Abstract:
Provided is an apparatus having a beam geometry changing device that changes the beam geometry of excitation light, and a control device that controls the beam geometry changing device, with favorable precision, which the apparatus performs a resonance angle scan and to detect a target material.

Description:
FIELD OF INVENTION 
       [0001]    The present invention relates to a surface plasmon-enhanced fluorescence measuring apparatus based on a principle of Surface Plasmon-Enhanced Fluorescence Spectroscopy (SPFS). 
       PRIOR ART 
       [0002]    Detection of, for example, a minimal analyte in a live body has been carried out based on the principle of Surface Plasmon-Enhanced Fluorescence Spectroscopy (SPFS). The Surface Plasmon-Enhanced Fluorescence Spectroscopy (SPFS) is that under a condition in which a laser light (exciting light) radiated from a light source is subject to attenuated total reflection (ATR) on a metal thin film surface, by creating a compression wave (surface plasmon) on the metal thin film surface, photon of the laser light (exciting light) radiated from the light source can be localized at a vicinity of the metal thin film whereby by exciting a fluorescent material in the vicinity of the metal thin film, the minimal analyte and/or an extremely low condensation analyte is detected. 
         [0003]    In recent years, a surface plasmon-enhanced fluorescence measuring apparatus has been developed based on the principle of the above Surface Plasmon-Enhanced Fluorescence Spectroscopy (SPFS), and the technologies thereof are disclosed, for example, in Patent document 1: U.S. Pat. No. 3,294,605 and Patent Document 2: Unexamined Japanese Patent Application Publication No. 2008-102117. 
         [0004]    As  FIG. 11  shows, the surface plasmon-enhanced fluorescence measuring apparatus  10  has a basic configuration provided with a chip structural  108  having a metal thin film  102  on a surface of a dielectric member  106  and a reaction layer  104  on a surface thereof. 
         [0005]    On the dielectric member  106  side of the chip structural  108  a light source  112  is disposed wherein the exciting light b 1  from the light source  112  is introduced into the dielectric member  106  and radiated towards the metal thin film  102 , and a light receiving device  116  to receive a metal thin film reflected light b 2  which is radiated by the light source  112  and reflected by the metal thin film  102 . 
         [0006]    On the other hand, on a reaction layer side  104  of the chip structure  108 , there is disposed a light detection device  120  to received fluorescent light b 3  emitted from a fluorescent substance which has labeled the analyte. 
         [0007]    Incidentally, between the reaction layer  104  and the light detection device  120 , a light collection member  122  to collect the fluorescent light b 3  efficiently and a filter  124  to select a necessary fluorescent light by eliminating light other than the fluorescent light b 3  are disposed. 
         [0008]    To use the surface plasmon-enhanced fluorescence measuring apparatus  10 , on a surface of the metal thin film there is immobilized in advance a first antibody to bind specifically to an antigen such as DNA, representing a detection object, included in the analyte. The analyte and a second antibody to bind specifically to the analyte are fed subsequently to the reaction layer  104  in contact with the metal thin film  102 , so that the second antibody is trapped on the reaction layer  104 . The trapped second antibody along with the analyte is labeled by the fluorescent substance. 
         [0009]    The light source  112  radiates the exciting light b 1  on reaction layer  104  where the second antibody is the trapped and the exciting light is introduced into the metal thin film  102  with a specific angle (resonance angle) θ 1 , whereby a compression wave (surface plasmon) occurs on the metal thin film. Incidentally, when the compression wave (surface plasmon) occurs, the exciting light b 1  and electronic oscillation in the metal thin film are coupled then a phenomenon of a light quantity reduction of metal thin film reflection light b 2  occurs. 
         [0010]    As a pair, the light receiving device  116  and the light source  112  rotate around a radiation area of the metal thin film  102  to change the incident angle to the metal thin film  102 . By changing the incident angle, a point at which the signal of the metal thin film reflection light b 2  received by a light receiving device  116  is changed (reduced) is found, whereby the resonance angle θ 1  where the compression wave (surface plasmon) occurs can be obtained. 
         [0011]    In accordance with the change of the incident angle, the signal of the metal thin film reflection light b 2  received by the light receiving device  116  changes (reduces). Corresponding to the reduction of the light quantity, the compression wave (surface plasmon) occurs locally at the vicinity of the metal thin film. When this occurs, an incident angle at which the light quantity is minimized or an angle near the above incident angle is obtained as the resonance angle θ 1 . 
         [0012]    By the above phenomenon of occurrence of the compression wave (surface plasmon), the fluorescent substance of the reaction surface  104  on the metal thin film  102  is effectively exited, whereby the light quantity of the fluorescent light b 3  emitted by the fluorescent substance increases. 
         [0013]    The enhanced fluorescent light b 3  is received by the light detection device  120  thought a converging member  122  and a filter  124 , whereby the analyte of infinitesimal quantity or extremely low concentration can be detected. 
         [0014]    As above, the surface plasmon-enhanced fluorescence measuring apparatus  10  is a high-sensitive measuring sensor capable of observing microscopic molecular activity particularly among biological molecules. 
       PRIOR ART DOCUMENT 
     Patent Document 
       [0000]    
       
         Patent Document 1: U.S. Pat. No. 3,294,605 
         Patent Document 2: Unexamined Japanese Patent Application Publication No. 2008-102117 
       
     
       DISCLOSURE OF THE INVENTION 
     Problems to be Solved by the Invention 
       [0017]    In a scanning process where the incident angle of the exciting light with respect to the metal thin film  102  is changed to obtain the resonance angle, the radiation area of the exciting light is preferred to be inside of a reaction area of the reaction layer  104 . On the other hand, in a detection process to detect the analyte by radiating the exciting light, it is preferred that the entire reaction area is irradiated so as to capture a signal from the labeling agent with a high sensitivity. Therefore, it was difficult to satisfy both of them. 
         [0018]    The present invention has one aspect to solve the above problems and an object of the present invention is to perform both of the resonance angle scanning and the detection of the labeled substance with high accuracy. 
       Means to Solve the Problems 
       [0019]    The above object is achieved by the followings. 
         [0000]    1. A surface plasmon-enhanced fluorescence measuring apparatus which, by radiating an exciting light on one side of a metal thin film so as to enhance an electric field on the metal thin film, excites a fluorescent substance on a reaction layer formed on another side of the metal thin film and, by a detection device, detects an enhanced fluorescent light from the excited fluorescent substance, the surface plasmon-enhanced fluorescence measuring apparatus, comprising: a beam geometry changing section to change a beam geometry of an exciting light; and a control section to control the beam geometry changing section,
 
2. The surface plasmon-enhanced fluorescence measuring apparatus of item 1, wherein the control section controls the beam geometry changing section so as to change the beam geometry of the exciting light so as to magnify or reduce a radiation area of the exciting light with respect to the metal thin film, wherein the radiation area in a detection process to detect the fluorescent substance is greater than the radiation area in a resonance angle scanning process to detect a resonance angle.
 
3. The surface plasmon-enhanced fluorescence measuring apparatus of item 2, wherein the radiation area in the resonance angle scanning process occupies a part of a reaction layer area and the radiation area in the detection process occupies the reaction layer area.
 
4. The surface plasmon-enhanced fluorescence measuring apparatus of item 2 or 3, further comprising a flow path in which an analyte solution and a reagent solution including the fluorescent goods sent to the reaction layer, wherein the control section controls in a way that the radiation area of the exciting light, which is radiated to detect a fluid sending status in a fluid sending process to send the analyte solution and the reagent solution to the reaction layer, is greater than the radiation area in the detection process.
 
5. The surface plasmon-enhanced fluorescence measuring apparatus of item 2, further comprising a flow path in which an analyte solution and a reagent solution including the fluorescent goods are sent to the reaction layer, wherein the control section controls the beam geometry changing section so that the radiation area in the resonance angle scanning process occupies a part of a reaction layer area, the radiation area in the detection process coincides with the reaction layer area and the radiation area in a fluid sending process to send an analyte solution and a reagent solution to the reaction layer occupies a reaction layer area and an outside of the reaction layer area.
 
6. The surface plasmon-enhanced fluorescence measuring apparatus of any one of item 1 to 5, wherein the surface plasmon-enhanced fluorescence measuring apparatus controls in a way that the beam geometry of the exciting light is changed by the beam geometry changing section in accordance with change of the incident angle so as to cancel change of the radiation area of the exciting light on the metal thin film due to the change of an incident angle of the exciting light into the metal thin film, whereby the radiation area of the exciting light on the metal thin film remains constant.
 
       Effect of the Invention 
       [0020]    According to the present invention, by providing the beam geometry changing section to change the beam geometry of the exciting light and the control section to control the above section, the resonance angle scanning and the detection of the labeled substance agent can be accurately preformed. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]      FIG. 1  is a schematic diagram of a surface plasmon-enhanced fluorescent measuring apparatus related to an embodiment. 
           [0022]      FIG. 2   a  is across-sectional view of a vicinity of a microchip  14 . 
           [0023]      FIG. 2   b  is a top view of a vicinity of a microchip  14 . 
           [0024]      FIG. 3  is a schematic diagram showing a beam geometry changing section  130   b  using a diaphragm mechanism. 
           [0025]      FIG. 4  is a schematic diagram showing a beam geometry changing section  130   b  using a beam expander. 
           [0026]      FIG. 5  is a schematic diagram showing a beam geometry changing section  130   b  using a zoom lens. 
           [0027]      FIG. 6  shows a flow chart showing a control flow executed by the control device  13 . 
           [0028]      FIG. 7   a  is a schematic diagram showing a positional relation between an exciting light b 13  and a reaction layer  104  in a resonance angle scanning process. 
           [0029]      FIG. 7   b  is a schematic diagram showing a cross-sectional shape of the exciting light b 12  after beam geometry changing in the resonance angle scanning process. 
           [0030]      FIG. 8   a  is a schematic diagram showing a positional relation between an exciting light b 13  and a reaction layer  104  in a detection process to detect an analyte. 
           [0031]      FIG. 8   b  is a schematic diagram showing a cross-sectional shape of the exciting light b 12  after beam geometry changing in the detection process. 
           [0032]      FIG. 9   a  and  FIG. 9   b  are exemplary modifications of  FIG. 8   a  and  FIG. 8   b  respectively. 
           [0033]      FIG. 10   c  is an exemplary modification using a dielectric member  106 . 
           [0034]      FIG. 10   a  and  FIG. 10   b  are corresponding to  FIG. 7   a  and  FIG. 7   b  respectively. 
           [0035]      FIG. 11  is a schematic view of a conventional surface plasmon-enhanced fluorescent measuring apparatus. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0036]    The present invention will be described based on the embodiments without being limited thereto. 
         [0037]      FIG. 1  and  FIG. 2  are schematic views of a surface plasmon-enhanced fluorescent measuring apparatus. 
         [0038]    The surface plasmon-enhanced fluorescent measuring apparatus is to detect fluorescent light generated by an excited fluorescent substance by creating a compression wave (surface plasmon) by radiating an exciting light on a metal thin film, thus the surface plasmon-enhanced fluorescent measuring apparatus enables detection of the fluorescent light with an ultrahigh accuracy though the detection sensitivity is increased. 
         [0039]    [Surface Plasmon Enhanced Fluorescent Measuring Apparatus  10  and Analyte Detection Method] 
         [0040]    As  FIG. 1  shows, the surface plasmon-enhanced fluorescent measuring apparatus  10  of the present invention is provided with a chip structure  108  having a metal thin film  102 , a reaction layer  104  formed on one surface side of the metal thin film  102  and a dielectric member  106  formed on the other surface side. 
         [0041]    On an dielectric member  106  side of the chip structure  108 , a light source  112  to emit an exciting light wherein the exciting light is introduced into the dielectric member  106  and radiated towards the metal thin film  102 , and a light receiving device  116  to receive a metal thin film reflection light b 2  which is the light from the light source  112  reflected by the metal thin film  102 . 
         [0042]    A laser light is preferable as the light radiated from the light source  112  and a gas laser or a solid-state laser having wavelength of 200 to 1000 nm and a semiconductor laser having the wave length of 385 to 800 nm are preferable. Beam geometry of the exciting light b 1  radiated from the light source  112  is changed by the beam geometry changing section  130 . Hereinafter, in case the exciting light b 1  is distinguished between before and after shape changing by the beam geometry changing section  130 , the exciting lights are called exciting light b 11  and exiting light b 12  respectively. Also, the exciting light passing through the inside of the dielectric member  106  and radiated onto the metal thin film  102  is called an exciting light b 13 . Further a collective term of the above lights is an exciting light b 1 . A radiation area on the metal thin film  102  irradiated by the exciting light b 13  is called a radiation area b 131  (refer to  FIG. 7  and so forth). A configuration of the beam geometry changing section  130  will be described later. 
         [0043]    On the other hand, on the reaction layer  104  side of the chip structure  108 , a light detection device  120  to receive the fluorescent light b 3  created in the reaction layer  104  is disposed. 
         [0044]    As the light detection device  120 , a photomultiplier tube having ultrahigh sensitivity or a CCD image sensor capable of multiple-point measuring is preferable to be used. 
         [0045]    The control device  13  is provided with a CPU and a memory. By executing programs stored in the memory each of devices such as the light beam  112 , the beam geometry changing section  130 , the light receiving device  116  and the light detection device  120  are controlled. 
         [0046]    Incidentally, between the reaction layer  104  of the chip structure  108  and the light detection device  120  a converging member  122  to and a filter  124  are disposed. The converging member  122  is to converge the light effectively and the filter  124  is configured to selectively transmit the fluorescent light b 3  among the lights by reducing transmission of lights having different wavelength from the fluorescent light b 3 . 
         [0047]    As the converging member  122 , discretional converging system can be used as far as the fluorescent signal can be converged efficiently on the light detection device  120 . As a simple converging system, an object lens marketed to be used in a microscope can be used. The magnification of the object lens is preferred to be 10 to 100. 
         [0048]    On the other hand, as a filter  124  an optical filter and a cut filter can be used. As the optical filter, a natural density filter (ND filter), a diaphragm lens are cited. Further a the cut filter, for example, a dichroic filter and color filter are cited, which removes various kinds of noises such as outside light (illumination light outside the apparatus), exciting light (transmissive element of the exciting light), stray light (scattering element of the exciting light at various portions), scattering light of the plasmon (scattering light originated from the light source caused by the structure or foreign maters on a surface of the plasmon exciting sensor) and self fluorescence of enzyme fluorescent substance. 
         [0049]    In the analyte detection method using the above surface plasmon-enhanced fluorescent measuring apparatus  10 , a SAM (Self-Assembled Monolayer) to which a first antibody is coupled or a polymer film are provided on a surface of the metal thin film on a side in contact with the reaction layer  104 . The first antibody is couple on one surface of the Self-Assembled Monolayer film or the polymer film, and on the other surface of the Self-Assembled Monolayer or the polymer film is fixed onto a surface of the metal thin film  102  directly or indirectly. As the Self-Assembled Monolayer film, for example, a film formed by substituted aliphatic thiol such as HOOC—(CH 2 ) 11 —SH, and as the polymer material, for example, polyethylene glycol and MPC polymer are cited. The above materials can be prepared at the point of use or a substrate formed by coupling the above materials in advance can be used. Also, a polymer having reactive group (or functional group substitutional for reactive group) reacting with the first antibody can be immobilized on the substrate directly then the first antibody can be immobilized thereon. When coupling the antibody and the polymer using various kinds of reactive groups, amidation condensation reaction through succinimidyl and addition reaction through maleimide are popular. 
         [0050]    In a solution “sending process”, a solution (hereinafter called analyte solution as well) containing an antigen of an analyte representing a target substances and a reagent solution containing a secondary antibody are sent to the reacting layer  104  configured as above. The antigen can be trapped by the immobilized first antibody. Further, by reacting the reagent solution containing the secondary antibody labeled by the fluorescent substance with the above solution, the trapped antigen is labeled. Incidentally, the secondary antibody can react with the antigen in advance then the first antibody can react with them afterwards. 
         [0051]    In the “detection process” to detect the analyte labeled by the fluorescent substance, the light source  112  radiates the exciting light b 1  onto the dielectric member  106  having the reacting layer  104  where the analyte is trapped. The exciting light b 1  is introduced to the metal thin film  102  at a specific incident angle (resonance angle θ 1 ) with respect to the metal thin film  102 , whereby, the compression wave (surface plasmon) occurs on the metal thin film. 
         [0052]    Incidentally, when the compression wave (surface plasmon) occurs on the metal thin film, the exciting light b 1  and the electronic oscillation in the metal thin film are coupled and the signal of the metal thin film reflection light b 2  is changed (light quantity reduces), whereby a point where the signal of the metal thin film reflection light b 2  received by the light receiving device  116  becomes minimum due to change of the signal (reduction of the light quantity) can be found. 
         [0053]    By the above compression wave (surface plasmon), the fluorescent substance created in the reaction layer  104  on the metal thin film  102  is effectively excited, whereby a light quantity of the fluorescent light b 3  emitted by the fluorescent substance increases. By the light detection device  120  to receive the fluorescent light b 3  via the converging member  122  and the filter  124 , the analyte of infinitesimal or extremely low condensation can be detected. 
         [0054]    Incidentally, as a material of the metal thin film of the chip structure  108  is preferable to be configured with a metal selected from a group consist of gold, silver, aluminum, copper and platinum and more preferable to be configured with gold and furthermore an alloy of the above metals. 
         [0055]    The above metals are suitable for the metal thin film  102  because the electric field enhancement is strengthen by the compression wave (surface plasmon). 
         [0056]    As manufacturing methods of the metal thin film  102 , for example, a spattering method, a vapor deposition method (a resistance heat vapor deposition method, an electron beam vapor deposition method and so forth), an electrolytic plating method, and a non-electrolytic plating method are cited. In particular among them the spattering method and the vapor deposition method are preferred since adjustment of metal thin film forming conditions is easy. 
         [0057]    Further the thickness of the metal thin film  102  is preferable in the following ranges: 
         [0058]    Gold: 5 to 500 nm 
         [0059]    Silver: 5 to 500 nm 
         [0060]    Aluminum: 5 to 500 nm 
         [0061]    Cupper: 5 to 500 nm 
         [0062]    Platinum: 5 to 500 nm 
         [0063]    Ally of the above metals: 5 to 500 nm 
         [0064]    From view point of electric filed enhancement, the following ranges are preferred: 
         [0065]    Gold: 20 to 70 nm 
         [0066]    Silver: 20 to 70 nm 
         [0067]    Aluminum: 10 to 50 nm 
         [0068]    Cupper: 20 to 70 nm 
         [0069]    Platinum: 20 to 70 nm 
         [0070]    Ally of the above metals: 10 to 70 nm 
         [0071]    The compression wave (surface plasmon) can easily occur if the thickness of the metal thin film falls  102  within the above ranges. As far as the thickness of the metal thin film  102  falls within the above ranges, the size (lateral×longitudinal) of the metal thin film is not limited. 
         [0072]      FIG. 2   a  is a cross-section of the chip structure  108  and  FIG. 2   b  is a partial top view of the chip structure  108 . As the figures show, the reaction layer  104  is disposed on a surface layer of the metal thin film  102  and on a lower layer side in a flow path  143  disposed in a quartz substrate  142 . The quartz substrate  142  is supported by fixing member  141 . A reagent solution consisting of the secondary antibody labeled by the fluorescent substance and an analyte solution consisting of the analyte are sent to the flow path  143  by an unillustrated pump. 
         [0073]    As the analyte, blood, serum, plasma, urine, nasal fluid, saliva, feces, and body cavity fluid (spinal fluid, ascites fluid and pleural effusion) are cited. The analyte concluded in the analyte solution is, for example, nucleic acid (single-strand or double strand DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), nucleotide, nucleotide and molecular modification of nucleotide), protein (polypeptide, and oligopeptide), amino acid (modified amino acid is included), glucide (oligosaccharide, polysaccharide chain and sugar chain), lipid, or molecular modification and complex thereof are cited. Specifically, the analyte can be carcinoembryonic antibody such as AFP (a fetoprotein), tumor marker, signal transducer and (a) hormone without being limited thereto. 
         [0074]    The fluorescent substance is not limited as far as the substance emits fluorescent light b 3  by being irradiated by a predetermined exciting light b 1  or by using a field effect. The fluorescent light b 3  in the present Specification includes various kinds of luminescence such as phosphorescence. 
         [0075]    As the dielectric member  106 , a prism having an angle of 60 degree with a high deflective index can be used. As a high deflection material, various kinds of transparent inorganic substances, natural polymers and synthetic polymers can be used. From the view points of chemical stability, manufacturing stability and optical transparency, silica dioxide (SiO2), titanium dioxide (tiO2), polycarbonate (PC), cycloolefin polymer (COP), and optical polyester (OKP: manufactured by Osaka Gas Chemical Co., Ltd.) can be used. 
         [0076]    The above surface plasmon-enhanced fluorescent measuring apparatus  10  has an angle changing section (unillustrated) to adjust an optimum angle (resonance angle θ 1 ) for surface plasmon resonance by the exciting light b 1  to be radiated on the metal thin film  102  from the light source  112 . 
         [0077]    Here, the angle changing section (unillustrated) controlled by the control device  13  is able to change the angle in the range of 45 to 85° by rotation via a servo motor of the angle changing section around an radiation area synchronized with the light receiving device  116  and the light source  112  to seek conditions of attenuated total reflection (ATR) in the “resonance angle scanning process”. A resolution thereof is preferred to be not less than 0.01°. 
         [0078]    [Beam Geometry Modification Section] 
         [0079]      FIG. 3  to  FIG. 5  are schematic diagrams showing examples of beam geometry changing sections  130 . In the present invention, meaning of beam the shape changing includes not only changing the geometry of the beam but magnification and reduction of a beam diameter. The beam geometry changing sections  130   a ,  130   b  and  130   c  (the beam geometry changing  130  is a collective term of the above sections) are controlled by the control device  13 . 
         [0080]      FIG. 3  is a schematic diagram showing the beam geometry changing section  130   a  using a diaphragm mechanism. In the above figure, a gear wheel  131   g  is meshed with a rack gear  107 L formed on a diaphragm member drive rack  107 . The gear wheel  131   g  is beard by a boss formed at an unillustrated frame. The gear wheel  131   a  meshes with a gear wheel section  135   g  formed at an outer circumference of a diaphragm opening/closing ring  135 . An axle hole  132  is formed on a diaphragm fin  131  so that the diaphragm fin  131  is rotatably supported by a boss formed on a diaphragm fin supporting ring  136 . Also, a pin  133  is implanted on the diaphragm fin so that the diaphragm pin  133  is inserted in a groove section  134  formed on the diaphragm ring  135 . With the above configuration, by moving the diaphragm member drive rack  107  in an arrow direction in the figure, the diaphragm open/close ring  135  rotates via the gear wheel  131   g  whereby multiple diaphragm fins  131  rotate around the axis hole  132  to perform an aperture function as an iris diaphragm. The beam geometry changing section  130   a  can narrow the shape of the exciting light b 1  radiated from the light source  112 . Incidentally, in the example shown in  FIG. 3 , while the exciting light b 1  is narrowed in a shape of a circle centering around the light axis, the exciting light b 1  can be narrowed in a shape of a square centering around the light axis with four or two diaphragm fins  131 . 
         [0081]      FIG. 4  is a schematic diagram showing the beam geometry changing section  130   b  using a beam expander. In  FIG. 4 , the beam geometry changing section  130   b  to serve as the beam expander is configured with a first lens LS 1 , a second lens LS 2  formed by jointing a positive lens and a negative lens. In the beam geometry changing section  130   b  in  FIG. 4 , by moving at least the first lens or the second lens in the light axis direction via an unillustrated lens moving device, a beam diameter of the exciting light b 11  radiated form the light source  112  can be changed. 
         [0082]      FIG. 5  is a schematic diagram showing a beam geometry changing section  130   c  using a zoom lens. In  FIG. 5 , the beam geometry changing section  130   c , to serve as a zoom lens, configured with a first lens LS 3  and a second lens LS 4  having a shorter focal length than that of the first lens LS 3 , can change the beam diameter with be a predetermined magnification percentage or a reduction percentage in accordance with the ratio of the focal length. Incidentally, there can be a configuration that one of the above lenses is configured with a lens group and one lens in the lens group is moved in the light axis direction by a moving mechanism, wherein the magnification and reduction percentages can be changed by changing the focal length of the lens group. 
         [0083]    Also, the beam geometry changing section  130  can be a combination of configurations in  FIG. 3  and  FIG. 5 . Also in  FIG. 4  and  FIG. 5 , while a spherical lens is used, a cylindrical lens can be used to change the magnification and reduction percentages. The configurations of the beam geometry changing section shown in  FIG. 3  to  FIG. 5  are examples without the present invention being limited thereto and the other configurations to change the beam geometry capable of changing the cross-sectional area, the beam diameter, and the beam geometry can be used. 
         [0084]    [Beam Geometry Control Flow] 
         [0085]    Control of beam geometry changing will be described with reference to the  FIG. 6  to  FIG. 8 . 
         [0086]      FIG. 6  is a flow chart showing a control flow executed by the control device  13 . 
         [0087]    [Beam Geometry in Resonance Angle Scanning Process] 
         [0088]    In Step S 11  in  FIG. 6 , the beam geometry changing section  130  conducts geometry changing with respect to the exciting light b 1  so that the beam geometry becomes a desired beam geometry set in advance. In Step S 12 , the resonance angle scanning process is executed with the beam geometry changed in Step S 11 . Here, the beam geometry changed in Step S 11  is an optimum beam geometry for the resonance angle scanning process, which will be described with reference to  FIG. 7 . 
         [0089]      FIG. 7   a  is a schematic diagram showing a positional relation between a reaction layer  104  and a radiation area b 131  by the exciting light b 13  on the metal thin film  102  in the resonance angle scanning process, and  FIG. 7   b  is a schematic diagram showing a cross-sectional shape of the exciting light b 12  after beam geometry changing in the resonance angle scanning process. As mentioned above, in the resonance angle scanning process executed in Step S 12 , the incident angle to the metal thin film  102  swings while synchronizing the light receiving device  116  and the light source  112 , so as to seek an incident angle (positions of the light receiving device  116  and the light source  112 ) where attenuated total reflection occurs. 
         [0090]    The resonance angle θ 1  changes with a chemical condition of the surface of the metal thin film  102 . In order to obtain the resonance angle θ 1  with accuracy in the resonance angle scanning process, the exciting light b 13  has to be radiated on an area of the reaction layer  104  where the first antibody is immobilized on the surface of the metal thin film  102 . Because in case the exciting light b 13  is radiated in a way that the radiation area b 131  locates partially or totally at outside the reaction layer  104  on the metal thin film  102 , the resonance angle θ 1  can not be obtained accurately. In the present embodiment, the beam geometry of the exciting light b 12  is optimized in advance by the beam geometry changing section  130  so that the radiation area b 131  of the exciting light b 13  falls within the area of the reaction layer  104  in a changing range of the incident angle in the resonance angle scanning process. 
         [0091]    In the present embodiment, (1) The metal thin film  102  is formed on a surface of the dielectric member having the refraction index of n=1.515. The dielectric member  106  is a prism of 60° and 25 mm on a side. A size of the reaction layer  104  is 1.0 mm (Y direction)×3.0 mm (X direction). (2) The exciting light b 1  is a laser light having a wavelength of 633 nm. A scanning range of the incident angle of the exciting light b 1  into the metal thin film  102  in the resonance angle scanning process is 79°±3°. The incident angle referred here is an incident angle where refraction by introducing into the dielectric member does not occur. Hereinafter, when the term of “incident angle” is used simply, it means the above mentioned incident angle. The incident angle of exciting light b 13  into the metal thin film  102  after the optical path is bent on the surface of the dielectric member  106  in the incident angle of 79°±3° is 70.48° to 74.32° in calculation. 
         [0092]    In  FIG. 7   a  the radiation area b 131  of the exciting light b 13  at incident angles 82°, 76° and 79° which are an upper limit, a lower limit and an intermediate of scanning range are denoted by x 1 , x 3  and x 2  respectively. As  FIG. 7   a  shows in accordance with change of the incident angle, the size of and the radiation position of the radiation area b 131  of the exciting light b 13  on the surface of the metal thin film  102  change. 
         [0093]    In the present embodiment, in the scanning rage of the incident angle in order that the radiation area b 131  of the exciting light b 13  falls within the area (1.0×3.0 mm) of the reaction layer  104 , it is calculated that the shape of the exciting light b 12  is adequate to be a circle of 40.3 mm. In Step S 11 , the control device  13  controls the beam geometry changing section  130  to conduct geometry changing to achieve the above geometry. Incidentally, with respect to the exciting light b 12  of φ0.3 mm, the size of the radiation area b 131  of the exciting light b 13  on the surface of the metal thin film  102  is 1.161 mm (X direction diameter) at x 1 (82°), and 0.918 mm (X direction diameter) at x 3 (76°). 
       Other Embodiment 
       [0094]    As above, in accordance with change of the incident angle of the exciting light, the size and the radiation position of the radiation area b 131  of the exciting light b 13  on the surface of the metal thin film changes. In another example it is possible to control that the size of the radiation area b 131  of the exciting light b 13  on the metal thin film surface  102  remains constant by changing the geometry of the beam of the exciting light b 12  via the beam geometry changing section  130  in accordance with change of the incident angle so as to cancel the change of the size. For example, in case the incident angle is changed in the range from x  1 (82°) to x  3 (76°), the beam geometry of the exciting light b 12  is changed in accordance with the change of the incident angle so that the diameter in x direction is a constant value of 0.90 mm. 
         [0095]    In the above manner, by maintaining the size of the area to be irradiated, an effect due to change of the size of the radiation area with respect to the light quantity of the fluorescent light b 3  can be inhibited. 
         [0096]    [Beam Geometry in Fluid Sending Process] 
         [0097]    In the fluid sending process, as described in the forgoing, the analyte solution and the reagent solution including the second antibody labeled by the fluorescent substance are sent to the reaction layer  104 . As mentioned above, since the first antibody is immobilized on the metal thin film  102  in contact with the reaction layer  104 , by sending the analyte solution and the reagent solution, the antigen included in the analyte solution to specifically react with the first antibody is trapped, and the second antibody to react specifically with the antigen is trapped. 
         [0098]    In Step S 21  in  FIG. 6 , the beam geometry change section  130  conducts geometry change with respect to the exciting light b 1  so that the beam geometry is changed to be a beam geometry set in advance. In Step S 22 , using the beam geometry changed in the Step S 21 , fluid sending control in the fluid sending process is conducted. Fluid sending control is to (detect) observe a condition of fluid sending that whether or not the analyte solution and the reagent solution are sent normally. In fluid sending control, for example, whether or not the fluid is sent normally or whether or not bubbles are created in the flow path or not is detected. 
         [0099]    In fluid sending control, besides the reaction layer  104 , the flow path  143  at vicinity thereof also has to be observed. In the fluid sending control, the beam geometry has to be set so that the radiation area covers at least entire area of the reaction layer  104  or the radiation area covers the entire reaction layer  104  and an outside thereof. Thus the beam geometry is set in a way that the radiation area (on the metal thin film surface  102 ) of the fluid sending control (Step S 22 ) is wider than the that of a detection process (Step S 32 ) to be described (Step S 21 ). Whereby, besides the reaction layer  104 , the fluid sending condition of the fluid in the vicinity flow path  143  can be controlled. 
         [0100]    Incidentally, in the fluid sending, detection is not necessarily via the fluorescent light b 3  generated by the surface plasmon on the surface of the metal thin film  102 . The detection can be carried out by detecting reflection light or scattering light on the surface of the metal thin film  102  via the light receiving device  116 . In the above case, the exciting light b 1  does not have to be introduced with the resonance angle θ 1 . Also, since there can be a problem that by giving a strong energy to the fluorescent substance at a time other than the detection, discoloration of a pigment causes reduction of sensitivity and quantitative accuracy, the detection carried out by the reflection light or the scattering light is more preferable than detection (observation) by the electric filed enhanced by the surface plasmon. The intensity or the wavelength of the exciting light b 1  can be changed. 
         [0101]    [Beam Geometry in Detection Process] 
         [0102]    In Step S 31  in  FIG. 6 , the beam geometry changing section  130  conducts geometry changing with respect to the exciting light b 1  so as to form a beam geometry set in advance. In Step S 32 , using the beam geometry changed in the Step S 31 , detection process is executed. Here the beam geometry set in Step S 31  is an optimum beam geometry for the detection process. The followings are descriptions with reference to  FIG. 8 . 
         [0103]      FIG. 8   a  is a schematic diagram showing a positional relation between the reaction layer  104  and the radiation area b 131  of the exciting light b 13  on the metal thin film  102  in the detection process to detect the analyte.  FIG. 8   b  is a schematic diagram showing a cross-section of the exciting light b 12  after beam geometry changing in the detection process. 
         [0104]    As described above, in the resonance angle scanning process, the size of the exciting light b 13  has to be reduced so that the exciting light b 13  is not radiated outside the reaction layer  104 . On the other hand in the detection process, in order to detect the fluorescent substance, which has labeled the analyte trapped across the entire area of the reaction layer  104 , it is preferred that the exciting light b 13  is radiated across the reaction layer  104  as wide as possible. 
         [0105]    In the present embodiment, the control device  13  controls the beam geometry changing section  130  so that the radiation area of the detection process is wider than that in the resonance angle scanning process. Specifically, in the resonance angle scanning process, a partial area of the reaction layer  104  is irradiated, and the geometry of the exciting light b 12  is determined in a way that at the incident angle (resonance angle) into the metal thin film  102  determined in the resonance angle scanning process, the radiation area b 131  of the exciting light b 13  in the detection process substantially coincides with an entire area of the reaction layer  104  or the radiation area b 131  substantially coincides with the area of the reaction layer  104 . In  FIG. 8   b  the beam geometry changing section  130  changes the geometry of the cross-section of the exciting light b 12  to a circle of Ø10 mm. With the above geometry, as  FIG. 8   a  shows, it is revealed that the radiation area b 131  of the exciting light b 13  covers most of the reaction layer  104 . Incidentally, in accordance with the value of the incident angle (resonance angle) determined in the resonance angle scanning process, the position of the radiation area differs, thus the beam geometry is formed so as to radiate the most of the reaction layer  104  even at the upper and lower limits in the scanning range of the incident angle. Also, it is preferred that with a configuration to move the chip structure  108  in the Z axis direction by the position adjusting mechanism  14  shown in  FIG. 1 , by adjusting in the Z axis direction via the position adjusting mechanism  14  in accordance with the resonance angle θ 1 , the center of the radiation area of the exciting light introduced with the resonance angle θ 1  coincides with the center of the reaction layer  104 . 
         [0106]    According to the present embodiment, the control section  13  controls the beam geometry changing section  130  to adequately change the beam geometry of the exciting light, whereby both the resonance angle scanning and the detection of the labeling substance can be accurately conducted. 
         [0107]    [Exemplary Modification] 
         [0108]      FIG. 9  shows an exemplary modification of one in  FIG. 8 . In the embodiment shown by  FIG. 9 , the beam geometry changing section  130  is a diaphragm mechanism shown in  FIG. 3 , which can form the beam geometry to be a square shape. In the example shown by  FIG. 9   b , the beam geometry changing section  130  forms the cross-section of the exciting light b 12  to be a square 1.0 mm on a side. Whereby, as  FIG. 9   a  shows, in case the reaction layer  104  (reaction area) is an rectangle, by forming the beam geometry similar to the shape thereof the radiation area b 131  of the exciting light b 13  in the detection process can cover the entire area of the reaction layer  104  easily, and the signals from the fluorescent substance in a wider area than that in  FIG. 8  can be detected unfailingly. 
       Other Embodiment 
       [0109]      FIG. 10   a  to  FIG. 10   c  show, an embodiment where the dielectric member  106  in a shape of a half circle is used as an exemplary modification. In the embodiment shown by  FIG. 10 , the radiation position of the exciting light b 13  on the metal thin film  102  can be fixed irrespective of the incident angle as described later. 
         [0110]    As  FIG. 10   c  shows, in case the dielectric member  106  in the shape of the half circle is used, in the resonance angle scanning process, even if the incident angle is changed by moving the light source  112  and the light receiving device  116 , by radiating the exciting light b 1  towards the center of the half circle of the dielectric member  106 , the exciting light b 1  is always radiated onto the surface of the dielectric member  106  form a normal line direction. Whereby, bending of the light path on the surface of the dielectric member  106  does not occur, therefore as  FIG. 10   a  shows, the center of the radiation area b 131  of the exciting light b 13  on the reaction layer  104  always stays at the same position even if the radiation area b 131  is changed as x 1  to x 3  due to change of the incident angle. 
         [0111]    In  FIG. 7   a  considering change of the radiation position of the exciting light b 13  and change of size in the radiation area, the beam geometry has been set. However, in the embodiment shown by  FIGS. 10   a  to  10   c , change of the size only has to be considered. As a result, as  FIG. 10   b  shows the beam geometry changed by the beam geometry changing section  130  can be set to be a circle of approximately Ø 0.8 mm (dielectric member  106  incident position) whose length in the X direction is extended compared with the one in  FIG. 7   b . Incidentally, in case the dielectric member  106  in the shape of the half circle is used, the exciting light b 12  has to be a diverging light so that the incident light (exciting light b 13 ) inside the dielectric member  106  is a parallel light. 
         [0112]    As  FIG. 1  shows, in an embodiment in which a triangle dielectric member  106  is used, by interlocking the incident angle and the position of the chip structure  108  calculated from a size and a refraction index of the dielectric member via the position adjusting mechanism  14 , the radiation area, which moves in accordance with the incident angle, can be maintained at a center of a reaction place (reaction layer  104 ), and the beam geometry can be controlled considering only change of the area size of the radiation area of the exciting light due to change of the incident angle. 
         [0113]    Further, as the former embodiment and the other embodiments by applying a configuration that the beam geometry changing section  130  changes the beam geometry of the exciting light b 12  in accordance with the change of the incident angle of the exciting light, control that the size and the center position of the radiation area b 131  of the exciting light b 1  on the metal thin film  102  remain constant is possible. 
       DESCRIPTION OF THE SYMBOLS 
       [0000]    
       
         
           
               10  Enhanced fluorescent measuring apparatus 
               13  Control device 
               14  Position adjusting mechanism 
               130 ,  130   a ,  130   b  and  130   c  Beam geometry changing section 
             b 1 , b 11 , b 12  and b 13  Exciting light 
             b 131  Radiation area 
             b 2  Metal thin film reflection light 
             b 3  Fluorescent light 
               102  Metal thin film 
               104  Reaction layer 
               106  Dielectric member 
               18  Chip structure