect to the reflection interface of the prism. Using an evanescent wave (enhanced electric field) that oozes from the reflection interface when reflected, a fluorescent substance labeled (attached) to a substance to be detected (hereinafter also simply referred to as “analyte”) is excited. Then, the analyzer detects the specimen by detecting the amount of fluorescent light generated by the excitation of the fluorescent substance.

  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 an analysis The optical measurement unit 40 that measures the intensity of the light generated in the chip 50 and the components of the analyzer 10 such as the chip holding unit 12, the excitation light emitting unit 20, and the optical 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 types of 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. This pretreatment unit receives a reagent chip (not shown), performs pretreatment (blood cell separation, dilution, mixing, etc.) of blood or the like injected into this reagent chip to generate a sample solution. Injection into the analysis chip 50. The reagent chip is provided with a plurality of storage units, and in each storage unit, a reagent, a diluting solution, a cleaning solution, and the like are individually sealed 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, a sample solution containing a specimen, a cleaning solution, and the like in contact with the metal film 55 on the metal film 55. And a flow path member 57 that forms a flow path 58 that flows while flowing. The analysis chip 50 of this embodiment is replaced every time a sample is detected (analyzed).

  The prism 51 has an incident surface 52 on which the excitation light α from the excitation light emitting unit 20 is incident on the inside of the prism 51 and a film formation surface on which a metal film 55 that reflects the excitation light α incident on the inside of the prism 51 is formed. (Reflection surface) 53 and an emission surface 54 on which the excitation light α reflected by the metal film 55 is emitted to the outside of the prism 51 are included on the surface. The prism 51 is made 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. The exit surface 54 is formed on the optical surface in the same manner as the entrance surface 52 so that the light of the S wave component of the excitation light α reflected by the metal film 55 does not stay inside the prism 51.

The excitation light emitting unit 20 of the present embodiment emits a beam-like excitation light α that is flat (specifically, an outline of a cross section orthogonal to the optical axis is elliptical). Then, in the prism 51, after the excitation light α enters the prism 51 from the incident surface 52 so that the minor axis direction of the cross section coincides with the inclination direction of the film forming surface 53 with respect to the incident surface 52, When the excitation light α is incident on the metal film 55 at an incident angle θ ₁ at which the intensity of the electric field (enhanced electric field) due to surface plasmon resonance is maximized, the prism is an angle formed by the incident surface 52 and the film formation surface 53. The angle θ _A is such that the irradiation region of the excitation light α on the metal film 55 is circular or substantially circular (see FIG. 3). From the refractive index of the material of the prism 51, the thickness and refractive index of the metal film 55, the refractive index of the sample solution, etc., the resonance angle at which the electric field enhancement due to surface plasmon resonance is maximized can be predicted. Therefore, the prism angle θ _A is determined based on the design value. At this time, the variation of each parameter, the concentration of the measurement object, since the resonance angle has a certain width, the irradiation area in the design center value prism angle theta _A so that a true circle is set. Further, the prism 51 of the present 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 may have any shape that includes the incident surface 52, the film forming surface 53, and the exit surface 54 on the surface of the prism 51. That is, in the prism 51, the excitation light α incident inside from the incident surface 52 is totally reflected by the metal film 55 on the film formation surface 53, and this excitation light α (specifically, the S wave component of the excitation light α) is reflected. Any shape may be used as long as it is emitted from the exit surface 54 without being irregularly reflected inside the prism 51.

  The metal film 55 is a metal thin film formed (formed) on the film formation surface 53 of the prism 51. The metal film 55 of this embodiment is made of gold. The metal film 55 has an evanescent wave generated by reflecting the excitation light α incident on the prism 51 on the film formation surface 53 on which the metal film 55 is formed so as to satisfy the total reflection condition with respect to the film formation surface 53. Amplifies (enhanced electric field). That is, the metal film 55 is provided on the film formation surface 53, and surface plasmon resonance occurs in the metal film 55, so that the excitation light α is totally reflected on the surface without the metal film 55 (film formation surface 53). The formed evanescent wave is amplified as compared with the case where a wave is generated (that is, an enhanced electric field (enhanced electric field) is 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, but may be any metal that causes surface plasmon resonance. For example, the metal film 55 may be silver, copper, aluminum or the like (including an alloy).

  A capturing body (physiologically active substance) 56 for capturing a specific antigen is fixed to 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 channel member 57 is provided on the film formation surface 53 (specifically, the metal film 55) of the prism 51, thereby forming a channel 58 through which the sample solution flows in cooperation with the film formation surface 53. The flow path member 57 is formed of a transparent resin. The flow path member 57 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 the 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 solution pretreated in the pretreatment unit is injected (supplied) into the flow path 58. The analysis chip 50 into which the sample solution 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. The analysis chip 50 conveyed to the chip holding unit 12 is held by the chip holding unit 12 so as to be in a predetermined posture with respect to the analyzer 10.

  The chip holding unit 12 holds the analysis chip 50 so that the analysis chip 50 is in a predetermined posture with respect to the analyzer 10 when detecting the sample. The predetermined posture means that the excitation light α emitted from the excitation light emitting unit 20 enters the prism 51 from the incident surface 52 so as to satisfy the total reflection condition with respect to the film formation surface 53, and this incident excitation light. The posture is such that α is reflected by the metal film 55 on the film formation surface 53. The chip holding unit 12 holds the analysis chip 50 in a detachable manner. The chip holding unit 12 of this embodiment holds the analysis chip 50 so that the prism 51 is positioned below the flow path member 57.

  In the analyzing apparatus 10, light absorption is performed near the emission surface 54 of the analysis chip 50 held by the chip holding unit 12 so that the influence of the light emitted from the emission surface 54 does not reach the light measurement unit 40. A body (not shown) is arranged.

  The excitation light emitting unit 20 causes the excitation light α to 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 in the chip holding unit 12. Specifically, the excitation light emitting unit 20 emits linearly polarized excitation light α, 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. And having.

  The light source unit 21 includes a light source unit unit 23 including an excitation light source (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.

  The light source unit 23 collimates the excitation light α emitted from the excitation light source 22. Further, the light source unit 23 adjusts and maintains the posture of the excitation light source 22 so that the excitation light α is incident on the metal film 55 of the prism 51 from the short axis side. Specifically, the light source unit unit 23 drives the excitation light source 22, the light source holding unit 122 that holds the excitation light source 22 so that the attitude of the excitation light source 22 with respect to the prism 51 can be changed, and the attitude of the excitation light source 22. And a temperature adjustment circuit 25 that performs temperature adjustment (temperature adjustment) of the excitation light source 22.

  The excitation light source 22 of this embodiment is a laser diode (semiconductor laser element). As described above, the excitation light source 22 emits the beam-like excitation light α as shown in FIG. 3 (specifically, the contour of the cross section orthogonal to the optical axis is elliptical).

  The light source holding unit 122 holds the excitation light source 22 so that the excitation light α when entering the prism 51 from the incident surface 52 can be rotated about its optical axis as a rotation center. As shown in FIGS. 4A to 5B, the light source holding unit 122 of the present embodiment parallels the circuit board (laser drive circuit board) 124 to which the excitation light source 22 is attached and the excitation light α. A collimating lens 125, a lens barrel 126 for holding the collimating lens 125, and a substrate mounting table 127 fixed to the analyzer 10.

  The lens barrel 126 is fixed to the circuit board 124 so that the optical axis of the held collimating lens 125 coincides with the optical axis of the excitation light α emitted from the excitation light source 22. The lens barrel 126 of the present embodiment has a cylindrical outer peripheral surface 126a. The lens barrel 126 is fixed to the circuit board 124 so that the optical axis of the excitation light α coincides with the optical axis of the collimating lens 125 held by the lens barrel 126 (FIG. 4B). reference).

  The board mount 127 holds the circuit board 124 to which the lens barrel 126 is fixed so as to be rotatable about the optical axis of the excitation light α (FIGS. 4A and 5). A) and an arrow ε in FIG. 5B). In the present embodiment, the circuit board 124 is mounted on the upper surface of the substrate mounting table 127 (FIG. 4B) with the lens barrel 126 inserted in the hole 128 that penetrates from the upper surface 127a to the lower surface 127b of the substrate mounting table 127. ) Is in sliding contact with 127a. The hole 128 has a lens barrel holding portion 128a having an inner peripheral surface having an inner diameter corresponding to the outer peripheral surface 126a of the lens barrel 126, and an excitation light passage portion 128b having an inner diameter smaller than that of the lens barrel holding portion 128a. . The circuit board 124 is provided with a plurality of guided portions (arc-shaped slits in the present embodiment) 124a at intervals on a circumference centered on the optical axis of the excitation light α. And the guide member (screw in this embodiment) B is attached to the board | substrate mounting stand 127 so that the inside of each slit 124a may be passed. As a result, the circuit board 124 can be rotated about the optical axis of the excitation light α (see FIGS. 5A and 5B).

  The posture change driving unit 123 is connected to the control processing unit 14 and changes the posture of the excitation light source 22 with respect to the prism 51 based on an instruction signal from the control processing unit 14. Specifically, the posture change drive unit 123 forms the excitation light α when the excitation light α is incident on the metal film 55 so as to satisfy the total reflection condition (shallow angle with respect to the film formation surface 53). The posture of the excitation light source 22 is changed so that the outline of the irradiation area on the surface 53 is approximately circular (see FIG. 3). The posture change drive unit 123 of this embodiment is configured by an actuator, a motor, or the like. The posture change driving unit 123 rotates the circuit board 124 to which the lens barrel 126 is fixed and held by the board mounting base 127 (see FIGS. 5A and 5B). As a result, the posture of the excitation light source 22 is changed so that the outline of the irradiation region of the excitation light α on the film formation surface 53 is approximately circular.

  In the present embodiment, the light source holding unit 122 and the attitude change driving unit 123 are configured to be able to rotate the excitation light α when entering the prism 51 from the incident surface 52 with the optical axis as the rotation center. However, the present invention is not limited to this. For example, in an analyzer that is not provided with the posture change driving unit 123, an operator or the like rotates the circuit board (light source holding unit) 124, and the excitation light α becomes a total reflection condition with respect to the film formation surface 53. As described above, when the light is incident on the prism 51, the outline of the irradiation region on the film formation surface 53 of the excitation light α is made to be approximately circular. In this state, the circuit board 124 is fixed to the board mounting base 127 with screws.

  The temperature adjustment circuit 25 is a regression circuit for adjusting the temperature of the excitation light source (laser diode) 22. Specifically, the temperature adjustment circuit 25 monitors the light amount branched from the light beam of the excitation light α after collimation by a photodiode or the like (not shown), and thereby the wavelength and light amount of the emitted excitation light α are determined. The temperature of the excitation light source 22 is adjusted so as to be constant. This is because the wavelength of light emitted from the excitation light source 22 and the emission energy vary depending on the temperature.

  The first wave shaping unit 24 waves the excitation light α emitted from the light source unit 23 by a plurality of filters (optical filters), and makes the excitation light α have an excitation wavelength with 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. The first NDF 28 is a so-called neutral density filter. That is, the first NDF 28 adjusts the amount of the excitation light α emitted from the light source unit 21 by dimming 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. The 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 (diameter changing optical system) 32 that adjusts the contour shape of the beam of the excitation light α, and the excitation light α. The incident path adjusting unit 35 changes the incident path into the prism 51 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 on 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 the present embodiment has a step motor, and drives the step motor based on an instruction signal from the control processing unit 14 to rotate the half-wave plate 33. When the half-wave plate 33 rotates in this way, the polarization direction of the excitation light α linearly polarized in the first wave rectifier 24 rotates. As a result, the amount of the P wave component and the amount of the S wave component in the excitation light α incident on the metal film 55 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 a condition in which the degree is the maximum) to a condition that does not exude at all (that is, a condition in which no enhanced electric field is formed in the vicinity of the surface 55a of the metal film 55).

  The shaping optical system 32 is constructed by a plurality of optical lenses 32a, 32a,. The shaping optical system 32 has a beam size (beam diameter) of the excitation light α and a contour shape of the beam so that the contour of the irradiation region of the excitation light α in the metal film 55 becomes a circle (substantially circular) having a predetermined diameter. Adjust. Specifically, the shaping optical system 32 has a plurality of optical lenses 32a, 32a,... Arranged so that their optical axes coincide with each other, and is excited by changing the distance between the optical lenses 32a, 32a. The beam diameter of the light α is changed. The shaping optical system 32 is configured so that the excitation light α after changing the beam diameter also becomes parallel light. The shaping optical system 32 of this embodiment is a so-called laser beam expander.

  The shaping optical system 32 is not limited to the above configuration. For example, the shaping optical system may be a prism or a cylindrical lens that changes magnification only in one axial direction. Further, the irradiation region of the excitation light α in the metal film 55 of the present embodiment is adjusted to a size that is included in the measurement region in the light measurement unit 40 and substantially coincides with the irradiation region (FIG. 6A and FIG. 6). (See FIG. 6B). Specifically, on the metal film 55, the measurement area is smaller than the area where the capturing body 56 is fixed. Then, the shaping optical system 32 adjusts the beam diameter of the excitation light α so that the irradiation area is slightly smaller than the measurement area.

  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. The reflecting member 36 of this embodiment is a reflecting mirror. In this reflecting member 36, a dielectric multilayer film (in which a phase shift, dimming, etc. do not occur between the excitation light α before entering the reflection surface 36a and the excitation light α after being reflected by the reflection surface 36a. Specifically, a dielectric multilayer film) in which neither the P wave component nor the S wave component has the wavelength dependency at the excitation light wavelength is formed on the reflecting surface 36a. Thereby, the detection accuracy and sensitivity of the sample in the analyzer 10 are improved.

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

  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 having.

  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 reflective surface with respect to a surface (the paper surface in FIG. 1) including the optical path of the excitation light α incident on the reflective member 36 and the optical path of the excitation light α after being reflected by the reflective member 36. The reflecting member 36 is supported so that 36a is in an orthogonal posture. The rotation drive mechanism is configured such that the reflecting surface 36a rotates along the surface including the optical path while maintaining the orthogonal posture of the reflecting member 36 (the posture in which the reflecting surface 36a is orthogonal to the surface including the optical path). Further, the reflecting member 36 is rotated based on the instruction signal from the control processing unit 14. 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 rotation motor is set so that the torque is sufficiently large. The rotary motor of this embodiment is a high-resolution step motor. The rotary motor rotates the reflecting member 36 in accordance with an instruction signal from the control processing unit 14 so as to have a predetermined angular interval (that is, stepwise in the rotation direction). The predetermined angular interval is related to the resolution in adjusting the orientation 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 reciprocates the stage 37a in the optical axis direction of the excitation light α from the light source unit 21, that is, in the vertical direction. Specifically, in the reciprocating drive mechanism, a step motor is controlled by an instruction signal from the control processing unit 14, and a stage 37a on which the rotational drive mechanism and the reflecting member 36 are mounted by a screw feed mechanism driven by the step motor. Reciprocates vertically. In other words, the reciprocating drive mechanism follows the instruction signal from the control processing unit 14 and keeps 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, while keeping the reflecting member 36 at a predetermined position on the optical axis. 36 is moved 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, Have In addition, the light measurement unit 40 has an intensity (in this embodiment, the light generated in the metal film 55 of the analysis chip 50 and a region adjacent to the metal film 55 (hereinafter, also simply referred to as “light generated in the metal film 55”). Light intensity).

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

  As shown in FIG. 7, 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 (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 same wavelength (excitation wavelength) as the excitation light α. As a result, the second BPF 46 causes the light receiving unit 41 to emit light having a wavelength other than the wavelength of the fluorescence (the light generated when the fluorescent substance labeled on the specimen is excited by the enhanced electric field) (for example, leakage light from the excitation light emitting unit 20). And plasmon scattered light, diffused light, etc.) can be prevented from entering. That is, the second BPF 46 removes 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 is a light receiving unit (PMT in the present embodiment) for detecting weak light (fluorescence in the present embodiment) by dimming plasmon scattered light or diffused light guided in the measurement optical system 42. ) 41, it is possible to measure plasmon scattered light and the like. Specifically, the amount of light measured to obtain 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. . For this reason, when the common light receiving unit 41 is used, the second NDF 47 attenuates the light measured to obtain the incident angle θ ₁ of the excitation light α at which the enhancement electric field is maximized, thereby damaging the light receiving unit 41. Is prevented.

  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. That is, 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 passing through the second BPF 46 or the second NDF 47 is shifted, so that optical measurement is performed. The measurement accuracy in the part 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. 7), 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 arrow δ in FIG. 7), thereby changing the position of each filter 46, 47. Change over. As a result, the positions of the two filters 46 and 47 are 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, it is not limited to this. If the amount of light to be measured does not exceed the allowable amount of the light receiving unit 41, the second NDF 47 may not be provided in the second wave shaping unit 43. Further, a configuration in which the light receiving unit is switched by the light to be measured, that is, a configuration in which the analyzer switches between the light receiving unit 41 for receiving the fluorescence and the light receiving unit for receiving the light having a light quantity larger than the fluorescence. Even when the second NDF 47 is provided, the second NDF 47 may not be provided.

  In this embodiment, the position switching unit 48 reciprocates the holding frame 49 to switch the positions of the filters 46 and 47. However, the present invention is not limited to this. For example, the disc-shaped holding frame holds the filters 46 and 47 so that the second BPF 46 and the second NDF 47 are aligned on the same plane, and the position switching unit rotates the intermediate position between the second BPF 46 and the second NDF 47. A disc-shaped holding frame may be rotated about the center. Also with this configuration, the positions of the filters 46 and 47 are switched. The position switching unit may have two drive 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 the sample, the control processing unit 14 changes the posture of the excitation light source 22 by the posture change driving unit 123. For example, the light source unit 21, the polarization direction adjusting unit 31, and the incident path adjusting unit. 35 and the light measuring unit 40 and the like are controlled. Thereby, in the said analyzer 10, a resonance angle scanning process, an optimal position scanning process, a birefringence measurement process, an excitation fluorescence measurement process, etc. are performed.

  The control processing unit 14 calculates based on the output signal sent from the light measuring unit 40 (specifically, the light receiving unit 41) when analyzing the sample in the analyzer 10, and this light measuring unit 40. Analysis for fluorescence measured by. For example, the control processing unit 14 counts the number of fluorescences per unit area detected by the light measurement unit 40, calculates the amount of increase in fluorescence over time, and the like.

  The calculation result by 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 in the control processing unit 14 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. The display unit 16 may be a combination of screen display and printout.

  The analysis of the sample in the analyzer 10 having the above configuration will be described below with reference to FIGS. Details of control of each component of the analyzer 10 by the control processing unit 14 and computations performed in the control processing unit 14 will also be described.

  FIG. 8 is a flowchart showing a basic sequence for analyzing a sample in the analyzer 10. FIG. 9 is a flowchart showing a resonance angle scanning sequence. FIG. 10 is a flowchart showing the optimum position scanning sequence. FIG. 11 is a flowchart showing a birefringence measurement sequence. FIG. 12 is a flowchart showing an 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 performs preprocessing (blood cell separation, dilution, mixing, etc.) of the set reagent chip by the preprocessing unit, thereby generating a sample solution. Next, when the analysis chip 50 is installed in the pretreatment unit, the control processing unit 14 injects the sample solution after the pretreatment into the flow channel 58 of the analysis chip 50 by the pretreatment unit. The capturing body 56 fixed on the surface captures the specimen (specific antigen). That is, the preprocessing unit reacts the capturing body 56 with the specimen. In the present embodiment, the capturing body 56 captures a specimen labeled with a fluorescent substance (in this embodiment, a fluorescent dye) (see FIG. 6A), but is not limited thereto. For example, the fluorescent substance may be injected into the analysis chip 50 after the capturing body 56 captures the specimen, whereby the fluorescent substance may be labeled with respect to the specimen captured by the capturing body 56.

  The analysis chip 50 subjected to the reaction is transported to the chip holding unit 12 and is 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 and the like in blood because the surface plasmon resonance conditions 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, the temperature of the excitation light source 22 is normally constantly maintained by the temperature adjustment 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. Then, 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 an incident angle (excitation incident angle θ ₁ ) 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, the control processing unit 14 drives the reflecting member 36 by the reflecting member driving unit 37, thereby causing the excitation light α to be incident on the metal film 55 of the prism 51 included in the analysis chip 50 (excitation incident angle). A scan of θ ₁ ) is performed. Specifically, the incidence of excitation light in which the intensity of the enhanced electric field (evanescent wave) based on surface plasmon resonance is maximized due to the material, shape, prism filling liquid (sample solution) refractive index, etc. of the prism 51 included in the analysis chip 50. The angle θ ₁ is fixed. 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. For this reason, the control processing unit 14 causes the excitation light α to be 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. Then, based on the amount of light generated in the metal film 55 at this time, the excitation incident angle θ ₁ in the analysis chip 50 is obtained.

  More specifically, first, the control processing unit 14 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 means that the excitation light α is reflected at a specific position of the metal film 55 (in the measurement region of the light measurement unit 40 in the present embodiment) when the excitation light α is emitted from the excitation light emitting unit 20. In this state, the position of the reflecting member 36 and the direction of the reflecting surface 36a are such that the incident angle θ at which the evanescent wave does not ooze out in the region near the surface 55a of the metal film 55.

  In this state, the control processing unit 14 measures the amount of light generated in the metal film 55 by the light measurement unit 40. Then, the control processing unit 14 acquires the measurement result from 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. 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. Light having the same wavelength as the excitation wavelength is enhanced by surface plasmon resonance generated in the metal film 55. For this reason, the amount of light having the same wavelength as the excitation wavelength is sufficiently larger than the amount of excitation fluorescence emitted by excitation of the fluorescent substance labeled on the specimen. Therefore, the control processing unit 14 retracts the second BPF 46 from the optical path of the measurement optical system 42 by the position switching unit 48, so that the light receiving unit 41 receives the light having the excitation wavelength, and thereby the light generated in the metal film 55. The amount of light is accurately measured.

  In this embodiment, the light receiving unit 41 that measures excitation fluorescence with a small amount of light also measures surface plasmon scattered light, diffused light, and the like that have a much larger amount of light than the excitation fluorescence. For this reason, in the analyzer 10 of the present embodiment, the position switching unit 48 retracts the second BPF 46 to the retracted position and moves the second NDF 47 to the filtering position, whereby the same light receiving unit (PMT in the present embodiment) 41 is used. It is possible to measure the light quantity of both lights (scattered light and the like and excitation fluorescence).

  In the state where the excitation light α is emitted from the light source unit 21, the control processing unit 14 adjusts the position of the reflection member 36 by the incident path adjustment unit 35. Specifically, the control processing unit 14 rotates the direction of the reflecting surface 36a by the rotation driving mechanism so as not to shift the irradiation position of the excitation light α on the metal film 55 (Step S24) and reflects by the reciprocating driving mechanism. The position of the member 36 is moved (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 36 a at that position so as to enter the prism 51 and reach a specific position of the metal film 55. A table in which the orientation of the surface 36a is associated is stored in advance. Then, the control processing unit 14 controls the rotational drive mechanism and the reciprocating mechanism based on this table to move the reflecting member 36. Thus, even if the position of the reflecting member 36 is changed and the direction of the reflecting surface 36a is adjusted by the reciprocating drive mechanism and the rotational drive mechanism that are not mechanically linked to each other, the irradiation of the excitation light α on the metal film 55 is performed. Only the incident angle θ of the excitation light α with respect to the metal film 55 can be changed without changing the position. In step S24 and step S25, after one of the steps is performed first, the other step may be performed, or both steps may be performed simultaneously.

  The light measurement unit 40 measures the amount of 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).

  As described above, the control processing unit 14 measures the amount of light 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.

The control processing unit 14 uses the light measurement unit 40 to measure the amount of light 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 emission of the excitation light α from the light source unit 21 is stopped (step S27). Then, the control processing unit 14 selects the maximum value and the minimum value of the stored light amount, and stores them (step S28). Further, the control processing unit 14 drives the reflecting member 36 by the reflecting member driving unit 37 so as to be in the position of the reflecting member 36 and the direction of the reflecting surface 36a when the maximum light amount is obtained (step S29).

  In this state, the control processing unit 14 changes the posture of the excitation light source 22 by the posture change driving unit 123 to make the outline of the irradiation region of the excitation light α in the metal film 55 circular or substantially circular.

<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. 13A). The control processing unit 14 causes the light source unit 21 to emit excitation light α when the reflecting member 36 is in this position, and measures the amount of light generated in the metal film 55 at this time by the light measuring unit 40, 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 movement of the reflecting member 36, the control processing unit 14 emits excitation light from the light source unit 21, measures the amount of light generated in 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 36 has reached the lower end position depending on whether or not the light amount has decreased by 50% compared to the maximum light amount among the respective light amounts stored by the measurement result by the light measuring unit 40. 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. 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. 13A), the measurement is performed by the light measurement unit 40. The amount of light emitted increases gradually. Further, when the reflecting member 36 moves downward and the entire irradiation region of the excitation light α is completely included in the measurement region of the light measurement unit 40 (see position C in FIG. 13A), The amount of light generated in the metal film 55 measured by the light measuring 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. 13A). The amount of light measured by the light measurement unit 40 decreases. That is, vignetting (light loss) occurs. The control processing unit 14 stores the light quantity of these lights in association with the vertical position of the reflecting member 36 when the light quantity is measured, and the measured light falls by 50% of the maximum light quantity. Judge whether or not. Then, the control processing unit 14 determines that the reflecting member 36 has reached the lower end position when the amount of light measured by the light measuring unit 40 falls by 50% of the maximum amount of light.

  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. 13A). The control processing unit 14 selects the position of the reflecting member 36 when the light amount measured by the light measurement unit 40 falls by 50% of the maximum light amount. Then, the control processing unit 14 calculates the center position (see the position Ce in FIG. 13A) and stores it (step S36). The control processing unit 14 moves the reflecting member 36 by the reciprocating drive mechanism to move the irradiation region to the obtained center position Ce (see step S37, FIG. 13B).

  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. However, since the fluorescence has the same level as the excitation fluorescence emitted from the fluorescent substance labeled on the sample when the concentration of the sample in the sample solution is low, it becomes noise in the measurement of the 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 the excitation light α reflected by the metal film 55 almost disappears when surface plasmon resonance occurs, autofluorescence from the incident side (left side in FIG. 13A) of the measurement region of the light measurement unit 40 is generated. It becomes a problem. The amount of autofluorescence is proportional to the optical path length. For this reason, for each analysis of the specimen, it is necessary to adjust the irradiation position of the excitation light α so that the optical path length on the incident side in the measurement region of the light measurement unit 40 is constant. Therefore, as described above, the excitation light in the measurement region of the light measurement unit 40 is adjusted so that the irradiation position of the excitation light α is always centered in the measurement region of the light measurement unit 40 for each analysis of the specimen. The optical path length on the incident side of α is constant. As a result, the difference in autofluorescence for each analysis chip 50 (prism 51) is suppressed, and the analysis accuracy of the sample can be improved.

  Further, in the present embodiment, the beam diameter of the excitation light α is adjusted so that the range (irradiation region) where surface plasmon resonance occurs in the metal film 55 by irradiation of the excitation light α is included in the measurement region of the light measurement unit 40. As a result, the beam diameter is smaller (see FIG. 14B) than in the case where the region other than the measurement region in the metal film 55 is irradiated with the excitation light α (see FIG. 14A). For this reason, the light quantity of the autofluorescence measured by the light measurement part 40 is suppressed. As a result, a decrease in the SN ratio of the signal obtained when light (excitation fluorescence) due to surface plasmon resonance generated in the metal film 55 is measured is suppressed.

<Birefringence measurement process>
Next, the control processing unit 14 measures the birefringence because birefringence occurs when the excitation light α travels through the prism 51 (step S4). Then, the control processing unit 14 improves the measurement accuracy of the specimen by taking the birefringence into consideration when measuring the excitation fluorescence from the fluorescent substance labeled on the specimen. 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 through the prism 51 due to the birefringence, and only the P wave is desired to enter the metal film 55, the S wave component is generated in the excitation light α by the phase rotation due to the birefringence. Arise. 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 corrects this decrease, so that the detection accuracy and sensitivity of the specimen in the analyzer 10 are improved.

  Specifically, the control processing unit 14 emits the excitation light α from the light source unit 21, and the light measurement unit 40 measures the amount of light generated in the metal film 55. At this time, the half-wave plate 33 is in an initial state (step S41). Then, the control processing unit 14 rotates the half-wave plate 33 by the rotation drive unit, and changes the light amount measured by the light measurement unit 40 to the rotation position of the half-wave plate 33 (the rotation angle from the initial state). ) And stored (step S42 and step S43). When the control processing unit 14 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 are increased or decreased, respectively. As the P wave component and S wave component increase or decrease, the amount of light measured by the light measurement unit 40 increases or decreases. 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. As the P wave component increases in the excitation light α incident on the metal film 55, the amount of light measured by the light measurement unit 40 increases. On the other hand, the amount of light measured by the light measurement unit 40 decreases as the S wave component increases. 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 sequentially repeats step S42 and step S43 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 selects the maximum value DR _max and the minimum value DR _min from the stored light amount and the rotational position of the half-wave plate 33. (Step S45) 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 the minimum value DR). The second rotation position when _min is obtained) is selected and stored.

Next, the control processing unit 14 _calculates the following formulas (1) and (2) from the stored maximum value DR _max and minimum value DR _min and the amount of surface diffused light SK stored in the resonance angle scanning process. ), The major axis rotation amount θ _i due to birefringence in the prism 51 and the correction coefficient K are derived and stored (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).

<Excitation fluorescence measurement process>
Next, the control processing unit 14 irradiates the reflection member 36 in the state in which the first positioning and the 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. The fluorescent substance labeled on the specimen captured by the capturing body 56 of the metal film 55 is excited by the enhanced electric field based on the surface plasmon resonance, thereby emitting fluorescence (excitation fluorescence). 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 7). 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. At this time, the control processing unit 14 performs measurement with the light measurement unit 40, and stores the output (dark noise DN) from the light measurement unit 40 at this time (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 generated in the vicinity of the metal film 55 and caused by the enhanced electric field 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 the light quantity generated in the metal film 55 when the half-wave plate 33 is obtained in the first rotation position obtained in the birefringence measurement step (that is, when the excitation light α is incident on the metal film 55 is the maximum value DR _max. This is because the intensity of the enhanced electric field in the vicinity of the metal film 55 is the highest because the rotation is made to the rotation position.

Next, the control processing unit 14 determines the second rotation position obtained in the birefringence step (that is, the amount of light generated in the metal film 55 when the excitation light α is incident on the metal film 55 becomes the minimum value DR _min. The half-wave plate 33 is rotated by the rotation drive unit 34 to the rotation 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 .

From the stored measurement maximum light quantity S _max , measurement minimum light quantity S _min , and dark noise DN, the control processing unit 14 calculates the prism as shown in the following expressions (3-1) to (8). The autofluorescence light amount h and the excitation fluorescence light amount H are derived and stored (step S58). 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、

また、

より、

そして、式（４）と式（６）とにより、

が得られ、複屈折測定工程において求められた補正係数Ｋが用いられて、

が導出される。First,

Than,

Also,

Than,

And by the formula (4) and the formula (6),

And the correction coefficient K obtained in the birefringence measurement step is used,

Is derived.

In the case where the prism 51 is formed of birefringence or a fine material (including resin), the control processing unit 14 obtains the amount H of excitation fluorescence by the following approximate expression (9).

Furthermore, in the case of a measurement system with very little dark noise DN, the control processing unit 14 obtains the excitation light quantity H by the following approximate expression (10).

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

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

  According to the analysis apparatus 10 of the present embodiment, the excitation light emitting unit so that the surface plasmon resonance range (irradiation range) generated in the metal film 55 by the irradiation of the excitation light α is included in the measurement region of the light measurement unit 40. By changing (adjusting) the beam diameter of the excitation light α emitted by 20, a region other than the measurement region in the metal film 55 is irradiated with the excitation light α (see FIG. 14A). The beam diameter becomes small (see FIG. 14B). For this reason, the amount of autofluorescence generated in the prism 51 is suppressed. As a result, a decrease in the SN ratio of a signal obtained when light (excitation fluorescence) due to surface plasmon resonance generated in the metal film 55 is measured can be suppressed.

  Moreover, when the shaping optical system 32 constructed by the plurality of optical lenses 32a, 32a,... Changes the beam diameter, a light shielding member provided with a pinhole such as a light shielding mask changes the beam diameter. The unevenness of the light amount of the excitation light α in the metal film 55 and the increase in size of the apparatus for suppressing the unevenness of the light amount are prevented. Specifically, when the beam diameter is changed by a light shielding mask or the like, the amount of light in the irradiation region of the metal film 55 due to interference between the diffracted light generated when the excitation light α passes through the pinhole and the excitation light α. Unevenness occurs. However, unlike the present invention, a shading mask or the like is not used, and the shaping optical system 32 constructed by the optical lens 32a changes the beam diameter, thereby suppressing the generation of diffracted light.

  In the analysis apparatus 10 of the above-described embodiment, the light source holding unit 122 and the posture change driving unit 123 are provided, so that the diode laser that emits the excitation light α having an elliptical cross-sectional shape orthogonal to the optical axis. Even when the excitation light source 22 is used, the contour of the irradiation region in the metal film 55 can be the same circle or substantially circular as the contour of the measurement region. As a result, surface plasmon resonance occurs in substantially the entire measurement region in the metal film 55, and the specimen is effectively analyzed.

In addition, according to the present embodiment, since the analysis chip 50 having the prism angle θ _A described above is used, the excitation light α having a beam diameter such that a circular or substantially circular irradiation region is included in the measurement region is incident on the incident surface. By making it enter into the prism 51 from 52, it becomes possible to generate surface plasmon resonance over substantially the entire measurement region while suppressing the amount of autofluorescence generated in the prism 51. As a result, a decrease in the signal-to-noise ratio of the signal obtained when the excitation fluorescence is measured is suppressed, and the sample is analyzed with high sensitivity and high accuracy. In addition, when the excitation light α is incident on the metal film 55 at an incident angle θ where the irradiation region is circular or substantially circular, the intensity of the electric field in the vicinity of the metal film is maximized, so that the amount of excitation fluorescence increases. This makes it possible to detect excitation fluorescence with higher sensitivity and higher accuracy.

  In addition, according to the present embodiment, the influence of autofluorescence in the prism 51 is removed from the measurement result, thereby ensuring a wide dynamic range.

  Specifically, when the light generated in the metal film 55 is measured while changing the polarization direction of the excitation light α with respect to the metal film 55, when the light amount (intensity) of the detected light is reflected by the metal film 55, The rotational position of the half-wave plate 33 when the S wave component (component that does not contribute to surface plasmon resonance) is the largest in the excitation light α and the P wave component (component that contributes to surface plasmon resonance) in the excitation light. The rotational position of the half-wave plate 33 when the number is the largest is obtained. Then, the half-wave plate 33 is adjusted 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, and the light measurement unit 40 measures the excitation fluorescence. Thus, the amount of autofluorescence in the prism 51 is obtained. Thereby, the measurement result of the excitation fluorescence by the light measurement unit 40 when the half wave plate 33 is adjusted and the polarization state of the excitation light α enters the metal film 55 becomes the state where the P wave component becomes the largest. Therefore, the influence of autofluorescence can be removed, and excitation fluorescence can be extracted. As a result, in the analyzer 10, the influence of autofluorescence is suppressed, excitation fluorescence is detected with high accuracy, and a wide dynamic range is ensured.

  In addition, according to the present embodiment, the incident angle θ of the excitation light α with respect to the metal film 55 is changed by driving the reflecting member 36, and therefore the reflection position of the excitation light α on the metal film 55 due to the change of the incident angle θ. Deviation is suppressed. That is, according to the above configuration, the incident angle θ of the excitation light α with respect to the metal film 55 is changed by changing and adjusting the position of the reflecting member 36 and the direction of the reflecting surface 36a. For this reason, the number of movable parts, the weight of the movable part, and the like can be suppressed as compared with the conventional case where the entire excitation optical system including the light source and the plurality of lenses is moved to adjust the incident angle θ. As a result, drive errors and backlash in the movable part are suppressed. As a result, the displacement of the reflection position of the excitation light α on the metal film 55 due to the change in the incident angle θ of the excitation light α is suitably suppressed.

  Moreover, since one excitation light α is incident on the prism 51 and an enhanced electric field based on surface plasmon resonance is generated in the vicinity of the metal film 55, a plurality of excitation lights α, α,. An increase in the amount of autofluorescent light as in the case of simultaneous incidence into the prism 51 is prevented. Thereby, in the signal obtained when the light generated in the vicinity of the metal film 55 by the surface plasmon resonance is measured, a decrease in the SN ratio due to autofluorescence is suppressed.

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 deviation occurs between the absorption peak in the angle spectrum of the reflected light on the metal film 55 and the electric field intensity peak of the enhanced electric field. However, the intensity peak of light (light generated in the metal film 55) resulting from surface plasmon resonance coincides with the electric field intensity peak of the enhanced electric field. Therefore, the reflective member 36 is adjusted so that the position of the reflective member 36 and the direction of the reflective surface 36a when the intensity of light generated in the metal film 55 is maximized are incident on the metal film 55. The excitation light α becomes the excitation incident angle θ _{1 at} which the electric field strength of the enhanced electric field is the largest.

In addition, according to the present embodiment, it is possible to detect a sample with high sensitivity and high accuracy. Specifically, the amount of light generated in the metal film 55 by surface plasmon resonance is measured, whereby excitation light to the metal film 55 that maximizes the intensity of the enhanced electric field formed in the vicinity of the surface 55a of the metal film 55. An α incident angle (excitation incident angle) θ ₁ can be obtained with high accuracy. At this time, the light generated in the metal film 55 is light having an excitation wavelength such as plasmon scattered light or surface diffused light. Therefore, when the position of the second BPF 46 that blocks this wavelength component is switched to the retracted position, the increase or decrease in the amount of light generated in the metal film 55 based on the surface plasmon resonance is accurately measured. In addition, at the time of detection of the specimen, the second BPF 46 is placed on the optical path of the measurement optical system 42, so that light components having excitation wavelengths such as plasmon scattered light and surface diffused light are measured from the light measured by the light measurement unit 40. Is removed. Thereby, the excitation fluorescence emitted from the fluorescent substance labeled on the specimen is accurately measured. Therefore, the signal-to-noise ratio of the signal obtained by measurement is high, and the specimen can be detected with high accuracy and high sensitivity.

  The surface plasmon resonance fluorescence analyzer of the present invention and the analysis chip used in the analyzer are not limited to the above embodiment, and various modifications can be made without departing from the scope of the present invention. Of course.

  In the above-described embodiment, the excitation light source 22 that emits the flat beam-like excitation light α is used. However, the present invention is not limited to this. For example, an excitation light source that emits beam-shaped excitation light having a circular cross-sectional outline perpendicular to the optical axis may be used. In this case, the shaping optical system constructed by a plurality of optical lenses, polarizing elements, or the like so that the outline of the irradiation region in the metal film 55 is circular or substantially circular is used as the excitation light beam shape (perpendicular to the optical axis). The profile of the cross section). However, a light-shielding mask (light-shielding member provided with a pinhole) or the like that causes light diffraction is not used in the optical element that constructs the shaping optical system so that unevenness in the amount of light does not occur in the irradiation region.

The specific shape of the analysis chip 50 is not limited. The analysis chip 50 of the above embodiment includes the prism 51 and the flow path member 57, and the metal film 55 is formed on the film formation surface 53 of the prism 51. However, the present invention is not limited to this. For example, as shown in FIG. 15, the analysis chip includes a flow path member 57 and a prism portion 60, and the prism portion 60 includes a transparent plate-like substrate 61 and a surface of one surface 61 </ b> A of the substrate 61. You may have the metal film 55 provided on top. Specifically, the substrate 61 is arranged on the surface of the predetermined surface 53 of the prism 51 so that one surface 61A of the substrate 61 faces the side opposite to the prism 51, and thereby the prism portion 60 is constructed. . The analysis chip 50A is constructed by the flow path member 57 coming into contact with the prism portion 60 from the one surface 61A side of the substrate 61. At this time, in the prism portion 60, the substrate 61 with the metal film 55 facing away from the prism 51 is disposed on the surface of the predetermined surface 53 of the prism 51. At this time, the substrate 61 is disposed on the prism 51 via the matching oil m. In this case, the prism angle θ _A is an angle formed by one surface 61A of the substrate 61 and the incident surface 52 of the prism 51.

[Outline of the embodiment]
The above embodiment is summarized as follows.

  The surface plasmon resonance fluorescence analyzer according to this embodiment is a surface plasmon resonance fluorescence analyzer that analyzes a specimen by measuring fluorescence caused by surface plasmon resonance generated in a metal film provided on a prism portion. , An excitation light emitting part that makes beam-like excitation light enter the prism part so as to be reflected by the metal film, and light generated in a region adjacent to the metal film when the excitation light is reflected by the metal film A light measurement unit for measuring The excitation light emitting unit includes an excitation light source that emits the excitation light, and a diameter changing optical system that is constructed by a plurality of optical lenses and is capable of changing a beam diameter of the excitation light. The optical system is configured so that the excitation light irradiation region in the metal film when the excitation light is reflected by the metal film is included in the light measurement region in the metal film of the light measurement unit. The beam diameter is changed.

  According to this configuration, the beam diameter of the excitation light is such that the range of surface plasmon resonance generated in the metal film by irradiation of the excitation light is included in the measurement region for measuring light due to this resonance. Therefore, the beam diameter can be reduced (see FIG. 14B) as compared with the case where the region other than the measurement region of the light measurement unit in the metal film is irradiated with excitation light (see FIG. 14A). . Thereby, the light quantity of the autofluorescence which generate | occur | produces in a prism part is suppressed. As a result, a decrease in the signal-to-noise ratio of a signal obtained when light due to surface plasmon resonance generated in the metal film is measured can be suppressed.

  Moreover, the excitation light on the metal film as in the case where a light shielding member provided with a pinhole such as a light shielding mask changes the beam diameter by the diameter changing optical system constructed by a plurality of optical lenses changing the beam diameter. And an increase in the size of the apparatus for suppressing the unevenness in the amount of light can be prevented. Specifically, when the beam diameter is changed by a light shielding mask or the like, unevenness in the amount of light in the irradiation region of the metal film is caused by interference between the diffracted light generated when the excitation light passes through the pinhole and the excitation light. Arise. However, as in the present invention, the generation of diffracted light can be suppressed by changing the beam diameter by a diameter changing optical system constructed by an optical lens (that is, an optical system that does not use a light shielding mask or the like).

  In the surface plasmon resonance fluorescence analyzer according to the present embodiment, the excitation light emitting unit changes the posture with respect to the prism unit so that the excitation light when entering the prism unit rotates about its optical axis. A light source holding unit that holds the excitation light source, and an attitude change driving unit that drives the excitation light source to change the attitude with respect to the prism unit, and the measurement region of the light measurement unit includes the metal The film has a circular contour, and the excitation light source emits excitation light having an elliptical cross-sectional shape orthogonal to the optical axis, and the posture change drive has a circular or substantially circular contour of the irradiation region. The excitation light source may be driven in such a posture.

  According to such a configuration, even when a light source that emits excitation light having an elliptical cross-sectional shape orthogonal to the optical axis is used, the contour of the irradiation region in the metal film is the same or substantially circular as the contour of the measurement region. Become. As a result, surface plasmon resonance occurs in substantially the entire region of the excitation light measurement region in the metal film, and the specimen is effectively analyzed. As a result, a small and inexpensive light source such as a semiconductor laser can be used as the excitation light source.

  The analysis chip used in the surface plasmon resonance fluorescence analyzer according to the present embodiment is an analysis chip used in a surface plasmon resonance fluorescence analyzer that analyzes a sample by measuring fluorescence caused by surface plasmon resonance. A prism portion including a metal film on which the surface plasmon resonance occurs, a reflecting surface on which the metal film is provided, and an incident surface on which excitation light for generating the surface plasmon resonance enters the surface. And comprising. Then, in the prism portion, the excitation light having an elliptical cross section perpendicular to the optical axis has a prism shape from the incident surface in a state where the minor axis direction of the cross section coincides with the inclination direction of the reflecting surface with respect to the incident surface. A prism angle that is an angle formed by the incident surface and the reflecting surface when the excitation light is incident on the metal film at an incident angle at which the intensity of the electric field due to the surface plasmon resonance is maximized after entering the portion Is an angle at which the irradiation region of the excitation light in the metal film is circular or substantially circular.

  By analyzing the specimen using this analysis chip, excitation light having a beam diameter such that a circular or substantially circular irradiation region is included in the measurement region is incident on the prism portion from the incident surface. Surface plasmon resonance occurs in substantially the entire area of the measurement region while suppressing the amount of autofluorescence generated in the unit. As a result, a decrease in the signal-to-noise ratio of the signal obtained when light due to surface plasmon resonance generated in the metal film is measured is suppressed, and the specimen is analyzed with high sensitivity and high accuracy. In addition, since the intensity of the electric field near the metal film is maximized when the excitation light is incident on the metal film at an incident angle at which the irradiation region is circular or substantially circular, the amount of light caused by surface plasmon resonance increases. This makes it possible to detect this light with higher sensitivity and accuracy.

  This application is based on Japanese Patent Application No. 2011-001059 filed on Jan. 6, 2011, the contents of which are included in the present application.

  As described above, the surface plasmon resonance fluorescence analyzer according to the present invention and the analysis chip used in this analyzer are useful for analyzing a specimen contained in a sample solution using surface plasmon resonance, This is suitable for suppressing a decrease in the signal-to-noise ratio of a signal obtained by measuring light caused by surface plasmon resonance generated in the metal film.

Claims (3)

A surface plasmon resonance fluorescence analyzer for analyzing a specimen by measuring fluorescence caused by surface plasmon resonance generated in a metal film provided on a prism part,
An excitation light emitting part that makes beam-like excitation light enter the prism part so as to be reflected by the metal film;
A light measurement unit that measures light generated in a region adjacent to the metal film by the excitation light being reflected by the metal film;
The excitation light emitting unit includes an excitation light source that emits the excitation light, and a diameter changing optical system that is constructed by a plurality of optical lenses and that can change a beam diameter of the excitation light,
The diameter changing optical system includes an irradiation region of the excitation light in the metal film when the excitation light is reflected by the metal film, so that the light measurement region in the metal film of the light measurement unit is included in the light measurement region. A surface plasmon resonance fluorescence analyzer characterized by changing a beam diameter of the excitation light. The excitation light emitting unit includes a light source holding unit that holds the excitation light source so that the posture with respect to the prism unit can be changed so that the excitation light when entering the prism unit rotates about its optical axis. And an attitude change drive unit that drives the excitation light source to change the attitude with respect to the prism unit,
The measurement region of the light measurement unit has a circular outline in the metal film,
The excitation light source emits excitation light having an elliptical cross-sectional profile orthogonal to the optical axis,
2. The surface plasmon resonance fluorescence analyzer according to claim 1, wherein the posture change drive drives the excitation light source in a posture in which an outline of the irradiation region is circular or substantially circular. An analysis chip used in a surface plasmon resonance fluorescence analyzer for analyzing a specimen by measuring fluorescence caused by surface plasmon resonance,
A metal film in which the surface plasmon resonance occurs;
A reflecting surface on which the metal film is provided, and a prism portion including an incident surface on the surface, on which excitation light for generating surface plasmon resonance enters, and
In the prism portion, excitation light having an elliptical cross-sectional outline orthogonal to the optical axis matches the minor axis direction of the cross-section with the inclination direction of the reflecting surface with respect to the incident surface, and enters the prism portion from the incident surface. When the excitation light is incident on the metal film at an incident angle at which the intensity of the electric field due to the surface plasmon resonance is maximized after being incident, a prism angle that is an angle formed by the incident surface and the reflecting surface is An analysis chip for use in a surface plasmon resonance fluorescence analyzer characterized in that an irradiation region of an excitation light metal film is circular or substantially circular.

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