WO2015152198A1 - Procédé d'analyse de fluorescence par résonance plasmonique de surface, dispositif d'analyse de fluorescence par résonance plasmonique de surface, et procédé d'alignement - Google Patents

Procédé d'analyse de fluorescence par résonance plasmonique de surface, dispositif d'analyse de fluorescence par résonance plasmonique de surface, et procédé d'alignement Download PDF

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Publication number
WO2015152198A1
WO2015152198A1 PCT/JP2015/060042 JP2015060042W WO2015152198A1 WO 2015152198 A1 WO2015152198 A1 WO 2015152198A1 JP 2015060042 W JP2015060042 W JP 2015060042W WO 2015152198 A1 WO2015152198 A1 WO 2015152198A1
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Prior art keywords
capture region
light
detected
excitation light
metal film
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PCT/JP2015/060042
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English (en)
Japanese (ja)
Inventor
正貴 松尾
平山　博士
好正 濱野
野田　哲也
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コニカミノルタ株式会社
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Priority to JP2016511907A priority Critical patent/JP6399089B2/ja
Publication of WO2015152198A1 publication Critical patent/WO2015152198A1/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
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons

Definitions

  • the present invention relates to a surface plasmon resonance fluorescence analysis method and a surface plasmon resonance fluorescence analysis apparatus for detecting a substance to be detected contained in a sample liquid by using surface plasmon resonance (SPR), and to be included in the sample liquid.
  • the present invention relates to an alignment method for aligning an analysis chip when detecting a detected substance to be detected.
  • a prism in which a metal film is arranged on a predetermined surface is used. Then, when the metal film is irradiated with excitation light through the prism at an angle at which surface plasmon resonance occurs, localized field light (enhanced electric field) can be generated on the surface of the metal film.
  • This localized field light excites a fluorescent substance that labels the target substance captured on the metal film, so that the presence or amount of the target substance can be determined by detecting the fluorescence emitted from the fluorescent substance. Can be detected.
  • the SPFS method in order to detect with high sensitivity and high accuracy, it is necessary to align the position of the analysis chip with high accuracy. In order to accurately detect the amount (concentration) of the substance to be detected, it is necessary to adjust the incident angle of the excitation light with high accuracy. However, if the position of the analysis chip is shifted, the incident angle of the excitation light Cannot be adjusted with high accuracy. In order to detect a substance to be detected with high sensitivity, it is preferable that the shape and position of the irradiation spot of the excitation light coincide with the shape and position of the reaction field on the metal film, but the position of the analysis chip is shifted. In this case, the shape and position of the irradiation spot of the excitation light cannot be adjusted with high accuracy. On the other hand, requiring the user to align the position of the analysis chip with high accuracy is not preferable from the viewpoint of usability (ease of use).
  • Patent Document 1 discloses that two detection holes are formed in an analysis chip (flow cell) in detection using the SPR method. The user can adjust the position of the analysis chip by using the position confirmation hole.
  • Patent Document 2 in detection using a fluorescent substance, an analysis chip (biochip) is irradiated with illumination light having a wavelength different from that of excitation light, and reflected light or transmitted light of the illumination light is detected and analyzed. It is disclosed to specify the position of the chip. By using illumination light having a wavelength different from that of the excitation light, the position of the analysis chip can be specified while preventing fading of the fluorescent material.
  • Patent Document 1 The alignment method described in Patent Document 1 has a problem that the manufacturing cost of the analysis chip increases because two position confirmation holes must be formed. In addition, there is a problem that usability is poor because the user must perform alignment.
  • Patent Document 2 has a problem that the manufacturing cost of the analyzer increases because a light source different from the excitation light source, a wavelength limiting filter, and the like must be added.
  • An object of the present invention is to provide a surface plasmon resonance fluorescence analysis method, a surface plasmon resonance fluorescence analysis apparatus, and an alignment method capable of aligning an analysis chip with high accuracy while preventing an increase in manufacturing costs of the analysis chip and the analysis device. Is to provide.
  • a surface plasmon resonance fluorescence analysis method is a method in which a fluorescent substance that labels a target substance is emitted by being excited by localized field light based on surface plasmon resonance.
  • a surface plasmon resonance fluorescence analysis method for detecting the presence or amount of the substance to be detected the prism having an entrance surface, an exit surface and a film formation surface, and disposed on the film formation surface, An analysis chip having a capture region on which the capture body for capturing the substance to be detected is immobilized and a metal film including a non-capture region on which the capture body is not immobilized is immobilized on a transport stage.
  • the surface plasmon resonance fluorescence analyzer emits a fluorescent substance that labels a substance to be detected by being excited by localized field light based on surface plasmon resonance.
  • a surface plasmon resonance fluorescence analyzer that detects the presence or amount of the substance to be detected by detecting the detected fluorescence, and is disposed on the film formation surface, a prism having an entrance surface, an exit surface, and a film formation surface And an detachable holding of an analysis chip including a capture region in which a capture body for capturing the substance to be detected is immobilized on the surface thereof and a metal film including a non-capture region in which the capture body is not immobilized
  • An excitation light irradiation unit that irradiates the back surface of the film with excitation light through the incident
  • a position adjusting unit that specifies a position of an end of the capture region of the analysis chip and moves the chip holder by the transfer stage to move the capture region of the analysis chip to a detection position; and the capture A fluorescence detection unit that detects fluorescence emitted from a fluorescent substance that labels the substance to be detected captured by the body.
  • the alignment method uses surface plasmon resonance to detect the presence or amount of a detection target substance contained in a specimen.
  • the present invention it is possible to achieve highly accurate alignment of the analysis chip without bothering the user. Therefore, according to the present invention, highly sensitive and highly accurate detection of a substance to be detected can be realized while preventing an increase in manufacturing cost and a decrease in usability.
  • FIG. 1 is a diagram schematically showing a configuration of an SPFS apparatus according to Embodiment 1 of the present invention.
  • FIG. 2 is a flowchart showing an operation procedure of the SPFS apparatus shown in FIG.
  • FIG. 3 is a flowchart showing the steps in the alignment step (S140) shown in FIG. 4A and 4B are schematic diagrams for explaining a step (S141) of obtaining position information of the end of the capture region in the analysis chip.
  • FIG. 5A is a graph showing an example of the detection result of plasmon scattered light by the light receiving sensor
  • FIG. 5B is a schematic diagram showing the positional relationship between the capture region and the non-capture region, and the irradiation spot.
  • FIG. 6A and 6B are graphs showing examples of detection results of plasmon scattered light by the light receiving sensor.
  • FIG. 7 is a diagram schematically showing the configuration of the SPFS apparatus according to Embodiment 2 of the present invention.
  • 8A and 8B are schematic diagrams for explaining the step (S141) of obtaining position information of the end of the capture region in the analysis chip.
  • FIG. 9A is a graph showing an example of the detection result of the reflected light by the reflected light receiving sensor
  • FIG. 9B is a schematic diagram showing the positional relationship between the capture region and the non-capture region and the irradiation spot.
  • 10A and 10B are graphs illustrating examples of detection results of reflected light by the reflected light receiving sensor.
  • FIG. 1 is a schematic diagram showing a configuration of a surface plasmon resonance fluorescence analyzer (SPFS apparatus) 100 according to Embodiment 1 of the present invention.
  • the SPFS device 100 includes an excitation light irradiation unit 110, a response light detection unit 130 (fluorescence detection unit), a liquid feeding unit 140, a transport unit 150, and a control unit 160.
  • the SPFS apparatus 100 is used with the analysis chip 10 mounted on the chip holder 154 of the transport unit 150. Therefore, the analysis chip 10 will be described first, and then each component of the SPFS device 100 will be described.
  • the analysis chip 10 includes a prism 20 having an incident surface 21, a film formation surface 22 and an emission surface 23, a metal film 30 formed on the film formation surface 22, and a flow disposed on the film formation surface 22 or the metal film 30. And a road lid 40. Usually, the analysis chip 10 is replaced for each analysis.
  • the prism 20 is made of a dielectric that is transparent to the excitation light ⁇ .
  • the prism 20 has an incident surface 21, a film forming surface 22, and an exit surface 23.
  • the incident surface 21 causes the excitation light ⁇ from the excitation light irradiation unit 110 to enter the prism 20.
  • a metal film 30 is disposed on the film formation surface 22.
  • the excitation light ⁇ incident on the inside of the prism 20 is reflected on the back surface of the metal film 30. More specifically, the excitation light ⁇ is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30.
  • the emission surface 23 emits the excitation light ⁇ reflected by the back surface of the metal film 30 to the outside of the prism 20.
  • the shape of the prism 20 is not particularly limited.
  • the shape of the prism 20 is a column having a trapezoidal bottom surface.
  • the surface corresponding to one base of the trapezoid is the film formation surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the emission surface 23.
  • the incident surface 21 is formed so that the excitation light ⁇ does not return to the excitation light irradiation unit 110.
  • the light source of the excitation light ⁇ is a laser diode (hereinafter also referred to as “LD”)
  • LD laser diode
  • the angle of the incident surface 21 is set so that the excitation light ⁇ does not enter the incident surface 21 perpendicularly in the scanning range centered on the ideal enhancement angle.
  • the angle between the incident surface 21 and the film formation surface 22 and the angle between the film formation surface 22 and the emission surface 23 are both about 80 °.
  • the resonance angle (and the enhancement angle near the pole) is generally determined by the design of the analysis chip 10.
  • the design factors are the refractive index of the prism 20, the refractive index of the metal film 30, the film thickness of the metal film 30, the extinction coefficient of the metal film 30, the wavelength of the excitation light ⁇ , and the like.
  • the resonance angle and the enhancement angle are shifted by the detection target substance immobilized on the metal film 30, but the amount is less than several degrees.
  • the prism 20 has a considerable amount of birefringence.
  • Examples of the material of the prism 20 include resin and glass.
  • the material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.
  • the metal film 30 is disposed on the film formation surface 22 of the prism 20.
  • an interaction (surface plasmon resonance) occurs between the photon of the excitation light ⁇ incident on the film formation surface 22 under the total reflection condition and free electrons in the metal film 30, and is locally on the surface of the metal film 30. In-situ light can be generated.
  • the material of the metal film 30 is not particularly limited as long as it is a metal that can cause surface plasmon resonance.
  • Examples of the material of the metal film 30 include gold, silver, copper, aluminum, and alloys thereof.
  • the metal film 30 is a gold thin film.
  • the method for forming the metal film 30 is not particularly limited. Examples of the method for forming the metal film 30 include sputtering, vapor deposition, and plating.
  • the thickness of the metal film 30 is not particularly limited, but is preferably in the range of 30 to 70 nm.
  • the capture region A1 and the non-capture region A2 are disposed on at least a part of the surface of the metal film 30 that does not face the prism 20 (the front surface of the metal film 30) (see FIG. 5B).
  • a capturing body for capturing the substance to be detected is immobilized in the capturing region A1. By immobilizing the capturing body, it becomes possible to selectively detect the substance to be detected.
  • the plan view shape of the capture region A1 is not particularly limited. Examples of the shape of the capture region A1 in plan view include a circle and a polygon. In the present embodiment, the capture region A1 has a circular shape in plan view (see FIG. 5B).
  • the type of capturing body is not particularly limited as long as it can capture the substance to be detected.
  • the capturing body is an antibody specific to the substance to be detected or a fragment thereof.
  • the non-capturing region A2 is a region other than the capturing region A1 where the capturing body is not immobilized.
  • the non-capturing region A ⁇ b> 2 may be the metal film 30 itself, or may have a blocking layer that prevents the detection target substance from being adsorbed on the surface of the metal film 30.
  • the flow path lid 40 is disposed on the metal film 30.
  • the flow path lid 40 may be disposed on the film formation surface 22.
  • a channel groove is formed on the back surface of the channel lid 40, and the channel lid 40 forms a channel 41 through which a liquid flows together with the metal film 30 (and the prism 20).
  • the liquid include a sample solution containing a substance to be detected, a labeled solution containing an antibody labeled with a fluorescent material, and a washing solution.
  • the capture region A1 and the non-capture region A2 of the metal film 30 are exposed in the flow channel 41. Both ends of the channel 41 are respectively connected to an inlet and an outlet (not shown) formed on the upper surface of the channel lid 40.
  • the channel lid 40 is preferably made of a material that is transparent to the fluorescence ⁇ and plasmon scattered light ⁇ emitted from the metal film 30.
  • An example of the material of the flow path lid 40 includes a resin.
  • the other portion of the channel lid 40 may be formed of an opaque material.
  • the flow path lid 40 is bonded to the metal film 30 or the prism 20 by, for example, adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.
  • the excitation light ⁇ enters the prism 20 from the incident surface 21.
  • the excitation light ⁇ incident on the prism 20 is incident on the metal film 30 at a total reflection angle (an angle at which surface plasmon resonance occurs).
  • localized field light (generally also referred to as “evanescent light” or “near field light”) is applied to the metal film 30.
  • This localized field light excites a fluorescent substance that labels the substance to be detected present on the metal film 30 and emits fluorescence ⁇ .
  • the SPFS device 100 detects the presence or amount of the substance to be detected by detecting the amount of fluorescent ⁇ emitted from the fluorescent substance.
  • the SPFS device 100 includes the excitation light irradiation unit 110, the response light detection unit 130, the liquid feeding unit 140, the transport unit 150, and the control unit 160.
  • the excitation light irradiation unit 110 emits excitation light ⁇ to the analysis chip 10 (the back surface of the metal film 30) held by the chip holder 154.
  • the excitation light irradiation unit 110 emits only the P wave toward the incident surface 21 so that the incident angle with respect to the metal film 30 becomes an angle that causes surface plasmon resonance.
  • the “excitation light” is light that directly or indirectly excites the fluorescent material.
  • the excitation light ⁇ is light that generates localized field light on the surface of the metal film 30 that excites the fluorescent material when the metal film 30 is irradiated through the prism 20 at an angle at which surface plasmon resonance occurs. is there.
  • the excitation light ⁇ is also used for positioning the analysis chip 10.
  • the excitation light irradiation unit 110 includes a configuration for emitting the excitation light ⁇ toward the prism 20 and a configuration for changing the incident angle of the excitation light ⁇ with respect to the back surface of the metal film 30.
  • the excitation light irradiation unit 110 includes a light source unit 111, a first angle adjustment mechanism 112, and a light source control unit 113.
  • the light source unit 111 emits the collimated excitation light ⁇ having a constant wavelength and light amount so that the shape of the irradiation spot S (see FIG. 5B) on the back surface of the metal film 30 is substantially circular.
  • the light source unit 111 includes, for example, a light source of excitation light ⁇ , a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).
  • the type of the light source is not particularly limited, and is, for example, a laser diode (LD).
  • Other examples of light sources include light emitting diodes, mercury lamps, and other laser light sources.
  • the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like.
  • the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like.
  • the light emitted from the light source is not linearly polarized light, the light emitted from the light source is converted into linearly polarized light by a polarizer or the like.
  • the beam shaping optical system includes, for example, a collimator, a band pass filter, a linear polarization filter, a half-wave plate, a slit, and a zoom means.
  • the beam shaping optical system may include all of these or a part thereof.
  • the collimator collimates the excitation light ⁇ emitted from the light source.
  • the band-pass filter turns the excitation light ⁇ emitted from the light source into narrowband light having only the center wavelength. This is because the excitation light ⁇ from the light source has a slight wavelength distribution width.
  • the linear polarization filter turns the excitation light ⁇ emitted from the light source into completely linearly polarized light.
  • the half-wave plate adjusts the polarization direction of the excitation light ⁇ so that the P-wave component is incident on the metal film 30.
  • the slit and zoom means adjust the beam diameter, contour shape, and the like of the excitation light ⁇ so that the shape of the irradiation spot S on the back surface of the metal film 30 is a circle of a predetermined size.
  • the APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light ⁇ with a photodiode (not shown) or the like. The APC mechanism controls the input energy by a regression circuit, thereby controlling the output of the light source to be constant.
  • the temperature adjustment mechanism is, for example, a heater or a Peltier element.
  • the wavelength and energy of the light emitted from the light source may vary depending on the temperature. For this reason, the wavelength and energy of the light emitted from the light source are controlled to be constant by keeping the temperature of the light source constant by the temperature adjusting mechanism.
  • the first angle adjusting mechanism 112 adjusts the incident angle of the excitation light ⁇ to the back surface of the metal film 30 (the interface between the prism 20 and the metal film 30 (film formation surface 22)).
  • the first angle adjusting mechanism 112 irradiates the excitation light ⁇ at a predetermined incident angle toward a predetermined position on the back surface of the metal film 30 via the prism 20 with the optical axis of the excitation light ⁇ and the chip holder 154. Rotate relatively.
  • the first angle adjusting mechanism 112 rotates the light source unit 111 around an axis orthogonal to the optical axis of the excitation light ⁇ (an axis perpendicular to the paper surface of FIG. 1).
  • the position of the rotation axis is set so that the position of the irradiation spot on the metal film 30 hardly changes even if the incident angle is changed.
  • the angle at which the maximum amount of plasmon scattered light ⁇ can be obtained is the enhancement angle.
  • the basic incident condition of the excitation light ⁇ is determined by the material and shape of the prism 20 of the analysis chip 10, the film thickness of the metal film 30, the refractive index of the liquid in the flow channel 41, etc.
  • the optimal incident condition varies slightly depending on the type and amount of the fluorescent material and the shape error of the prism 20. For this reason, it is preferable to obtain an optimal enhancement angle for each measurement.
  • a suitable emission angle of the excitation light ⁇ with respect to the normal line of the metal film 30 is about 70 °.
  • the light source control unit 113 controls various devices included in the light source unit 111 to control the emission of the emitted light (excitation light ⁇ ) of the light source unit 111.
  • the light source control unit 113 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • the response light detection unit 130 detects the fluorescence ⁇ generated by the irradiation of the excitation light ⁇ on the back surface of the metal film 30 when detecting the detection target substance, and the back surface of the metal film 30 when positioning the analysis chip 10 and measuring the enhancement angle.
  • the plasmon scattered light ⁇ generated by the irradiation of the excitation light ⁇ is detected.
  • the response light detection unit 130 includes, for example, a light receiving unit 131, a position switching mechanism 132, and a sensor control unit 133.
  • the light receiving unit 131 is arranged in the normal direction of the metal film 30 of the analysis chip 10 (z-axis direction in FIG. 1).
  • the light receiving unit 131 includes a first lens 134, an optical filter 135, a second lens 136, and a light receiving sensor 137.
  • the first lens 134 is, for example, a condensing lens, and condenses light emitted from the metal film 30.
  • the second lens 136 is an imaging lens, for example, and forms an image of the light collected by the first lens 134 on the light receiving surface of the light receiving sensor 137.
  • the optical path between both lenses is a substantially parallel optical path.
  • the optical filter 135 is disposed between both lenses.
  • the optical filter 135 guides only the fluorescence component to the light receiving sensor 137 and removes the excitation light component (plasmon scattered light ⁇ ) in order to detect the fluorescence ⁇ with a high S / N ratio.
  • the optical filter 135 include an excitation light reflection filter, a short wavelength cut filter, and a band pass filter.
  • the optical filter 135 is, for example, a filter including a multilayer film that reflects a predetermined light component, but may be a colored glass filter that absorbs the predetermined light component.
  • the light receiving sensor 137 detects fluorescence ⁇ or plasmon scattered light ⁇ .
  • the light receiving sensor 137 has high sensitivity capable of detecting weak fluorescence ⁇ or plasmon scattered light ⁇ from a minute amount of a substance to be detected.
  • the light receiving sensor 137 is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).
  • the position switching mechanism 132 switches the position of the optical filter 135 on or off the optical path in the light receiving unit 131. Specifically, when the light receiving sensor 137 detects the fluorescence ⁇ , the optical filter 135 is disposed on the optical path of the light receiving unit 131, and when the light receiving sensor 137 detects the plasmon scattered light ⁇ , the optical filter 135 is inserted into the light receiving unit 131. Placed outside the optical path.
  • the position switching mechanism 132 includes, for example, a rotation driving unit and a known mechanism (such as a turntable or a rack and pinion) that moves the optical filter 135 in the horizontal direction by using a rotational motion.
  • the sensor control unit 133 controls detection of an output value of the light receiving sensor 137, management of sensitivity of the light receiving sensor 137 based on the detected output value, change of sensitivity of the light receiving sensor 137 for obtaining an appropriate output value, and the like.
  • the sensor control unit 133 is configured by, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • the liquid feeding unit 140 supplies a sample liquid, a labeling liquid, a cleaning liquid, and the like into the flow path 41 of the analysis chip 10 held by the chip holder 154.
  • the liquid feeding unit 140 includes a chemical liquid chip 141, a syringe pump 142, and a liquid feeding pump drive mechanism 143.
  • the chemical solution chip 141 is a container for storing a liquid such as a sample solution, a labeling solution, or a cleaning solution.
  • a liquid such as a sample solution, a labeling solution, or a cleaning solution.
  • a plurality of containers are usually arranged according to the type of liquid, or a chip in which a plurality of containers are integrated is arranged.
  • the syringe pump 142 includes a syringe 144 and a plunger 145 that can reciprocate inside the syringe 144.
  • the plunger 145 By the reciprocating motion of the plunger 145, the liquid is sucked and discharged quantitatively. If the syringe 144 can be replaced, the syringe 144 need not be cleaned. For this reason, it is preferable from the viewpoint of preventing contamination of impurities.
  • the syringe 144 is not configured to be replaceable, it is possible to use the syringe 144 without replacing it by adding a configuration for cleaning the inside of the syringe 144.
  • the liquid feed pump driving mechanism 143 includes a driving device for the plunger 145 and a moving device for the syringe pump 142.
  • the drive device of the syringe pump 142 is a device for reciprocating the plunger 145, and includes, for example, a stepping motor.
  • the drive device including the stepping motor is preferable from the viewpoint of managing the remaining liquid amount of the analysis chip 10 because it can manage the liquid feeding amount and the liquid feeding speed of the syringe pump 142.
  • the moving device of the syringe pump 142 freely moves the syringe pump 142 in two directions, ie, an axial direction (for example, a vertical direction) of the syringe 144 and a direction crossing the axial direction (for example, a horizontal direction).
  • the moving device of the syringe pump 142 is configured by, for example, a robot arm, a two-axis stage, or a turntable that can move up and down.
  • the liquid feeding unit 140 determines the position of the tip of the syringe 144. It is preferable to further have a device for detecting.
  • the liquid feeding unit 140 sucks various liquids from the chemical liquid chip 141 and supplies them to the flow path 41 of the analysis chip 10. At this time, by moving the plunger 145, the liquid reciprocates in the flow path 41 in the analysis chip 10, and the liquid in the flow path 41 is stirred. As a result, it is possible to achieve a uniform concentration of the liquid and promotion of a reaction (for example, an antigen-antibody reaction) in the flow channel 41. From the viewpoint of performing such an operation, the analysis chip 10 and the syringe 144 are protected by a multilayer film, and the analysis chip 10 and the syringe 144 can be sealed when the syringe 144 penetrates the multilayer film. It is preferable to be configured.
  • the liquid in the channel 41 is again sucked by the syringe pump 142 and discharged to the chemical liquid chip 141 and the like.
  • reaction with various liquids, washing, and the like can be performed, and the detection target substance labeled with the fluorescent substance can be arranged in the capture region A1 in the flow path 41.
  • the transport unit 150 transports and fixes the analysis chip 10 to the measurement position or the liquid feeding position.
  • the “measurement position” is a position where the excitation light irradiation unit 110 irradiates the analysis chip 10 with the excitation light ⁇ , and the response light detection unit 130 detects the fluorescence ⁇ or the plasmon scattered light ⁇ generated accordingly.
  • the “liquid feeding position” is a position where the liquid feeding unit 140 supplies a liquid into the flow channel 41 of the analysis chip 10 or removes the liquid in the flow channel 41 of the analysis chip 10.
  • the transfer unit 150 includes a transfer stage 152 and a chip holder 154. The chip holder 154 is fixed to the transfer stage 152 and holds the analysis chip 10 in a detachable manner.
  • the shape of the chip holder 154 is a shape that can hold the analysis chip 10 and does not obstruct the optical path of the excitation light ⁇ .
  • the chip holder 154 is provided with an opening through which the excitation light ⁇ passes.
  • the transfer stage 152 moves the chip holder 154 in one direction (x-axis direction in FIG. 1) and in the opposite direction.
  • the transfer stage 152 is driven by, for example, a stepping motor.
  • the control unit 160 controls the first angle adjustment mechanism 112, the light source control unit 113, the position switching mechanism 132, the sensor control unit 133, the liquid feed pump drive mechanism 143, and the transport stage 152. Further, the control unit 160 specifies the position of the end of the capture region A1 in the analysis chip 10 held by the chip holder 154 based on the detection result of the response light detection unit 130, and uses the transfer stage 152 to insert the chip holder 154. To move the capture region A1 of the analysis chip 10 to an appropriate measurement position.
  • the control unit 160 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • FIG. 2 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 100.
  • 3 to 5 are diagrams for explaining the alignment process.
  • FIG. 3 is a flowchart showing the steps in the alignment step (S140) shown in FIG.
  • the analysis chip 10 is installed in the chip holder 154 of the SPFS device 100 (S100).
  • the control unit 160 operates the transfer stage 152 to move the analysis chip 10 to the liquid feeding position (S110).
  • control unit 160 operates the liquid feeding unit 140 to introduce the sample liquid in the chemical liquid chip 141 into the flow path 41 of the analysis chip 10 (S120).
  • the substance to be detected is captured on the metal film 30 by the antigen-antibody reaction (primary reaction).
  • the sample liquid in the flow path 41 is removed, and the flow path 41 is cleaned with a cleaning liquid.
  • the humectant is washed by washing the flow channel 41 before introducing the sample solution so that the capturing body can appropriately capture the substance to be detected. Remove.
  • control unit 160 operates the transfer stage 152 to move the analysis chip 10 to the vicinity of the measurement position (S130).
  • control unit 160 operates the excitation light irradiation unit 110, the response light detection unit 130, and the transport stage 152 to obtain the position information of the end of the capture area A1, and capture area based on the obtained position information.
  • the position of A1 (analysis chip 10) is adjusted (S140). As shown in FIG. 3, in this step, first, the analysis chip 10 held by the chip holder 154 is directly below the capture region A1 and the non-capture region A2, and has the same shape as the capture region A1 and the non-capture region A2.
  • FIG. 4A is a schematic diagram for explaining the step (S141) of obtaining position information of the end of the capture region A1
  • FIG. 4B shows the relationship between the incident angle of the excitation light ⁇ and the light amount of the plasmon scattered light ⁇ .
  • the fluctuation of the light quantity of the plasmon scattered light ⁇ with respect to is shown.
  • FIG. 5A is a graph schematically showing the relationship between the movement distance of the irradiation spot S of the excitation light ⁇ and the amount of plasmon scattered light ⁇
  • FIG. 5B shows the capture region A1 and the non-capture region A2, and the irradiation It is a schematic diagram which shows the positional relationship with the spot S.
  • the dependence characteristic of the excitation light ⁇ on the incident angle is used. That is, in a state where the incident angle of the excitation light ⁇ is fixed, the irradiation spot S of the excitation light ⁇ is scanned on the back surface of the metal film 30, and the position of the end of the capture region A1 is detected by the fluctuation of the plasmon scattered light ⁇ . To do.
  • the excitation stage ⁇ is moved by driving the transport stage 152 and moving the analysis chip 10.
  • the irradiation spot is scanned on the back surface of the metal film 30.
  • the incident angle of the excitation light ⁇ is such that the amount of plasmon scattered light ⁇ emitted from the capture region A1 and the amount of plasmon scattered light ⁇ emitted from the non-capture region A2 are not the same amount (FIG. 4B). (Incident angle at a position where L1 and L2 do not intersect).
  • the incident angle of the excitation light ⁇ is an enhancement angle in the capture region A1 described later or an enhancement angle in the non-capture region A2.
  • the fluctuation of the amount of plasmon scattered light ⁇ with respect to the incident angle of the excitation light ⁇ as shown in FIG. 4B is caused by the excitation light ⁇ on any part of the back surface of the metal film 30 corresponding to the capture region A1 and the non-capture region A2. Can also be obtained by irradiation.
  • the back surface of the metal film 30 corresponding to the capture region A1 and the back surface of the metal film 30 corresponding to the non-capture region A2 are irradiated with the excitation light ⁇ at the same incident angle. Then, the amount of plasmon scattered light ⁇ emitted from the capture region A1 and the non-capture region A2 is different (see FIG. 4B).
  • the point (point b) at which the amount of plasmon scattered light ⁇ begins to increase in FIG. 5A is the point where the tip in the scanning direction of the irradiation spot S reaches the boundary between the capture region A1 and the non-capture region A2 in FIG. I understand that.
  • the amount of plasmon scattered light ⁇ incident on the light receiving sensor 137 gradually increases (points b to c in FIG. 5A). This is because, in the irradiation spot S, the ratio of the portion corresponding to the capture region A1 gradually increases with respect to the portion corresponding to the non-capture region A2. That is, on the back surface of the metal film 30, it can be seen that the irradiation spot S has shifted from the portion corresponding to the non-capturing region A2 to the portion corresponding to the capturing region A1.
  • the amount of plasmon scattered light ⁇ incident on the light receiving sensor 137 becomes constant (points c to d in FIG. 5A). This is because all of the irradiation spot S is located within the range corresponding to the capture region A1.
  • the amount of plasmon scattered light ⁇ incident on the light receiving sensor 137 gradually decreases (points d to e in FIG. 5A). This is because, in the irradiation spot S, the ratio of the portion corresponding to the capture region A1 gradually decreases with respect to the portion corresponding to the non-capture region A2. That is, on the back surface of the metal film 30, it can be seen that the irradiation spot S has shifted from the portion corresponding to the capture region A1 to the portion corresponding to the non-capture region A2.
  • any one of b, c, d, and e may be used to specify the position of the end of the capture region A1.
  • b and d indicate points where the tip of the irradiation spot S in the scanning direction has reached the end of the capture region A1. Therefore, if the irradiation spot S diameter of the excitation light ⁇ is taken into consideration, the position of the end of the capture region A1 can be specified, and as a result, the center position of the capture region A1 can be specified.
  • C and e indicate points where the rear end in the scanning direction of the irradiation spot S of the excitation light ⁇ has reached the end of the capture region A1. Therefore, if the irradiation spot S diameter of the excitation light ⁇ is taken into consideration, the position of the end of the capture region A1 can be specified, and as a result, the center position of the capture region A1 can be specified.
  • the position of the end of the capture region A1 can be specified without considering the irradiation spot S diameter of the excitation light ⁇ , and the result
  • the center position of the capture region A1 can be specified as In this case, from the viewpoint of suppressing the influence of the irradiation spot S diameter of the excitation light ⁇ , it is preferable to specify the end of the capture region A1 using the intermediate value of the light amount of the plasmon scattered light ⁇ .
  • the back surface of the analysis chip 10 is irradiated with the excitation light ⁇ , and the plasmon scattered light ⁇ emitted from the capture region A1 and the non-capture region A2 is detected, whereby the position of the capture region A1 in the analysis chip 10 is determined. Can be identified.
  • the control unit 160 operates the excitation light irradiation unit 110 and the response light detection unit 130 to irradiate the analysis chip 10 disposed at an appropriate measurement position with the excitation light ⁇ and has the same wavelength as the excitation light ⁇ .
  • Plasmon scattered light ⁇ is detected to detect the enhancement angle (S150).
  • the controller 160 operates the response light detection unit 130 to detect the plasmon scattered light ⁇ while operating the excitation light irradiation unit 110 to scan the incident angle of the excitation light ⁇ with respect to the metal film 30. .
  • control unit 160 operates the position switching mechanism 132 to arrange the optical filter 135 outside the optical path of the light receiving unit 131. And the control part 160 determines the incident angle of the excitation light (alpha) when the light quantity of the plasmon scattered light (gamma) is the maximum as an enhancement angle.
  • control unit 160 operates the excitation light irradiation unit 110 and the response light detection unit 130 to irradiate the analysis chip 10 disposed at an appropriate measurement position with the excitation light ⁇ , and outputs the output value ( Optical blank value) is recorded (S160).
  • the controller 160 operates the first angle adjustment mechanism 112 to set the incident angle of the excitation light ⁇ to the enhancement angle.
  • control unit 160 controls the position switching mechanism 132 to arrange the optical filter 135 in the optical path of the light receiving unit 131.
  • control unit 160 operates the transfer stage 152 to move the analysis chip 10 to the liquid feeding position (S170).
  • the controller 160 operates the liquid feeding unit 140 to introduce a liquid (labeled liquid) containing a secondary antibody labeled with a fluorescent substance into the flow channel 41 of the analysis chip 10 (S180).
  • a liquid labeled liquid
  • the detection target substance captured on the metal film 30 is labeled with a fluorescent substance by an antigen-antibody reaction (secondary reaction).
  • secondary reaction antigen-antibody reaction
  • control unit 160 operates the transport stage 152 to move the analysis chip 10 to the appropriate measurement position determined in S140 (S190).
  • control unit 160 operates the excitation light irradiation unit 110 and the response light detection unit 130 to irradiate the analysis chip 10 disposed at an appropriate measurement position with the excitation light ⁇ and is captured by the capturing body.
  • Fluorescent ⁇ released from the fluorescent substance that labels the substance to be detected is detected (S200).
  • the control unit 160 subtracts the optical blank value from the detection value, and calculates the fluorescence intensity that correlates with the amount of the substance to be detected.
  • the detected fluorescence intensity is converted into the amount and concentration of the substance to be detected as necessary.
  • the detection of the enhancement angle (S150) may be performed before the primary reaction (S120). In this case, determination of the measurement position of the analysis chip 10 (S130 and S140) is performed before the primary reaction (S110 and S120). When the incident angle of the excitation light ⁇ is determined in advance, the detection of the enhancement angle (S150) may be omitted. Also in this case, the determination of the measurement position of the analysis chip 10 (S130 and S140) is performed before the measurement of the optical blank value (S160). Thus, the determination of the measurement position of the analysis chip 10 (S130 and S140) is preferably performed before the first optical measurement (detection of an enhancement angle, measurement of an optical blank value, or detection of fluorescence).
  • the step of labeling the detected substance with a fluorescent substance is performed (2 Process method).
  • the timing for labeling the substance to be detected with a fluorescent substance is not particularly limited.
  • a labeling solution may be added to the sample solution to label the target substance with a fluorescent substance in advance.
  • the sample solution and the labeling solution may be simultaneously injected into the flow channel 41 of the analysis chip 10. In the former case, by injecting the sample solution into the flow channel 41 of the analysis chip 10, the target substance labeled with the fluorescent substance is captured by the capturing body.
  • both the primary reaction and the secondary reaction can be completed by introducing the sample solution into the flow channel 41 of the analysis chip 10 (one-step method).
  • the detection of the enhancement angle (S150) is performed before the antigen-antibody reaction, and further, the measurement position of the analysis chip 10 is determined (S130 and S140).
  • the timing of performing the alignment step (S140) may not be before the primary reaction (S120) as long as it is before the fluorescence emitted from the fluorescent substance labeled with the substance to be detected is detected.
  • the alignment step (S140) may be performed after the primary reaction (S120), or after the primary reaction (S120) and before the secondary reaction (S180).
  • the incident angle of the excitation light ⁇ may be an enhancement angle (see P1 in FIG. 4B) in the capture region A1 where the amount of detected plasmon scattered light ⁇ is maximum.
  • the enhancement angle here is not a precise enhancement angle but a theoretical angle determined by the design of the analysis chip 10.
  • FIG. 6A when the enhancement angle of the excitation light is the enhancement angle in the capture region A1, it can be seen that the amount of fluctuation of the plasmon scattered light ⁇ is larger than when the incident angle is an arbitrary angle. (See FIGS. 5B and 6A). Therefore, in the step of acquiring the position information of the end portion of the capture region A1, the end portion of the capture region A1 can be detected more accurately.
  • the incident angle of the excitation light ⁇ may be an enhancement angle (see P2 in FIG. 4B) in the non-capturing region A2 where the amount of the detected plasmon scattered light ⁇ is maximum.
  • the amount of plasmon scattered light ⁇ received by the light receiving sensor 137 is increased.
  • the back surface of the metal film 30 corresponding to the capture region A1 and the back surface of the metal film 30 corresponding to the non-capture region A2 are irradiated with the excitation light ⁇ at the same incident angle. Then, the amount of plasmon scattered light ⁇ emitted from the capture region A1 and the non-capture region A2 is different (see FIG. 4B). Therefore, it can be seen that the point where the amount of plasmon scattered light ⁇ starts to increase corresponds to the boundary between the capture region A1 and the non-capture region A2.
  • the amount of plasmon scattered light ⁇ incident on the light receiving sensor 137 gradually decreases (points b to c in FIG. 6B). This is because, in the irradiation spot S, the ratio of the portion corresponding to the capture region A1 gradually increases with respect to the portion corresponding to the non-capture region A2. That is, on the back surface of the metal film 30, it can be seen that the irradiation spot S has shifted to the portion corresponding to the capture region A1 because it corresponds to the non-capture region A2.
  • the amount of plasmon scattered light ⁇ incident on the light receiving sensor 137 becomes constant (points c to d in FIG. 6B). This is because all of the irradiation spot S is located within the range corresponding to the capture region A1.
  • the irradiation spot S when the irradiation spot S is continuously scanned as it is, the amount of the plasmon scattered light ⁇ incident on the light receiving sensor 137 increases (points d to e in FIG. 6B). This is because, in the irradiation spot S, the ratio of the portion corresponding to the capture region A1 gradually decreases with respect to the portion corresponding to the non-capture region A2. That is, it can be seen that, on the back surface of the metal film 30, the irradiation spot S has shifted to the portion corresponding to the non-capturing region A2 because it corresponds to the capturing region A1.
  • the enhancement angle of the excitation light is the enhancement angle in the trapping region A1
  • the amount of fluctuation of the plasmon scattered light ⁇ is larger than when the incident angle is an arbitrary angle (FIGS. 5A and 6B). reference). Therefore, in this case as well, the end of the capture region A1 can be detected more accurately in the step of acquiring the position information of the end of the capture region A1.
  • the SPFS device 200 according to the second embodiment is different from the SPFS device 100 according to the first embodiment in that it includes a reflected light detection unit 270 that detects the reflected light ⁇ of the excitation light ⁇ . Therefore, the same constituent elements as those of the SPFS apparatus 100 according to Embodiment 1 are denoted by the same reference numerals, and description thereof is omitted.
  • FIG. 7 is a diagram illustrating a configuration of the SPFS apparatus 200 according to the second embodiment.
  • the SPFS device 200 according to the second embodiment includes a reflected light detection unit in addition to the excitation light irradiation unit 110, the response light detection unit 130, the liquid feeding unit 140, the transport unit 150, and the control unit 160. 270.
  • the reflected light detection unit 270 is disposed on the opposite side of the excitation light irradiation unit 110 across the normal to the metal film 30 passing through the irradiation spot S.
  • the reflected light detection unit 270 includes a reflected light detection sensor 271 and a second angle adjustment mechanism 272.
  • the reflected light detection sensor 271 detects the reflected light ⁇ of the excitation light ⁇ .
  • the type of the reflected light detection sensor 271 is not particularly limited as long as the reflected light ⁇ can be detected.
  • the reflected light detection sensor 271 is a photodiode (PD).
  • the size of the light receiving surface of the reflected light detection sensor 271 is preferably larger than the beam diameter of the reflected light ⁇ .
  • the reflected light detection sensor 271 is disposed at a position where the reflected light ⁇ of the excitation light ⁇ is incident.
  • the second angle adjustment mechanism 272 is configured in the same manner as the first angle adjustment mechanism 112, and the angle of the light receiving surface of the reflected light detection sensor 271 is interlocked with the first angle adjustment mechanism 112 according to the angle of the reflected light ⁇ . Adjust.
  • the detection operation of the SPFS device 200 (a surface plasmon resonance fluorescence analysis method according to an embodiment of the present invention and an alignment method according to an embodiment of the present invention) will be described. Note that the detection operation of the SPFS device 200 according to the second embodiment is the same as that of the first embodiment except for the alignment step, and therefore only the alignment step will be described in the present embodiment.
  • FIG. 8 is a schematic diagram for explaining a process of obtaining position information of the end of the capture region A1.
  • FIG. 8A is a schematic diagram for explaining the process of obtaining the position information of the end of the capture region A1
  • FIG. 8B schematically shows the relationship between the incident angle of the excitation light ⁇ and the light quantity of the reflected light ⁇ . It is the shown graph.
  • the solid line (L3) in FIG. 8B shows the variation in the amount of reflected light ⁇ with respect to the incident angle of the excitation light ⁇ in the capture region A1
  • the broken line (L4) shows the change in the incident angle of the excitation light ⁇ in the non-capture region A2. The fluctuation of the amount of reflected light ⁇ is shown.
  • FIG. 8A is a schematic diagram for explaining the process of obtaining the position information of the end of the capture region A1
  • FIG. 8B schematically shows the relationship between the incident angle of the excitation light ⁇ and the light quantity of the reflected light ⁇ . It is the
  • FIG. 9A is a graph schematically showing the relationship between the moving distance of the irradiation spot S of the excitation light ⁇ and the light amount of the reflected light ⁇
  • FIG. 9B shows the capturing area A1, the non-capturing area A2, and the irradiation spot. It is a schematic diagram which shows the positional relationship with S. Note that points a to f indicated by arrows in FIG. 9A correspond to a to f shown in FIG. 9B.
  • the incident of reflected light ⁇ obtained by scanning the back surface of the metal film 30 with the irradiation spot S of the excitation light ⁇ in a state where the incident angle of the excitation light ⁇ is fixed Take advantage of angle dependence. That is, in a state where the incident angle of the excitation light ⁇ is fixed, the irradiation spot S of the excitation light ⁇ is scanned on the back surface of the metal film 30, and the end of the capture region A1 is detected by the fluctuation of the reflected light ⁇ . Specifically, as shown in FIG.
  • the transport stage 152 is driven and the analysis chip 10 is moved.
  • the incident angle of the excitation light ⁇ is such that the amount of reflected light emitted from the capture region A1 and the amount of reflected light emitted from the non-capture region are not the same amount (L3 and L4 in FIG. (Incident angle at a position not intersecting).
  • the incident angle of the excitation light ⁇ is a resonance angle in the capture region A1 described later or a resonance angle in the non-capture region A2.
  • the amount of reflected light ⁇ emitted from the emission surface 23 is different (see FIG. 8B).
  • This case is an example when excitation light is incident at an incident angle of P3 in FIG. 8B. Therefore, it can be seen that the point at which the amount of the reflected light ⁇ starts to fall corresponds to the boundary between the capture region A1 and the non-capture region A2.
  • the amount of reflected light ⁇ incident on the reflected light detection sensor 271 gradually decreases. This is because, in the irradiation spot S, the ratio of the portion corresponding to the capture region A1 gradually increases with respect to the portion corresponding to the non-capture region A2. That is, on the back surface of the metal film 30, it can be seen that the irradiation spot S is in the process of moving from the portion corresponding to the non-capturing region A2 to the portion corresponding to the capturing region A1.
  • the amount of the reflected light ⁇ incident on the reflected light detection sensor 271 becomes constant. This is because all of the irradiation spot S is located within the range corresponding to the capture region A1.
  • the amount of the reflected light ⁇ incident on the reflected light detection sensor 271 gradually increases. This is because, in the irradiation spot S, the ratio of the portion corresponding to the capture region A1 gradually decreases with respect to the portion corresponding to the non-capture region A2. That is, on the back surface of the metal film 30, it can be seen that the irradiation spot S has shifted from the portion corresponding to the capture region A1 to the portion corresponding to the non-capture region A2.
  • any one of the points b to e and b to e can be used as in the first embodiment. Good.
  • the position of the end of the capture region A1 in the analysis chip 10 can be specified by irradiating the back surface of the analysis chip 10 with the excitation light ⁇ and detecting the reflected light ⁇ emitted from the emission surface 23. it can.
  • the incident angle of the excitation light ⁇ may be a resonance angle (see P3 in FIG. 8B) in the capture region A1 where the amount of the detected reflected light ⁇ is minimized.
  • the resonance angle here is not an accurate resonance angle but a theoretical angle determined by the design of the analysis chip 10.
  • FIG. 10A when the incident angle of the excitation light is the resonance angle in the capture region A1, it can be seen that the amount of reflected light ⁇ varies more greatly than when the incident angle is an arbitrary angle. (See FIGS. 9A and 10A). Therefore, in the step of acquiring the position information of the end portion of the capture region A1, the end portion of the capture region A1 can be detected more accurately.
  • the incident angle of the excitation light ⁇ may be a resonance angle (see P4 in FIG. 8B) in the non-capturing region A2 where the amount of the detected reflected light ⁇ is minimized.
  • the resonance angle of the excitation light is the resonance angle in the non-trapping region A2
  • the variation in the amount of reflected light ⁇ is larger than when the incident angle is an arbitrary angle. (See FIGS. 9A and 10B). Therefore, in this case as well, the end of the capture region A1 can be detected more accurately in the step of acquiring the position information of the end of the capture region A1.
  • the SPFS apparatuses 100 and 200 in which the transfer stage 152 moves only in the X direction in FIGS. 1 and 7 have been described.
  • the transfer stage has an X-direction moving mechanism that moves the chip holder 154 in the X direction and a Y-direction moving mechanism that moves the chip holder 154 in the Y direction.
  • the irradiation spot S can be scanned in a plurality of directions, so that the detection accuracy of the end of the capture region A1 can be further increased.
  • the SPFS device has been described.
  • the method for aligning the analysis chip 10 according to the present invention can be applied to an analysis device other than the SPFS device such as an SPR device. Note that. When applied to the SPR device, the reflected light detection unit 270 is unnecessary, and therefore the analysis chip 10 can be aligned without changing the device configuration.
  • the surface plasmon resonance fluorescence analysis method, the surface plasmon resonance fluorescence analysis apparatus, and the analysis chip positioning method according to the present invention can detect a substance to be detected with high reliability, and are useful for clinical examinations, for example.

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Abstract

La présente invention concerne un procédé d'analyse de fluorescence par résonance plasmonique de surface, dans lequel une puce d'analyse ayant un prisme et un film métallique comprenant une région de capture sur laquelle un corps de capture pour capturer une substance à détecter est fixé et une région non-capture sur laquelle le corps de capture n'est pas fixé est placée dans un support de puce. Ensuite, une lumière d'excitation est irradiée sur la surface arrière du film métallique correspondant à la région de capture et à la région non-capture de la puce d'analyse placée dans le support de puce, la lumière diffusée plasmonique émise depuis la région de capture et la région non-capture ou la lumière d'excitation réfléchie est détectée, et des informations de position pour la partie d'extrémité de la région de capture sont ainsi obtenues. De plus, le support de puce est déplacé sur la base des informations de position, et la région de capture est déplacée à une position de détection. Finalement, la lumière d'excitation est irradiée sur la surface arrière du film métallique, et la fluorescence émise par une substance récente est détectée.
PCT/JP2015/060042 2014-03-31 2015-03-31 Procédé d'analyse de fluorescence par résonance plasmonique de surface, dispositif d'analyse de fluorescence par résonance plasmonique de surface, et procédé d'alignement WO2015152198A1 (fr)

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