Abstract:
Biomolecules are specifically captured with magnetic particles and the biomolecules are labeled with fluorescence. A magnetic field generator, for attracting the magnetic particles to the support substrate, is provided on the reverse face of the support substrate, and an adhesion layer is provided on the surface of the support substrate to hold the magnetic particles. First, a dispersing solution for the magnetic particles is placed on the surface of the support substrate with the magnetic field in an off state. Next, the magnetic field is turned on, and the magnetic particles in solution are attracted to the support substrate surface. The magnetic particles colliding with the support substrate adhere to the adhesion layer of the support substrate surface, and then the magnetic field is turned off. Thus, aggregations can be broken up while the magnetic particles are held, and a magnetic particle layer on the support substrate can be a single layer.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a biomolecule analysis method using magnetic particulates and a biomolecule analyzer. 
       BACKGROUND ART 
       [0002]    In recent years, in the field of cancer diagnosis, various cancer markers have been investigated in order to know a sign of cancer onset in an early stage, and practical applications is progressing. The cancer marker is a secretory biological factor derived from cancer cells, and increases with the progression of the cancer and appears in blood and/or urine. For example, proteins such as hormones and cytokines, and nucleic acid such as micro-RNA are known. In the early stage cancer, the amount of these cancer markers is small and difficult to detect these markers; it is the same situation when there exists the cancer marker of originally low expression level. At present, the mainstream of the high sensitive method for detecting the cancer markers is in the immunoassay method using an antibody, and the techniques such as the ELISA method and the nanoparticulate assay are known. Recently, although it is still in a study stage, as a further high sensitive immunoassay method, a digital ELISA which is capable of detecting by a single molecule has been developed (Non-Patent Literature 1). When the cancer marker in blood is detected, the amount of blood which can be collected from patient is limited; therefore, it is required to detect by capturing a trace amount of the cancer marker contained therein as much as possible. For example, if the detection is performed with 50 μl of plasma, since the cancer marker in the early stage cancer is within the concentration range from 10 −16  to 10 −12  M, the detection sensitivity which can determine quantitatively 3000 molecules of the target molecule present in 50 μl of the plasma is necessary. Thus, detection device with very high sensitivity which is capable of detecting the cancer markers of low concentrations is required. 
       CITATION LIST 
     Non Patent Literature 
       [0003]    Non-Patent Literature 1: Rissin D M et al., Nature Biotechnology, Jun 28(6); p595-599 (2010) 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0004]    In order to determine quantitatively a trace amount of biomolecules, the biomolecules are captured specifically with magnetic particulates, and the biomolecules are labeled with fluorescence. The detection is performed by immobilizing the biomolecules captured on the magnetic particulates to a supporting substrate. The magnetic particulates capturing the biomolecules suspending in a liquid are attracted to the surface of the supporting substrate by a magnetic field. Since the magnetic particulates themselves are magnetized in the presence of a magnetic field, the particulates pull each other on the surface of the supporting substrate and, therefore, the magnetic particulates aggregate. When the magnetic particulates aggregate, the magnetic particulates would overlap in the direction perpendicular to the surface of a plate on the support base. In addition, the aggregated particulates would enfold the fluorescent labels internally. 
         [0005]    There is a problem that the magnetic particulates overlapped in this way or the magnetic particulates enfolding fluorescent labels internally will not be counted accurately at the time of fluorescence observation. 
       SOLUTION TO PROBLEM 
       [0006]    In order to attract the magnetic particulates to the supporting substrate, a magnetic field generator, which can switch on/off, on the back surface of the supporting substrate and an adhesive layer, which holds the magnetic particulates, on the front surface of the supporting substrate are provided. First, a dispersed solution of the magnetic particulates is placed on the front surface of the supporting substrate with the magnetic field of the front surface of the supporting substrate being off. Next, the magnetic field is turned on and the magnetic particulates in the solution are attracted onto the front surface of the supporting substrate. The magnetic particulates collided onto the supporting substrate are forced to adhere to the adhesion layer of the front surface of the supporting substrate and, further, the magnetic field is turned off. 
       ADVANTAGEOUS EFFECTS OF INVENTION 
       [0007]    According to the present invention, the majority of magnetic particulates in the liquid can be attracted to the surface of the support base, and held by the adhesive layer. Further, by turning off the magnetic field, the aggregation among the magnetic particulates can be resolved, and the magnetic particulate layer on the supporting substrate can be made single-layered. By making the magnetic particulate layer single-layered, focusing to the fluorescent dyes on the support base can be performed easily, and by preventing the engulfment of fluorescent dyes due to the aggregation of the magnetic particulates, quantitativity is improved. Since the capture rate of biomolecules, which are the analysis object, can be improved significantly by the biomolecule analysis using the present invention, detection with higher sensitivity than in the prior art is possible. Further, because the device having a complex structure is not required, it is very simple in comparison with the prior art and a significant improvement of throughput can be obtained by combining with the automatic control unit. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0008]    [ FIG. 1 ]  FIG. 1  is a figure for explaining one example of the analysis method of the present example; 
           [0009]    [ FIG. 2 ]  FIG. 2  is a figure for explaining one example of the configuration of the device to be used in the analysis method of the present example; 
           [0010]    [ FIG. 3 ]  FIG. 3  is a figure for explaining one example of the configuration of the device to be used in the analysis method of the present example; 
           [0011]    [ FIG. 4 ]  FIG. 4  is a figure for explaining one example of a method for capturing the antigenic molecules and a method for fluorescence-labeling the antigenic molecules in the present example; 
           [0012]    [ FIG. 5 ]  FIG. 5  is a figure for explaining one example of a method for capturing the nucleic acid fragments and a method for fluorescence-labeling the nucleic acid fragments in the present example; 
           [0013]    [ FIG. 6 ]  FIG. 6  is a figure for explaining one example of the configuration of a biomolecule analyzer of the present example; 
           [0014]    [ FIG. 7 ]  FIG. 7  is a figure for explaining one example of a method for immobilizing the magnetic particulates using a magnet, in the present example; and 
           [0015]    [ FIG. 8 ]  FIG. 8  is a microscopic image of the magnetic particulates immobilized using a magnet, in the present example. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0016]    In the nucleic acid analysis device according to one example of the present invention, biomolecules of the analysis object are captured by magnetic particulates, a device has a smooth supporting substrate for presenting the particulates two-dimensionally, and there exists an adhesive layer for immobilizing the magnetic particulates with antibodies onto the surface of the supporting substrate. As a supporting substrate, a thin quartz glass substrate or a silicon substrate which transmits the magnetic force well is suitable, and according to the type of the adhesive layer, a quartz glass substrate or a silicon substrate deposited with a metal thin film may be used properly. The adhesive surface for immobilizing the magnetic particulates modified with protein is a hydrophobic adhesive layer or a biotin-introduced adhesive layer and, as a hydrophobic adhesive agent, a self-assembled film of alkyl group is used. In addition, here is disclosed use of photoresponsive azobenzene as adhesive which can switch reversibly the strength of adhesive force of the adhesive layer. As to a functional group used for immobilizing to the supporting substrate, a silanol group is used if the substrate is quartz or an oxidation-treated silicon substrate; a thiol group is used if the substrate is a gold-deposited substrate; and a phosphate group is used if the substrate is a titanium-oxide-deposited substrate. A magnetic field generator for attracting the magnetic particulates with biomolecules to the adhesive layer is installed just beneath the supporting substrate and, furthermore, the magnetic field generator is equipped with a function of switching on/off or the intensity levels of the magnetic field. The magnetic field generator is selected from an electromagnet, a movable permanent magnet, a movable electromagnet, and a permanent magnet or an electromagnet equipped with a movable magnetic field shielding plate between the supporting substrate and the magnet. The present device is used while installing the supporting substrate on a movable stage so that fluorescence observation can be performed by scanning the entire surface of the supporting substrate with an imaging device fixed. 
         [0017]    An observation procedure will be explained. At the beginning, a reaction solution containing the magnetic particulates capturing the objective biomolecules is placed on the supporting substrate, a magnetic field is generated next by turning on the magnetic field generator so that all magnetic particulates in the reaction solution are attracted on the support base, and the magnetic particulates contacting with the adhesive layer are immobilized to the supporting substrate. The magnetic particulates which did not contact with the adhesive layer for the first time are increased in collision opportunities of the magnetic particulates with the adhesive layer by repeating the on/off or up-and-down of the magnetic field. After immobilizing the magnetic particulates to the adhesive layer, suspending unreacted fluorescence-labeled antibodies are rinsed away by flushing a cleaning solution. A photograph is taken while irradiating the excitation light to the magnetic particulates immobilized on the supporting substrate and to the fluorescence-labeled antigen bound thereto. By counting the number of observed bright spots, the concentration of the objective biomolecules is determined. 
         [0018]    As for the observation procedure using a device having a function of changing reversibly the adhesive force of the adhesive layer, on the occasion of immobilizing the magnetic particulates, while azobenzene is changed to a cis-form by irradiation of ultraviolet light, a certain amount of the reaction solution is poured under the state where the magnetic field is turned off. After that, the magnetic particulates in the reaction solution are attracted onto the support base by generating a magnetic field, and the magnetic particulates contacting with the adhesive layer are immobilized on the supporting substrate. After immobilizing the magnetic particulates to the adhesive layer, floating unreacted fluorescence-labeled antibodies are rinsed away by flushing a cleaning solution. A photograph is taken while irradiating the excitation light to the magnetic particulates immobilized on the supporting substrate and to the fluorescence-labeled antigen bound thereto, and the concentration of the objective biomolecules is determined by counting the number of observed bright spots. After completion of observation, by applying visible light or heat to the supporting substrate, azobenzene is made hydrophilic, and the magnetic particulates are peeled off from the supporting substrate. Irradiation of ultraviolet light and visible light is configured to be able to be implemented on the entire surface of the supporting substrate by preparing light sources and a wavelength separation filter aside from the detection system. As a method for applying the heat, heated hot water is passed through a flow path. When cleaning has completed, by returning to the cis-form again by irradiation of ultraviolet light, immobilization and observation are carried out in the same way by pouring a new sample. In order to reduce the drag-in of the sample at the second run or later, repeated washing is performed. Otherwise, the contamination is removed by changing the wavelength of the fluorescent dye and the detection filter used for labeling in each time of the measurement. 
         [0019]    In the Examples, it is disclosed that the biomolecules of the analysis object are peptides, proteins, or a group of nucleic acid fragments. A method of immunological analysis which is characterized in that antigens of the analysis object are prepared, the magnetic particulates bound to antibodies against the antigens and the phosphor-labeled antibodies are coupled with the antigens of the analysis object, and the labeling phosphors are detected, will be disclosed. Alternatively, a nucleic acid analysis method characterized in that the nucleic acid fragment group of the analysis object is prepared, nucleic acid molecules which have known nucleotide sequences and labeled with phosphors are hybridized with the nucleic acid fragment group of the analysis object, and the phosphors labeled on the hybridized nucleic acid molecules are detected will be disclosed. 
         [0020]    In addition, in an Example, a nucleic acid analysis method which is characterized in that, in the biomolecule analysis method, the phosphor labels are the particulates containing plural kinds of phosphors with different mixing ratios for each type of biomolecules of the analysis object will be disclosed. In addition, in an Example, a biomolecule analysis method which is characterized in that, in the biomolecule analysis method, by using the same phosphor label for the molecular species other than specific molecular species, counting the number of fluorescent bright spots for each molecule, and calculating a ratio of the number of bright spots of each specific molecular species to the total number of bright spots, the abundance of the each specific molecular species is evaluated, will be disclosed. In addition, in an Example, a biomolecule analysis method which is characterized in that, in the nucleic acid analysis method, a step of labeling a biomolecule group of the analysis object with a common phosphor and subjecting biomolecules labeled with a phosphor having different fluorescence wavelength or fluorescence intensity from the phosphor to binding reaction specifically is included, and by calculating a ratio between the numbers of bright spots of the former and the latter phosphor, the abundance of each type of biomolecules of the analysis object is evaluated, will be disclosed. 
         [0021]    In addition, in an Example, a biomolecule analyzer characterized by including a device for immobilizing biomolecules of the analysis object as deploying two-dimensionally, a biomolecule analyzer installed with the device, and a means for measuring the fluorescence of the phosphor, will be disclosed. 
         [0022]    Hereinafter, the above-described and the other novel features and effects of the present invention will be explained with reference to the figures. Here, for thorough understandings of the present invention, detailed descriptions are made on specific embodiments; however, the present invention is not limited to the contents described herein. 
       Example 1 
       [0023]    One example of the analysis method and the configuration of the device of the present Example are explained with reference to  FIG. 1 . In the beginning, biomolecules to be detected are captured by magnetic particulates in advance, and further labeled with fluorescent labels. All of these reactions are carried out under a normal temperature in a buffer solution, and either one of the reaction of magnetic particulates with biomolecules and the reaction of fluorescence labelling substances with biomolecules may be carried out first, or may be carried out at the same time. The preparation method of magnetic particulates to capture and the method of fluorescence labeling will be described in detail in Examples 3 and 4. 
         [0024]    In the present Example, the reaction method is explained with reference to  FIG. 1  by taking as an example a method that the biomolecules to be detected are antigens  101 , antigens  101  are captured by antibody-bound magnetic particulates  102 , and further they are labeled with fluorescence-labeled antibodies  103 . First, the antibody-bound magnetic particulates  102  are placed in a reaction vessel  110 , and stirred well. To this solution, a solution containing the antigens  101  to be detected is added and mixed well, and incubated for a few minutes. At this time, the reaction is accelerated by either rotating the reaction vessel up and down or by agitating with shaking. As to the ratio of mixing, the antibody-bound magnetic particulates  102  are mixed so as to be excessive as compared with the antigens  101 . Further, to this reaction solution, the fluorescence-labeled antibodies  103  are added, and incubated for an additional few minutes. In the mixed solution thus reacted, a large amount of unreacted magnetic particulates  102  and fluorescence-labeled antibodies  103  are present, and a small amount of a mixture of magnetic particulates  102  and antigens  101  and fluorescence-labeled antibodies  103  are present therein. Hereinafter, this mixed solution is referred to as a reaction solution A. The reaction solution A is deployed two-dimensionally on the supporting substrate, and the amount of antigens is determined by detecting the fluorescence. 
         [0025]    Next, a configuration of a device for detection of fluorescence is explained with reference to  FIG. 1 . The present device has a smooth supporting substrate  104  for presenting the magnetic particulates  102  two-dimensionally, and an adhesive layer  105  for immobilizing the magnetic particulates  102  is present on the surface of the supporting substrate  104 . As a supporting substrate  104 , a thin quartz glass substrate or a thin silicon substrate which transmits the magnetic force well is suitable; according to a type of the adhesive layer  105 , a quartz glass substrate deposited with a metal thin film or a silicon substrate may be used selectively. Since the magnetic particulates  102  modified with protein are well adsorbed on a hydrophobic layer, the adhesive layer  105  is formed by immobilizing an alkyl group to the surface of the supporting substrate  104 . The alkyl chain with a functional group can be used as an adhesive agent. As to the functional group, a silanol group is used if the material of the supporting substrate  104  is quartz or an oxidized silicon substrate, a thiol group is used if the material is a gold-deposited substrate, and a phosphate group is used if the material is a titanium-oxide-deposited substrate. When the reaction is carried out, the functional group is immobilized to the supporting substrate, and the alkyl chains crowd with the alkyl-chain hydrophobic interaction and form a self-assembled membrane (SAM) on the surface of the supporting substrate  104 . By this method, the surface of the supporting substrate  104  can be made hydrophobic uniformly. A magnetic field generator  106  for attracting the magnetic particulates  102  to the adhesive layer  105  has been installed just below the supporting substrate  104 . Further, the magnetic field generator  106  is equipped with a function for switching the on/off or the intensity levels of the magnetic field. This device is used as being installing on a movable stage  107  so as to be able to scan the entire surface of the supporting substrate  104 . 
         [0026]    Hereunder, the role of the device and the observation procedure are explained. At the beginning, the reaction solution A is placed on the supporting substrate  104  using a pipette  108 . By making side walls surrounding the supporting substrate  104  all around, the amount of solution per area of the supporting substrate can be adjusted strictly, and a uniform liquid thickness can be obtained as compared with a method of placing a droplet directly as is. Namely, a uniform immobilization of the magnetic particulates  102  can be achieved. In the state that the magnetic field generator  106  is turned on, because the magnetic particulates  102  are attracted from a spot where they are introduced, the magnetic particulates will be immobilized non-uniformly. Therefore, the magnetic field generator  106  is turned off when the reaction solution A is introduced, and after introduction, the magnetic field is generated by turning on the magnetic field generator  106  to attract all of the magnetic particulates  102  in the reaction solution A onto the support base  104 . The magnetic particulates  102  contacting with the adhesive layer  105  are immobilized to the supporting substrate  104 . Since the antibody-bound magnetic particulates  102  used in this study are paramagnet, they can be magnetized by applying a magnetic field from the outside. However, if each magnetic particulate  102  is magnetized, the magnetic particulates attract each other and form an aggregate like a string of beads. Therefore, if the observation is performed in a state that the magnetic field is applied from the outside, since the magnetic particulates  102  have a steric structure toward the direction perpendicular to the supporting substrate  104 , focusing becomes difficult on observation or, by enfolding the fluorescence-labeled antibody  103  inside the aggregate, excitation light becomes non-uniform and the original quantitative performance is impaired. Therefore, by turning off the magnetic field to resolve aggregation of magnetic particulates  102 , only the magnetic particulate  102  immobilized to the adhesive layer  105  is presented on the supporting substrate  104  and a magnetic particulate layer of a single layer can be formed. In this case, the magnetic particulates which did not contact with the adhesive layer  105  are not presented on the supporting substrate  104 ; however, if the collision chance is increased by repeating the on/off or the up-and-down of the magnetic field, most magnetic particulates  102  can be immobilized to the adhesive layer  105 . After immobilizing the magnetic particulates  102  to the adhesive layer  105 , all floating unreacted fluorescence-labeled antibodies  103  can be washed away by flushing the cleaning solution. In this regard, however, removal of the unreacted fluorescence-labeled antibodies  103  may be carried out in the reaction vessel  110  in advance. A photograph is taken while irradiating excitation light to the magnetic particulates  102  immobilized in a single layer on the supporting substrate  104  and the fluorescence-labeled antigens  101  bound thereto. The antigen concentration can be determined by counting the number of observed bright spots. 
       Example 2 
       [0027]    Next, a configuration of a device for fluorescence detection in combination with a flow channel is explained with reference to  FIG. 2 . In the same manner as in Example 1, the present device has a smooth supporting substrate  203  for presenting magnetic particulates  201  in two-dimension, and an adhesive layer  204  for immobilizing the magnetic particulates  201  on the surface of the supporting substrate  203  and a magnetic field generator  205  which is capable of switching the on/off or the intensity levels of the magnetic field for attracting the magnetic particulates  201  to the adhesive layer  204  just below the supporting substrate  203  are installed. Further, in all the directions of the supporting substrate  203 , side walls  206  are installed, and further covered thereon with a smooth transparent cover member  207 . For example, PDMS (polydimethylsiloxane) may be used as the material of the side wall  206 , and the quartz can be used for the material of the cover member  207 . Tubes  208  for inserting and removing the solution are mounted at two places of the side wall. Silicone rubber can be used as the material of the tubes  208 . The magnetic field generator  205  for attracting the magnetic particulates  201  to the adhesive layer  204  is installed just below the supporting substrate  203 . The present device is used as installed on a movable stage  209  so as to be able to scan the entire surface of the supporting substrate  203 . Alternatively, the scanning is performed by adding a moving mechanism to a side with an objective lens  210 . 
         [0028]    Hereunder, the role of the device and the observation procedure are explained. 
         [0029]    At the beginning, a certain amount of the reaction solution A is placed in the state that the magnetic field is off. Then by generating the magnetic field, the magnetic particulates  201  in the reaction solution A are attracted onto the supporting substrate  203 . The magnetic particulates  201  contacted with the adhesive layer  204  are immobilized to the supporting substrate  203 . After immobilizing the magnetic particulates  201  to the adhesive layer  204 , all floating unreacted fluorescent dyes  202  can be washed away by flushing a cleaning solution  211 . A photograph is taken while irradiating excitation light to the magnetic particulates  201  immobilized in a single layer on the supporting substrate  203  and the fluorescent dyes  202  bound thereto. The concentration of the biomolecules can be determined by counting the number of observed bright spots. 
       Example 3 
       [0030]    Next, a configuration of a device having a function of changing reversibly the adhesive force of an adhesive layer  304  is explained with reference to  FIG. 3 . In the same manner as in Example 2, the present device has a smooth supporting substrate  303  for presenting magnetic particulates  302  in two-dimension, and the adhesive layer  304  for immobilizing the magnetic particulates  302  on the surface of the supporting substrate  303  and a magnetic field generator  305  which is capable of switching the on/off or the intensity levels of the magnetic field to attract the magnetic particulates  302  to the adhesive layer  304  just below the supporting substrate  303  are installed. Further, in all the directions of the supporting substrate  303 , side walls  306  are installed, and further covered thereon with a smooth transparent cover member  307 . Tubes for inserting and removing a solution are mounted at two places of the side walls  306 . The magnetic field generator  305  for attracting the magnetic particulates  302  to the adhesive layer  304  is installed just below the supporting substrate  303 . Furthermore, the adhesive layer  304  is formed with a material which can change reversibly its adhesive force. For example, a substance which changes wettability of the surface from hydrophobic to hydrophilic or from hydrophilic to hydrophobic with use of temperature or heat is used as an adhesive agent. By changing reversibly the adhesion force of the adhesive layer  304 , after observation, the supporting substrate  303  can be washed after peeling off the magnetic particulates  302  from the supporting substrate  303  immobilizing thereon the magnetic particulates  302 , and further by recovering the adhesion force again, a new sample can be observed. Namely, the supporting substrate  303  is reused many times. As to the adhesive agent which achieves reuse, a photoresponsive alkyl azobenzene can be used. The azobenzene has a structure in which two benzene rings are attached to two azo groups and is isomerized to the cis form when irradiated with ultraviolet light  308  and isomerized to the more stable trans form by applying visible light  310  or heat. For example, an alkyl azobenzene with a functional group, in which an azobenzene and a functional group that reacts with the substrate side are bound to one end and the other of the alkyl chain, respectively, is prepared. The functional group to be used is a silanol group if the substrate is quartz or an oxidized silicon substrate, a thiol group if the substrate is a gold-deposited substrate, and a phosphate group if the substrate is a titanium-oxide-deposited substrate. When the reaction is performed, the functional group is immobilized to the supporting substrate and the alkyl chains crowd with the alkyl-chain hydrophobic interaction to form a self-assembled membrane (SAM) on the surface of the supporting substrate. By this method, the azobenzene can be introduced uniformly on the substrate. On the occasion of immobilizing the magnetic particulates  302 , the azobenzene should be in the cis form by irradiation for approximately 5 minutes with the ultraviolet light  308 . 
         [0031]    Hereunder, the role of the device and the observation procedure is explained. At the beginning, a certain amount of the reaction solution A is placed with the magnetic field off. Then, by generating a magnetic field, the magnetic particulates  302  in the reaction solution A are attracted onto the support base. The magnetic particulates  302  contacting with the adhesive layer  304  are immobilized on the supporting substrate  303 . After immobilizing the magnetic particulates  302  to the adhesive layer  304 , all floating unreacted fluorescence-labeled antibodies can be washed away by flushing a cleaning solution  309 . A photograph is taken while irradiating excitation light to the magnetic particulates  302  which have been immobilized onto the supporting substrate  203  in a single layer and the fluorescence-labeled antigens bound thereto. The antigen concentration can be determined by counting the number of observed bright spots. 
         [0032]    After completion of observation, by applying visible light  310  or heat to a supporting substrate  303  on which the magnetic particulates  302  have been immobilized, the azobenzene is made hydrophilic, and the magnetic particulates  302  are peeled off from the supporting substrate  303 . Irradiation of visible light  310  is set to be able to irradiate the entire surface of the supporting substrate  303  by preparing a light source and a wavelength separation filter aside from the detection system. In order to apply heat, hot water heated to 35° C. or higher is passed through the flow channel. In either method of making hydrophilic, higher removing effect of the magnetic particulates  302  than that obtained by washing with an aqueous solution containing a surface active agent can be obtained. When cleaning has completed, the azobenzene is returned to the cis form again by irradiation with the ultraviolet light  308  for approximately 5 minutes, and immobilization and observation are carried out in the same way by placing a new sample. 
         [0033]    When immobilizing and removing are carried out repeatedly in this way, there is a possibility that fluorescent dyes  301  immobilized in the previous time may remain, and would be added in on the second and/or subsequent quantification. In order to prevent this, washing conditions with which the magnetic particulates  302  can be removed completely or until down to an acceptable amount are determined in advance. When a trace of sample is quantified, a difference of a few bright spots may affect quantitative results significantly. At that time, it is possible to completely remove contaminations by changing the wavelength of the fluorescent dyes  301  to be used for labeling and the detection filter in each of measurement times. 
       Example 4 
       [0034]    A sample preparation method in the present Example is explained with reference to  FIG. 4 . When biomolecules to be detected are antigens  401 , they are captured in advance by magnetic particulates  403  bound to antibodies  402 , and further labeled with fluorescence-labeled antibodies  405 . All of these reactions are carried out in a buffer for reaction (Tris buffer (pH 8.0), 50 mM NaCl, 0.1% Tween 20) under a normal temperature. Either one of the reaction between the particulate bound to the antibody  402  and the antigen  401  and the reaction between the fluorescence-labeled antibody  405  and the antigen  401  may be carried out first, or they may be carried out at the same time. Any kind can be used as an antigen  401 ; however, as an antibody  402 , it is preferable to select one with high specifity against the antigen  401 . For example, when PSA (prostate specific antigen) which is a tumor marker for prostate cancer is selected as an antigen  401 , a PSA antibody is required. For the PSA antibody, one for binding with a magnetic particulate and another for being labeled with a fluorescent dye are required respectively. The antibodies may be either a polyclonal antibody or a monoclonal antibody, and are selected appropriately according to the type of the antigen  401 . 
         [0035]    When the antibodies  402  are bound to the magnetic particulates  403 , Protein A modified or Protein G modified magnetic particulates  403  which can bind with the antibody  402  while maintaining the activity of the antibody  402  is prepared. Such magnetic particulates  403  are commercially available and can be easily obtained. For example, Protein A modified Adembeads which are paramagnetic particulates (φ300 nm) from Ademtech Inc. can be used. The Protein A modified magnetic particulates  403  are mixed with about 10 times or more of PSA antibodies and incubated. Then, after the magnetic particulates  403  are captured with a magnet, the solution is removed, and they are suspended in a clean buffer. This operation is repeated until the unreacted antibodies  402  are removed. The magnetic particulates  403  modified with streptavidin can be used as the magnetic particulates  403 . For example, streptavidin-modified Adembeads (φ100 nm, φ200 nm, φ300 nm) from Ademtech Inc. can be used. When these magnetic particulates  403  are used, the antibodies  402  are bound to the surfaces of the magnetic particulates  403  by using biotinylated antibodies  402 . The diameter of the magnetic particulates  403  to be used is desirably selected from those which are smaller than the excitation wavelength, thereby an increase in background due to scattered light of the magnetic particulates  403  can be prevented. 
         [0036]    Various types of fluorescence-labeled antibodies are commercially available. For example, FITC, Alexa®, CY5, and the like are well known. However, in the present study, in order to enable detection of the antigen  401  of at least one molecule, it is desirable to use a fluorescent dye with high brightness and a long quenching time. As such a fluorescent dye, for example, a dendrimer-type fluorescent dye, in which fluorescent dyes of several hundred molecules are bonded to one branched carbon chain, or a quantum dot  404  can be used. 
         [0037]    In the present Example, a fluorescence labeling method of antibodies using quantum dots  404  is explained. The quantum dots  404  are the semiconductor fine particulates with a diameter of a few nanometers to several tens of nanometers. They have a long lifetime and high brightness as compared to the conventional fluorescent dyes, and emit fluorescence of different wavelengths according to the particle sizes. Quantum dots  404  are commercially available from several manufacturers, and some are modified with various functional groups. As the quantum dots  404  that can bind any fluorescence-labeled antibodies  405 , for example, Qdot® antibody labeling kit from Invitrogen Corporation can be utilized. In this kit, the quantum dots  404  introduced with a thiol group on the surface and the fluorescence-labeled antibodies  405  with the disulfide bond reduced are mixed and reacted, thereby the fluorescence-labeled antibodies  405  can be labeled with the quantum dots  404 . 
         [0038]    Using the magnetic particulates  403  bound to antibodies  402 , which are prepared as described above, and the fluorescence-labeled antibodies  405 , capture and detection of antigens  401  are carried out. First, the magnetic particulates  403  bound to antibodies  402  are placed in the reaction vessel and stirred well. To this solution, a solution containing the antigens  401  to be detected is added and mixed well; then it is incubated for 1 hour. At this time, the reactions between the antigens  401  and the antibodies  402  are accelerated by either rotating the vessel up and down or by agitating with shaking. As to the ratio of mixing, the magnetic particulates  403  bound to antibodies  402  are mixed with the antigens  401  so as to be excessive as compared with the antigens  401 . Specifically, to an assumed amount of the antigens, 100 to 10,000 times the amount of the magnetic particulates  403  bound to antibodies  402  is placed. Then, to this reaction solution, the fluorescence-labeled antibodies are placed, and it is incubated for an additional few minutes. The fluorescence-labeled antibodies  405  are also placed to be a 100 to 10,000 times more amount so as to be in excess to the amount of the antigens. By placing each in an excessive amount, collision frequencies with the antigens  401  increase and the capture rate and the fluorescence labeling index of the antigens  401  can be improved. The mixture reacted as described above is observed in the same manner as in the reaction solution of Example 3 and the antigens  401  are determined quantitatively. 
       Example 5 
       [0039]    The sample preparation method in the present study is explained with reference to  FIG. 5 . When biomolecules to be detected are nucleic acid fragments, sample nucleic acid fragments  501  of the detection objects are captured by magnetic particulates  507 , and further labeled with nucleic acid fragments  505  bound to fluorescent dyes  506  having sequences complementary to the sample nucleic acid fragments  501 . This is a specific reaction by hybridization, and is carried out in a buffer for reaction (PBS buffer (pH 7.4), 50 mM to 1 M NaCl, 0.1% Tween 20). Either one of the reaction between the magnetic particulate  507  attached with nucleic acid and the sample nucleic acid fragment  501  and the reaction between the nucleic acid fragment  505  attached with the fluorescent dye  506  and the sample nucleic acid fragment  501  may be carried out first, or they may be carried out at the same time. For the nucleic acid fragments  505 , single-stranded DNAs or single-stranded RNAs can be used. Details are explained while taking a micro-RNA as an example of a concrete analysis object. 
         [0040]    A micro-RNA is a single-stranded nucleic acid fragment of about 20 mer. The simplest detection method is a method of direct fluorescence labeling of the sample nucleic acid fragment  501 . In this case, a biotinylated sample nucleic acid fragment  501  is prepared by binding a biotinylated dUTP to the 3′ terminal side of the sample nucleic acid fragment  501 . An avidin-labeled quantum dot is reacted to this sample nucleic acid fragment  501 . In the same way, a magnetic particulate  507  attached with a complementary strand to the sample nucleic acid fragment  501  is prepared, and allowed to react with the sample nucleic acid fragment  501  which has been reacted with the quantum dot. The amount of the sample nucleic acid fragments  501  captured by the magnetic particulates  507  is measured from the fluorescent dyes  504 . 
         [0041]    When the amount of specific micro-RNAs among the total amount of overall micro-RNAs is to be measured, nucleic acid fragments each of which contains a consensus sequence  503  and a capture sequence  508  should be added to the ends of the micro-RNAs of the sample nucleic acid fragments  501  by ligation in advance. The consensus sequence  503  refers to a sequence for fluorescence-labeling of all micro-RNA irrespective of the type, and the capture sequence refers to a sequence for being captured by magnetic particulates  507 . As to the consensus sequence and the capture sequence, a GC content and a base length can be synthesized arbitrarily in accordance with the Tm value of the sample to be measured. Hereinafter, the nucleic acid fragment containing a consensus sequence and a capture sequence is referred to as a tag fragment  502 . First, a complementary strand to the consensus sequence labeled with a fluorescent dye  504  and a complementary strand to a sample nucleic acid fragment  501  labeled with a fluorescent dye  506  are prepared. These are mixed and hybridized with the sample nucleic acid fragment  501  added with a tag molecule  502 . At this time, when plural types of sample nucleic acid fragments  501  are present, the strands complementary to sample nucleic acid fragments  501  are prepared for respective types, and labeled with fluorescent labels of different wavelengths, respectively. The sample nucleic acid fragments  501  fluorescence-labeled in this way are captured with magnetic particulates  507  bound with the capture sequence. This reaction is also performed by hybridization. The magnetic particulates  507  capturing the fluorescence-labeled sample nucleic acid fragments  501  are immobilized to the adhesion layer on the supporting substrate, and observation of the bright spots is performed in the same manner as in Examples 1 and 2. At this time, the number of fluorescent bright spots of the consensus sequence side corresponds to the total number of micro-RNAs, and the number of fluorescent bright spots of the sample side corresponds to the number of molecules of the  sample nucleic acid fragments  501 . Judging the ratio of the numbers of both the bright spots as the abundance ratio of each type of the nucleic acid sample molecules is also effective when the expression level of only a particular nucleic acid molecule is desired to be examined. 
         [0042]    The magnetic particulates  507  are commercially available and can be obtained easily. For example, streptavidin-modified magnetic particulates from Ademtech Inc., which are paramagnetic particulates, can be used. For example, streptavidin-modified Adembeads (φ100 nm, φ200 nm, φ300 nm) from Ademtech Inc. can be used. When these magnetic particulates are used, the nucleic acids are bound to avidin  509  on the surface of the magnetic particulates  507  using a biotinylated nucleic acid fragments. The diameter of the magnetic particulates  507  to be used is desirably selected from those which are smaller than the excitation wavelength, thereby an increase in background due to scattered light of the magnetic particulates  507  can be prevented. Various types of fluorescent dyes are commercially available. For example, FITC, Alexa®, and CY5 are well known. However, in the present study, in order to enable detection of the sample nucleic acid fragments of at least one molecule, it is desirable to use a fluorescent dye with high brightness and a long quenching time. As such a fluorescent dye, for example, a dendrimer-type fluorescent dye, in which fluorescent dyes of several hundred molecules are bonded to one branched carbon chain, or a quantum dot  404  can be used. In the present Example, a fluorescence labeling method of nucleic acid fragments using quantum dots is explained. For example, streptavidin-modified Qdot® from Invitrogen is available. Bindings between the quantum dots and the nucleic acid fragments may be performed by mixing and followed by incubation for more than 30 minutes, and after reaction unreacted nucleic acid fragments are removed with a spin column of 50 kDa cut-off. 
         [0043]    Using the magnetic particulates  507  bound with the capture sequence and a fluorescent dye-labeled nucleic acid fragments labeled by fluorescent dyes that are prepared as described above, the sample nucleic acid fragments  501  are captured. First, the magnetic particulates  507  bound with the capture sequence are placed in a reaction vessel and stirred well. To this solution, a solution containing the sample nucleic acid fragments  501  to be detected is added, mixed well, and then incubated for 6 hours. At this time, the hybridization reaction is accelerated by either rotating the reaction vessel up and down or by agitating with shaking. As to the ratio of mixing, the magnetic particulates  507  are mixed with the nucleic acid fragments so as to be excessive as compared with the objective nucleic acid fragments. Specifically, to an assumed number of the sample molecules, 100 to 10,000 times the amount of the magnetic particulates  507  are placed. The reaction solution treated as described above is observed in the same manner as in Examples 1 and 2. 
       Example 6 
       [0044]    In the present Example, one example of a preferred configuration of the biomolecule analyzer is explained with reference to  FIG. 6 . The biomolecule analyzer of the present Example is equipped with a means for supplying the biomolecule solution of the analysis object and the cleaning solution to a supporting substrate  601  for deploying the biomolecules two-dimensionally, a means for attracting the magnetic particulates on the supporting substrate, a means for holding the magnetic particulates on the supporting substrate, a means for controlling the temperature for heating the cleaning solution in the supporting substrate, a means for irradiating the light to the supporting substrate, an emission detection means for measuring the fluorescence of the phosphor of the fluorescence-labeled molecule, and a means for scanning the supporting substrate  601 . 
         [0045]    More specifically, by placing the supporting substrate  601  on a movable stage  604  and sticking the flow path member provided with the flow channel together thereon, a reaction chamber  605  is formed. As to the flow path member, for example, PDMS (polydimethylsiloxane) can be used. A liquid feed pump  607  is connected at an inlet  606 , and the biological sample solution and the cleaning solution are supplied sequentially to the supporting substrate  601  from a solution tank  602  of the biological sample of the analysis object and a cleaning solution tank  603 , and discarded to a waste liquid tank  612  after use. Once the biological sample solution enters the reaction chamber, a magnetic field generator  608  generates a magnetic field to attract the magnetic particulates onto the surface of the supporting substrate  601 . The attracted magnetic particulates contact with the adhesive layer on the surface of supporting substrate and are immobilized. Washing is carried out by introducing the cleaning solution into the reaction chamber  605  from the liquid feed pump  607 . After washing, fluorescence detection is carried out. An excitation light source  609  can be selected appropriately according to a type of a phosphor to be used. For example, in the case where the quantum dots are used as a fluorescent dye for fluorescent labeling, the light source can be accommodated with 532 nm (YAG laser) or a mercury lamp. The excitation light emitted from the excitation light source  609  is passed through an excitation filter  616  and a lens  617 , is led to an objective lens  611  by a dichroic minor  610 , and irradiates onto the supporting substrate  601 . The fluorescent light emitted from fluorescence-labeled molecules on the support base  601  travels on the same light path as the excitation light in an opposite direction, passes through the dichroic mirror  610  after collected by the objective lens  611 , and is imaged on a photosurface of a two-dimensional CCD camera  614  by an imaging lens  613 . Scattered light of the excitation light is removed by an absorption filter  615 . In order to improve quantitative performance, it is necessary to increase the number of bright spots for observation. The bright spots can be increased by operating the movable stage  604  so that the entire surface of the supporting substrate  601  is scanned. By assembling a biomolecule analyzer with the liquid feed pump  607 , the inlet  606 , the excitation light source  609  and the fluorescence detection unit, the magnetic field generator  608 , and the movable stage  604  as described above, automatic analysis can be performed and thereby speed improvement can be achieved. 
       Example 7 
       [0046]    In the present Example, with regard to an arrangement of a magnet and a supporting substrate to be used for immobilizing magnetic particulates  701 , one of the configurations that are desirable to be used in combination with Examples 1 to 5 is explained with reference to  FIG. 7 . Since the equipment to be used in the present invention scans the surface of a smooth supporting substrate  702 , the more magnetic particulates  701  which are immobilized to a unit area, the more advantageous it is in terms of throughput. On the other hand, if there too many magnetic particulates  701  per area, they are multi-layered and the magnetic particulates  701  that do not fit within the depth of focus of the objective lens increase so that the number of detection decreases. Therefore, in order to implement an efficient detection, it is desirable that the magnetic particulates  701  are to form a thin layer while being immobilized at high density. Accordingly, a method for immobilizing the magnetic particulates  701  with use of a powerful magnet  703  having magnetic field lines  704  parallel to the supporting substrate  702  was devised. 
         [0047]    The arrangement method follows. As shown in  FIG. 7 , at first, the magnetic particulates  701  were put on the supporting substrate  702 ; then, the supporting substrate  702  was arranged on the magnetic field generator  703 . The directions and the intensities of the magnetic field lines  704  had been examined in advance and the supporting substrate  702  was arranged so that the directions of the magnetic field lines  704  in the central part of the surface of the supporting substrate  702  were parallel. Further, by performing the immobilizing reaction for a few seconds, the magnetic particulates  701  could be immobilized in a form of lines as shown in  FIG. 8 . As a result of extensive studies on positions of the above arrangement, it turned out that, by arranging the supporting substrate  702  on the surface of the magnet  703  and also at a central part as much as possible between both poles, using the magnet  703  larger than the supporting substrate  702 , and further using the magnet  703  having high magnetic flux densities, more uniform and quicker immobilization can be achieved. 
         [0048]    The principle of immobilization follows. The normal magnet forms magnetic field lines  704  which draw arcs between both poles. At that time, in the central part of the surface of the magnet  703  belt-shaped magnetic field lines  704  that connect in parallel between both poles are formed. Since the magnetic particulates  701  used in the present Example have a paramagnetic property, by applying a magnetic field from the outside, each particulate was magnetized and formed a bead-like linear chain with other magnetic particulates  701  according to the parallel magnetic field lines  704  ( FIG. 8 ). In this occasion, the parallel magnetic force generated in the center of the magnet  703  is very weak compared to the magnetic force that arises at the both poles. Therefore, it is desirable that the arranged magnetic particulates  701  are placed with sufficient distances from the poles so as not to be pulled toward either pole. Namely, it is desirable to be located in the central part between the both poles, and a bias of immobilization can be reduced by placing with sufficient distances. The term “sufficient” mentioned here can be achieved, for example, by making the distance from the center of the supporting substrate  702  which is a 4-mm square to each pole be 40 mm In addition, the parallel magnetic field generated in the center of the magnet  703  requires a magnetic force component perpendicular to the magnet  703  for immobilizing the magnetic particulates  701  on the supporting substrate  702 . Therefore, it is possible to increase the immobilization rate by choosing the magnet  703  with a relatively strong magnetic force. This is also advantageous in that the magnetic particulates  701  are immobilized at a high density; specifically, a surface magnetic flux density of 0.1 T or higher is desirable. In the present Example, a neodymium magnet of 0.5 T in φ8 cm and a supporting substrate  702  of a 4-mm square were used. When the magnetic particulates are attracted in the vicinity of the poles of the magnet  703 , chains of the magnetic particulates would extend in a direction perpendicular to the supporting substrate. In fact, when the equal amount (20 pM) of the magnetic particulates  701  (φ300 nm) is introduced in the same volume (4×4×0.13 mm) and a parallel magnetic flux and a perpendicular magnetic flux are applied with respect to the supporting substrate  702 , respectively, the magnetic particulate density that fits within the same depth of focus on the surface of the supporting substrate  702  was about 8 times higher in the parallel magnetic flux. Hereby, it could be confirmed that the parallel magnetic flux was effective in high-density immobilization of the magnetic particulates. 
       REFERENCE SIGNS LIST 
       [0000]    
       
           101 ,  401  antigen 
           102 ,  201 ,  302 ,  403 ,  507  magnetic particulate 
           103 ,  405  fluorescence-labeled antibody 
           104 ,  203 ,  303 ,  406 ,  601  supporting substrate 
           105 ,  204 ,  304 ,  407 ,  511  adhesive layer 
           106 ,  205 ,  305 ,  608  magnetic field generator 
           107 ,  209 ,  604  movable stage 
           108  pipette 
           109 ,  210 ,  611  objective lens 
           110  reaction vessel 
           202 ,  301 ,  504 ,  506  fluorescent dye 
           206 ,  306  side wall 
           207 ,  307  cover material 
           208  tube 
           211 ,  309  cleaning solution 
           308  ultraviolet light 
           310  visible light 
           311  capturing molecule 
           402  antibody 
           404  quantum dot 
           501  sample nucleic acid fragment 
           502  tag molecule 
           503  consensus sequence 
           505  nucleic acid fragment 
           508  capture sequence 
           509  avidin 
           510  support base 
           602  biological sample solution tank 
           603  cleaning solution tank 
           605  reaction chamber 
           606  inlet 
           607  liquid feed pump 
           609  excitation light source 
           610  dichroic minor 
           612  waste liquid tank 
           613  imaging lens 
           614  two-dimensional CCD camera 
           615  absorption filter 
           616  excitation filter 
           617  lens 
           701  magnetic particulate 
           702  supporting substrate 
           703  magnet 
           704  magnetic field line (magnetic flux)