WO2012042807A1 - Dispositif de fluorimétrie par résonance plasmonique de surface et procédé de fluorimétrie par résonance plasmonique de surface - Google Patents

Dispositif de fluorimétrie par résonance plasmonique de surface et procédé de fluorimétrie par résonance plasmonique de surface Download PDF

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Publication number
WO2012042807A1
WO2012042807A1 PCT/JP2011/005364 JP2011005364W WO2012042807A1 WO 2012042807 A1 WO2012042807 A1 WO 2012042807A1 JP 2011005364 W JP2011005364 W JP 2011005364W WO 2012042807 A1 WO2012042807 A1 WO 2012042807A1
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Prior art keywords
light
metal film
unit
excitation light
excitation
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PCT/JP2011/005364
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English (en)
Japanese (ja)
Inventor
和田　滋
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コニカミノルタホールディングス株式会社
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Priority to JP2012536191A priority Critical patent/JP5835223B2/ja
Publication of WO2012042807A1 publication Critical patent/WO2012042807A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Definitions

  • the present invention relates to a surface plasmon resonance fluorescence analysis apparatus and a surface plasmon resonance fluorescence analysis method for detecting a specimen contained in a sample liquid using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • a surface plasmon resonance fluorescence analysis (surface plasmon excitation enhanced fluorescence spectroscopy: SPFS) method is known as a method for detecting a sample (a substance to be detected) with high sensitivity in bioassay for detecting proteins, DNA, and the like.
  • the SPFS method in a prism in which a metal film made of gold, silver or the like is formed on a predetermined surface, excitation light is incident on the metal film from the prism side so as to satisfy the total reflection condition on the predetermined surface, When excitation light is reflected on the metal film, light (evanescent wave) that exudes from the metal film is used.
  • light evanescent wave
  • the SPFS method when the excitation light is totally reflected on the metal film, an evanescent wave that exudes from the metal film causes an analyte contained in the sample liquid that flows on the surface of the metal film or attached to the specimen. Fluorescence (excitation fluorescence) emitted by the excited fluorescent substance (labeling substance) is analyzed. Thereby, the presence or the amount of the specimen can be detected.
  • excitation light is incident from the prism side toward the metal film so as to satisfy the total reflection condition with respect to the predetermined surface, and the incident angle of the excitation light with respect to the metal film is changed. Then, the intensity of the reflected light is measured. Since the surface plasmon resonance occurs in the metal film when the amount of reflected light received in this measurement is minimized, the incident angle of the excitation light to the metal film when the amount of received light is minimized is the metal. The excitation light incident angle sufficiently enhances the electric field near the surface of the film.
  • An object of the present invention is to provide a surface plasmon resonance fluorescence analyzer and a surface plasmon resonance fluorescence analysis method that can detect a specimen with higher accuracy.
  • the first optical filter that blocks the light having the wavelength of the excitation light is retracted from between the light receiving unit and the metal film.
  • the first optical filter is inserted between the light receiving unit and the metal film. Therefore, according to the present invention, it is possible to provide a surface plasmon resonance fluorescence analyzer and a surface plasmon resonance fluorescence analysis method that can detect a specimen with higher accuracy.
  • 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.
  • FIG. 4 is a diagram for explaining a measurement optical system of the surface plasmon resonance fluorescence analyzer.
  • FIG. 5 is a flowchart showing a basic sequence for analyzing a sample in the surface plasmon resonance fluorescence analyzer.
  • FIG. 6 is a flowchart showing the resonance angle scanning sequence of FIG.
  • FIG. 7 is a flowchart showing the optimum position scanning sequence of FIG.
  • FIG. 8 is a flowchart showing the birefringence measurement sequence of FIG.
  • FIG. 9 is a flowchart showing the excitation fluorescence measurement sequence of FIG.
  • FIG. 10A is a diagram for explaining the second positioning of the reflecting member in the surface plasmon resonance fluorescence analyzer
  • FIG. 10B shows the irradiation region moved to the center position of the measurement region. It is a figure which shows a state.
  • the surface plasmon resonance fluorescence analyzer (hereinafter also simply referred to as “analyzer”) 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.
  • an evanescent wave enhanced electric field
  • analyte a fluorescent substance labeled (attached) to a substance to be detected
  • the analyzer detects the specimen by detecting the amount of fluorescent light generated by the excitation of the fluorescent substance.
  • 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.
  • the pretreatment unit accepts a reagent chip (not shown), performs pretreatment (blood cell separation, dilution, mixing, etc.) of blood injected into the reagent chip to generate a sample liquid, 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.
  • the analysis chip 50 includes a prism 51, a metal film 55 formed on the surface of the prism 51, and a sample solution or a cleaning solution containing a specimen 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 is formed such that an incident surface 52 through which the excitation light ⁇ from the excitation light emitting unit 20 is incident on the inside of the prism 51 and a metal film 55 on which the excitation light ⁇ incident on the prism 51 is reflected are formed.
  • the surface includes a surface (predetermined surface) 53 and an emission surface 54 on which the excitation light ⁇ reflected by the metal film 55 is emitted to the outside of the prism 51.
  • the prism 51 is made of transparent glass or resin.
  • the exit surface 54 is a surface that first strikes after the excitation light ⁇ 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 ⁇ reflected by the metal film 55 does not stay inside the prism 51.
  • the prism 51 of this embodiment is made of a transparent resin 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 ⁇ 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 ⁇ (specifically, the S wave component of the excitation light ⁇ ) 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 ⁇ 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 ⁇ is totally reflected on the surface without the metal film 55 (film formation surface 53). Compared with the case where a wave is generated, the formed evanescent wave is amplified (that is, an 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.
  • the metal film 55 may be silver, copper, aluminum or the like (including an alloy).
  • a capturing body 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 flow path member 57 is provided on the film formation surface 53 (specifically, the metal film 55) of the prism 51, thereby forming a flow path 58 through which the sample liquid 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 bonded to the prism 51 by adhesive, welding such as laser welding or ultrasonic welding, pressure bonding using a clamp member, or the like.
  • the flow path 58 has a shape such that a region where the metal film 55 and the sample solution are in contact with each other is wider than a measurement region of the light measurement unit 40.
  • the sample liquid pretreated by the pretreatment unit is injected (supplied) into the flow path 58.
  • the analysis chip 50 into which the sample liquid has been injected is transported to the chip holding unit 12 when the reaction between the capturing body 56 fixed on the metal film 55 and the specimen (specific antigen) is completed.
  • 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 specimen.
  • the predetermined posture means that the excitation light ⁇ emitted from the excitation light emitting unit 20 is incident on the inside of the prism 51 from the incident surface 52 so as to satisfy the total reflection condition with respect to the film formation surface 53.
  • the posture is such that the light ⁇ is reflected by the metal film 55.
  • 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.
  • the analyzing apparatus 10 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 ⁇ 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.
  • the excitation light emitting unit 20 includes a light source unit 21 that emits linearly polarized excitation light, and an excitation optical system 30 that guides the excitation light ⁇ emitted from the light source unit 21 to the incident surface 52 of the prism 51.
  • the light source unit 21 includes a light source unit unit 23 including an excitation light source 22 and a first wave shaping unit 24 that waves the excitation light ⁇ emitted from the excitation light source 22.
  • the light source unit 21 of the present embodiment emits excitation light ⁇ downward.
  • the excitation light source 22 of this embodiment is a laser diode.
  • the light source unit 23 includes an excitation light source 22 and a temperature adjustment circuit 25 that performs temperature adjustment (temperature adjustment) of the excitation light source 22.
  • This light source unit 23 collimates the excitation light ⁇ emitted from the excitation light source 22 and also positions the excitation light source 22 so that the excitation light ⁇ enters the metal film 55 of the prism 51 from the short axis side. Adjust and hold. This is due to the following reason.
  • the excitation light ⁇ emitted from the excitation light source (laser diode) 22 has a flat shape even when it is comomitted, and the polarization direction is substantially biased in one direction. For this reason, the posture of the excitation light source 22 is adjusted, and this adjusted posture is maintained, so that the excitation light ⁇ is totally reflected on the film formation surface 53 (a shallow angle with respect to the film formation surface 53). Thus, when the light enters the prism 51, the outline shape of the irradiation region on the film formation surface 53 of the excitation light ⁇ becomes approximately circular (see FIG. 3).
  • the temperature adjustment circuit 25 is a regression circuit for adjusting the temperature of the excitation light source (laser diode) 22. Specifically, the temperature adjustment circuit 25 monitors the light amount branched from the light beam of the excitation light ⁇ after collimation by a photodiode or the like (not shown), and thereby the wavelength and light amount of the emitted excitation light ⁇ 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 ⁇ emitted from the light source unit 23 by a plurality of filters (optical filters), and makes the excitation light ⁇ have an excitation wavelength with a unique polarization direction.
  • the first harmonizing 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.
  • first BPF first bandpass filter
  • LPF linear polarization filter
  • first NDF first. 1 ND filter
  • 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 ⁇ 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 ⁇ from the light source unit 21 to the prism 51 of the analysis chip 50 held in the chip holding unit 12.
  • This excitation optical system 30 includes a polarization direction adjusting unit 31 that changes the polarization direction of the excitation light ⁇ , a shaping optical system 32 that adjusts the contour shape of the beam of the excitation light ⁇ , and the excitation light ⁇ into the prism 51.
  • An incident path adjusting unit 35 that changes the incident path to change the reflection position of the excitation light ⁇ on the metal film 55 and the incident angle ⁇ of the excitation light ⁇ with respect to the metal film 55.
  • the polarization direction adjusting unit 31 includes a half-wave plate 33 and a rotation driving unit 34 that rotates the half-wave plate 33.
  • the half-wave plate 33 is disposed on the optical path of the excitation optical system 30 and is used as a polarization rotator that continuously rotates the polarization direction of the excitation light ⁇ .
  • the rotation driving unit 34 rotates the polarization direction of the excitation light ⁇ 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 ⁇ linearly polarized in the first wave rectifier 24 rotates.
  • the rotation drive unit 34 rotates the half-wave plate 33 to maximize the evanescent wave in the metal film 55 (that is, the electric field enhancement of the enhanced electric field formed in the vicinity of the surface 55a of the metal film 55). It is possible to freely change the polarization direction from the condition where the degree is the maximum) to the condition where no exudation occurs (that is, the condition where no enhanced electric field is formed near the surface 55a of the metal film 55).
  • the shaping optical system 32 adjusts the beam size of the excitation light ⁇ and the contour shape of the beam by a slit, a zoom function or the like so that the contour of the irradiation region of the excitation light ⁇ in the metal film 55 becomes a circle having a predetermined diameter. .
  • region of the excitation light (alpha) in the metal film 55 of this embodiment is adjusted so that it may become smaller than the measurement area
  • the light measurement part 40 can measure the light resulting from surface plasmon resonance and the enhanced electric field based on this surface plasmon resonance.
  • the incident path adjustment unit 35 includes a reflection member 36 that reflects the excitation light ⁇ 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 ⁇ .
  • the reflecting member 36 of this embodiment is a reflecting mirror.
  • a dielectric multilayer film in which a phase shift, dimming, etc. do not occur between the excitation light ⁇ before entering the reflection surface 36a and the excitation light ⁇ after being reflected by the reflection surface 36a.
  • 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.
  • a non-reflective light absorbing material that absorbs the excitation light ⁇ without reflecting is attached to the back surface 36b of the reflecting member 36.
  • the non-reflective 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 ⁇ in FIG. 1).
  • 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 ⁇ incident on the reflective member 36 and the optical path of the excitation light ⁇ 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 rotates the reflecting surface 36a along the surface including the optical path while maintaining the orthogonal posture of the reflecting member 36 (the reflecting surface 36a is orthogonal to the surface including the optical path). As described above, the reflecting member 36 is rotated based on the instruction signal from the control processing unit 14.
  • the rotation drive mechanism has 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 to a position where the excitation light ⁇ from the light source unit 21 is incident on the back surface 36 b of the reflecting member 36 based on an instruction signal from the control processing unit 14.
  • 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 interval is related to the resolution in adjusting the direction of the reflecting surface 36a, and is appropriately set according to the performance of the apparatus.
  • the reciprocating drive mechanism linearly moves the stage 37a, that is, the reflecting member 36 along the optical axis of the excitation light ⁇ from the light source unit 21 (see arrow ⁇ in FIG. 1).
  • the reciprocating drive mechanism of the present embodiment reciprocates the stage 37a in the optical axis direction of the excitation light ⁇ from the light source unit 21, that is, in the vertical direction.
  • 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.
  • 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 ⁇ 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
  • 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.
  • a photomultiplier tube (PMT) having a high sensitivity and a high S / N ratio is used as the light receiving unit 41 in order to detect weak light such as fluorescence generated by exciting a fluorescent substance labeled on the specimen.
  • the light receiving unit 41 is not limited to the PMT, and may be a cooled CCD type image sensor or the like.
  • 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 rectifying 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 filters 46 and 47. Part 48.
  • the second band-pass filter (hereinafter simply referred to as “second BPF”) 46 blocks light of the wavelength of the excitation light ⁇ (excitation wavelength).
  • 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 can measure plasmon scattered light and the like. Specifically, the amount of light to be measured for obtaining the incident angle ⁇ 1 of the excitation light ⁇ that maximizes the enhanced electric field is much larger than the amount of excitation fluorescence measured at the time of detection of the specimen.
  • the second NDF 47 reduces the light to be measured in order to obtain the incident angle ⁇ 1 of the excitation light ⁇ that maximizes the enhanced electric field, thereby preventing the light receiving unit 41 from being damaged. Can be removed.
  • 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 condenser lens 44 and the imaging lens 45, and each of the filters 46 and 47 is orthogonal to the optical axis of the parallel light or the substantially parallel light between the lenses 44 and 45 and the parallel. It is a position that crosses light or substantially parallel light.
  • 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.
  • the retracted position is a position deviated from the optical path in the measurement optical system 42.
  • the position switching unit 48 is configured so that the second NDF 47 is in the retracted position when the second BPF 46 is in the filtering position (see FIG. 4), and the second NDF 47 is in the filtering position when the second BPF 46 is in the retracted position. Switch each position.
  • the position switching unit 48 of the present embodiment reciprocates the holding frame 49 along the plane in which the second BPF 46 and the second NDF 47 are arranged (see the arrow ⁇ in FIG. 4), thereby changing the position of each filter 46, 47. Switch. 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 the instruction signal from the control processing unit 14.
  • 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.
  • the position switching unit 48 reciprocates the holding frame 49 to switch the positions of the filters 46 and 47.
  • 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.
  • 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 a sample, the control processing unit 14 controls the light source unit 21, the polarization direction adjustment unit 31, the incident path adjustment unit 35, the light measurement unit 40, and the like. 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. Further, when analyzing the sample, the control processing unit 14 calculates based on an output signal sent from the light measuring unit 40 (specifically, the light receiving unit 41), and measures the fluorescence measured by the light measuring unit 40.
  • the control processing unit 14 calculates based on an output signal sent from the light measuring unit 40 (specifically, the light receiving unit 41), and measures the fluorescence measured by the light measuring unit 40.
  • 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 like a printer.
  • the display unit 16 may be a combination of screen display and printout.
  • FIG. 5 is a flowchart showing a basic sequence for analyzing a sample in the analyzer 10.
  • FIG. 6 is a flowchart showing a resonance angle scanning sequence.
  • FIG. 7 is a flowchart showing the optimum position scanning sequence.
  • FIG. 8 is a flowchart showing a birefringence measurement sequence.
  • FIG. 9 is a flowchart showing an excitation fluorescence measurement sequence.
  • 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 such as blood by the preprocessing unit to generate a sample solution.
  • the control processing unit 14 injects the sample liquid after the pre-processing into the flow path 58 of the analysis chip 50 by the pre-processing unit.
  • the capturing body 56 fixed on the surface captures the specimen (specific antigen).
  • the preprocessing unit reacts the capturing body 56 with the specimen.
  • the capturing body 56 captures a specimen labeled with a fluorescent substance (in this embodiment, a fluorescent dye), but the present invention is not limited to this.
  • 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).
  • the excitation light source 22 (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.
  • the control processing unit 14 performs scanning (resonance angle scanning) under the optimum surface plasmon resonance condition in the analysis chip 50. Based on the scanning result, the control processing unit 14 causes the excitation light ⁇ to be incident on the metal film 55 at an incident angle (excitation incident angle ⁇ 1) at which the electric field strength of the enhanced electric field generated in the metal film 55 is the largest. Then, the reflecting member 36 is positioned (first positioning) (step S2).
  • control processing unit 14 drives the reflecting member 36 by the reflecting member driving unit 37, thereby causing the excitation light ⁇ to be incident on the metal film 55 of the prism 51 included in the analysis chip 50 (excitation incident angle).
  • a scan of ⁇ 1) is performed. More specifically, an enhanced electric field (evanescent wave) based on surface plasmon resonance is generated in the metal film 55 of the prism 51 depending on the material, shape, flow path filling liquid (sample liquid) refractive index, and the like of the prism 51 included in the analysis chip 50.
  • the incident angle ⁇ 1 of the excitation light having the highest intensity is determined.
  • the excitation light incident condition fluctuates due to the molecular weight of the sample captured by the capturing body 56, the substance constituting the molecule, the manufacturing error on the prism 51 side, and the like. Therefore, the control processing unit 14 causes the excitation light ⁇ to be incident on the metal film 55 so that the incident angle is less than ⁇ 10 ° with the excitation incident angle ⁇ 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 ⁇ 1 in the analysis chip 50 is obtained.
  • 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).
  • the half-wave plate 33 of the polarization direction adjusting unit 31 has the largest P wave component in the excitation light ⁇ incident on the metal film 55 when the excitation light ⁇ is emitted from the excitation light emitting unit 20. In addition, it is in a state (initial state) required 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 ⁇ 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 ⁇ is emitted from the excitation light emitting unit 20.
  • the position of the reflecting member 36 and the direction of the reflecting surface 36a are such that the incident angle ⁇ at which the evanescent wave does not ooze out in the region near the surface 55a of the metal film 55.
  • control processing unit 14 measures the amount of light generated in the metal film 55 by the light measuring 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 measuring unit 40 when the reflecting member 36 is at the maximum separation position is the surface diffused light SK in the prism 51.
  • the control processing unit 14 stores the incident angle ⁇ of the excitation light ⁇ with respect to the metal film 55 and the amount of light measured by the light measurement unit 40 in association with each other (step S23).
  • the light received by the light receiving unit 41 includes light having the same wavelength as the excitation wavelength of the excitation light ⁇ .
  • 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.
  • the light having the same wavelength as the excitation wavelength is enhanced by surface plasmon resonance generated in the metal film 55.
  • 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.
  • the light receiving unit 41 that measures excitation fluorescence with a small amount of light measures surface plasmon scattered light, diffused light, or the like that has a much larger amount of light than the excitation fluorescence.
  • the position switching unit 48 retracts the second BPF 46 to the retracted position and moves the second NDF 47 to the filtering position, so that the same light receiving unit (PMT in the present embodiment) 41 is provided. The amount of light (scattered light or the like and excitation fluorescence) can be measured.
  • the control processing unit 14 adjusts the position of the reflecting member 36 by the incident path adjusting unit 35 in a state where the excitation light ⁇ is emitted from the light source unit 21. 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 ⁇ on the metal film 55 (Step S24), and the reflection member by the reciprocating driving mechanism. The position 36 is moved (step S25). Specifically, the control processing unit 14 reflects the position of the reflecting member 36 and the excitation light ⁇ 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.
  • 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 ⁇ ( Step S26).
  • control processing unit 14 measures the amount of light by the light measurement unit 40 while changing the incident angle ⁇ so that the irradiation position on the metal film 55 is not shifted, and stores the measurement result.
  • the control processing unit 14 measures the amount of light in a predetermined scanning region (for example, an incident angle ⁇ of less than ⁇ 10 ° with the excitation incident angle ⁇ 1a based on the design as a center) by the light measuring unit 40, and then the light source The emission of the excitation light ⁇ from the 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).
  • a predetermined scanning region for example, an incident angle ⁇ of less than ⁇ 10 ° with the excitation incident angle ⁇ 1a based on the design as a center
  • the control processing unit 14 reflects the irradiation position (incident position) of the excitation light ⁇ 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).
  • 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 ⁇ is incident on the metal film 55 (see position A in FIG. 10A).
  • the control processing unit 14 causes the light source unit 21 to emit excitation light ⁇ when the reflecting member 36 is at 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.
  • 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 ⁇ from the light source unit 21.
  • 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 measurement 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 reflection member has reached the lower end position based 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 measurement unit 40. To do. Specifically, since the irradiation region of the excitation light ⁇ 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 ⁇ enters the measurement region of the light measurement unit 40 (see position B in FIG. 10A), the measurement is performed by the light measurement unit 40. The amount of light emitted increases gradually.
  • the control processing unit 14 stores the intensity of these lights and the vertical position of the reflecting member 36 when the intensity is measured in association with each other, 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.
  • control processing unit 14 determines 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 value of the stored light amount (step S35). At this time, there are two values to be selected (see position B and position D in FIG. 10A). 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. 10A) and stores it (step S36). The control processing unit 14 moves the reflection member 36 by the reciprocating drive mechanism to move the irradiation region to the obtained center position Ce (see step S37, FIG. 10B).
  • the amount of autofluorescence for each analysis chip 50 measured by the light measurement unit 40 can be made constant.
  • fluorescence is generated inside the prism 51 by the excitation light ⁇ 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.
  • the fluorescence 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.
  • the autofluorescence is weak, most of the light in the vicinity of the irradiation region of the excitation light ⁇ in the metal film 55 can cause noise in the measurement of the excitation fluorescence. Since the excitation light ⁇ reflected by the metal film 55 is almost eliminated when surface plasmon resonance occurs, autofluorescence from the incident side (left side in FIG. 10A) 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 ⁇ so that the optical path length on the incident side in the measurement region of the light measurement unit 40 is constant.
  • the excitation light in the measurement region of the light measurement unit 40 is adjusted so that the irradiation position of the excitation light ⁇ 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 ⁇ is constant.
  • the control processing unit 14 measures the birefringence (step S4). Then, the control processing unit 14 improves the measurement accuracy of the specimen by considering the birefringence when measuring the excitation fluorescence from the fluorescent substance labeled on the specimen. Specifically, birefringence occurs when light is transmitted 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 formed. Therefore, the degree of birefringence differs depending on the individual prism 51.
  • phase rotation occurs in the excitation light ⁇ traveling through the prism 51 due to birefringence and only the P wave is desired to be incident on the metal film 55, an S wave component is generated in the excitation light ⁇ due to the phase rotation due to the birefringence. .
  • the amount of excitation fluorescence excited by the enhanced electric field decreases in accordance with the amount of S wave component generated by this birefringence. Therefore, the detection accuracy and sensitivity of the sample in the analyzer 10 are improved by the control processing unit 14 correcting this decrease.
  • control processing unit 14 emits the excitation light ⁇ from the light source unit 21, and the light measurement unit 40 measures the amount of light generated in the metal film 55.
  • the half-wave plate 33 is in an initial state (step S41).
  • 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).
  • the control processing unit 14 rotates the half-wave plate 33, the P wave component and the S wave component of the excitation light ⁇ incident on the metal film 55 are increased or decreased, respectively.
  • 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 ⁇ , 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 ⁇ 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 repeats step S42 and step S43 in order until the maximum value DRmax and the minimum value DRmin of the light amount of light measured by the light measurement unit 40 are obtained (step S44).
  • the control processing unit 14 selects the maximum value DRmax and the minimum value DRmin 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 DRmax is obtained and the minimum value DRmin are obtained). Are selected and stored.
  • control processing unit 14 uses the following formulas (1) and (2) from the stored maximum value DRmax and minimum value DRmin and the amount of surface diffused light SK stored in the resonance angle scanning step. Then, the major axis rotation amount ⁇ i due to the birefringence in the prism 51 and the correction coefficient K are derived and stored (step S46).
  • control processing unit 14 rotates the half-wave plate 33 to the rotation position when the maximum value DRmax is obtained by the rotation driving unit 34. Accordingly, the excitation light ⁇ 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).
  • 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 ⁇ .
  • the excitation light ⁇ 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 to emit fluorescence (excitation fluorescence).
  • the control process part 14 measures excitation fluorescence by the light measurement part 40 (step S5).
  • 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 retreat position (step S51: see FIGS. 1 and 4). Then, the control processing unit 14 uses the rotation driving mechanism of the reflecting member driving unit 37 so that the excitation light ⁇ 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 ⁇ 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 ⁇ 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 ⁇ from the light source unit 21, measures the amount of excitation fluorescence caused by the enhanced electric field generated in the vicinity of the metal film 55 by the light measurement unit 40, and stores this (step S55). . Thereby, the control processing unit 14 obtains the measurement maximum light quantity (first light quantity value) Smax. This is because 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 ⁇ is incident on the metal film 55 is the maximum value DRmax. This is because the intensity of the enhanced electric field in the vicinity of the metal film 55 is maximized.
  • 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 ⁇ is incident on the metal film 55 becomes the minimum value DRmin.
  • the half-wave plate 33 is rotated by the rotation drive unit 34 to the rotation position) (step S56).
  • the control process part 14 measures the light quantity of excitation fluorescence by the light measurement part 40, and memorize
  • the control processing unit 14 Based on the stored measurement maximum light quantity Smax, measurement minimum light quantity Smin, and dark noise DN, the control processing unit 14 performs self-measurement in the prism as shown in the following formulas (3-1) to (8).
  • the fluorescence light quantity h and the excitation fluorescence light quantity H are derived and stored (step S58).
  • the amount of excitation fluorescence when Smax is obtained is H1
  • the amount of autofluorescence is h1
  • the amount of excitation fluorescence when Smin is obtained is H2
  • the amount of autofluorescence is h2.
  • the control processing unit 14 obtains the amount H of excitation fluorescence by the following approximate expression (9).
  • control processing unit 14 obtains the amount H of excitation fluorescence by the following approximate expression (10).
  • 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.
  • control processing unit 14 returns the reflecting member 36 to the initial position (step S7), and ends the series of measurements.
  • 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 ⁇ incident angle (excitation incident angle) ⁇ 1 can be obtained with high accuracy.
  • 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.
  • 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 obtained by measurement has a high S / N ratio, and it is possible to detect a sample with high accuracy and high sensitivity.
  • the influence of autofluorescence in the prism 51 is removed from the measurement result, thereby ensuring a wide dynamic range.
  • the half-wave plate 33 is adjusted so that the polarization direction of the excitation light ⁇ 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.
  • the amount of autofluorescence in the prism 51 is obtained.
  • the incident angle ⁇ of the excitation light ⁇ with respect to the metal film 55 can be changed by driving the reflecting member 36, the reflection position of the excitation light ⁇ on the metal film 55 by changing the incident angle ⁇ is changed. Deviation is suppressed. That is, according to the above configuration, the incident angle ⁇ of the excitation light ⁇ 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 incident angle ⁇ is adjusted by moving the entire excitation optical system including a light source and a plurality of lenses.
  • the analyzer 10 causes one excitation light ⁇ to enter the prism 51 to generate an enhanced electric field based on surface plasmon resonance in the vicinity of the metal film 55, a plurality of excitation lights having different incident angles ⁇ as in the prior art. This prevents the amount of autofluorescence from increasing as in the case where ⁇ , ⁇ ,... Thereby, in the signal obtained by measuring the light generated in the vicinity of the metal film 55 by surface plasmon resonance, a decrease in the SN ratio due to autofluorescence is suppressed.
  • the incident angle ⁇ of the excitation light ⁇ with respect to the metal film 55 is set with high accuracy so that the electric field strength of the enhanced electric field due to surface plasmon resonance is maximized.
  • the absorption peak in the angle spectrum of the reflected light in the metal film 55 and the peak of the electric field intensity of the enhanced electric field are shifted from each other, but light (light generated in the metal film 55) generated due to surface plasmon resonance is shifted.
  • the intensity peak coincides with the electric field intensity peak of the enhanced electric field. Therefore, the excitation light ⁇ to the metal film 55 is adjusted by adjusting the reflection member 36 so that the position of the reflection member 36 and the direction of the reflection surface 36a when the light amount of light generated in the metal film 55 becomes maximum. Is the excitation incident angle ⁇ 1 at which the electric field strength of the enhanced electric field is the largest.
  • the surface plasmon resonance fluorescence analysis apparatus and the surface plasmon resonance fluorescence analysis method of the present invention are not limited to the above-described embodiments, and various changes can be made without departing from the scope of the present invention. is there.
  • the analysis chip 50 is replaced every time the sample is detected, but the present invention is not limited to this. That is, the analyzer 10 may be an analyzer in which an analysis chip is incorporated in a part of the analyzer 10 and a sample is detected by repeatedly using one analysis chip.
  • the control processing unit 14 determines the excitation incident angle ⁇ 1, and the light generated in the metal film 55 while continuously changing the incident angle ⁇ of the excitation light ⁇ to the metal film 55.
  • strength in the said embodiment, light quantity
  • the control processing unit 14 measures the amount of light generated in the metal film 55 at each incident angle ⁇ while changing the incident angle ⁇ intermittently (for example, in increments of 1 °) within the first range.
  • the incident angle ⁇ is set to be smaller than the first scan (for example, the incident angle is set in increments of 0.1 °).
  • a second scan that measures the amount of light generated in the metal film 55 intermittently or while continuously changing the incident angle ⁇ may be performed.
  • the control processing unit 14 narrows down the second scanning range by the first scanning in discrete steps, and then finely scans within the narrowed range, whereby the light generated in the metal film 55 becomes the maximum light amount.
  • the incident angle ⁇ 1 may be obtained. Note that the second range is included in the first range.
  • the control processing unit 14 changes the incident angle ⁇ in increments of 1 ° within an incident angle ⁇ range (first range) of less than ⁇ 10 ° centered on the excitation incident angle ⁇ 1a based on the design. .
  • the control processing unit 14 measures the amount of light generated in the metal film 55 at each incident angle ⁇ (first scanning), and stores the result in association with the corresponding incident angle ⁇ .
  • the control processing unit 14 increments by 0.1 ° within the range (second range) of the incident angle ⁇ within ⁇ 1 ° around the incident angle ⁇ at which the maximum light quantity is obtained in the first scanning.
  • the incident angle ⁇ is continuously changed.
  • the control processing unit 14 measures the amount of light generated in the metal film 55 (second scanning), and stores the result in association with the corresponding incident angle ⁇ .
  • the control processing unit 14 sets the incident angle ⁇ when the maximum amount of light is obtained in the second scanning as the excitation incident angle ⁇ 1. In this way, the excitation incident angle ⁇ 1 at which the enhanced electric field is the largest can be efficiently obtained as compared with the case where the entire first range is finely scanned.
  • the first scanning and the second scanning are performed, and the chip holding unit 12 holds the plurality of analysis chips 50 in order, and analyzes the specimen one after another.
  • the first scan may be performed only for the first analysis chip 50, and only the second scan may be performed for the second and subsequent analysis chips 50.
  • the control processing unit 14 applies the first analysis chip 50 to the first analysis chip 50 in the same manner as described above, for example, the range of the incident angle ⁇ less than ⁇ 10 ° with the excitation incident angle ⁇ 1a based on the design as the center (first In the range), the incident angle ⁇ is changed in increments of 1 °.
  • the control processing unit 14 measures the amount of light generated in the metal film 55 at each incident angle ⁇ (first scanning), and stores the result in association with the corresponding incident angle ⁇ . Then, the control processing unit 14 increments by 0.1 ° within the range (second range) of the incident angle ⁇ within ⁇ 3 ° around the incident angle ⁇ at which the maximum light quantity is obtained in the first scanning.
  • the incident angle ⁇ is continuously changed.
  • control processing unit 14 measures the amount of light generated in the metal film 55 (second scanning), and stores the result in association with the corresponding incident angle ⁇ . Then, the control processing unit 14 sets the incident angle ⁇ when the maximum amount of light is obtained in the second scanning as the excitation incident angle ⁇ 1 in the first analysis chip 50.
  • the control processing unit 14 performs 0 for the second analysis chip 50 within the second scan range obtained in the first scan to the first analysis chip 50 without performing the first scan. Change the incident angle ⁇ in increments of 1 ° or continuously. At this time, the control processing unit 14 measures the amount of light generated in the metal film 55 (second scanning), and stores the result in association with the corresponding incident angle ⁇ . Then, the control processing unit 14 sets the incident angle ⁇ when the maximum amount of light is obtained in the second scanning as the excitation incident angle ⁇ 1 in the second analysis chip 50. Thus, for the second and subsequent analysis chips 50, the control processing unit 14 performs only a scan (second scan) within the second range obtained by the first analysis chip 50, Analysis time can be shortened.
  • the surface plasmon resonance fluorescence analyzer is a surface plasmon resonance fluorescence analyzer that measures fluorescence emitted from a fluorescent substance attached to a specimen by being excited by an electric field based on surface plasmon resonance, and has a predetermined surface.
  • An analysis chip including a prism having a metal film formed thereon is reflected by a chip holding unit that can be detachably attached, and a metal film of the prism included in the analysis chip held by the chip holding unit. In this way, an excitation light emitting part that makes excitation light enter the prism, and the excitation light is reflected by the metal film, so that the intensity of the light generated on the surface opposite to the prism in the metal film is measured.
  • the light measuring unit includes a light receiving unit that receives light generated in the metal film, a measurement optical system that guides light generated in the metal film to the light receiving unit, and a first light that blocks light having a wavelength of the excitation light.
  • the surface plasmon resonance fluorescence analyzer is a surface plasmon resonance fluorescence analyzer that measures the fluorescence emitted when a fluorescent substance attached to a specimen is excited by an electric field based on surface plasmon resonance, and a metal is formed on a predetermined surface.
  • a light measuring unit capable of measuring the intensity of light generated on the surface of the film opposite to the prism, and a control unit for controlling the excitation light emitting unit and the light measuring unit.
  • the light measuring unit includes a light receiving unit that receives light generated in the metal film, a measurement optical system that guides light generated in the metal film to the light receiving unit, and a first light that blocks light having a wavelength of the excitation light.
  • the intensity of the electric field (enhanced electric field) formed in the vicinity of the surface of the metal film by measuring the intensity of light generated on the surface of the metal film (surface opposite to the prism) by surface plasmon resonance.
  • the angle of incidence of the excitation light on the metal film that maximizes can be obtained with high accuracy.
  • the first optical filter that blocks this wavelength component is switched to the retracted position. The increase / decrease in the intensity of light generated on the surface side of the metal film based on plasmon resonance can be measured with high accuracy.
  • the first optical filter is placed on the optical path of the measurement optical system, so that the component of the plasmon scattered light or the surface diffused light that is the excitation wavelength is changed from the light measured in the light measurement unit.
  • the fluorescence emitted by the fluorescent substance attached to the specimen excited by the enhanced electric field can be accurately measured.
  • 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 light measurement unit includes a second optical filter that attenuates and emits incident light
  • the position switching unit includes the first optical filter at a filtering position. The position of each optical filter can be switched so that sometimes the second optical filter is in the retracted position, and the second optical filter is in the filtering position when the first optical filter is in the retracted position. It is preferable.
  • the light receiving portion of the light measuring portion it is possible to prevent the light receiving portion of the light measuring portion from being damaged by the light generated on the surface side of the metal film measured in order to obtain the incident angle of the excitation light that maximizes the enhanced electric field.
  • the amount of light measured to obtain the incident angle of the excitation light that maximizes the enhanced electric field is much larger than the amount of fluorescence measured when detecting the specimen. For this reason, when a common light receiving unit is used, the light measured to obtain the incident angle of the excitation light that maximizes the enhanced electric field is attenuated by the second optical filter, thereby damaging the light receiving unit. Is prevented.
  • the measurement optical system is a two-group conjugate optical system in which light traveling between groups becomes parallel light or substantially parallel light, and the filtering position is set by the first optical filter and the second optical filter in the group. It is preferably a position that is perpendicular to the optical axis of the parallel light or substantially parallel light between them and crosses the parallel light or substantially parallel light.
  • the specimen is detected with high accuracy. That is, if the first optical filter or the second optical filter is inclined with respect to the optical axis of parallel light or the like traveling between the groups, the light that has passed through the first optical filter or the second optical filter Since the optical axis is shifted, the measurement accuracy in the optical measurement unit is lowered.
  • the surface plasmon resonance fluorescence analysis method is a surface plasmon resonance fluorescence analysis method for measuring light emitted when a fluorescent substance attached to a specimen is excited by an electric field based on surface plasmon resonance, A prism having a metal film formed on the surface thereof, a preparatory step of flowing a sample solution containing the specimen on the metal film, and excitation light for generating surface plasmon resonance in the metal film in the prism
  • the incident angle of the excitation light to the metal film is changed while the excitation light is reflected at a specific position of the metal film, and the excitation light is reflected by the metal film.
  • the resonance angle scanning step of measuring the intensity of light generated on the surface of the metal film opposite to the prism Based on the resonance angle scanning step of measuring the intensity of light generated on the surface of the metal film opposite to the prism, and the light measurement result in the resonance angle scanning step. While irradiating a specific position of the metal film with the excitation light so as to have an incident angle, the light generated on the surface opposite to the prism of the metal film is reflected by the excitation light.
  • the measuring step includes a position switching step of placing the first optical filter between the light receiving unit and the metal film.
  • the intensity of light generated on the surface side of the metal film by surface plasmon resonance is measured, so that the intensity of the electric field (enhanced electric field) formed near the surface of the metal film is maximized.
  • the incident angle of the excitation light can be obtained with high accuracy.
  • the light generated on the surface side of the metal film is light having an excitation wavelength such as plasmon scattered light or surface diffused light. For this reason, when the first optical filter that blocks this wavelength component is withdrawn from between the light receiving portion and the metal film, the intensity of light generated on the surface side of the metal thin film on the surface side based on surface plasmon resonance can be accurately increased or decreased. Measured.
  • the first optical filter is inserted between the light receiving unit and the metal film, so that the components of plasmon scattered light and surface diffused light, which are excitation wavelengths, are received from the light received by the light receiving unit. Excluded.
  • the fluorescence emitted when the fluorescent substance attached to the specimen is excited by the enhanced electric field is accurately measured.
  • 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 analysis apparatus and the surface plasmon resonance fluorescence analysis method according to the present invention cause the fluorescent material contained in the specimen to emit light using the electric field of the evanescent wave generated by the surface plasmon resonance, and this fluorescence It is useful for measuring a sample by detecting the sample, and is suitable for more accurately detecting the sample.

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Abstract

Selon la présente invention, un liquide de test est amené à s'écouler sur un film métallique (55) sur un prisme (51), et une lumière d'excitation (α) est émise dans le prisme (51). L'intensité de la lumière de longueur d'onde d'excitation qui se produit au niveau du film métallique (55) est mesurée tout en faisant varier l'angle d'incidence (θ) et en maintenant un état dans lequel la lumière d'excitation (α) qui entre dans le prisme (51) est réfléchie en une position prescrite sur le film métallique (55). La lumière d'excitation (α) est rayonnée sur une position prescrite sur le film métallique (55) de façon à obtenir un angle d'incidence (θ1) sur la base des résultats de ces mesures, tandis qu'une unité de réception de lumière (41) reçoit et mesure l'intensité de la fluorescence excitée produite au niveau du film métallique (55). Lorsque la lumière à la longueur d'onde d'excitation est mesurée, un filtre optique (46) qui arrête la lumière ayant la longueur d'onde de la lumière d'excitation est rétracté depuis l'espace entre l'unité de réception de lumière (41) et le film métallique (55). Lorsque la fluorescence d'excitation est mesurée, le filtre optique (46) est inséré entre l'unité de réception de lumière (41) et le film métallique (55).
PCT/JP2011/005364 2010-09-30 2011-09-26 Dispositif de fluorimétrie par résonance plasmonique de surface et procédé de fluorimétrie par résonance plasmonique de surface WO2012042807A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015059803A (ja) * 2013-09-18 2015-03-30 コニカミノルタ株式会社 表面プラズモン共鳴蛍光分析装置および表面プラズモン共鳴蛍光分析方法
WO2016147937A1 (fr) * 2015-03-17 2016-09-22 コニカミノルタ株式会社 Dispositif de détection

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JP2004527741A (ja) * 2001-03-14 2004-09-09 バイアコア アーベー 全反射分光法のための装置および方法
JP2008268063A (ja) * 2007-04-23 2008-11-06 Sanyo Electric Co Ltd エバネッセント波発生装置及びそれを用いた観察装置
JP2009204476A (ja) * 2008-02-28 2009-09-10 Fujifilm Corp センシング装置
JP2010091553A (ja) * 2008-09-09 2010-04-22 Konica Minolta Holdings Inc 生体分子の検出方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004527741A (ja) * 2001-03-14 2004-09-09 バイアコア アーベー 全反射分光法のための装置および方法
JP2008268063A (ja) * 2007-04-23 2008-11-06 Sanyo Electric Co Ltd エバネッセント波発生装置及びそれを用いた観察装置
JP2009204476A (ja) * 2008-02-28 2009-09-10 Fujifilm Corp センシング装置
JP2010091553A (ja) * 2008-09-09 2010-04-22 Konica Minolta Holdings Inc 生体分子の検出方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015059803A (ja) * 2013-09-18 2015-03-30 コニカミノルタ株式会社 表面プラズモン共鳴蛍光分析装置および表面プラズモン共鳴蛍光分析方法
WO2016147937A1 (fr) * 2015-03-17 2016-09-22 コニカミノルタ株式会社 Dispositif de détection
JPWO2016147937A1 (ja) * 2015-03-17 2017-12-28 コニカミノルタ株式会社 検出装置
US10267735B2 (en) 2015-03-17 2019-04-23 Konica Minolta, Inc. Surface plasmon-field enhanced fluorescence detection device

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