US20180313756A1 - Method for surface plasmon resonance fluorescence analysis and device for surface plasmon resonance fluorescence analysis - Google Patents

Method for surface plasmon resonance fluorescence analysis and device for surface plasmon resonance fluorescence analysis Download PDF

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Publication number
US20180313756A1
US20180313756A1 US15/771,879 US201615771879A US2018313756A1 US 20180313756 A1 US20180313756 A1 US 20180313756A1 US 201615771879 A US201615771879 A US 201615771879A US 2018313756 A1 US2018313756 A1 US 2018313756A1
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Prior art keywords
excitation light
analysis chip
light
fluorescence
chip
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English (en)
Inventor
Nobuhiro Yamauchi
Yuichi KYOGOKU
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Konica Minolta Inc
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Konica Minolta Inc
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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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/13—Moving of cuvettes or solid samples to or from the investigating station
    • 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
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • 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
    • 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
    • G01N21/84—Systems specially adapted for particular applications
    • G01N2021/845—Objects on a conveyor
    • G01N2021/8455—Objects on a conveyor and using position detectors

Definitions

  • the present invention relates to a surface plasmon resonance fluorescence analysis method and a surface plasmon resonance fluorescence analysis apparatus that detect a detection object substance in a sample solution with use of surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the SPFS uses a prism including a metal film disposed on a predetermined surface.
  • a prism including a metal film disposed on a predetermined surface.
  • excitation light from an excitation light irradiating section through the prism at an angle that causes a surface plasmon resonance
  • localized light enhanced electric field
  • a fluorescence material labelling the detection object substance captured on the metal film is excited, and therefore the presence or the amount of the detection object substance can be detected by detecting the fluorescence emitted from the fluorescence material.
  • the SPFS apparatus is provided with a light receiving sensor for measuring excitation light, and whether the excitation light irradiating section is in a normal condition is checked from the viewpoint of preventing detection errors due to a lowered power of an excitation light irradiating section.
  • a light receiving sensor for example, a photodiode (PD)
  • a high-power for example, 1 m W/mm 2 or greater
  • a light quantity adjustment means such as a neutral density (ND) filter and a light diffusion plate are used together with the light receiving sensor.
  • the above-mentioned light quantity adjustment means is influenced by aging, dust in the use environment, and the like. Therefore, to perform precise measurement, maintenance of the light quantity adjustment means such as replacing and cleaning of the light quantity adjustment means has to be periodically performed to eliminate the influence.
  • An object of the present invention is to provide a surface plasmon resonance fluorescence analysis method and a surface plasmon resonance fluorescence analysis apparatus that can correctly determine whether an excitation light irradiating section is in a normal condition without separately using a light quantity adjustment means in the case where a high-power light source is used.
  • a surface plasmon resonance fluorescence analysis method is a method of detecting presence or an amount of a detection object substance by detecting fluorescence that is emitted by a fluorescence material labelling the detection object substance when the fluorescence material is excited by localized light based on a surface plasmon resonance, the surface plasmon resonance fluorescence analysis method including: acquiring output information of an excitation light irradiating section by applying excitation light from the excitation light irradiating section to an analysis chip installed in a chip holder, and by detecting excitation light reflected by the analysis chip, the analysis chip including a prism including an incidence surface and a film formation surface, a metal film disposed on the film formation surface, and a capturing body fixed on the metal film.
  • a surface plasmon resonance fluorescence analysis apparatus detects presence or an amount of a detection object substance by detecting fluorescence that is emitted by a fluorescence material labelling the detection object substance when the fluorescence material is excited by localized light based on a surface plasmon resonance
  • the surface plasmon resonance fluorescence analysis apparatus including: a chip holder for detachably holding an analysis chip including a prism including an incidence surface and a film formation surface, a metal film disposed on the film formation surface, and a capturing body fixed on the metal film; an excitation light irradiating section that applies excitation light to the analysis chip held by the chip holder; an excitation light detecting section that detects excitation light reflected by the analysis chip; a fluorescence detecting section that detects fluorescence emitted from the fluorescence material labelling the detection object substance captured by the capturing body; and a processing section that acquires output information of the ex
  • the present invention it is possible to correctly determine whether an excitation light irradiating section is in a normal condition without separately using a light quantity adjustment means in the case where a high-power light source is used, and it is thus possible to prevent detection errors due to abnormality in the excitation light irradiating section.
  • the task and the cost for maintenance such as replacing and cleaning of a light quantity adjusting component can be reduced.
  • FIG. 1 schematically illustrates a configuration of an SPFS apparatus according to Embodiments 1 and 2 of the present invention
  • FIG. 2 is a flowchart of an exemplary procedure of a first operation of the SPFS apparatus according to Embodiment 1 of the present invention
  • FIG. 3 is a flowchart of steps in a position adjustment step (step S 120 ) illustrated in FIG. 2 ;
  • FIGS. 4A to 4C are schematic views for describing a step of acquiring position information of an analysis chip and output information of an excitation light irradiating unit (step S 1201 );
  • FIGS. 5A and 5B are graphs showing exemplary detection results of reflection light by a first light receiving sensor
  • FIGS. 6A and 6B are schematic views for describing a modification of the step of acquiring the position information of the analysis chip and the output information of the excitation light irradiating unit (step S 1201 );
  • FIG. 7 is a sectional view illustrating another exemplary analysis chip
  • FIGS. 8A and 8B are schematic views for describing a step of disposing the analysis chip at a measurement position (step S 1204 );
  • FIGS. 9A and 9B are schematic views for describing the step of disposing the analysis chip at the measurement position (step S 1204 );
  • FIG. 10 is a flowchart of an exemplary procedure of a second operation of the SPFS apparatus according to Embodiment 1 of the present invention.
  • FIG. 11A is a flowchart of steps in a position adjustment step (step S 220 ) illustrated in FIG. 10
  • FIG. 11B is a flowchart of steps in a calculation and correction step (step S 255 ) illustrated in FIG. 10 ;
  • FIG. 12 is a flowchart of an exemplary procedure of an operation of the SPFS apparatus according to Embodiment 2 of the present invention.
  • FIG. 13 is a flowchart of steps in a step of adjusting the position and the output (step S 320 ) illustrated in FIG. 12 ;
  • FIG. 14 is a schematic view for describing a step of acquiring output information of the excitation light irradiating unit (step S 3201 ) in the case where a PD including four light reception surfaces is used;
  • FIG. 15 is a graph for describing another method for acquiring information relating to the power of the excitation light irradiating unit.
  • FIGS. 16A and 16B are graphs showing a relationship between the incident angle and the reflectivity of excitation light.
  • Embodiment 1 a surface plasmon resonance fluorescence analysis apparatus (SPFS apparatus) that can acquire, as output information of an excitation light irradiating unit (excitation light irradiating section), information relating to the power of the excitation light irradiating unit is described.
  • SPFS apparatus surface plasmon resonance fluorescence analysis apparatus
  • FIG. 1 is a schematic view illustrating a configuration of SPFS device 100 according to Embodiment 1 of the present invention.
  • SPFS apparatus 100 includes excitation light irradiating unit (excitation light irradiating section) 110 , excitation light detecting unit (excitation light detecting section) 120 , fluorescence detecting unit (fluorescence detecting section) 130 , liquid feeding unit 140 , conveyance unit 150 and control processing section (processing section) 160 .
  • SPFS device 100 is used in a state where analysis chip 10 is attached to chip holder 154 of conveyance unit 150 . For such a configuration, analysis chip 10 is described first, and each component of SPFS device 100 is described after the description of analysis chip 10 .
  • Analysis chip 10 includes prism 20 including incidence surface 21 , film formation surface 22 and emission surface 23 , metal film 30 formed on film formation surface 22 , and channel closure 40 disposed on film formation surface 22 or metal film 30 . Normally, analysis chip 10 is replaced for each analysis. Analysis chip 10 is preferably a structure with each side of several millimeters to several centimeters, but may be a smaller structure or a larger structure that are not categorized as “chip.”
  • Prism 20 is a dielectric that is transparent to excitation light ⁇ .
  • Prism 20 includes incidence surface 21 , film formation surface 22 and emission surface 23 .
  • Incidence surface 21 allows a large part of excitation light ⁇ from excitation light irradiating unit 110 to enter prism 20 , and reflects a part of excitation light ⁇ from excitation light irradiating unit 110 .
  • the proportion in light quantity (reflectivity) of excitation light ⁇ that is reflected by incidence surface 21 depends on the refractive index of prism 20 , the refractive index of the gas around prism 20 , and the incident angle of excitation light ⁇ to incidence surface 21 (see the reference experiment described later).
  • prism 20 is surrounded by air, and accordingly the reflectivity is substantially constant when the same the material of prism 20 and the same the incident angle of excitation light ⁇ are used.
  • the material of prism 20 is a resin whose refractive index is 1.4 to 1.6, the reflectivity is approximately 4.2%.
  • Metal film 30 is disposed on film formation surface 22 .
  • Excitation light ⁇ having entered prism 20 is reflected by the rear surface of metal film 30 .
  • excitation light ⁇ is reflected by an interface (film formation surface 22 ) between prism 20 and metal film 30 .
  • Emission surface 23 is a surface through which excitation light ⁇ reflected by metal film 30 is emitted out of prism 20 .
  • the shape of prism 20 is not limited.
  • the shape of prism 20 has a columnar shape whose bottom surface has a trapezoidal shape.
  • the surface corresponding to one leg is incidence surface 21
  • the surface corresponding to the other leg is emission surface 23 .
  • the trapezoid serving as the bottom surface is an isosceles trapezoid. With such a configuration, incidence surface 21 and emission surface 23 are symmetrical, and the S wave component of excitation light ⁇ does not easily remain in prism 20 .
  • Incidence surface 21 is formed such that excitation light ⁇ does not return to excitation light irradiating unit 110 .
  • excitation light ⁇ returns to a laser diode (hereinafter also referred to as “LD”) in the case where the light source of excitation light ⁇ is the LD, the excitation state of the LD is disturbed, and the wavelength and the output of excitation light ⁇ are varied.
  • the angle of incidence surface 21 is set within a scanning range around the ideal enhancement angle such that that excitation light ⁇ is not perpendicularly incident on incidence surface 21 .
  • the “enhancement angle” is an incident angle at which the quantity of scattering light (hereinafter referred to as “plasmon scattering light”) 6 having a wavelength equal to that of excitation light ⁇ emitted upward of analysis chip 10 is maximized when the incident angle of excitation light ⁇ with respect to metal film 30 is scanned.
  • plasma scattering light the quantity of scattering light (hereinafter referred to as “plasmon scattering light”) 6 having a wavelength equal to that of excitation light ⁇ emitted upward of analysis chip 10 is maximized when the incident angle of excitation light ⁇ with respect to metal film 30 is scanned.
  • each of the angle between incidence surface 21 and film formation surface 22 and the angle between film formation surface 22 and emission surface 23 is approximately 80 degrees.
  • the enhancement angle (and the resonance angle in the close vicinity thereof) largely depends on the design of analysis chip 10 .
  • the design factors are the refractive index of prism 20 , the refractive index of metal film 30 , the film thickness of metal film 30 , the extinction coefficient of metal film 30 , the wavelength of excitation light ⁇ , and the like.
  • the enhancement angle and the resonance angle are shifted by the detection object substance fixed on metal film 30 , the shift amount is smaller than several degrees.
  • the “resonance angle” is an incident angle at which the quantity of reflection light (not illustrated in the drawing) that is reflected by film formation surface 22 and emitted from emission surface 23 is minimized when the incident angle of excitation light ⁇ with respect to metal film 30 is scanned.
  • Prism 20 has a birefringence property to a certain degree.
  • the material of prism 20 include a resin and glass.
  • the resin of prism 20 include polymethylmethacrylate (PMMA), polycarbonate (PC), and cycloolefin polymer.
  • the material of prism 20 is a resin that has a refractive index of 1.4 to 1.6 and causes a small birefringence.
  • Metal film 30 is disposed on film formation surface 22 of prism 20 .
  • interaction surface plasmon resonance
  • the photon of excitation light ⁇ incident on film formation surface 22 under the total reflection condition and the free electron in metal film 30 , and thus localized-field light can be generated on the surface of metal film 30 .
  • the material of metal film 30 is not limited as long as a surface plasmon resonance can be caused.
  • Examples of the material of metal film 30 include gold, silver, copper, aluminum, and their alloys.
  • metal film 30 is a thin film formed of gold.
  • the formation method for metal film 30 is not limited.
  • Examples of the formation method for metal film 30 include sputtering, deposition, and plating preferably, the thickness of metal film 30 is, but not limited to, 30 to 70 nm.
  • a capturing body for capturing a detection object substance is fixed on the surface of metal film 30 that faces away from prism 20 (the front surface of metal film 30 ). By fixing the capturing body, the detection object substance can be selectively detected.
  • the capturing body is uniformly fixed in a predetermined region (reaction site) on metal film 30 .
  • the type of the capturing body is not limited as long as the detection object substance can be captured.
  • the capturing body is an antibody that can be specifically coupled with the detection object substance or a fragment of the antibody. In the reaction site, reactions such as a coupling between the capturing body and the detection object substance (primary reaction), and fluorescent labeling of the detection object substance (secondary reaction) are performed.
  • Channel closure 40 is disposed on metal film 30 .
  • channel closure 40 may be disposed on film formation surface 22 .
  • a channel groove is formed on the rear surface of channel closure 40 .
  • channel closure 40 forms channel 41 through which liquid flows.
  • the capturing body fixed on metal film 30 is exposed to the interior of channel 41 .
  • Both ends of channel 41 are respectively connected to an inlet and an outlet, which are formed on the top surface of channel closure 40 and are not illustrated in the drawing.
  • channel closure 40 is formed of a material that is transparent to light (fluorescence ⁇ and plasmon scattering light ⁇ ) emitted from metal film 30 .
  • the material of channel closure 40 include a resin.
  • Other parts of channel closure 40 may be formed of an opaque material as long as the material is transparent to the above-mentioned light.
  • Channel closure 40 is joined to metal film 30 or prism 20 by bonding with a double-sided tape, an adhesive agent and the like, laser welding, ultrasound welding, pressure bonding with a clamp member and the like, for example.
  • the type of the liquid that flows through channel 41 is not limited.
  • the liquid include a sample containing a detection object substance, a fluorescent labeling solution containing a fluorescence material, and a buffer solution.
  • the types of the sample and the detection object substance are not limited.
  • the sample include bodily fluids such as blood, serum, plasma, urine, nasal mucus, saliva, and semen, and their diluted solutions.
  • the detection object substance include nucleic acid (such as DNA and RNA), protein (such as polypeptides and oligopeptides), amino acid, glucide, lipid and their modifier molecules.
  • excitation light ⁇ enters prism 20 from incidence surface 21 .
  • a part of excitation light ⁇ is reflected by incidence surface 21 and becomes reflection light ⁇ .
  • Excitation light ⁇ having entered prism 20 hits metal film 30 at a total reflection angle (an angle at which a surface plasmon resonance is caused).
  • a total reflection angle an angle at which a surface plasmon resonance is caused.
  • SPFS device 100 detects the presence or the amount of the detection object substance.
  • SPFS apparatus 100 can acquire the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 by detecting the quantity (intensity) of reflection light ⁇ .
  • SPFS apparatus 100 includes excitation light irradiating unit (excitation light irradiating section) 110 , excitation light detecting unit (excitation light detecting section) 120 , fluorescence detecting unit 130 , liquid feeding unit 140 , conveyance unit 150 and control processing section (processing section) 160 .
  • Excitation light irradiating unit 110 applies excitation light ⁇ to analysis chip 10 held by chip holder 154 .
  • excitation light irradiating unit 110 emits only P wave with respect to metal film 30 toward incidence surface 21 such that the incident angle to metal film 30 is an angle at which a surface plasmon resonance is caused.
  • excitation light is light that directly or indirectly excites the fluorescence material.
  • excitation light ⁇ is light that generates localized light that excites the fluorescence material on the surface of metal film 30 when it is emitted to metal film 30 through prism 20 at an angle that causes a surface plasmon resonance.
  • SPFS apparatus 100 uses excitation light ⁇ also for positioning of analysis chip 10 and for detection of abnormality in excitation light irradiating unit 110 .
  • Excitation light irradiating unit 110 includes a configuration for emitting excitation light ⁇ toward prism 20 , and a configuration for scanning the incident angle of excitation light ⁇ to the rear surface of metal film 30 .
  • excitation light irradiating unit 110 includes light source unit 111 , angle adjusting mechanism 112 and light source controlling section 113 .
  • Light source unit 111 emits collimated excitation light ⁇ having a constant wavelength and a constant quantity (intensity) such that the irradiation spot on the rear surface of metal film 30 has a substantially circular shape.
  • Light source unit 111 includes, for example, a light source of excitation light ⁇ , a beam shaping optical system, and a temperature adjusting mechanism (not illustrated).
  • the light source be a high-power light source from the viewpoint of the use of a light detector that does not have high sensitivity such as a photodiode (PD) as second light receiving sensor 137 .
  • the light source is a laser diode (LD).
  • Other examples of the light source include a light-emitting diode, a mercury lamp, and other laser light sources.
  • the light emitted from the light source is converted to a beam by a lens, a mirror, a slit and the like.
  • the light emitted from the light source is converted to monochromatic light by a diffraction grid and the like.
  • the light emitted from the light source is converted to linear polarized light by a polarizer and the like.
  • the beam shaping optical system includes a collimator, a band pass filter, a linear polarization filter, a half-wave plate, a slit, a zooming unit and the like, for example.
  • the beam shaping optical system may include one or more of the above-mentioned components.
  • the collimator collimates excitation light ⁇ emitted from the light source.
  • the band pass filter changes excitation light ⁇ emitted from the light source to narrowband light composed only of a central wavelength.
  • excitation light ⁇ from the light source has a slight wavelength distribution width.
  • the linear polarization filter changes excitation light ⁇ emitted from the light source to completely linearly polarized light.
  • the half-wave plate adjusts the polarization direction of excitation light ⁇ such that the P wave component hits on metal film 30 .
  • the slit and the zooming unit adjust the beam diameter, the outline shape and the like of excitation light ⁇ such that the shape of the irradiation spot on the rear surface of metal film 30 has a circular shape of a predetermined size.
  • the temperature adjusting mechanism is composed of a heater, a Peltier device, or the like, for example.
  • the wavelength and the energy of the light emitted from the light source can vary depending on the temperature. Therefore, the temperature of the light source is maintained at a constant value by the temperature adjusting mechanism to control the wavelength and the energy of the light emitted from the light source at a constant value.
  • Angle adjusting mechanism 112 adjusts the incident angle of excitation light ⁇ to metal film 30 (the interface between prism 20 and metal film 30 (film formation surface 22 )). Angle adjusting mechanism 112 relatively rotates the optical axis of excitation light ⁇ and chip holder 154 to emit excitation light ⁇ at a predetermined incident angle toward a predetermined position in metal film 30 through prism 20 .
  • angle adjusting mechanism 112 turns light source unit 111 around an axis (an axis perpendicular to the plane of FIG. 1 ) orthogonal to the optical axis of excitation light ⁇ .
  • the position of the rotation axis is set such that the position of the irradiation spot on metal film 30 is not substantially moved when the incident angle is scanned.
  • the enhancement angle is an angle at which a maximum quantity of plasmon scattering light ⁇ is obtained, among the incident angles of excitation light ⁇ with respect to metal film 30 .
  • the incident angle of excitation light ⁇ is set to the enhancement angle or an angle approximately equal to the enhancement angle, fluorescence ⁇ having a high intensity can be detected.
  • the basic incident condition of excitation light ⁇ is determined by the material and the shape of prism 20 of analysis chip 10 , the film thickness of metal film 30 , the refractive index of the liquid in the channel and the like
  • the optimum incident condition slightly varies depending on the type and the amount of the fluorescence material in the channel, shaping errors of prism 20 and the like. Therefore, it is preferable to determine the optimum enhancement angle in each measurement.
  • the preferable emission angle of excitation light ⁇ to the normal of metal film 30 is approximately 70 degrees.
  • Light source controlling section 113 controls the components included in light source unit 111 to control the output of emission light (excitation light ⁇ ) of light source unit 111 .
  • Light source controlling section 113 is composed of a publicly known computer, microcomputer, or the like including a computation device, a control device, a storage device, and an inputting device, for example.
  • Excitation light detecting unit 120 detects reflection light ⁇ generated by irradiation of analysis chip 10 with excitation light ⁇ for the purpose of the detection of abnormality in excitation light irradiating unit 110 and the positioning of analysis chip 10 at the time of optical measurement (for example, detection of the enhancement angle, measurement of the optical blank value, detection of fluorescence ⁇ and the like).
  • excitation light detecting unit 120 detects reflection light ⁇ for the purpose of positioning of analysis chip 10 and the detection of abnormality in excitation light irradiating unit 110 .
  • the first optical measurement is the detection of the enhancement angle, and it is therefore preferable to detect reflection light ⁇ before the detection of the enhancement angle.
  • reflection light ⁇ is detected before the measurement of the optical blank value.
  • reflection light ⁇ is detected before the detection of fluorescence ⁇ .
  • the detection of reflection light ⁇ for detecting abnormality in excitation light irradiating unit 110 , and the detection of reflection light ⁇ for positioning of analysis chip 10 may be simultaneously performed, or separately performed. In the present embodiment, the detection of reflection light ⁇ for detecting abnormality in excitation light irradiating unit 110 and the detection of reflection light ⁇ for positioning of analysis chip 10 are simultaneously performed.
  • Excitation light detecting unit 120 includes first light receiving sensor 121 and first sensor controlling section 122 .
  • First light receiving sensor 121 detects reflection light ⁇ of excitation light ⁇ .
  • the type of first light receiving sensor 121 is not limited as long as reflection light ⁇ of excitation light ⁇ can be detected.
  • first light receiving sensor 121 is a photodiode (PD), a position detection device (PSD), or the like.
  • the size of the light reception surface of first light receiving sensor 121 is greater than the beam diameter of excitation light ⁇ .
  • the length of each side of the light reception surface of first light receiving sensor 121 is preferably 3 mm or greater.
  • First light receiving sensor 121 is disposed at a position where reflection light ⁇ of excitation light ⁇ reaches.
  • first light receiving sensor 121 is disposed at a position where reflection light ⁇ from incidence surface 21 reaches.
  • first light receiving sensor 121 is disposed at a position where reflection light ⁇ of excitation light ⁇ emitted at an angle equal to or approximately equal to the angle for the detection of fluorescence ⁇ reaches.
  • the irradiation position (irradiation direction) of excitation light ⁇ slightly varies depending on variation of the incident angle, and therefore, by setting the incident angle of excitation light ⁇ for the positioning of analysis chip 10 and the incident angle of excitation light ⁇ for the detection of fluorescence ⁇ to values equal to or approximately equal to each other, the accuracy of the positioning in the detection of fluorescence ⁇ can be enhanced.
  • the emission angle of excitation light ⁇ to the normal of metal film 30 (straight line along the z-axis direction in FIG. 1 ) is approximately 70 degrees
  • reflection light ⁇ from incidence surface 21 advances in a direction approximately parallel to the travelling direction of the conveyance stage (x-axis direction in FIG. 1 ). Accordingly, first light receiving sensor 121 is disposed at a position where the reflection light ⁇ that travels in parallel reaches (see FIG. 4C ).
  • First sensor controlling section 122 controls the detection of the output value of first light receiving sensor 121 , the management of the sensitivity of first light receiving sensor 121 according to the detected output value, the changing of the sensitivity of first light receiving sensor 121 for acquiring an appropriate output value, and the like.
  • First sensor controlling section 122 is composed of a publicly known computer, microcomputer, or the like including a computation device, a control device, a storage device, and an inputting device, for example.
  • Fluorescence detecting unit 130 detects fluorescence ⁇ generated by irradiation of metal film 30 with excitation light ⁇ . In addition, as necessary, fluorescence detecting unit 130 also detects plasmon scattering light ⁇ generated by irradiation of metal film 30 with excitation light ⁇ . Fluorescence detecting unit 130 includes light receiving unit 131 , position switching mechanism 132 and second sensor controlling section 133 , for example.
  • Light receiving unit 131 is disposed in the normal direction of metal film 30 of analysis chip 10 (the z-axis direction in FIG. 1 ).
  • Light receiving unit 131 includes first lens 134 , optical filter 135 , second lens 136 and second light receiving sensor 137 .
  • First lens 134 is, for example, a condenser lens, and condenses the light emitted from metal film 30 .
  • Second lens 136 is, for example, an image forming lens, and images the light condensed by first lens 134 on the light reception surface of second light receiving sensor 137 .
  • the light paths between the lenses are substantially parallel to each other.
  • Optical filter 135 is disposed between first lens 134 and second lens 136 .
  • optical filter 135 allows only the fluorescence component of the light incident on optical filter 135 to pass therethrough, while removing the excitation light component (plasmon scattering light ⁇ ). With this configuration, it is possible to guide only the fluorescence component to second light receiving sensor 137 to detect fluorescence ⁇ with a high S/N ratio.
  • the type of optical filter 135 include an excitation light reflection filter, a short wavelength cut filter and a band pass filter.
  • optical filter 135 include a filter including a multi-layer film that reflects a predetermined light component and a color glass filter that absorbs a predetermined light component.
  • Second light receiving sensor 137 detects fluorescence ⁇ and plasmon scattering light ⁇ emitted from analysis chip 10 .
  • Examples of second light receiving sensor 137 include a photodiode (PD), a photomultiplier tube (PMT) and an avalanche photodiode (APD).
  • PD photodiode
  • PMT photomultiplier tube
  • APD avalanche photodiode
  • Position switching mechanism 132 switches the position of optical filter 135 between a position on the light path and a position outside the light path in light receiving unit 131 .
  • optical filter 135 is disposed on the light path of light receiving unit 131 when second light receiving sensor 137 detects fluorescence ⁇
  • optical filter 135 is disposed at a position outside the light path of light receiving unit 131 when second light receiving sensor 137 detects plasmon scattering light ⁇ .
  • Position switching mechanism 132 is composed of a rotation driving section, and a publicly known mechanism (such as a turntable and a rack-and-pinion) that laterally moves optical filter 135 by utilizing a rotational movement, for example.
  • Second sensor controlling section 133 controls the detection of the output value of second light receiving sensor 137 , the management of the sensitivity of second light receiving sensor 137 according to the detected output value, the changing of the sensitivity of second light receiving sensor 137 for acquiring an appropriate output value and the like.
  • Second sensor controlling section 133 is composed of a publicly known computer, microcomputer, or the like including a computation device, a control device, a storage device, and an inputting device, for example.
  • Liquid feeding unit 140 supplies sample solution, labeling solution, washing solution and the like into channel 41 of analysis chip 10 held by chip holder 154 .
  • Liquid feeding unit 140 includes chemical liquid chip 141 , syringe pump 142 and liquid feed pump driving mechanism 143 .
  • Chemical liquid chip 141 is a vessel for housing liquid such as sample solution, labeling solution, and washing solution. Normally, as chemical liquid chip 141 , a plurality of vessels corresponding to the types of the liquid are disposed, or a chip composed of a plurality of integrated vessels is disposed.
  • Syringe pump 142 is composed of syringe 144 , and plunger 145 that can reciprocate in syringe 144 . Through reciprocation of plunger 145 , suction and discharge of the liquid is quantitatively performed. In the case where syringe 144 is replaceable, washing of syringe 144 is unnecessary. Such a configuration is preferable from the viewpoint of preventing entry of impurities. In the case where syringe 144 is not replaceable, it is possible to use syringe 144 without replacing syringe 144 by additionally providing a configuration for washing the interior of syringe 144 .
  • Liquid feed pump driving mechanism 143 includes a device for driving plunger 145 , and a device for moving syringe pump 142 .
  • the driving device for syringe pump 142 is a device for reciprocating plunger 145 , and includes a stepping motor, for example.
  • a driving device including a stepping motor can manage the liquid feed amount of syringe pump 142 and the liquid feed speed, and is therefore preferable from the viewpoint of managing the amount of the residual liquid of analysis chip 10 .
  • the device for moving syringe pump 142 freely moves syringe pump 142 in the axial direction (for example, a vertical direction) of syringe 144 and a direction (for example, a lateral direction) crossing the axial direction, for example.
  • the device for moving syringe pump 142 is composed of a robot arm, a biaxial stage or a vertically movable turntable, for example.
  • liquid feeding unit 140 further includes a device that detects the position of an end of syringe 144 from the viewpoint of adjusting the relative heights of syringe 144 and analysis chip 10 to constant values, and managing the amount of the residual liquid in analysis chip 10 at a constant value.
  • Liquid feeding unit 140 sucks various kinds of liquid from chemical liquid chip 141 , and supplies the liquid into channel 41 of analysis chip 10 . At this time, when plunger 145 is moved, the liquid reciprocates in channel 41 in analysis chip 10 , thus agitating the liquid in channel 41 . In this manner, the density of the liquid can be uniformized, and reaction (for example, antigen-antibody reaction) in channel 41 can be facilitated. From the view point of performing the above-mentioned operations, it is preferable that analysis chip 10 and syringe 144 be configured such that the inlet of analysis chip 10 is protected with a multi-layer film and that the inlet can be tightly sealed when syringe 144 penetrates the multi-layer film.
  • the liquid in channel 41 is again sucked by syringe pump 142 , and ejected to chemical liquid chip 141 and the like.
  • reaction, washing and the like of various kinds of liquid can be performed, and a detection object substance labeled with a fluorescence material can be placed at a reaction site in channel 41 .
  • Conveyance unit 150 conveys analysis chip 10 to an installation position, a measurement position or a liquid feeding position, and fixes analysis chip 10 .
  • the “installation position” is a position for installing analysis chip 10 to SPFS apparatus 100 (i.e. chip holder 154 ).
  • the “measurement position” is a position where fluorescence detecting unit 130 detects fluorescence ⁇ that is generated when excitation light irradiating unit 110 applies excitation light ⁇ to analysis chip 10 .
  • the “liquid feeding position” is a position where liquid feeding unit 140 supplies liquid into channel 41 of analysis chip 10 , or removes the liquid in channel 41 of analysis chip 10 .
  • Conveyance unit 150 includes conveyance stage 152 and chip holder 154 .
  • Conveyance stage 152 moves chip holder 154 in a specific direction (x-axis direction in FIG. 1 ) and a direction opposite to the specific direction. Conveyance stage 152 is driven by a stepping motor and the like, for example.
  • Chip holder 154 is fixed to conveyance stage 152 so as to detachably hold analysis chip 10 .
  • Chip holder 154 has a shape that can hold analysis chip 10 and does not block the light paths of excitation light ⁇ , reflection light ⁇ , fluorescence ⁇ and the like.
  • chip holder 154 is provided with an opening through which these lights pass.
  • Control processing section 160 controls angle adjusting mechanism 112 , light source controlling section 113 , first sensor controlling section 122 , position switching mechanism 132 , second sensor controlling section 133 , liquid feed pump driving mechanism 143 and conveyance stage 152 .
  • control processing section 160 acquires the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 . Then, on the basis of the acquired position information, control processing section 160 specifies the position of analysis chip 10 held by chip holder 154 .
  • preliminarily acquired output information that serves as a reference of excitation light irradiating unit 110 is stored.
  • control processing section 160 compares the acquired output information with the output information that serves as a reference to determine whether excitation light irradiating unit 110 is in a normal condition.
  • the output information of excitation light irradiating unit 110 is information relating to the power of excitation light irradiating unit 110 and/or information relating to the irradiation direction of excitation light ⁇ .
  • the output information that serves as a reference is the output information of excitation light irradiating unit 110 in a normal condition, and is, for example, excitation light irradiating unit 110 in the initial state.
  • the output information of excitation light irradiating unit 110 is information relating to the power of excitation light irradiating unit 110 .
  • Control processing section 160 is composed of a publicly known computer, microcomputer, or the like including a computation device, a control device, a storage device, and an inputting device, for example.
  • SPFS apparatus 100 surface plasmon resonance fluorescence analysis method according to the present embodiment (also referred to simply as “analysis method”)) is described.
  • SPFS apparatus 100 can perform two operations described below, for example.
  • SPFS apparatus 100 acquires information relating to the power of excitation light irradiating unit 110 as the output information of excitation light irradiating unit 110 . Then, when it is determined that the power of excitation light irradiating unit 110 is abnormal, SPFS apparatus 100 adjusts the power of excitation light irradiating unit 110 .
  • FIG. 2 is a flowchart of an exemplary procedure of a first operation of SPFS apparatus 100 .
  • FIG. 3 is a flowchart of steps in a step of adjusting the position and the output (step S 120 ) illustrated in FIG. 2 .
  • an abnormality detection step step S 1202 is included in the step of adjusting the position and the output (step S 120 ).
  • analysis chip 10 is installed at an installation position of SPFS apparatus 100 (step S 100 ). To be more specific, analysis chip 10 is installed in chip holder 154 of SPFS apparatus 100 .
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a liquid feeding position (step S 105 ).
  • control processing section 160 operates liquid feeding unit 140 to introduce the sample solution in chemical liquid chip 141 into channel 41 of analysis chip 10 (step S 110 ).
  • channel 41 the detection object substance is captured on metal film 30 by an antigen-antibody reaction (primary reaction).
  • the sample solution in channel 41 is removed, and the interior of channel 41 is washed with the washing solution. It is to be noted that when moisturizing agent is present in channel 41 of analysis chip 10 , the interior of channel 41 is washed prior to the introduction of the sample solution to remove the moisturizing agent so that the capturing body can properly capture the detection object substance.
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a position near the measurement position (step S 115 ).
  • control processing section 160 operates excitation light irradiating unit 110 , excitation light detecting unit 120 and conveyance stage 152 to acquire the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 .
  • Control processing section 160 adjusts the position of analysis chip 10 on the basis of the acquired position information.
  • control processing section 160 determines whether excitation light irradiating unit 110 is in a normal condition on the basis of the acquired output information. Then, when it is determined that the power of excitation light irradiating unit 110 is abnormal, control processing section 160 adjusts the power of excitation light irradiating unit 110 on the basis of the acquired output information (step S 120 ).
  • step S 1201 analysis chip 10 held by chip holder 154 is irradiated with excitation light ⁇ , and reflection light ⁇ of excitation light ⁇ reflected by the surface of analysis chip 10 is detected, thereby acquiring the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 (information relating to the power) (step S 1201 ).
  • control processing section 160 determines whether the power of excitation light irradiating unit 110 (i.e. light source unit 111 ) is normal (step S 1202 ).
  • information relating to the power of excitation light irradiating unit 110 of the initial state is stored.
  • the intensity of reflection light ⁇ , from the surface of analysis chip 10 , of excitation light ⁇ emitted from excitation light irradiating unit 110 of the initial state (hereinafter referred to as simply as “initial value”) is stored.
  • Control processing section 160 compares the detection value of reflection light ⁇ detected in step S 121 with the initial value preliminarily stored in control processing section 160 to determine whether the power of excitation light irradiating unit 110 is normal. For example, the difference between the initial value and the detection value is greater than a predetermined reference value (threshold), control processing section 160 determines that the power of excitation light irradiating unit 110 is abnormal.
  • the reference value (threshold) is appropriately set in accordance with the accuracy required for the analysis.
  • control processing section 160 adjusts the output of excitation light irradiating unit 110 to a desired power (step S 1203 ).
  • control processing section 160 continues the subsequent operations.
  • control processing section 160 adjusts the position of analysis chip 10 (step S 1204 ).
  • control processing section 160 can specify the degree of the positional displacement of analysis chip 10 from the measurement position on the basis of the position information.
  • control processing section 160 operates conveyance stage 152 to move chip holder 154 , and dispose analysis chip 10 at an appropriate measurement position.
  • FIGS. 4A and 4B are schematic views for describing a step of acquiring the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 (step S 1201 ).
  • the excitation light ⁇ is reflected by channel closure 40 and travels toward the lower side (conveyance stage 152 side).
  • reflection light ⁇ from the surface of analysis chip 10 is not incident on first light receiving sensor 121 of excitation light detecting unit 120 .
  • excitation light ⁇ from light source unit 111 reaches the boundary (hereinafter referred to as “edge”) between prism 20 and channel closure 40 .
  • edge the boundary between prism 20 and channel closure 40 .
  • excitation light ⁇ (reflection light ⁇ ) reflected by channel closure 40 does not enter first light receiving sensor 121
  • excitation light ⁇ (reflection light ⁇ ) reflected by incidence surface 21 enters first light receiving sensor 121 .
  • a part of reflection light ⁇ from analysis chip 10 enters first light receiving sensor 121 .
  • FIGS. 5A and 5B are graphs showing examples of detection results of reflection light ⁇ by first light receiving sensor 121 .
  • the intensity of reflection light ⁇ was measured with first light receiving sensor 121 while moving analysis chip 10 by conveyance stage 152 in one direction (x-axis direction) in units of 100 ⁇ m.
  • the beam diameter of excitation light ⁇ is approximately 1 to 1.5 mm.
  • FIG. 5A shows only detection results, and FIG. 5B shows also three approximation lines.
  • the intensity of reflection light ⁇ incident on first light receiving sensor 121 is almost constant and maximized.
  • the reason for this is that the entirety of reflection light ⁇ enters first light receiving sensor 121 (see FIG. 4C ).
  • the inclined part (moving distance: approximately 1,000 ⁇ m to approximately 2,000 ⁇ m) in the graph corresponds to the edge.
  • the width of the inclined part corresponds to the beam diameter (approximately 1 mm to approximately 1.5 mm) of excitation light ⁇ in the x-axis direction.
  • the horizontal part in the former half (moving distance: 0 to approximately 1,000 ⁇ m), the inclined part (moving distance: approximately 1,000 ⁇ m to approximately 2,000 ⁇ m), and the horizontal part in the latter half (moving distance: greater than approximately 2,000 ⁇ m) are each approximated by a straight line.
  • point A is the intersection of the approximation straight line of the horizontal part in the former half and the approximation straight line of the inclined part.
  • Point B is the intersection of the approximation straight line of the inclined part and the approximation straight line of the horizontal part in the latter half.
  • Point C is the middle point between point A and point B.
  • Point A corresponds to a minimum value of the intensity of reflection light ⁇ .
  • Point B corresponds to a maximum value of the intensity of reflection light ⁇ .
  • Point C corresponds to an intermediate value of the intensity of reflection light ⁇ .
  • points A to C may be used to specify the position of analysis chip 10 .
  • Point A and point B indicate points where the end of the beam of excitation light ⁇ reaches the edge. Accordingly, the position of the edge can be specified by considering the beam diameter of excitation light ⁇ , and as a result, the position of analysis chip 10 can be specified.
  • point C indicates a point where the center of the beam of excitation light ⁇ reaches the edge.
  • point C is utilized, the position of the edge can be specified without considering the beam diameter of excitation light ⁇ , and as a result, the position of analysis chip 10 can be specified. Accordingly, from the viewpoint of suppressing the influence of the beam diameter of excitation light ⁇ , it is preferable to use the intermediate value of the intensity of reflection light ⁇ of excitation light ⁇ for specifying the position of analysis chip 10 .
  • point B may be used to acquire information relating to the power of excitation light irradiating unit 110 .
  • Point B is a maximum value of the intensity of reflection light ⁇ of excitation light ⁇ emitted from excitation light irradiating unit 110 , and is an output value of excitation light ⁇ emitted from irradiation light irradiating unit 110 . Accordingly, by comparing the initial value and the maximum value of the intensity of reflection light ⁇ , whether the power of excitation light irradiating unit 110 (light source unit 111 ) is lowered can be determined.
  • the position of analysis chip 10 can be specified by irradiating analysis chip 10 with excitation light ⁇ , and detecting reflection light ⁇ of excitation light ⁇ reflected by the surface of analysis chip 10 .
  • first light receiving sensor 121 is disposed at a position where reflection light ⁇ from channel closure 40 reached but reflection light ⁇ from incidence surface 2 does not reach as illustrated in FIG. 6A , an effect similar to those of the exemplary cases illustrated in FIGS. 4A to 4C is achieved (the intensity of reflection light ⁇ is high when the moving distance is short, and the intensity of reflection light ⁇ is low when the moving distance is long).
  • incidence surface 21 since the position of incidence surface 21 is an important factor, it is preferable to irradiate incidence surface 21 and a surface adjacent to incidence surface 21 (in the present embodiment, the rear surface of channel closure 40 ) of analysis chip 10 with excitation light ⁇ when two adjacent surfaces of analysis chip 10 are irradiated with excitation light ⁇ . Then, incidence surface 21 of prism 20 and the bottom surface of prism 20 may be irradiated with excitation light ⁇ as illustrated in FIG. 6B . In the example illustrated in FIG. 6B , however, analysis chip 10 is closer to light source unit 111 relative to the measurement position at the time of specifying the position of analysis chip 10 (step S 120 ).
  • chip holder 154 is required to be moved in an opposite direction by conveyance stage 152 when moving analysis chip 10 to the measurement position (step S 142 ).
  • This operation of conveyance stage 152 in two directions might lead to reduction in operation accuracy in comparison with the case where conveyance stage 152 is operated in only one direction.
  • chip holder 154 is not required to be moved in opposite directions by conveyance stage 152 in the exemplary cases illustrated in FIGS. 4A to 4C and FIG. 6A .
  • step S 1201 it is preferable to move chip holder 154 by conveyance stage 152 only in a direction (x-axis direction) toward the light source of excitation light ⁇ (light source unit 111 ) in the step of acquiring the position information of analysis chip 10 (step S 1201 ) and the step of moving the analysis chip to the measurement position (step S 1204 ).
  • the “two adjacent surfaces of analysis chip 10 ” include two substantially adjacent surfaces.
  • analysis chip 10 ′ including prism 20 , metal film 30 disposed on film formation surface 22 of the prism, spacer 42 disposed on metal film 30 , and channel closure 40 disposed on spacer 42 is used as illustrated in FIG. 7 .
  • the shape of channel 41 is defined by spacer 42
  • channel closure 40 is a transparent flat plate. Strictly speaking, in this case, since the side surface of spacer 42 extends between incidence surface 21 of prism 20 and the bottom surface of channel closure 40 , incidence surface 21 and the bottom surface of channel closure 40 are not adjacent to each other.
  • incidence surface 21 and the bottom surface of channel closure 40 can be considered to be substantially adjacent to each other. Accordingly, in this case, reflection light ⁇ from incidence surface 21 and the bottom surface of channel closure 40 that are substantially adjacent to each other is detected to detect the edge.
  • joining members such as adhesive agent and a double-sided tape, metal film 30 , and the like are also negligible.
  • the thickness of the above-mentioned members (for example, spacer 42 ) that are negligible in detection of reflection light ⁇ is equal to or smaller than 1 ⁇ 5 of the beam diameter of excitation light ⁇ , preferably, equal to or smaller than 1/10 of the beam diameter of excitation light ⁇ .
  • the most part of (4 ⁇ 5 or more, or 9/10 or more) of reflection light ⁇ from the surface of analysis chip 10 ′ is reflection light ⁇ from incidence surface 21 or the bottom surface of channel closure 40 , and is utilized for the position detection.
  • the position of analysis chip 10 ′ can be specified without being influenced by spacer 42 .
  • a member such as spacer 42 , a joining member and metal film 30 ) whose thickness is equal to or smaller than 1 ⁇ 5 of the beam diameter of excitation light ⁇ is negligible in detection of reflection light ⁇ . That is, incidence surface 21 and the bottom surface of channel closure 40 of analysis chip 10 ′ can be regarded as two substantially adjacent surfaces.
  • FIGS. 8A and 8B are schematic views for describing a step (step S 1204 ) of disposing analysis chip 10 to an appropriate measurement position.
  • step S 1204 the position of the edge is specified as illustrated in FIG. 8A .
  • the distance between the position of the edge and the region to which excitation light ⁇ should be applied in the rear surface of metal film 30 (the region on the rear side of the reaction site) is determined.
  • FIG. 8B by controlling conveyance stage 152 to move chip holder 154 by a predetermined distance, analysis chip 10 can be disposed at an appropriate measurement position.
  • analysis chip 10 can be disposed at an appropriate measurement position.
  • a case is assumed in which the position of the edge is specified as illustrated in FIG. 9A .
  • the position of analysis chip 10 is shifted in the x-axis direction in comparison with the case (indicated with the broken line in the drawing) where analysis chip 10 is not shifted in the z-axis direction.
  • analysis chip 10 can be disposed at an appropriate measurement position by moving chip holder 154 by a predetermined distance by conveyance stage 152 on the basis of the position of the edge.
  • control processing section 160 operates excitation light irradiating unit 110 and fluorescence detecting unit 130 to irradiate analysis chip 10 disposed at an appropriate measurement position with excitation light ⁇ , and detects plasmon scattering light ⁇ whose wavelength is identical to that of excitation light ⁇ , thereby detecting the enhancement angle (step S 125 ).
  • control processing section 160 operates excitation light irradiating unit 110 to scan the incident angle of excitation light ⁇ to metal film 30 , while operating fluorescence detecting unit 130 to detect plasmon scattering light ⁇ .
  • control processing section 160 operates position switching mechanism 132 to dispose optical filter 135 at a position outside the light path of light receiving unit 131 . Then, control processing section 160 determines, to be the enhancement angle, the incident angle of excitation light ⁇ at which the quantity of the plasmon scattering light ⁇ is maximized.
  • control processing section 160 operates excitation light irradiating unit 110 and fluorescence detecting unit 130 to irradiate analysis chip 10 disposed at an appropriate measurement position with excitation light ⁇ , and records an output value (optical blank value) of second light receiving sensor 137 (step S 130 ).
  • control processing section 160 operates angle adjusting mechanism 112 to set the incident angle of excitation light ⁇ to the enhancement angle.
  • control processing section 160 controls position switching mechanism 132 to dispose optical filter 135 at a position on the light path of light receiving unit 131 .
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a liquid feeding position (step S 135 ).
  • control processing section 160 operates liquid feeding unit 140 to introduce liquid (labeling solution) containing a secondary antibody labeled with a fluorescence material into channel 41 of analysis chip 10 (step S 140 ).
  • liquid (labeling solution) containing a secondary antibody labeled with a fluorescence material into channel 41 of analysis chip 10
  • secondary reaction an antigen-antibody reaction (secondary reaction)
  • a detection object substance captured on metal film 30 is labeled with the fluorescence material.
  • the labeling solution in channel 41 is removed, and the interior of the channel is washed with washing solution.
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to an appropriate measurement position determined in step S 120 (step S 145 ).
  • control processing section 160 operates excitation light irradiating unit 110 and fluorescence detecting unit 130 to irradiate analysis chip 10 disposed at an appropriate measurement position with excitation light ⁇ , and detects fluorescence ⁇ emitted from the fluorescence material labelling the detection object substance captured by the capturing body (step S 150 ).
  • control processing section 160 subtracts the optical blank value from the detection value of fluorescence ⁇ to calculate the intensity of the fluorescence correlating with the amount of the detection object substance (step S 155 ).
  • the intensity of the calculated fluorescence is converted to the amount, the concentration, and the like of the detection object substance as necessary.
  • the presence or the amount of the detection object substance in the sample solution can be detected.
  • SPFS apparatus 100 acquires information relating to the power of excitation light irradiating unit 110 as the output information of excitation light irradiating unit 110 .
  • SPFS apparatus 100 corrects the detection value of fluorescence ⁇ in accordance with the value of the power of excitation light irradiating unit 110 .
  • FIG. 10 is a flowchart of an exemplary procedure of the second operation of SPFS apparatus 100 .
  • FIG. 11A is a flowchart of steps in the position adjustment step (step S 220 ) illustrated in FIG. 10
  • FIG. 11B is a flowchart of steps in the step of calculating the fluorescence intensity correlating with the amount of the detection object substance (step S 255 ) illustrated in FIG. 10 .
  • an abnormality detection step step S 2552
  • step S 255 is included in the step of calculating the amount of the detection object substance and the like (step S 255 ).
  • analysis chip 10 is installed at an installation position of SPFS apparatus 100 (step S 200 ). To be more specific, analysis chip 10 is installed in chip holder 154 of SPFS apparatus 100 .
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a liquid feeding position (step S 205 ).
  • control processing section 160 operates liquid feeding unit 140 to introduce the sample solution in chemical liquid chip 141 into channel 41 of analysis chip 10 (step S 210 ).
  • channel 41 a detection object substance is captured on metal film 30 by an antigen-antibody reaction (primary reaction). Thereafter, the sample solution in channel 41 is removed, and the interior of channel 41 is washed with the washing solution. It is to be noted that when moisturizing agent is present in channel 41 of analysis chip 10 , the interior of channel 41 is washed prior to the introduction of the sample solution to remove the moisturizing agent so that the capturing body can appropriately capture the detection object substance.
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a position near the measurement position (step S 215 ).
  • control processing section 160 operates excitation light irradiating unit 110 , excitation light detecting unit 120 and conveyance stage 152 to acquire the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 . On the basis of the acquired position information, control processing section 160 adjusts the position of analysis chip 10 (step S 220 ).
  • step S 2201 analysis chip 10 held by chip holder 154 is irradiated with excitation light ⁇ , and reflection light ⁇ of excitation light ⁇ reflected by the surface of analysis chip 10 is detected, thereby acquiring the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 (information relating to the power) (step S 2201 ).
  • the output information acquired at this time is recorded in control processing section 160 .
  • control processing section 160 adjusts the position of analysis chip 10 (step S 2202 ).
  • the step of acquiring the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 (step S 2201 ) is similar to step S 1201
  • the step of disposing analysis chip 10 at an appropriate measurement position on the basis of the acquired position information (step S 2202 ) is similar to step S 1204 , and therefore, the descriptions thereof will be omitted.
  • control processing section 160 detects the enhancement angle as in step S 125 (step S 225 ).
  • control processing section 160 measures and records the optical blank value as in step S 130 (step S 230 ).
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a liquid feeding position (step S 235 ).
  • control processing section 160 labels the detection object substance captured on metal film 30 with the fluorescence material as in step S 140 (step S 240 ).
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to an appropriate measurement position determined in step S 220 (step S 245 ).
  • control processing section 160 detects fluorescence ⁇ emitted from the fluorescence material labelling the detection object substance captured by the capturing body as in step S 150 (step S 250 ).
  • control processing section 160 calculates the amount, the concentration and the like of the detection object substance (step S 255 ).
  • the optical blank value is subtracted from the detection value of fluorescence ⁇ , and the fluorescence intensity correlating with the amount of the detection object substance is calculated (step S 2551 ).
  • control processing section 160 determines whether the power of excitation light irradiating unit 110 is normal (step S 2552 ).
  • control processing section 160 the detection value of reflection light ⁇ detected in step S 2201 and the initial value of reflection light ⁇ are stored.
  • Control processing section 160 compares the detection value and the initial value to determine whether the power of excitation light irradiating unit 110 is normal.
  • control processing section 160 corrects the fluorescence intensity calculated in step S 2551 (step S 2553 ).
  • control processing section 160 calculates the degree of the reduction of the power of excitation light irradiating unit 110 , and multiplies the fluorescence intensity calculated in step S 2551 by a correction coefficient (for example, 100/70 when the power of excitation light irradiating unit 110 is lowered to 70%) configured to make up the power reduction. In this manner, control processing section 160 corrects the calculated fluorescence intensity.
  • the corrected fluorescence intensity is converted to the amount, the concentration, and the like of the detection object substance as necessary.
  • the presence or the amount of the detection object substance in the sample solution can be detected.
  • the surface plasmon resonance fluorescence analysis method and the surface plasmon resonance fluorescence analysis apparatus (SPFS apparatus 100 ) can acquire the output information (information relating to the power) of excitation light irradiating unit 110 by detecting reflection light ⁇ of excitation light ⁇ .
  • Reflection light ⁇ that is reflected at the surface (in the present embodiment, incidence surface 21 ) of analysis chip 10 is approximately several percent of excitation light ⁇ from excitation light irradiating unit 110 , and therefore first light receiving sensor 121 is not saturated.
  • the analysis method and SPFS apparatus 100 can correctly determine whether excitation light irradiating unit 110 is in a normal condition, and can prevent detection errors due to abnormality in excitation light irradiating unit 110 , without separately using a light quantity adjustment means.
  • the task and the cost for maintenance such as replacing and cleaning of a light quantity adjusting component can be reduced.
  • analysis chip 10 is replaced for each analysis and excitation light ⁇ from excitation light irradiating unit 110 is always reflected by a clean surface, there is no influence of the aging of the reflecting surface, dust, and the like. Also in view of this, it is possible to correctly determine whether excitation light irradiating unit 110 is in a normal condition.
  • Embodiment 2 an SPFS apparatus that can acquire information relating to the power of excitation light irradiating unit 110 and the irradiation direction of excitation light ⁇ as output information of the excitation light irradiating unit (excitation light irradiating section) is described.
  • SPFS apparatus 200 according to Embodiment 2 is different from SPFS apparatus 100 according to Embodiment 1 only in configuration of first light receiving sensor 221 of excitation light detecting unit 220 .
  • first light receiving sensor 221 in SPFS apparatus 200 is described below, and the components identical to those of SPFS apparatus 100 according to Embodiment 1 are denoted with the same reference numerals, and, the description thereof will be omitted.
  • analysis chip 10 used in SPFS apparatus 200 according to Embodiment 2 is identical to analysis chip 10 used in SPFS apparatus 100 according to Embodiment 1, and therefore the description thereof will be omitted.
  • FIG. 1 is a schematic view illustrating a configuration of a surface plasmon resonance fluorescence analysis apparatus (SPFS apparatus) 100 according to Embodiment 2 of the present invention.
  • SPFS apparatus 100 includes excitation light irradiating unit (excitation light irradiating section) 110 , excitation light detecting unit (excitation light detecting section) 220 , fluorescence detecting unit (fluorescence detecting section) 130 , liquid feeding unit 140 , conveyance unit 150 and control processing section (processing section) 160 .
  • Excitation light detecting unit 220 includes first light receiving sensor 221 and first sensor controlling section 122 .
  • First light receiving sensor 221 detects the quantity and the position of reflection light ⁇ .
  • light receiving sensor 221 is a position detection device (PSD) or a photodiode (PD) including a plurality of light reception surfaces.
  • PSD position detection device
  • PD photodiode
  • the number of the light reception surfaces of first light receiving sensor 221 is preferably, but not limited to, four or more.
  • first light receiving sensor 221 is a PD including four light reception surfaces.
  • first light receiving sensor 221 is a PSD
  • the size of the light reception surface of first light receiving sensor 221 is set to a size greater than the beam diameter of excitation light ⁇ from the viewpoint of correctly detecting the irradiation position of excitation light ⁇ .
  • the length of one side of the light reception surface of first light receiving sensor 221 is preferably 3 mm or greater.
  • First light receiving sensor 221 is disposed at a position where reflection light ⁇ of excitation light ⁇ reaches.
  • first light receiving sensor 221 is disposed at a position where reflection light ⁇ from incidence surface 21 reaches.
  • first light receiving sensor 221 is disposed at a position where reflection light ⁇ of excitation light ⁇ emitted at an angle for the detection of fluorescence ⁇ or an angle approximately equal to the angle for the detection of fluorescence ⁇ reaches.
  • the irradiation position of excitation light ⁇ slightly varies depending on variation of the incident angle, and therefore, by setting the incident angle of excitation light ⁇ for positioning of analysis chip 10 and the incident angle of excitation light ⁇ for detection of fluorescence ⁇ to values equal to or approximately equal to each other, the accuracy of positioning at the time of detection of fluorescence ⁇ can be enhanced.
  • the emission angle of excitation light ⁇ to the normal of metal film 30 (straight line along the z-axis direction in FIG. 1 ) is approximately 70 degrees
  • reflection light ⁇ from incidence surface 21 advances in a direction approximately parallel to the travelling direction of the conveyance stage (x-axis direction in FIG. 1 ). Accordingly, first light receiving sensor 221 is disposed at a position where the reflection light ⁇ that travels in parallel reaches.
  • SPFS apparatus 200 acquires information relating to the power of excitation light irradiating unit 110 and the irradiation direction of excitation light ⁇ as output information of excitation light irradiating unit 110 . Then, when it is determined that the power of excitation light irradiating unit 110 is abnormal, SPFS apparatus 200 adjusts the power of excitation light irradiating unit 110 . In addition, when it is determined that the irradiation direction of excitation light ⁇ is abnormal, excitation light irradiating unit 110 stops the subsequent operations.
  • FIG. 12 is a flowchart of an exemplary procedure of an operation of SPFS apparatus 200 .
  • FIG. 13 is a flowchart of steps in the step of adjusting the position and the output illustrated in FIG. 12 (step S 320 ).
  • the abnormality detection step (step S 3202 and step S 3203 ) is included in the step of adjusting the position and the output (step S 320 ).
  • analysis chip 10 is installed at an installation position of SPFS apparatus 200 (step S 300 ). To be more specific, analysis chip 10 is installed in chip holder 154 of SPFS apparatus 200 .
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a liquid feeding position (step S 305 ).
  • control processing section 160 operates liquid feeding unit 140 to introduce the sample solution in chemical liquid chip 141 into channel 41 of analysis chip 10 (step S 310 ).
  • channel 41 a detection object substance is captured on metal film 30 by an antigen-antibody reaction (primary reaction). Thereafter, the sample solution in channel 41 is removed, and the interior of channel 41 is washed with the washing solution. It is to be noted that when moisturizing agent is present in channel 41 of analysis chip 10 , the interior of channel 41 is washed prior to the introduction of the sample solution to remove the moisturizing agent so that the capturing body can appropriately capture the detection object substance.
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a position near the measurement position (step S 315 ).
  • control processing section 160 operates excitation light irradiating unit 110 , excitation light detecting unit 220 and conveyance stage 152 to acquire the position information of analysis chip 10 and the output information of excitation light irradiating unit 110 .
  • Control processing section 160 adjusts the position of analysis chip 10 on the basis of the acquired position information, and adjusts the output of excitation light irradiating unit 110 on the basis of the acquired output information (step S 320 ).
  • control processing section 160 stops the subsequent steps.
  • control processing section 160 determines whether the irradiation direction of excitation light ⁇ from excitation light irradiating unit 110 is normal (step S 3202 ).
  • information relating to the irradiation direction of excitation light ⁇ of excitation light irradiating unit 110 in the initial state is stored.
  • information relating to the irradiation position of reflection light ⁇ with respect to first light receiving sensor 221 (hereinafter referred to also simply as “initial position”) is stored.
  • control processing section 160 compares the position of the irradiation spot on first light receiving sensor 221 of reflection light ⁇ detected in step S 3201 with the initial position preliminarily stored in control processing section 160 to determine whether the irradiation direction of excitation light ⁇ from excitation light irradiating unit 110 is normal.
  • control processing section 160 When it is determined that the irradiation direction of excitation light ⁇ is normal, control processing section 160 continues the subsequent operations, whereas when it is determined that the irradiation direction of excitation light ⁇ is shifted from the desired direction (abnormal), control processing section 160 terminates the operation.
  • step S 3203 The step of detecting abnormality in the power (step S 3203 ), the step of adjusting the power of excitation light irradiating unit 110 (step S 3204 ), the step of disposing analysis chip 10 at an appropriate measurement position on the basis of the position information of analysis chip 10 (step S 3205 ) are respectively similar to steps S 1202 to S 1204 of Embodiment 1, and therefore the descriptions thereof will be omitted.
  • FIG. 14 is a schematic view for describing the step (step S 3201 ) of acquiring the output information of excitation light irradiating unit 110 (information relating to the power and the irradiation direction of excitation light ⁇ ) in the case where a PD including four light reception surfaces is used.
  • the dotted area indicates an irradiation spot of excitation light ⁇ applied to light reception surfaces 221 a to 221 d of first light receiving sensor 221 .
  • first light receiving sensor 221 is a PD including four light reception surfaces, 221 a to 221 d .
  • photoelectric currents I a to I d corresponding to the quantity of applied reflection light ⁇ are generated in light reception surfaces 221 a to 221 d , respectively.
  • the irradiation positions of excitation light ⁇ in light reception surfaces 221 a to 221 d in the plane direction can be specified.
  • control processing section 160 can acquire information relating to the irradiation direction of excitation light ⁇ .
  • the irradiation position of reflection light ⁇ in the vertical direction in FIG. 14 can be specified on the basis of the ratio of (I a +I b ):(I c +I d ), and the irradiation positions of reflection light ⁇ in the lateral direction in FIG. 14 can be specified on the basis of the ratio of (I a +I c ):(I b +I d ).
  • control processing section 160 in control processing section 160 , photoelectric current values I a to I d output from light reception surfaces 221 a to 221 d of first light receiving sensor 221 are stored as the initial position, and control processing section 160 can determine whether the irradiation direction of excitation light ⁇ is shifted by comparing photoelectric current values I a to I d measured in step S 3201 with preliminarily stored photoelectric current values I a to I d .
  • control processing section 160 can measure the intensity of reflection light ⁇ on the basis of the sum (I a +I b +I c +I d ) of photoelectric currents I a to I d output from light reception surfaces 221 a to 221 d , and can acquire information relating to the power of excitation light irradiating unit 110 .
  • Control processing section 160 can determine whether the power of excitation light irradiating unit 110 is lowered by comparing the sum (I a +I b +I c +I d ) of photoelectric currents I a to I d measured in step S 3201 with the sum (I a +I b +I c +I d ) of preliminarily stored photoelectric currents I a to I d .
  • step S 3202 the order of the step of detecting abnormality in the irradiation direction of excitation light ⁇ (step S 3202 ) and the step of detecting abnormality in the power of excitation light irradiating unit 110 (step S 3203 ) is not limited to the above-mentioned order, and the step of detecting abnormality in the irradiation direction of excitation light ⁇ (step S 3202 ) may be performed after the step of detecting abnormality in the power (step S 3203 ).
  • the output information of excitation light irradiating unit 110 (information relating to power and the irradiation direction of excitation light ⁇ ) can be acquired.
  • a PD including a plurality of divided light reception surfaces reflection light ⁇ incident on a region between adjacent light reception surfaces (region where excitation light ⁇ is not received) is not detected, and consequently the quantity of reflection light ⁇ cannot be wholly detected.
  • the quantity of reflection light ⁇ can be wholly detected since the light reception surface of a PSD is one continuous surface. It is therefore preferable to use a PSD as first light receiving sensor 221 from the viewpoint of more correctly detecting the quantity of reflection light ⁇ .
  • control processing section 160 detects the enhancement angle as in step S 125 (step S 325 ).
  • control processing section 160 measures and records the optical blank value as in step S 130 (step S 330 ).
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to a liquid feeding position (step S 335 ).
  • control processing section 160 labels the detection object substance captured on metal film 30 with the fluorescence material as in step S 140 (step S 340 ).
  • control processing section 160 operates conveyance stage 152 to move analysis chip 10 to an appropriate measurement position determined in step S 320 (step S 345 ).
  • control processing section 160 detects fluorescence ⁇ emitted from the fluorescence material labelling the detection object substance captured by the capturing body as in step S 150 (step S 350 ).
  • control processing section 160 subtracts the optical blank value from the detection value of fluorescence ⁇ to calculate the fluorescence intensity correlating with the amount of the detection object substance (step S 355 ).
  • the intensity of the detected fluorescence is converted to the amount, the concentration, and the like of the detection object substance as necessary.
  • the presence or the amount of the detection object substance in the sample solution can be detected.
  • the analysis method and the SPFS apparatus according to Embodiment 2 can detect abnormality in the irradiation direction of excitation light ⁇ from excitation light irradiating unit 110 while achieving the effect of Embodiment 1.
  • FIG. 15 is a graph for describing another method for acquiring information relating to the power of the excitation light irradiating unit.
  • the information relating to the power of excitation light irradiating unit 110 may be acquired on the basis of the inclination of a straight line between point A and point C (a ratio of a variation of a reflection light intensity to a variation of a moving distance of analysis chip 10 ) instead of the point B in FIG. 5B .
  • the inclination of the straight line between point A and point C is stored as the information relating to the power of excitation light irradiating unit 110 .
  • the black circle (•) indicates a detection result of reflection light by the first light receiving sensor in the case where excitation light irradiating unit 110 of the initial state is used
  • the white circle ( ⁇ ) indicates a detection result of reflection light by the first light receiving sensor in the case where excitation light irradiating unit 110 in a low power state is used.
  • a result of the case where the power of excitation light irradiating unit 110 is lowered to 1 ⁇ 2 of the power of the initial state is illustrated.
  • the maximum value of reflection light ⁇ obtained with excitation light irradiating unit 110 in a low power state is halved.
  • the inclination of the inclined part (moving distance: approximately 2320 to approximately 2380 ⁇ m) is also halved.
  • the information relating to the power of excitation light irradiating unit 110 can be acquired also on the basis of a ratio of a variation of a reflection light intensity to a variation of a moving distance of analysis chip 10 .
  • the order of the steps of the analysis method is not limited to Embodiments 1 and 2.
  • the position of analysis chip 10 may be adjusted before the power of excitation light irradiating unit 110 is adjusted, or after the power of excitation light irradiating unit 110 is adjusted, or, while the power of excitation light irradiating unit 110 is adjusted.
  • the enhancement angle may be detected (steps S 125 , S 225 , S 325 ) before the primary reaction (steps S 110 , S 210 , S 310 ).
  • the adjustment of the position (position and output) (steps S 120 , S 220 , S 320 ) is also performed before the primary reaction (steps S 110 , S 210 , S 310 ).
  • step S 135 , S 235 , S 335 it is possible to omit the step of moving analysis chip 10 to the liquid feeding position (steps S 135 , S 235 , S 335 ) that is performed between the primary reaction (steps S 120 , S 220 , S 320 ) and the secondary reaction (steps S 140 , S 240 , S 340 ) in the flowcharts of FIGS. 2, 10, and 12 .
  • step S 105 , S 205 , S 305 the positional accuracy in the step of moving analysis chip 10 to the liquid feeding position (steps S 105 , S 205 , S 305 ) prior to the primary reaction (steps S 120 , S 220 , S 320 ) is enhanced, and, in the primary reaction (steps S 120 , S 220 , S 320 ) and the secondary reaction (steps S 140 , S 240 , S 340 ), an end of syringe 144 of liquid feeding unit 140 can be more surely inserted into analysis chip 10 .
  • the timing when the detection object substance is labeled with the fluorescence material is not limited.
  • the labeling solution may be added to the sample solution to preliminarily label the detection object substance with the fluorescence material before the sample solution is introduced into the channel of analysis chip 10 .
  • the sample solution and the labeling solution may be simultaneously injected into the channel of analysis chip 10 .
  • both the primary reaction and the secondary reaction can be completed (one step method).
  • the detection of the enhancement angle step S 125 , S 225 , S 325
  • the adjustment of the position is performed (steps S 120 , S 220 , S 320 ) before the detection of the enhancement angle.
  • the surface plasmon resonance fluorescence analysis method and the surface plasmon resonance fluorescence analysis apparatus according to the embodiments of the present invention are not limited to this.
  • the surface plasmon resonance fluorescence analysis method and the surface plasmon resonance fluorescence analysis apparatus according to the embodiments of present invention may be used only for acquiring the output information.
  • FIGS. 16A and 16B are graphs showing a relationship between the incident angle and the reflectivity of excitation light.
  • FIG. 16A shows a result of the case where excitation light hits the surface of the prism from the outside of the prism
  • FIG. 16B shows a result of the case where excitation light hits the surface of the prism from the inside of the prism.
  • the solid line indicates p-polarized light
  • the broken line indicates s-polarized light.
  • the relationship between the refractive index of the prism and the reflectivity of p-polarized light of excitation light was calculated.
  • the incident angle of the excitation light to the incidence surface was set to 9 degrees (0.158 rad) such that the incident angle to the metal film is 71 degrees.
  • the reflectivity of the excitation light in the case where the refractive index is varied by ⁇ 0.002 from the reference refractive index, and the reflectivity of the excitation light in the case where the refractive index is varied by ⁇ 0.003 from the reference refractive index were calculated. Results of the calculation are shown in Table 1.
  • the refractive index variation is a variation in refractive index with respect to the reference refractive index 1.527.
  • the change rate of reflectivity [%] is a change rate with respect to a reference reflectivity obtained with a prism having a refractive index of 1.527.
  • the reflectivity of the p-polarized light of the excitation light was approximately 4.2%.
  • the variation of the refractive index was ⁇ 0.002
  • the change rate of the reflectivity was approximately ⁇ 0.61%
  • the variation of the refractive index was ⁇ 0.003
  • the change rate of the reflectivity was approximately ⁇ 0.91%. That is, it was confirmed that as long as the variation of the refractive index is approximately ⁇ 0.003, the change rate of the reflectivity is 1% or lower and the reflectivity is substantially constant.
  • abnormality in the power can be correctly detected by detecting reflection light of excitation light from the prism so as to acquire information relating to the power of the excitation light irradiating unit since, when the incident angle of the excitation light to the prism is small, the reflectivity of the excitation light is substantially constant as long as the prism is made of the same material.
  • the surface plasmon resonance fluorescence analysis method and the surface plasmon resonance fluorescence analysis apparatus according to the embodiments of the present invention can detect a detection object substance with high reliability, and therefore is suitable for laboratory test and the like, for example.

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