Abstract:
First, an analytical chip having a prism, a metal film that includes a trapping region having immobilized on the surface thereof a trapping element for trapping a substance to be analyzed, and a mark in which the scatter of emitted plasmon scattered light differs from the scatter of plasmon scattered light emitted from the surrounding region, is disposed in a chip holder. Next, the rear surface of the metal film is irradiated with excitation light, plasmon scattered light emitted from the proximity of the mark is detected, and, on the basis of the detected plasmon scattered light, location information for the trap region is obtained. Next, the portion of the rear surface of the metal film that corresponds to the trap region arranged at the detected location is irradiated with excitation light, and fluorescence emitted by a fluorescent substance is detected.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    Technical Field 
         [0002]    The present invention relates to a surface-plasmon enhanced fluorescence measurement method and a surface-plasmon enhanced fluorescence measurement device for detecting a detection-target substance contained in a sample solution, using surface plasmon resonance (SPR), and also relates to an analysis chip used in detection of the detection-target substance contained in the sample solution. 
         [0003]    Background Art 
         [0004]    Highly-sensitive and quantitative detection of a slight amount of a detection-target substance in measurement for detecting biological substances such as protein and DNA makes it possible to immediately figure out the condition of a patient and treat the patient. For this reason, there has been a demand for an analysis method and an analysis device for highly-sensitive and quantitative detection of weak light caused by a slight amount of the detection-target substance. As an exemplary method of detecting a detection-target substance with high sensitivity, a surface plasmon resonance fluorescence analysis (Surface Plasmon-field enhanced Fluorescence Spectroscopy (SPFS)) is known. 
         [0005]    SPFS uses a prism provided with a metal film disposed on a predetermined surface of the prism. Irradiation of the metal film with excitation light through the prism at an angle at which surface plasmon resonance occurs can generate localized light (enhanced electric field) on the surface of the metal film. This localized light excites a fluorescent substance used for labeling the detection-target substance captured on the metal film, therefore making it possible to detect the presence or amount of the detection-target substance through detection of the fluorescence emitted from the fluorescent substance. 
         [0006]    In SPFS, highly-sensitive and accurate detection requires accurate positioning of the analysis chip. Accurate detection of the amount (density) of the detection-target substance requires accurate adjustment of the incident angle of excitation light. However, when the analysis chip is shifted in position, accurate adjustment of the incident angle of the excitation light is impossible. In addition, the irradiation spot of the excitation light and the reaction site on the metal film preferably coincide with each other in shape and position for highly-sensitive detection of the detection-target substance. However, when the analysis chip is shifted in position, the irradiation spot of excitation light cannot be adjusted accurately in shape and position. Meanwhile, requiring users to accurately position the analysis chip is unfavorable in terms of usability. 
         [0007]    There have been proposed methods for positioning an analysis chip in methods for detecting a detection-target substance by irradiating the analysis chip with light although they are not SPFS. For example, Patent Literature (hereinafter, referred to as “PTL”) 1 discloses identifying the position of an analysis chip (biochip) through irradiation of the analysis chip with illumination light different from excitation light in wavelength and detection of reflection light or transmission light of the illumination light in detection using a fluorescent substance. The use of illumination light different from excitation light in wavelength makes it possible to identify the position of the analysis chip while preventing the fluorescent substance from being discolored. 
       CITATION LIST 
     Patent Literatures 
       [0008]    PTL 1 
         [0009]    Japanese Patent Application Laid-Open No. 2007-093250 
       SUMMARY OF THE INVENTION 
     Technical Problem 
       [0010]    The positioning method disclosed in PTL 1 has a problem that the manufacturing costs of analysis chips increase because the method requires addition of a light source different from the excitation light source, and a wavelength limiting filter, for example. 
         [0011]    An object of the present invention is to provide a surface-plasmon enhanced fluorescence measurement method, a surface-plasmon enhanced fluorescence measurement device, and an analysis chip each enabling accurate positioning of the analysis chip while preventing an increase in manufacturing costs of the analysis chip and surface-plasmon enhanced fluorescence measurement device. 
       Solution to Problem 
       [0012]    To solve the above-mentioned problems, a surface-plasmon enhanced fluorescence measurement method according to an embodiment of the present invention is a method in which fluorescence that is emitted from a fluorescent substance for labeling a detection-target substance when the fluorescent substance is excited by localized light based on surface plasmon resonance is detected to detect the presence or amount of the detection-target substance, the method including: installing an analysis chip to a chip holder fixed to a conveyance stage, the analysis chip including: a prism having an incidence surface, an emission surface, and a film-formation surface; a metal film disposed on the film-formation surface and including a capturing region having a surface to which a capturing body for capturing the detection-target substance is fixed; and one or more marks each of which is formed on a plane identical to that of the metal film and in which a scattered state of plasmon scattering light emitted from the mark is different from a scattered state of plasmon scattering light emitted from a region around the mark; irradiating a rear surface of the metal film corresponding to the mark in the analysis chip installed to the chip holder with excitation light through the incidence surface, detecting plasmon scattering light emitted from the vicinity of the mark, and obtaining position information of the capturing region based on the detected plasmon scattering light; moving the chip holder by the conveyance stage based on the position information to move the captured region to a detection position; and irradiating a rear surface of the metal film corresponding to the capturing region disposed at the detection position with excitation light and detecting fluorescence emitted from the fluorescent substance for labeling the detection-target substance captured by the capturing body. 
         [0013]    To solve the above-mentioned problems, a surface-plasmon enhanced fluorescence measurement device according to an embodiment of the present invention is a device configured to detect fluorescence that is emitted from a fluorescent substance for labeling a detection-target substance when the fluorescent substance is excited by localized light based on surface plasmon resonance and to detect the presence or amount of the detection-target substance, the device including: a chip holder configured to detachably hold an analysis chip including: a prism having an incidence surface, an emission surface, and a film-formation surface; a metal film disposed on the film-formation surface and including a capturing region having a surface to which a capturing body for capturing the detection-target substance is fixed; and a mark in which a scattered state of plasmon scattering light emitted from the mark is different from a scattered state of plasmon scattering light emitted from a region around the mark; a conveyance stage configured to move the chip holder; an excitation-light irradiating section configured to irradiate a rear surface of the metal film with excitation light through the incidence surface; a plasmon-scattering-light detecting section configured to detect plasmon scattering light emitted from the metal film; a position adjustment section configured to identify a position of the capturing region of the analysis chip held by the chip holder, based on a detection result of the plasmon-scattering-light detecting section on plasmon scattering light based on the excitation light with which a rear surface of the metal film corresponding to the mark is irradiated, and to move the chip holder via the conveyance stage to move the capturing region of the analysis chip to a detection position; and a fluorescence detection section configured to detect fluorescence emitted from a fluorescent substance for labeling the detection-target substance captured by the capturing body. 
         [0014]    An analysis chip according to an embodiment of the present invention is a chip configured to be used for detecting fluorescence that is emitted from a fluorescent substance for labeling a detection-target substance when the fluorescent substance is excited by localized light based on surface plasmon resonance and to be used for detecting the presence or amount of the detection-target substance, the chip including: a prism including: an incidence surface, an emission surface, and a film-formation surface; a metal film disposed on the film-formation surface of the prism and including a capturing region having a surface to which a capturing body for capturing the detection-target substance is fixed; and a positioning mark in which a scattered state of plasmon scattering light emitted from the positioning mark is different from a scattered state of plasmon scattering light emitted from a region around the positioning mark. 
       Advantageous Effects of Invention 
       [0015]    According to the present invention, accurate positioning of an analysis chip can be realized. Thus, according to the present invention, highly-sensitive and accurate detection of a detection-target substance is made possible while an increase in manufacturing costs is prevented. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIG. 1  is a diagram schematically illustrating a configuration of an SPFS device according to Embodiment 1 of the present invention; 
           [0017]      FIG. 2  is a diagram illustrating a positional relationship between a capturing region and a mark; 
           [0018]      FIG. 3  is a flowchart illustrating an operation procedure of the SPFS device illustrated in  FIG. 1 ; 
           [0019]      FIG. 4  is a flowchart illustrating steps in a positioning step (S 140 ) illustrated in  FIG. 3 ; and 
           [0020]      FIG. 5  is a schematic view for describing a step (S 141 ) of obtaining position information on an end portion of the capturing region in an analysis chip. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0021]    Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying drawings. 
         [0022]      FIG. 1  is a schematic view illustrating a configuration of surface-plasmon enhanced fluorescence measurement device (SPFS device)  100  according to an embodiment of the present invention.  FIG. 2  is a diagram illustrating a positional relationship between a capturing region and a mark. 
         [0023]    As illustrated in  FIG. 1 , SPFS device  100  includes excitation-light irradiation unit  110 , response-light detection unit  130 , liquid-feeding unit  140 , conveyance unit  150 , and control section  160 . SPFS device  100  is used in a state where analysis chip  10  is attached to chip holder  154  of conveyance unit  150 . Thus, a description will be given of analysis chip  10 , first, followed by a description of each component of SPFS device  100 . 
         [0024]    [Configuration of Analysis Chip] 
         [0025]    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 ; mark  50  disposed on film-formation surface  22  or metal film  30 ; and channel closure  40  disposed on film-formation surface  22  or metal film  30 . Usually, analysis chip  10  is replaced for each analysis. 
         [0026]    Prism  20  is composed of a dielectric which is transparent to excitation light α. Prism  20  includes incidence surface  21 , film-formation surface  22 , and emission surface  23 . Incidence surface  21  is a surface through which excitation light α from excitation-light irradiation unit  110  enters prism  20 . Metal film  30  is disposed on film-formation surface  22 . Excitation light α having entered prism  20  is reflected by the rear surface of metal film  30 . More specifically, excitation light α 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 α reflected by the rear surface of metal film  30  is emitted out of prism  20 . 
         [0027]    Prism  20  is not limited to any particular shape. In this embodiment, prism  20  is in a columnar shape having a trapezoid as its bottom surface. The surface of the columnar shape corresponding to one bottom side of the trapezoid is film-formation surface  22  while the surface thereof corresponding to one leg of the trapezoid is incidence surface  21 , and the surface thereof corresponding to the other leg of the trapezoid is emission surface  23 . Preferably, the trapezoid serving as the bottom surface is an isosceles trapezoid. This configuration makes incidence surface  21  and emission surface  23  symmetric and makes it harder for an S-wave component of excitation light α to stay in prism  20 . 
         [0028]    Incidence surface  21  is formed such that excitation light α does not return to excitation-light irradiation unit  110 . When the light source of excitation light α is a laser diode (hereinafter, may be referred to as “LD”), returning of excitation light α to the LD disturbs the excitation state of LD, causing the wavelength and the output of excitation light α to vary. Thus, the angle of incidence surface  21  is set within a scanning range around an ideal enhanced angle to prevent excitation light α from perpendicularly entering incidence surface  21 . In this embodiment, the angle between incidence surface  21  and film-formation surface  22  and the angle between film-formation surface  22  and emission surface  23  are each approximately 80 degrees. 
         [0029]    Note that, the design of analysis chip  10  substantially determines the resonance angle (and the enhanced angle in the close vicinity thereof). The design factors include 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 α, the refractive index of a measurement solution introduced into a channel during measurement, and the like. While the resonance angle and the enhanced angle shift due to the detection-target substance fixed to metal film  30 , the amount of shit in this case is less than several degrees. 
         [0030]    Prism  20  has a birefringence property to a certain degree. Examples of the material of prism  20  include a resin and glass. Preferably, the material of prism  20  is a resin having a refractive index of 1.4 to 1.6 and causing a small birefringence. 
         [0031]    Metal film  30  is disposed on film-formation surface  22  of prism  20 . Thus, interaction (surface plasmon resonance) occurs between photons of excitation light α incident on film-formation surface  22  under the total reflection condition and free electrons in metal film  30 , thus making it possible to generate localized light on the surface of metal film  30 . 
         [0032]    The material of metal film  30  is not limited to any particular one as long as a metal capable of causing surface plasmon resonance is employed. Examples of the material of metal film  30  include gold, silver, copper, aluminum, and their alloys. In this embodiment, metal film  30  is a gold thin film. The method of forming metal film  30  is not limited to any particular one. Examples of the method of forming metal film  30  include sputtering, vapor deposition, and plating. The thickness of metal film  30  is not limited to any particular thickness, but preferably is in a range from 30 to 70 nm. 
         [0033]    Capturing region A is disposed on at least a part of the surface of metal film  30  not facing prism  20  (top surface of metal film  30 ). A capturing body for capturing the detection-target substance is fixed to capturing region A. Fixing the capturing body to the region enables selectively detecting the detection-target substance. The planar view shape of capturing region A is not limited to any particular shape. Examples of the planar view shape of capturing region A include a circle and a polygon. Note that, in this embodiment, the planar view shape of capturing region A is a circle. The type of capturing body is not limited to a particular one as long as it is capable of capturing the detection-target substance. In this embodiment, the capturing body is an antibody specific to the detection-target substance or a fragment of the antibody. 
         [0034]    As illustrated in  FIG. 2 , mark  50  is disposed on film-formation surface  22  of prism  20 , or metal film  30 . Mark  50  serves as a reference for positioning capturing region A. Although a detailed description will be given hereinafter, the positioning of capturing region A is performed based on a scattered state of plasmon scattering light emitted from the vicinity of mark  50  and the surrounding region thereof. For this reason, as long as mark  50  is formed to cause the scattered state of plasmon scattering light emitted therefrom to be different from the scattered state of plasmon scattering light emitted from a region other than mark  50 , mark  50  is not limited to any particular one. Examples of mark  50  include a protrusion or a recess formed in film-formation surface  22 , an exposed shaped film, a patterned metal film, and a seal attached to metal film  30 . 
         [0035]    As described above, irradiation with excitation light α while metal film  30  is disposed on film-formation surface  22  generates plasmon scattering light γ. Thus, the intensity of plasmon scattering light γ generated at mark  50  decreases when mark  50  is exposed film-formation surface  22  or patterned metal film  30 , so that the position where the intensity of plasmon scattering light γ generated at mark  50  has decreased is detectable as mark  50 . 
         [0036]    Plasmon scattering light γ is generated when excitation light α enters metal film  30  on film-formation surface  22 , and the intensity of plasmon scattering light γ depends on the incident angle of excitation light α. As will be described hereinafter, in order to detect fluorescence β of high intensity, fluorescence β is measured at an angle where the largest light amount of plasmon scattering light γ can be obtained or at an angle close to this angle in actual measurement. When mark  50  is a protrusion or a recess formed in film-formation surface  22 , there exists a surface at an angle different from the angle of film-formation surface  22  and metal film  30  in analysis chip  10 . Since the intensity of plasmon scattering light γ depends on the incident angle of excitation light α, the position where the intensity of plasmon scattering light γ changes can be detected as mark  50 . 
         [0037]    Moreover, when a seal having a light blocking property is used as mark  50 , since plasmon scattering light γ generated from mark  50  decreases, the position where the intensity of plasmon scattering light γ decreases can be detected as mark  50 . Meanwhile, when a seal having no light blocking property is used as mark  50 , use of a material having a refractive index different from the refractive index of the liquid in channel  41  as the material of the seal makes it possible to detect the position where the intensity of plasmon scattering light γ changes, as mark  50 . The intensity of plasmon scattering light γ changes depending on 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 α, the refractive index of the measurement liquid introduced into the channel during measurement, and/or the like. Stated differently, since the intensity of plasmon scattering light γ differs depending on whether or not a seal is present on metal film  30 , or whether or not a measurement solution is present, the position where the intensity of plasmon scattering light γ changes can be detected as mark  50 . As described above, the use of plasmon scattering light γ to be detected makes it possible to detect the position of mark  50 . 
         [0038]    Mark  50  is preferably formed in a structure opposite to the traveling direction of excitation light α when metal film  30  is viewed in its normal direction. This is because irradiation of the portion of the opposite structure with excitation light α cause the scattered state of plasmon scattering light γ to change more significantly, thus making it possible to increase the detection accuracy for the position of mark  50 . The effect of forming this structure opposite to excitation light α is more effective than that of the protrusion or recess or the patterning shape of metal film  30  described above. 
         [0039]    Moreover, the position where mark  50  is formed is not limited to any particular position. Mark  50  may be disposed inside or outside of capturing region A when metal film  30  is viewed from its normal direction. In this embodiment, mark  50  is disposed at a position outside of capturing region A. Moreover, the position where mark  50  is formed may overlap with the light path of excitation light α or may be outside of the light path of excitation light α when metal film  30  is viewed from its normal direction. When mark  50  is disposed inside of capturing region A in particular, the positioning to be described hereinafter can be more surely performed because a distance between mark  50  and the measurement region during measurement is small. Meanwhile, disposing mark  50  outside of capturing region A is also preferable because mark  50  does not hinder detection of fluorescence when fluorescence β is detected from a fluorescent substance through irradiation with excitation light α within capturing region A after the positioning to be described, hereinafter. Furthermore, mark  50  may be disposed in a conveyance direction in which analysis chip  10  is conveyed with respect to capturing region A when metal film  30  is viewed from its normal direction. As a result, both mark  50  and capturing region A can be irradiated with excitation light α, using conveyance unit  150  of analysis chip  10 . 
         [0040]    Moreover, the number of marks  50  is not limited in particular. The number of marks  50  may be one or more. In this embodiment, the number of marks  50  is one. The area of mark  50  when metal film  30  is viewed from its normal direction is not limited in particular either. In this embodiment, the area of mark  50  is preferably of a size that fits into the irradiation spot of irradiation light when metal film  30  is viewed from its normal direction. When the area of mark  50  in planar view is larger than the area of the irradiation spot of irradiation light in planar view, it may become difficult to accurately identify the position of mark  50 . 
         [0041]    Moreover, when two or more marks  50  are disposed, marks  50  are formed in a direction orthogonal to the conveyance direction of analysis chip  10 , for example. In this case, disposing analysis chip  10  or excitation-light irradiation unit  110  or a drive mechanism in which excitation-light irradiation unit  110  and response light detection unit  130  are integrally driven (not illustrated) makes it possible to perform positioning in two directions (irradiation direction of excitation light α and its orthogonal direction), using two marks  50 . In another case where two or more marks  50  are disposed, two marks  50  are formed at positions opposite to each other with respect to capturing region A when metal film  30  is viewed in its normal direction, for example. Detecting the positions of two marks  5  makes it possible to figure out that capturing region A is positioned at a middle point between the two positions, so that, even when the detection accuracy for one of marks  50  is low, the position of capturing region A can be accurately detected. 
         [0042]    Channel closure  40  is disposed on metal film  30 . When metal film  30  is formed on only a part of film-formation surface  22  of prism  20 , channel closure  40  may be disposed on film-formation surface  22 . A channel groove is formed in the rear surface of channel closure  40 , and channel closure  40  forms channel  41  together with metal film  30  (and prism  20 ) through which liquid flows. Examples of the liquid include a sample solution containing the detection-target substance, a labeling solution containing an antibody labeled by a fluorescent substance, a washing solution and the like. Capturing body A of metal film  30  is exposed inside of channel  41 . Both ends of channel  41  are respectively connected to an inlet and outlet (not illustrated) formed in the top surface of channel closure  40 . When liquid is injected into channel  41 , the liquid makes contact with the capturing body of capturing region A. 
         [0043]    Channel closure  40  is preferably composed of a material transparent to fluorescence β and plasmon scattering light γ emitted from metal film  30 . Examples of the material of channel closure  40  include a resin. As long as the part of channel closure  40  used for taking out fluorescence β and plasmon scattering light γ to the outside is transparent to fluorescence β and plasmon scattering light γ, another part of channel closure  40  may be formed of an opaque material. Channel closure  40  is bonded to metal film  30  or prism  20  by adhesion using a double-sided tape, an adhesive and/or the like, laser welding, ultrasound welding, or pressure bonding using a clamp member and/or the like, for example. 
         [0044]    As illustrated in  FIG. 1 , excitation light α enters prism  20  from incidence surface  21 . Excitation light α having entered prism  20  is incident on metal film  30  at a total reflection angle (angle at which surface plasmon resonance occurs). Metal film  30  is thus irradiated with excitation light α at an angle which surface plasmon resonance occurs, and thus, localized light (which is also generally called “evanescent light” or “near-field light”) can be generated on metal film  30 . This localized light excites the fluorescent substance labeling the detection-target substance existing on metal film  30 , and fluorescence β is emitted. SPFS device  100  can detect the presence or amount of the detection-target substance through detection of the light amount of fluorescence β emitted from the fluorescent substance. 
         [0045]    [Configuration of SPFS Device] 
         [0046]    Next, the configuration elements of SPFS device  100  will be described. As described above, SPFS device  100  includes excitation-light irradiation unit  110 , response-light detection unit  130 , liquid-feeding unit  140 , conveyance unit  150  and control section  160 . 
         [0047]    Excitation-light irradiation unit  110  emits excitation light α to analysis chip  10  (rear surface of metal film  30 ) held by chip holder  154 . During measurement of fluorescence β, excitation-light irradiation unit  110  emits only P-wave with respect to metal film  30  toward incidence surface  21  such that the incident angle with respect to metal film  30  is an angle at which surface plasmon resonance occurs. The term “excitation light” used herein is light which directly or indirectly excites a fluorescent substance. For example, excitation light α is light generating localized light which excites a fluorescent substance on the surface of metal film  30  when excitation light α is emitted to metal film  30  through prism  20  at an angle at which surface plasmon resonance occurs. In SPFS device  100  according to this embodiment, excitation light α is used also for positioning of analysis chip  10 . 
         [0048]    Excitation-light irradiation unit  110  includes a configuration for emitting excitation light α toward prism  20 , and a configuration for changing the incident angle of excitation light α with respect to the rear surface of metal film  30 . In this embodiment, excitation-light irradiation unit  110  includes light source unit  111 , angle-adjustment mechanism  112  and light-source control section  113 . 
         [0049]    Light source unit  111  emits collimated excitation light α having a constant wavelength and a constant light amount 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 α, a beam-shaping optical system, an APC mechanism and a temperature adjustment mechanism (which are not illustrated). 
         [0050]    The light source is not limited to any particular type, and is a laser diode (LD), for example. Other examples of the light source include a light-emitting diode, a mercury lamp, and other laser light sources. When 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 and/or the like. In addition, when 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 grid or the like. Furthermore, when the light emitted from the light source is not linear polarization, the light emitted from the light source is converted into light of linear polarization by a polarizer and/or the like. 
         [0051]    The beam-shaping optical system includes a collimator, a bandpass 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 some or all of these components. The collimator collimates excitation light α emitted from the light source. The bandpass filter changes excitation light α emitted from the light source into narrowband light composed only of a central wavelength. This is because excitation light α from the light source has a slight wavelength distribution width. The linear polarization filter changes excitation light α emitted from the light source into complete linearly polarized light. The half-wave plate adjusts the polarization direction of excitation light α such that the P-wave component is incident on metal film  30 . The slit and the zooming unit adjust the beam diameter, the outline shape and/or the like of excitation light α such that the shape of the irradiation spot on the rear surface of metal film  30  has a circular shape having a predetermined size. 
         [0052]    The APC mechanism controls the light source so as to keep the output of the light source constant. More specifically, the APC mechanism detects the light amount of the light diverged from excitation light α by a photodiode (not illustrated) or the like. The APC mechanism then controls the input energy by a recurrent circuit to control the output of the light source to remain constant. 
         [0053]    The temperature adjustment mechanism is composed of a heater, a Peltier device, or the like, for example. The wavelength and energy of the light emitted from the light source may vary depending on the temperature. Therefore, keeping the temperature of the light source constant using the temperature adjustment mechanism controls the wavelength and energy of the light emitted from the light source to remain constant. 
         [0054]    Angle-adjustment mechanism  112  adjusts the incident angle of excitation light α to the rear surface of metal film  30  (the interface (film-formation surface  22 ) between prism  20  and metal film  30 ). Angle-adjustment mechanism  112  relatively turns the optical axis of excitation light α and chip holder  154  to emit excitation light α toward a predetermined position of the rear surface of metal film  30  at a predetermined incident angle through prism  20 . 
         [0055]    For example, angle-adjustment mechanism  112  turns light source unit  111  around an axis orthogonal to the optical axis of excitation light α (axis perpendicular to the sheet surface of  FIG. 1 ). At this time, the position of the turning axis is set such that the position of the irradiation spot on metal film  30  barely changes even when the incident angle is changed. Setting the position of the turning center at a position near the intersection of the optical axes of two rays of excitation light α at both ends of the scanning range of the incident angle (position between the irradiation position on film-formation surface  22  and incidence surface  21 ) makes it possible to minimize shifting of the irradiation position. 
         [0056]    In the incident angle of excitation light α with respect to the rear surface of metal film  30 , the angle at which the largest light amount of plasmon scattering light γ is obtainable is the enhanced angle. Setting the incident angle of excitation light α to the enhanced angle or an angle close to the enhanced angle enables measurement of fluorescence β of high intensity. While 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 channel  41  and the like determine the basic incident condition of excitation light α, the optimum incident condition slightly varies depending on the type and amount of detection-target object captured in channel  41 , non-specific adsorption of a foreign substance in the sample, shaping errors of prism  20  and the like. Therefore, the optimum enhanced angle is preferably determined for each measurement. In this embodiment, the favorable emission angle of excitation light α with respect to the normal of metal film  30  (straight line along the z-axis direction in  FIG. 1 ) is approximately 70 degrees. 
         [0057]    Light-source control section  113  controls various components included in light source unit  111  to control emission of emission light (excitation light α) of light source unit  111 . Light-source control section  113  is composed of a publicly known computer, microcomputer and/or the like including an arithmetic unit, a controller, a storage, an input unit, an output unit, and/or the like, for example. 
         [0058]    Response-light detection unit  130  detects fluorescence β generated by irradiation of the rear surface of metal film  30  with excitation light α during detection of a detection-target object, and plasmon scattering light γ generated by irradiation of the rear surface of metal film  30  with excitation light α during positioning of analysis chip  10  and during measurement of the enhanced angle. Response-light detection unit  130 , for example, includes light reception unit  131 , position-switching mechanism  132  and sensor control section  133 . 
         [0059]    Light reception unit  131  is disposed in the normal direction of metal film  30  of analysis chip  10  (the z-axis direction in  FIG. 1 ). Light reception unit  131  includes first lens  134 , optical filter  135 , second lens  136 , and light reception sensor  137 . 
         [0060]    First lens  134  is, for example, a condenser lens, and condenses the light emitted above 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 light reception sensor  137 . The light paths between the lenses are substantially parallel to each other. Optical filter  135  is disposed between the lenses. 
         [0061]    Optical filter  135  guides only a fluorescent component to light reception sensor  137  and removes the excitation light component (plasmon scattering light γ) in order to detect fluorescence β with a high S/N ratio. Examples of optical filter  135  include an excitation light reflection filter, a short wavelength cut filter, and a bandpass filter. Optical filter  135  is, for example, a filter including a multilayer film that reflects a predetermined light component, but may be a color glass filter that absorbs a predetermined light component. 
         [0062]    Light reception sensor  137  detects fluorescence β or plasmon scattering light γ. The sensitivity of light reception sensor  137  is so high that light reception sensor  137  can detect weak fluorescence β or plasmon scattering light γ from a slight amount of detection-target substance. Light reception sensor  137  is, for example, a photomultiplier tube (PMT), an avalanche photodiode (APD) or the like. 
         [0063]    Position-switching mechanism  132  switches the position of optical filter  135  between a position on the light path and a position outside of the light path in light reception unit  131 . More specifically, optical filter  135  is disposed on the light path of light reception unit  131  when light reception sensor  137  detects fluorescence β, and optical filter  135  is disposed outside the light path of light reception unit  131  when light reception sensor  137  detects plasmon scattering light γ. Position-switching mechanism  132  is composed of a turn driving section and a publicly known mechanism (such as a turntable and a rack-and-pinion) that moves optical filter  135  in a horizontal direction by utilizing turning movement, for example. 
         [0064]    Sensor control section  133  controls detection of an output value of light reception sensor  137 , management of the sensitivity of light reception sensor  137  according to the detected output value, change of the sensitivity of light reception sensor  137  for obtaining an appropriate output value, and the like. Sensor control section  133  is composed of a publicly known computer, microcomputer, and/or the like including an arithmetic unit, a controller, a storage, an input unit, and an output unit, for example. 
         [0065]    Liquid-feeding unit  140  supplies a sample solution, labeling solution, washing solution and/or 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-feeding pump driving mechanism  143 . 
         [0066]    Chemical-liquid chip  141  is a container for housing liquid such as a sample solution, labeling solution, and washing solution. Usually, as chemical-liquid chip  141 , a plurality of containers are disposed in accordance with types of liquid, or a chip formed by integrating a plurality of containers is disposed. 
         [0067]    Syringe pump  142  is composed of syringe  144  and plunger  145  capable of reciprocating in syringe  144 . The reciprocation of plunger  145  quantitatively makes suction and discharge of liquid. When syringe  144  is replaceable, washing of syringe  144  is unnecessary. This configuration is favorable in terms of preventing entry of impurities. When syringe  144  is not replaceable, adding a configuration to wash the inside of syringe  144  makes it possible to use syringe  144  without replacement of syringe  144 . 
         [0068]    Liquid-feeding pump driving mechanism  143  includes a driving unit of plunger  145  and a moving unit of syringe pump  142 . The driving unit of syringe pump  142  is a device for reciprocating plunger  145  and includes a stepping motor, for example. The driving unit including a stepping motor can manage the liquid feed amount and liquid feed speed of syringe pump  142 , so that it is favorable in term of management of the amount of residual liquid of analysis chip  10 . The moving unit of syringe pump  142 , for example, freely moves syringe pump  142  in two directions including the axial direction (e.g., vertical direction) of syringe  144  and a direction crossing the axial direction (e.g., horizontal direction). The moving unit of syringe pump  142  is composed of a robot arm, a biaxial stage or a vertically movable turntable, for example. 
         [0069]    Preferably, liquid-feeding unit  140  further includes a device that detects the position of a leading end of syringe  144  in terms of adjusting the relative heights of syringe  144  and analysis chip  10  to keep them constant, and managing the amount of residual liquid in analysis chip  10  to keep it constant. 
         [0070]    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, moving plunger  145  causes the liquid to reciprocate in channel  41  in analysis chip  10  to agitate the liquid in channel  41 . In this manner, uniformization of the density of liquid, acceleration of reaction (e.g., antigen-antibody reaction) in channel  41 , and/or the like can be achieved. From the view point of performing the above-mentioned operations, analysis chip  10  and syringe  144  are preferably configured such that an inlet of analysis chip  10  is protected with a multilayer film and can be sealed when syringe  144  penetrates through the multilayer film. 
         [0071]    The liquid in channel  41  is again sucked by syringe pump  142  and discharged to chemical-liquid chip  141  and/or the like. Repeating the above-mentioned operations carries out reaction, washing and the like using various kinds of liquid, thereby making it possible to dispose a detection-target substance labeled by a fluorescent substance in capturing region A in channel  41 . 
         [0072]    Conveyance unit  150  conveys analysis chip  10  to a measurement position or a liquid-feeding position and fixes analysis chip  10  thereto. The term “measurement position” herein refers to a position where excitation-light irradiation unit  110  irradiates analysis chip  10  with excitation light α, and response-light detection unit  130  detects fluorescence β or plasmon scattering light γ generated with the irradiation. In addition, the term “liquid-feeding position” herein refers to 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 . Chip holder  154  is fixed to conveyance stage  152  so as to detachably hold analysis chip  10 . Chip holder  154  has a shape capable of holding analysis chip  10  without blocking the light paths of excitation light α. For example, chip holder  154  is provided with an opening through which excitation light α passes. Conveyance stage  152  moves chip holder  154  in a certain direction (X-axis direction in  FIG. 1 ) and a direction opposite to the certain direction. Conveyance stage  152  is driven by a stepping motor or the like, for example. 
         [0073]    Control section  160  controls angle-adjustment mechanism  112 , light-source control section  113 , position-switching mechanism  132 , sensor control section  133 , liquid-feeding pump driving mechanism  143  and conveyance stage  152 . In addition, control section  160  functions also as a position adjustment section that identifies the position of the end portion of capturing region A in analysis chip  10  held by chip holder  154  and moves chip holder  154  by conveyance stage  152  to move capturing region A of analysis chip  10  to an appropriate measurement position on the basis of a detection result of response-light detection unit  130 . Control section  160  is composed of a publicly known computer, microcomputer, and/or the like including an arithmetic unit, a controller, a storage, an input unit, and an output unit, for example. 
         [0074]    Next, a detection operation of SPFS device  100  (the surface-plasmon enhanced fluorescence measurement method according to Embodiment 1 of the present invention) will be described.  FIG. 3  is a flowchart of an exemplary operation procedure of SPFS device  100 .  FIG. 4  is a flowchart illustrating steps in a position adjustment step (S 140 ). 
         [0075]    First, analysis chip  10  is installed in chip holder  154  of SPFS device  100  (S 100 ). Next, control section  160  operates conveyance stage  152  to move analysis chip  10  to a liquid-feeding position (S 110 ). 
         [0076]    Subsequently, control section  160  operates liquid-feeding unit  140  to introduce the sample solution in chemical-liquid chip  141  into channel  41  of analysis chip  10  (S 120 ). In channel  41 , the detection-target 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 a washing solution. Note that, when a 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 in order for the capturing body to appropriately capture the detection-target substance. 
         [0077]    Next, control section  160  operates conveyance stage  152  to move analysis chip  10  to a position near the measurement position (S 130 ). 
         [0078]    Next, control section  160  operates excitation-light irradiation unit  110 , response-light detection unit  130  and conveyance stage  152  to obtain the position information of the center of capturing region A and to adjust the position of capturing region A (analysis chip  10 ) on the basis of the obtained position information (S 140 ). In this step, the region having a shape identical to mark  50  (region of the rear surface of metal film  30 ) and positioned right below mark  50  in analysis chip  10  held by chip holder  154  is irradiated with excitation light α, and plasmon scattering light γ emitted from mark  50  is detected to obtain the position information of the end portion of capturing region A of analysis chip  10  (S 141 ). More specifically, scanning is performed on the irradiation spot on the rear surface of metal film  30  corresponding to mark  50  and the vicinity thereof to detect plasmon scattering light γ emitted from the vicinity of mark  50  and the other region (see  FIG. 5 ). Scattered states (light amounts) of plasmon scattering light γ to be emitted from mark  50  and from the region in the vicinity of mark  50  are different. Thus, the position of mark  50  is identified from variation in the light amount of obtained plasmon scattering light γ. Next, the position of the center of capturing region A is identified from a distance between the center portion of previously set mark  50  and the position of the center portion of capturing region A. Accordingly, the degree of shifting in position of capturing region A from the measurement position can be identified. Next, chip holder  154  is moved by conveyance stage  152  to dispose capturing region A of analysis chip  10  at an appropriate measurement position on the basis of the obtained position information (S 142 ). 
         [0079]    Next, control section  160  operates excitation-light irradiation unit  110  and response-light detection section  130  to irradiate analysis chip  10  disposed at an appropriate position with excitation light α, and to detect plasmon scattering light γ having a wavelength identical to that of excitation light α to detect the enhanced angle (S 150 ). More specifically, control section  160  operates excitation-light irradiation unit  110  to perform scanning of an incident angle of excitation light α with respect to metal film  30  and also operates response-light detection unit  130  to detect plasmon scattering light γ. At this time, controller  160  operates position-switching mechanism  132  to dispose optical filter  135  at a position outside of the light path of light reception unit  131 . Control section  160  then determines the incident angle of excitation light α at which the light amount of plasmon scattering light γ is largest to be the enhanced angle. 
         [0080]    Next, control section  160  operates excitation-light irradiation unit  110  and response-light detection section  130  to irradiate analysis chip  10  disposed at an appropriate measurement position with excitation light α and records an output value (optical blank value) of light reception sensor  137  (S 160 ). At this time, control section  160  operates angle-adjustment mechanism  112  to set the incident angle of excitation light α to the enhanced angle. Furthermore, control section  160  controls position-switching mechanism  132  to dispose optical filter  135  inside the light path of light reception unit  131 . 
         [0081]    Next, control section  160  operates conveyance stage  152  to move analysis chip  10  to the liquid-feeding position (S 170 ). 
         [0082]    Subsequently, control section  160  operates liquid-feeding unit  140  to introduce liquid (labeling solution) containing a secondary antibody labeled by a fluorescent substance into channel  41  of analysis chip  10  (S 180 ). In channel  41 , through an antigen-antibody reaction (secondary reaction), a detection-target substance captured on metal film  30  is labeled by the fluorescent substance. Thereafter, the labeling solution in channel  41  is removed, and the interior of channel  41  is washed with a washing solution. 
         [0083]    Next, control section  160  operates conveyance stage  152  to move analysis chip  10  to the appropriate measurement position determined in step S 140  (S 190 ). 
         [0084]    Next, control section  160  operates excitation-light irradiation unit  110  and response-light detection unit  130  to irradiate analysis chip  10  disposed at the appropriate measurement position with excitation light α and to detect fluorescence β emitted from the fluorescent substance labeling the detection-target substance captured by the capturing body (S 200 ). Control section  160  subtracts the optical blank value from the detection value to calculate the intensity of the fluorescence correlating with the amount of the detection-target substance. The intensity of the fluorescence thus detected is converted into the amount, density, and/or the like of the detection-target substance as appropriate. 
         [0085]    Through the above-mentioned procedure, the presence or amount of the detection-target substance in the sample solution can be detected. 
         [0086]    Note that, the detection of the enhanced angle (S 150 ) may be performed before the primary reaction (S 120 ). In this case, the determination of the measurement position of analysis chip  10  (S 130  and S 140 ) is also performed before the primary reaction (S 110  and S 120 ). Additionally, when the incident angle of excitation light α is determined in advance, detection of the enhanced angle (S 150 ) may be omitted. In this case as well, the determination of the measurement position of analysis chip  10  (S 130  and S 140 ) is also performed before the measurement of an optical blank value (S 160 ). As described above, the determination of the measurement position of analysis chip  10  (S 130  and S 140 ) is favorably performed before an optical measurement (detection of the enhanced angle, measurement of the optical blank value, or detection of the fluorescence) is performed for the first time. 
         [0087]    In the above description, the step in which a detection-target substance and a capturing body are caused to react with each other (primary reaction, S 120 ) is performed is followed by the step in which a detection-target substance is labeled by a fluorescent substance (secondary reaction, S 180 ) (two-step scheme). However, the timing at which the detection-target substance is labeled by the fluorescent substance is not limited to any particular timing. For example, a labeling solution may be added to a sample solution to label the detection-target substance by the fluorescent substance in advance prior to introduction of the sample solution into channel  41  of analysis chip  10 . Moreover, the sample solution and labeling solution may be injected into channel  41  of analysis chip  10  simultaneously. In the former case, injecting the sample solution into channel  41  of analysis chip  10  causes the capturing body to capture the detection-target substance labeled by the fluorescent substance. In the latter case, while the detection-target substance is labeled by the fluorescent substance, the detection-target substance is captured by the capturing body. In either case, the introduction of the sample solution into channel  41  of analysis chip  10  completes both the primary reaction and the secondary reaction (one-step scheme). When one-step scheme is adopted in the manner described above, detection of the enhanced angle (S 150 ) is performed before antigen-antibody reaction, and determination of the measurement position of analysis chip  10  (S 130  and S 140 ) is performed even before the detection of the enhanced angle (S 150 ). 
         [0088]    Moreover, the timing at which positioning step (S 140 ) is performed may not be before the primary reaction (S 120 ) as long as it is performed before detection of the fluorescence emitted from the fluorescent substance obtained by labeling the detection-target substance with fluorescence. For example, positioning step (S 140 ) may be performed before the primary reaction (S 120 ) or after the primary reaction (S 120 ) or after the primary reaction (S 120 ) but before the secondary reaction (S 180 ). 
         [0089]    For SPFS device  100  described above, a description has been given of SPFS device  100  in which conveyance stage  152  moves only in the X direction in  FIG. 1 , but a configuration in which conveyance stage  152  also moves in the Y direction (direction perpendicular to the sheet surface) in  FIG. 1  may be employed. In this configuration, the conveyance stage includes an X-direction moving mechanism to move chip holder  154  in the X direction and a Y-direction moving mechanism to move chip holder  154  in the Y direction. Furthermore, in an SPFS device including conveyance stage  152  movable in a planar direction, scanning of an irradiation spot can be performed in multiple directions, so that the detection accuracy for the end portion of capturing region A can be further improved. The Y-direction moving mechanism may include a driving mechanism configured to drive excitation-light irradiation unit  110 , or to drive excitation-light irradiation unit  110  and response-light detection unit  130  integrally. Note that, two marks  50  may be disposed in this case. 
         [0090]    This application is entitled to and claims the benefit of Japanese Patent Application No. 2014-111298, filed on May 29, 2014, the disclosure of which including the specification and drawings is incorporated herein by reference in its entirety. 
       INDUSTRIAL APPLICABILITY 
       [0091]    The surface-plasmon enhanced fluorescence measurement method and the surface-plasmon enhanced fluorescence measurement device, and the analysis chip according to the present invention enable detection of a detection-target substance with high reliability, and therefore are suitable for laboratory tests and/or the like, for example. 
       REFERENCE SIGNS LIST 
       [0092]      10  Analysis chip 
         [0093]      20  Prism 
         [0094]      21  Incidence surface 
         [0095]      22  Film-formation surface 
         [0096]      23  Emission surface 
         [0097]      30  Metal film 
         [0098]      40  Channel closure 
         [0099]      41  Channel 
         [0100]      50  Mark 
         [0101]      100  SPFS device 
         [0102]      110  Excitation-light irradiation unit 
         [0103]      111  Light source unit 
         [0104]      112  Angle-adjustment mechanism 
         [0105]      113  Light-source control section 
         [0106]      130  Response-light detection unit 
         [0107]      131  Light reception unit 
         [0108]      132  Position-switching mechanism 
         [0109]      133  Sensor control section 
         [0110]      134  First lens 
         [0111]      135  Optical filter 
         [0112]      136  Second lens 
         [0113]      137  Light reception sensor 
         [0114]      140  Liquid-feeding unit 
         [0115]      141  Chemical-liquid chip 
         [0116]      142  Syringe pump 
         [0117]      143  Liquid-feeding pump driving mechanism 
         [0118]      144  Syringe 
         [0119]      145  Plunger 
         [0120]      150  Conveyance unit 
         [0121]      152  Conveyance stage 
         [0122]      154  Chip holder 
         [0123]      160  Control section 
         [0124]    α Excitation light 
         [0125]    β Fluorescence 
         [0126]    γ Plasmon scattering light