Abstract:
In the conventional nucleic acid analysis devices and nucleic acid analyzers, there was no technique available for sequencing a single nucleic acid molecule easily and highly efficiently. The present invention enabled a highly efficient single molecule immobilization of nucleic acid with good reproductivity in a short time at a low price by providing small metallic bonding pads at predetermined positions on a support substrate, firmly fixing a hydrophobic linker on the bonding pads, and bonding on to the linker bulky microparticles onto which a single molecule of a nucleic acid sample fragment is immobilized. According to the present invention, in the nucleic acid analysis device which uses a nucleic acid analyzer, the nucleotide read length can be extended and many types of nucleic acid molecule to be analyzed can be analyzed at one time.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a nucleic acid analysis device and a nucleic acid analyzer comprising the device. 
       BACKGROUND ART 
       [0002]    As a means for detecting a target substance such as DNA, proteins, or the like, commonly used is a method in which a target substance is labeled with fluorescence, irradiated with a predetermined excitation light such as laser, or the like, and the fluorescence thus emitted is detected. As an application of the method, a parallel sequencer, wherein DNA or RNA is immobilized on a substrate to determine the nucleotide sequence thereof, has been proposed. At present, commercially available parallel sequencers have dramatically improved the number of nucleotide read and the number of parallel per analysis by arranging a large number of DNA fragments to be read. Most of the parallel sequencers read a nucleotide sequence targeting clusters of copied DNA strands. However, the cluster formation not only requires time and the cost of a reagent but a phenomenon (dephasing) in which the sequence reaction loses the synchronicity between the DNA strands also occurs and hence limits the read length. Further, it is not suitable for quantitative analysis because the deviation is caused between the DNAs easy to be amplified and hard to be amplified. Thus, as a system for solving these drawbacks, a single molecule DNA sequencing method has been proposed. In this system, the nucleotide sequence for every single DNA molecule can be determined which thus eliminates the need for the purification and amplification of a sample DNA in cloning, PCR, or the like, that have been the problem in the conventional art, and hence faster genome analysis and gene diagnosis can be expected. One of such systems is SMRT technology of Pacific Biosciences Inc. (see Non-Patent Literature 1). In the SMART technology, a substrate in which a large number of several tens-nm holes called zero-mode waveguidances (ZMW) are aligned is produced and a single molecule of polymerase is placed in each of the holes. Nucleotides labeled with fluorescent dyes are incorporated therein and the fluorescence detection is carried out while the nucleotides are allowed to elongate to obtain the sequence information of each fragment. Such a technique wherein the detection is carried out while allowing the elongation with the incorporation of nucleotides is usually called Sequencing by synthesis. However, when the ZMW is used, the single molecule placement of a polymerase depends on the probability and consequently holes in which a single polymerase molecule is placed account for theoretically up to about 30% out of a large number of holes produced. In Sequencing by synthesis, a continuous nucleotide elongation reaction is detected and thus the field of vision to be detected cannot be moved until one cycle of elongation reaction is completed. Accordingly, to measure many samples at one time, it is desirable to immobilize samples as high density as possible, which is a factor to determine the final sequencing performance. 
         [0003]    Various techniques have been reported for immobilizing on an analysis device a plurality of chemical substances such as nucleic acid, or the like, including DNA (see Patent Literatures 1 and 2). 
       CITATION LIST 
     Patent Literature 
       [0000]    
       
         Patent Literature 1: U.S. Patent Serial No. 2005/0014151 
         Patent Literature 2: JP Patent Publication (Kokai) No. 2010-172271 A 
       
     
       Non Patent Literature 
       [0000]    
       
         Non Patent Literature 1: Science 2009, Vol. 323. pp. 133-138 
       
     
       SUMMARY OF INVENTION 
     Technical Problem 
       [0007]    In Patent Literature 1, a chemical substance having a selective binding property is adsorbed on a device consisting of a metal material and quartz to achieve a selective immobilization of a biological molecule and form a pattern on the device surface. However, in the immobilization technique described in Patent Literature 1, a plurality of small molecules having a size of several nm is immobilized on a substrate produced by a semiconductor process, and in such a technique the immobilization of a single nucleic acid molecule in a high probability is theoretically not possible. On the other hand, as described in Patent Literature 2, there is a technique in which single molecules of a nucleic acid probe are captured in advance onto bulky microparticles, which are arbitrary arrayed on metallic bonding pads to immobilize the molecules onto the pads. As an example thereof, a molecule having a functional group selectively adsorbed by the bonding pad is used as the linker molecule for connecting the microparticle and the bonding pad, and biotin is added within the molecule. The microparticle is, on the other hand, conjugated with avidin to achieve the immobilization by the avidin-biotin binding. This technique is capable of immobilizing the microparticles one by one on the bonding pad and enables the immobilization of a single nucleic acid molecule. However, the drawback posed in this technique is that when biotin is incorporated in a small bonding pattern via a functional group, some of the bonding pads had a low density of incorporated biotin. This is presumably caused by a low amount of biotin incorporated per binding molecule and the inconsistent synthesis reaction of biotinylated binding molecules. It is difficult, not only limited to this example of biotin, to uniformly incorporate binding molecules in an sufficient amount for immobilizing microparticles on a small pattern, which is the portion formed on an analysis device for immobilizing a nucleic acid. 
         [0008]    An object of the present invention is to provide, to solve the problems posed by the conventional art, a method for arraying single molecules of a nucleic acid probe in a high density at predetermined immobilization positions easily and highly efficiently by using microparticles and a hydrophobic linker. 
       Solution to Problem 
       [0009]    The present inventors conducted extensive studies on a method which solves the problems posed in the conventional technique for binding a chemical substance to an analysis device, immobilizes single molecules of nucleic acid on an analysis device in a high probability, and uniformly on the portions at which the nucleic acid is immobilized, that is, immobilizes single molecules substantially throughout the entire portion. The present inventors have come up to an idea of using a hydrophobic interaction as a method for binding onto a substrate microparticles onto which a single probe molecule is immobilized in place of the specific binding between the specific molecules as in the method described in the above Patent Literature 2. It was difficult to predict that microparticles can be firmly bound on a substrate by the hydrophobic interaction, but the present inventors surprisingly found that the immobilization method, which employs the hydrophobic interaction using an SAM film (Self-Assembled Monolayer) of hydrophobic alkyl chain and hydrophobic nanosize microparticles, is suitable for immobilizing nanosize microparticles, and allowing the binding in a high density to a small pattern portion formed on an analysis device at which a nucleic acid is immobilized. As a result, the present inventors accomplished the single molecule immobilization method using microparticles, which can be carried out in a short time at a low cost with good consistency. According to the present invention, small metallic bonding pads are provided at predetermined positions on a support substrate, a hydrophobic linker having an alkyl chain as the main component is immobilized on the metallic bonding pads, whereby microparticles can be arrayed in a high efficiency on the linker. 
         [0010]    The present specification encompasses the contents described in the specification and/or drawings of Japanese Patent Application No. 2011-057091, which is the basis of priority of the present application. 
       Advantageous Effects of Invention 
       [0011]    The nucleic acid analysis device of the present invention has a flat support substrate having metallic bonding pads formed regularly, each of which has a single molecule of a probe immobilized on a microparticle, which is immobilized using a hydrophobic interaction on the pads, wherein single molecules of the probe are immobilized uniformly on the bonding pads in a high probability. More specifically, according to the present invention, a nucleic acid analysis device in which a probe molecule of small molecule is immobilized at predetermined positions in a high efficiency can be provided at a low cost, and a nucleic acid analysis such as determining a DNA sequence, or the like, can be carried out in a high throughput using the nucleic acid analysis device and the nucleic acid analyzer comprising the same of the present invention. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0012]    [ FIG. 1 ]  FIGS. 1   a  and  1   b  are drawings showing the structure of a nucleic acid analysis device of Example 1 wherein microparticles onto which a probe molecule is captured are arrayed.  FIG. 1   a  shows the state in which a microparticle is immobilized on a bonding pad, and  FIG. 1   b  shows the state in which the bonding pads on which microparticles are immobilized are regularly aligned on the substrate. 
           [0013]    [ FIG. 2 ]  FIGS. 2   a  and  2   b  are drawings showing the structure of a nucleic acid analysis device of Example 2 wherein microparticles onto which a nucleic acid polymerase is captured are arrayed.  FIG. 2   a  shows the state in which a microparticle is immobilized on a bonding pad, and  FIG. 2   b  shows the state in which the bonding pads on which microparticles are immobilized are regularly aligned on the substrate. 
           [0014]    [ FIG. 3 ]  FIG. 3  is a drawing showing the steps (a to f) for producing a nucleic acid analysis device of Example 3. 
           [0015]    [ FIG. 4 ]  FIGS. 4A and 4B  are drawings showing the steps for producing a nucleic acid analysis device of Example 4.  FIG. 4A  shows a method (steps a to e) in which a metal thin film is formed on a flat support substrate, a pattern is then formed by resist and a flat covering film is subsequently formed, and  FIG. 4B  shows a method (steps a to e) in which a flat covering film is formed on a flat support substrate and a pattern is formed by resist. 
           [0016]    [ FIG. 5 ]  FIG. 5  is a drawing showing the structure of a nucleic acid analyzer of Example 5. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0017]    The present example discloses a nucleic acid analysis device comprising a support substrate and microparticles having a probe molecule capable of capturing a nucleic acid to be detected, wherein each of the microparticles is immobilized separately from each other on the support substrate, 
         [0018]    the nucleic acid analysis device comprising hydrophobically treated bonding pads at positions at which the microparticles are immobilized on the substrate, 
         [0019]    wherein the microparticles are immobilized by a hydrophobic interaction on the bonding pads. 
         [0020]    Further, the present example discloses a nucleic acid analyzer for obtaining nucleotide sequence information of a nucleic acid sample, the analyzer comprising: a means for supplying a nucleotide having a fluorescent dye, and a nucleic acid sample to the nucleic acid analysis device produced by selecting only the microparticles having a single probe molecule and regularly immobilizing the selected microparticles on the bonding pad on the support substrate; a means for irradiating the nucleic acid analysis device with evanescent light, and a luminescence detector for measuring the fluorescence of the fluorescent dye incorporated into a nucleic acid chain through the nucleic acid elongation reaction induced by the coexistence of the nucleotide, the nucleic acid polymerase, and the nucleic acid sample on the nucleic acid analysis device. 
         [0021]    Furthermore, the present example discloses a nucleic acid analysis device comprising bonding pads at immobilization positions of the microparticles on the support substrate, wherein the microparticles and the bonding pads are bound by the hydrophobic interaction of alkyl chains, the diameter of the bonding pads are ⅕ to 5 times the size of the microparticles, and the support substrate surface has a thin film layer made of an organic polymer for preventing non-specific adsorption. 
         [0022]    Still furthermore, the present example discloses that a single probe molecule is immobilized on a single microparticle. 
         [0023]    Further, the present example discloses that the bonding pads are made of a material selected from the group consisting of gold, titanium, nickel and aluminum. 
         [0024]    Furthermore, the present example discloses that the organic polymer for preventing the non-specific adsorption of the microparticle is made of a material selected from polyethylene glycol (PEG), polyacrylamide, polymethoxy ethyl acrylate, 3-glycidoxypropylmethoxysilane (GOPS), and the like. 
         [0025]    Still furthermore, the present example discloses an evanescent field as a means for irradiating the above nucleic acid analysis device with light. 
         [0026]    Hereinbelow, the above and other novel features and effects of the present invention are described with reference to the drawings. A specific embodiment is described in detail for thorough understanding of the present invention, but the present invention is not limited to the content described herein. 
         [0027]    The structure of a device of the present example is described with reference to  FIG. 1 . 
         [0028]      FIG. 1   a  shows the state in which a bonding pad  102  is formed on a flat support substrate  101 , and a microparticle  103  on which a probe molecule  104  is immobilized is bound on the bonding pad by a hydrophobic interaction via a hydrophobic film formed of linear molecules  105  having an end functional group  106 , and  FIG. 1   b  shows the state in which a plurality of the microparticles  103  on which a probe molecule  104  is immobilized is regularly immobilized on the bonding pads  102  on the flat support substrate  101 . 
         [0029]    The bonding pads  102  on the flat support substrate  101  are formed regularly in a large number, and are formed, for example, in a grid pattern as shown in  FIG. 1   b.  The bonding pad  102  and the microparticle  103  are chemically bound via the linear molecule  105 . A thin film  107  for preventing a non-specific adsorption is formed in the region other than the region on which the bonding pads  102  are formed on the flat support substrate  101 . The thin film is called a non-specific adsorption prevention film. The end functional group  106  of the linear molecule  105  is preferably bound to the bonding pad  102  by the hydrophobic interaction established by an alkyl chain. The functional group  106  preferably has the weak interaction with the flat support substrate  101  or the thin film of non-specific adsorption prevention  107 , but has the firm binding to the bonding pad  102 , due to which the microparticle on which the probe molecule  104  is immobilized is immobilized only on the bonding pad  102 . More specifically, the microparticle  103  can be selectively bound to the bonding pad  102 . In the present invention, the binding of the microparticles  104  on the flat support substrate  101  in a regular pattern is called the formation of pattern (patterning), and the selective binding only to the bonding pad  102  region on the flat support substrate  101  is termed as having pattern selectivity. 
         [0030]    From this perspective, the flat support substrate  101 , the bonding pad  102  and the functional group  106  of the linear molecule  105  may be selected in the combination so that the firm binding is established between the bonding pad  102  and the functional group  106  but the firm binding is not established between the flat support substrate  101  and the functional group  106 . 
         [0031]    For the flat support substrate  101 , a quartz glass, sapphire, a silicon substrate, or the like, can be used. The size of the flat support substrate  101  can be suitably designed in accordance with the purpose of use, but, for example, tetragonal ones can be used. In the present invention, the flat support substrate is sometimes simply referred to as a substrate. 
         [0032]    Further, the bonding pad  102  can be composed of a metallic material selected from the group consisting of gold (Au), titanium (Ti), nickel (Ni) and aluminum (Al). The thin metallic film described above may further be formed on the metallic pad. The size of the bonding pad  102  depends on the purpose of use and the size of flat support substrate, but, for example, circular ones can be used. Also, the shape is not limited, and tetragons such as square, rectangles, and other irregular shapes may be used. 
         [0033]    The functional group  106  of the linear molecule  105  must be selected in consideration of the combination of the flat support substrate  101  and the bonding pad  102  as described above, and, for example, a sulfhydryl group (thiol group), an amino group, a carboxyl group, a phosphoric acid group, an aldehyde group, or the like, may be used. The hydroxyl group of sugar may also be used. Of these, the combination may be selected so that the firm binding is established between the functional group  106  and the bonding pad  102 , the firm binding is not established between the functional group  106  and the flat support substrate  101 , or no binding is established. Hereinbelow, metallic materials to which a sulfhydryl group, an amino group, a carboxyl group, a phosphoric acid group, an aldehyde group or the hydroxyl group of sugar is easily adsorbed. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Sulfhydryl group 
                 Au, Ni 
               
               
                   
                 Amino group 
                 Ti, Ni 
               
               
                   
                 Carboxyl group 
                 Ti 
               
               
                   
                 Phosphoric acid group 
                 Ti, Al 
               
               
                   
                 Aldehyde group 
                 Ti, Ni 
               
               
                   
                 Hydroxyl group of sugar 
                 Ti 
               
               
                   
                   
               
             
          
         
       
     
         [0034]    Also, of the above functional groups, the amino group, carboxyl group and aldehyde group have a low selectivity with quartz and hardly bind to quartz. 
         [0035]    Accordingly, the preferable combinations are, for example, a quartz glass used as the flat support substrate  101 , an amino group used as the functional group  106  and titanium or nickel used as the bonding pad  102 , a quartz glass used as the flat support substrate  101 , a carboxyl group used as the functional group  106  and titanium used as the bonding pad  102 , or a quartz glass used as the flat support substrate  101 , an aldehyde group used as the functional group  106  and titanium or nickel used as the bonding pad  102 . However, the combination is not limited thereto, and any combinations of the above materials can be used. For example, a quartz glass used as the flat support substrate  101 , the glycoside group of sugar used as the functional group  106  and titanium used as the bonding pad  102  can be used in combination. In the case of the combination of glycoside group of sugar and titanium, the binding of the glycoside group of sugar and titanium can be controlled by changing the pH, or the like. Consequently, after using the nucleic acid analysis device, for example, the pH is changed, the linear molecule  105  is removed from the bonding pad  102  to reuse the flat support substrate  101  including the bonding pad  102 . Also, as to be described later, the non-specific adsorption prevention film  107  is formed on the region other than the region on which the bonding pads  102  are formed on the flat support substrate  101 . The functional groups other than the glycoside group of sugar easily bind to the non-specific adsorption prevention film  107 . Accordingly, when a functional group other than the glycoside group of sugar is used as the functional group  106 , the non-specific adsorption prevention film must be formed after the functional group  106  of the linear molecule  105  is allowed to bind to the bonding pad  102 . The glycoside group of sugar, on the other hand, is not likely to bind, or does not bind, to the non-specific adsorption prevention film  107 . For this reason, when the glycoside group of sugar is used as the functional group, the order of the immobilization of the linear molecule on the bonding pad  102  and the formation of the non-specific adsorption prevention film is irrelevant. 
         [0036]    For the linear molecule  105 , an amphipathic molecule having the above functional group  106  at the end of a hydrophobic compound can be used. The linear molecule  105  has a hydrophobic moiety, due to which the interaction occurs between the linear molecule  105  and the microparticle  103  and serves to connect the microparticle  103  and the bonding pad  102 . More specifically, the linear molecule  105  functions as a hydrophobic linker to bind the bonding pad  102  and the microparticle  103 . Examples of the hydrophobic moiety of the linear molecule  105  include an alkyl chain. The alkyl chain is preferably a straight chain. The length of alkyl chain is not limited but, in terms of the carbon number, preferably 3 to 20, and more preferably 8 to 12. 
         [0037]    Examples of the linear molecule  105  having the functional group  106  satisfying the above include alkylphosphate, alkyl sulfonic acid, alkyl glycoside (sugar alkyl), and the like. Specific examples include propylphosphoric acid, butylphosphoric acid, pentyl phosphoric acid, hexyl phosphoric acid, heptyl phosphoric acid, octylphosphoric acid, nonylphosphoric acid, decylphosphoric acid, undecylphosphoric acid, dodecylphosphoric acid, tridecylphosphoric acid, tetradecylphosphoric acid, pentadecylphosphoric acid, hexadecylphosphoric acid, heptadecylphosphoric acid, octadecylphosphoric acid, nonadecylphosphoric acid, eicosyl phosphoric acid; propylsulfonic acid, butylsulfonic acid, pentylsulfonic acid, hexylsulfonic acid, heptylsulfonic acid, octylsulfonic acid, nonylsulfonic acid, decylsulfonic acid, undecylsulfonic acid, dodecylsulfonic acid, tridecylsulfonic acid, tetradecylsulfonic acid, pentadecylsulfonic acid, hexadecylsulfonic acid, heptadecylsulfonic acid, octadecylsulfonic acid, nonadecylsulfonic acid, eicosylsulfonic acid; propyl glycoside, butyl glycoside, pentyl glycoside, hexyl glycoside, heptyl glycoside, octyl glycoside, nonyl glycoside, decyl glycoside, undecyl glycoside, dodecyl glycoside, tridecyl glycoside, tetradecyl glycoside, pentadecyl glycoside, hexadecyl glycoside, heptadecyl glycoside, octadecyl glycoside, nonadecyl glycoside, eicosyl glycoside, and the like. The alkyl chain forms a high density self-assembled monolayer (SAM film) and imparts the hydrophobic property on the bonding pad. In the present invention, the SAM film formed by the alkyl chain is also called a hydrophobic film. The hydrophobic property used herein refers to a low wettability to water and has a contact angle of more than a certain degree. More specifically, when water contacts the SAM film formed by an alkyl chain, the contact angle of a water drop to the SAM film is more than a certain degree. Typically, the hydrophobic property refers to the case in which a contact angle is 90° C. or more. However, in the present invention, when a contact angle is 50° or more, the pattern selectivity becomes larger and it is verified that the sufficient immobilization force between the microparticle  103  and the bonding pads  102  is further attained. The hydrophobic interaction is the phenomenon in which hydrophobic molecules clump up together rather than distributing itself in water, and thus in the present invention which uses water as the main component of the reaction solution, the hydrophobicity even lower than that based on the typical definition is considered to be sufficiently functional from the perspective of imparting the firm binding between the microparticle  103  and the bonding pad  102 . For the above reason, the alkyl chain contained in the linear molecule  105  may have the hydrophobic property having a contact angle of 50° or more. The linear molecule may contain other functional groups or may have a structural modification as long as a contact angle is 50° or more. 
         [0038]    Further, the thin film for preventing the non-specific adsorption (non-specific adsorption prevention film)  107  is formed at a contact angle of 50° or more on the region other than the region on which the bonding pads  102  are formed on the flat support substrate  101 . The thin film  107  is preferably made of an organic polymer material which prevents the non-specific adsorption of other compounds to the region other than the region on which the bonding pads  102  for the microparticles  103  are formed on the flat support substrate  101 . Examples of the organic polymer material usable include polyethylene glycol (PEG), poly-L-lysine PEG (pLL-PEG), polyacrylamide, 3-glycidoxypropylmethoxysilane (GOPS), and the like. The method for forming the non-specific adsorption prevention film  107  is not limited and a known method is employed, but, for example, the non-specific adsorption prevention film  107  can be formed by the silane coupling using a PEG silane agent, or the like. The non-specific adsorption prevention film  107  is not formed on the bonding pad  102 . 
         [0039]    The microparticle  103  hydrophobically interacts with the alkyl chain of the linear molecule  105  and is immobilized on the bonding pad  102  via the linear molecule  105 . Thus, the material itself composing the microparticle  103  must be hydrophobic, or the microparticle  103  must be hydrophobically treated by being conjugated with a hydrophobic substance such as a hydrophobic protein, or the like. Examples of such a microparticle  103  include organic polymer microparticles such as resin microparticles including polystyrene, polypropylene, and the like, semiconductor microparticles such as quantum dots (semiconductor nano particles) made of a semiconductor material including cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium sulfide (CdS), zinc selenide (ZnSe), zinc oxide (ZnO), and the like, metallic microparticles such as gold microparticles, and the like, polymer microparticles such as silica microparticles, and the like. These microparticles are used without further treatment when made of a hydrophobic material, whereas the microparticles, when otherwise, may be conjugated with a hydrophobic conjugation substance. The term conjugation used herein means to bind a conjugation substance to the microparticle surface, more specifically, to cover a part of or throughout the entire microparticle surface with a conjugation substance. Proteins are preferably used to be the conjugation substances, and avidin which is also usable as the probe of microparticle is preferably used to be the conjugation protein. The average particle diameter of the microparticle  103  is preferably 10 nm to 1 μm, more preferably 40 nm. 
         [0040]    A single microparticle  103  is immobilized on a single bonding pad  102 . In other words, the microparticle  103  is spatially immobilized on the flat support substrate  101  so as not contact with each other. 
         [0041]    For this reason, the diameter of the bonding pad is ⅕ to 5 times the diameter of the microparticle, and preferably less than or equal to the diameter of the microparticle. 
         [0042]    The probe molecule is not limited, but a single strand nucleic acid molecule of DNA or RNA capable of capturing the nucleic acid to be detected or analyzed in the present invention may be used. The nucleic acid to be detected or analyzed by the probe molecule is captured by the hybridization of the probe molecule and the nucleic acid to be detected or analyzed. The size of probe molecule is not also limited, and the probe molecule may be an adaptor molecule such as adaptor DNA, or the like, and in such a case the nucleic acid molecule to be analyzed is immobilized to the microparticle by binding a nucleic acid fragment complementarily hybridizable to the adaptor DNA immobilized on the microparticle to the nucleic acid to be analyzed. 
         [0043]    The nucleic acid analysis device shown in  FIG. 1  consists of the above flat support substrate  101 , the bonding pad  102 , the microparticle  103  on which the probe molecule  104  is immobilized, the linear molecule  105  and the non-specific adsorption film  107 . 
         [0044]    The formation of the bonding pad  102  on the flat support substrate  101  is carried out by using a known thin film process used for forming a semiconductor. 
         [0045]    The binding of the linear molecule  105  to the bonding pad  102  is carried out by allowing a solution of the linear molecule  105  to contact the bonding pad  102 , and, for example, the flat support substrate  101  on which the bonding pads  102  are formed may be immersed in the solution of the linear molecule  105  for a predetermined period of time at a predetermined temperature. 
         [0046]    After binding the linear molecule  105  to the bonding pad  102 , the non-specific adsorption prevention film  107  is formed on the region other than the region on which the bonding pads  102  are formed on the flat support substrate  101 . The formation of the non-specific adsorption prevention film  107  is carried out by silane coupling using as the silane coupling agent a silane derivative of an organic polymer which is to be a material for the non-specific adsorption prevention film  107  and forming a thin silane film of the organic polymer. The silane film is not produced on the metallic bonding pad  102 , or produced in a very weak force. The silane film bound on the metallic bonding pad can be removed by, for example, washing using a surfactant such as SDS, or the like. 
         [0047]    The order of binding the linear molecule  105  for bonding to the bonding pad  102  and the formation of the non-specific adsorption prevention film  107  is not limited, but the order may be determined in consideration of the binding properties of the functional group  106  of the linear molecule  105  and the organic polymer such as PEG, or the like, to be used for forming the non-specific adsorption prevention film  107 . For example, when the functional group of the linear molecule  105  is other than the hydroxyl group of sugar, e.g., when the linear molecule  105  is alkylphosphate, it easily binds to an organic polymer such as PEG, or the like, and hence it is preferable to form the non-specific adsorption prevention film  107  after the linear molecule  105  is bound to the bonding pad  102 . On the other hand, when the functional group of the linear molecule  105  is the hydroxyl group of sugar, the linear molecule  105  can be bound to the bonding pad  102  after the non-specific adsorption prevention film  107  is formed since such a linear molecule does not easily bind to an organic polymer such as PEG, or the like. 
         [0048]    The immobilization of the probe molecule  104  to the microparticle  103  is carried out so that a single probe molecule is immobilized to a single microparticle. The method for immobilizing a single probe molecule to a single microparticle is described in Example 1. The method for binding the probe molecule  104  to the microparticle  103  is not limited and may be chemical or physical binding, but, for example, avidin or streptavidin is bound to the microparticle  103 , while biotin is bound to the probe molecule  104 , thereby achieving the binding using the avidin-biotin binding. Alternatively, the binding is attained by the adsorption. 
         [0049]    The microparticle  103  on which the probe molecule  104  is immobilized is made contact with the flat support substrate  101 , on which the bonding pads  102  are formed and the non-specific adsorption prevention film  107  is formed. Since the microparticle  103  has the hydrophobic surface or is conjugated with a hydrophobic conjugation substance, the hydrophobic interaction occurs between the hydrophobic linear molecule  105  bound on the bonding pad  102  and the microparticle  103 , whereby the microparticle  103  is immobilized on the bonding pad  102 . 
         [0050]    Thus, the flat support substrate  101  to which the probe molecule  104  is bound can be produced. The presence of the probe molecule  104  on the flat support substrate  101  can be confirmed using, for example, a laser microscope. 
       EXAMPLE 1 
       [0051]    Hereinbelow, an example is shown in which the microparticle  103  on which the probe molecule  104  is immobilized is actually immobilized on the nucleic acid analysis device. In this example, a nucleic acid analysis device was produced for use in which titanium bonding pads having a thickness of 10 nm and a diameter of 60 nm are arrayed in a grid pattern with a 1 μm interval on a quartz glass substrate having a thickness of 0.7 mm. 
         [0052]    For the method for forming the bonding pad  102  on the quartz glass substrate, the thin film process which has already been used practically in the semiconductors was applied. More specifically, after forming a titanium thin film by the vapor deposition-sputtering through a mask, and the vapor deposition-sputtering, a resist pattern was formed by lithography using EB, and the bonding pad was produced by dry and wet etchings. The interval among the bonding pads  102  may be arbitrary set, but is preferably 500 nm or more when an optical measurement is carried out as the detective means in consideration of the diffraction limit of optical detection. The formation method of the bonding pad is described in detail in Example 3. 
         [0053]    The linear molecule  105  for immobilizing the microparticle was reacted and bound on the bonding pad  102  of the nucleic acid analysis device. In the present example, dodecylphosphoric acid, which is alkylphosphate, was used as the linear molecule  105 . A solution in which a powder of dodecylphosphoric acid was dispersed in ultra pure water in a concentration of 0.3 mM was prepared, and the nucleic acid analysis device on which the bonding pads  102  produced by the above method were formed was immersed in the solution and treated at 90° C. for an hour or more. When treating using dodecylphospholic acid in a concentration of 0.3 mM, the solubility of dodecylphospholic acid is enhanced by adding the equimolar amount of ammonia. Alternatively, ammonium dodecyl phosphate may be directly dissolved in water. At this time, the linear molecule  105  did not bind to the region other than the bonding pads  102  on the quartz glass substrate due to the binding selectivity between the functional group  106  of the linear molecule  105  and the bonding pad  102 . 
         [0054]    Next, the non-specific adsorption prevention film  107  was formed on the quartz glass surface of the nucleic acid analysis device using a PEG silane agent. Using 2-methoxypolyethyleneoxypropyltrimethoxysilane (produced by Gelest, Inc., Mw: 2,000) as the PEG silane agent, an SAM film was produced to be the non-specific adsorption prevention film. In the production method, the PEG silane agent was dissolved in a toluene solvent so as to give 3 mM and triethylamine was added as a catalyst to give the final concentration of 1%. The nucleic acid analysis device was immersed in the mixed solution and reacted for 30 minutes at 60° C. The nucleic acid analysis device was washed using toluene and ethanol after taken out from the mixed solution and baked at 90° C. for 10 minutes using an electric furnace. The film thickness of silane film on the substrate was measured using a spectroscopic ellipsometer, and the result found that the silane film had a film thickness of 20 Å. It is also verified that the PEG silane film was not produced on the bonding pad to which dodecylphospholic acid was bound. 
         [0055]    Subsequently, the microparticle on which a single molecule of the nucleic acid probe was immobilized was reacted to the nucleic acid analysis device treated in the above at room temperature for 15 minutes or more. A polystyrene NeutrAvidin-conjugated bead (FluoSpheres (registered trademark) F8773 Invitrogen) having a diameter of 40 nm was used as the microparticle. Biotinylated DNA was used as the probe molecule for capturing a nucleic acid. 
         [0056]    A method for binding a single probe molecule to a single microparticle is described below. In the present example, a nucleic acid sample fragment, in which the end of polynucleotide composed of 50 to 100 bases was biotinylated and the 5′-end was fluorescence-labeled with Cy3, was used as the probe molecule. When the number of microparticle is increased 10 times the number of probe molecule for the reaction, the probe molecules are not captured in about 90% of the microparticles and a single probe molecule is captured by the avidin-biotin binding in about 9% of the microparticles. This result exactly matches the prediction results of when the Poisson distribution is assumed. Accordingly, when collecting only the microparticles which captured the probe molecule, 90% or more of the collected microparticles are those which captured only a single probe molecule. In this condition, the microparticles to which a single probe molecule is bound can be obtained in much higher purity using molecular weight separation, magnetic microparticle collection, electrophoresis separation using an electrical charge difference, or the like. The probe molecule  104  immobilized on the substrate  101  was confirmed using a total internal reflection laser microscope. After the immobilization reaction of the microparticles, YAG laser (532 nm) is incident to the substrate back side under the condition of total reflection while the substrate top is filled with water, and the projected fluorescence was condensed from the substrate top side through an objective lens for CCD camera observation. A single molecule of Cy3 can be confirmed when Cy3 is excited with a laser, caused to light up and quenches in a single stage. When Cy3 is more than 2 molecules, a multistage quenching occurs. The single molecule immobilization rate was calculated by this method and the results revealed that a single molecule of the probe molecule  104  was immobilized on 70% or more of the bonding pads. Further, when 500 pieces of the bonding pads of the nucleic acid analysis device were observed at random using a scanning electron microscope (SEM), the microparticles were revealed to have been immobilized in an immobilization rate of about 90%. 
         [0057]    In accordance with the method shown in Example 1 of Patent Literature 2, when using PVPA (polyvinyl phosphonic acid) in which 30 molecules of biotin were incorporated in a single molecule, microparticles conjugated with streptavidin in a 500 nM density was reacted therewith for 2 hours to immobilize the microparticles on the substrate by the streptavidin-biotin binding, the microparticle has an immobilization density on the substrate of 30 to 50 pieces/μm 2 . In contrast with this method, when microparticles in the same density were reacted using dodecylphospholic acid for 2 hours to immobilize the microparticles on a substrate by the hydrophobic interaction in accordance with the method of the present example, the microparticle had an immobilization density of 80 to 100 pieces/μm 2 . The immobilization amount of the microparticle was substantially saturated in the 2 hour-reaction. Consequently, the method wherein dodecylphospholic acid was used according to the present example has a higher density as the bonding molecule on the substrate. Further, it was revealed that when dodecylphospholic acid was used, the immobilization reaction of the microparticles reached 60 to 80 pieces/μm 2  in 10 minutes and substantially saturated in 30 minutes from the start. The reaction rate is about 5 times the rate in the case where the biotin-incorporated PVPA is used. This is presumably due to the influence of the electrical charge difference at the metal portion on which the linear molecule  105  is attached. Both of the linear molecules use a phosphoric acid group as the functional group, and thus the bonding pads are negatively charged. For this reason, the electric repulsion is caused against the microparticles which are also similarly negatively charged. However, the dodecyl phosphoric acid has the alkyl chain moiety whose film thickness reaches 2 to 3 nm and hence the negative electric charge at the outermost surface is reduced and the above electric repulsion is also decreased, whereby the immobilization reaction rate when the microparticles are bound to the bonding pads is thought to be enhanced. As described above, the present example demonstrated the result that it is more advantageous to use dodecyl phosphoric acid as the linker for binding the microparticles to the substrate, that is, to bind the microparticles to the substrate using the hydrophobic interaction. Further, the dodecyl phosphoric acid preparation which only requires the dissolution in water can be carried out in an extremely short time at an inexpensive price and furthermore has advantageously good reproductivity, and thus the method according to the present invention in which the microparticles are bound to the substrate using the hydrophobic interaction is very effective. 
         [0058]    There are some systems conceivable for the method for detecting information on a nucleic acid sample from the nucleic acid analysis device of the present example, but the method which uses the fluorometric detection is preferable in light of sensitivity and convenience. First, a nucleic acid sample is supplied to the nucleic acid analysis device and the probe molecule  104  is allowed to capture the nucleic acid sample. Next, a nucleotide having a fluorescent dye is supplied, and when the probe molecule  104  is a DNA probe, a nucleic acid polymerase is supplied. The fluorescent dye incorporated into the nucleic acid chain during the nucleic acid elongation reaction occurred on the device is subjected to the fluorescence measurement. In this case, the so-called sequential elongation reaction system, in which one kind of nucleotide is supplied, unreacted nucleotides are washed and fluorescence imaged, a different kind of nucleotide is supplied and the same procedure is repeated thereafter, is easily carried out. The fluorescent dye is quenched after the fluorescence observation or the nucleotide in which the fluorescent dye is attached to the phosphoric acid moiety is used to cause the sequential reaction, whereby the nucleotide sequence information of the nucleic acid sample can be obtained. Alternatively, the so-called real time reaction system can also be carried out by supplying 4 kinds of nucleotide having different fluorescent dyes respectively without washing to cause a sequential nucleic acid elongation reaction and continuously carrying out the fluorescence observation. In this case, when the nucleotide in which the fluorescent dye is attached to the phosphoric acid moiety is used, the phosphoric acid moiety is cut off after the elongation reaction and thus the fluorescence measurement is continuously carried out without quenching to obtain the nucleotide sequence information of the nucleic acid sample. 
       EXAMPLE 2  
       [0059]    In Example 2, an example is shown wherein octyl glucoside, which is an alkyl glycoside non-ionic surfactant, is used as the linear molecule  105  and a semiconductor quantum dot is used as the microparticle  103 . In this example, the nucleic acid analysis device had the same components as in Example 1, provided that the flat support substrate  101  had the titanium bonding pad  102  having a diameter of 20 nm. Examples of the advantage of using octyl glucoside include that the linear molecule  105  for bonding the microparticles can be reacted even after the production of the non-specific adsorption prevention film  107  and the linear molecule  105  is removable by changing the pH, or the like. Alkylphosphate comparatively easily adheres to PEG and it is hence required to treat the bonding pad  102  with alkylphosphate before forming the non-specific adsorption prevention film  107  for binding alkylphosphate to the bonding pad  102 . On the other hand, when alkyl glycoside such as octyl glucoside is used, alkyl glucoside is bound on the bonding pad  102  after forming the non-specific adsorption prevention film  107  by producing a PEG silane film since alkyl glucoside is hardly adsorbed to the non-specific adsorption prevention film  107 , whereby the hydrophobic patterning is enabled. Consequently, when alkyl glucoside is used as the linear molecule  105 , alkyl glucoside is removable from the substrate after the nucleic acid analysis device is used, thereby being beneficial for repeatedly using the nucleic acid analysis device. 
         [0060]    In the present example, using 2-methoxypolyethyleneoxypropyltrimethoxysilane (produced by Gelest, Inc., Mw: 2,000) as the PEG silane agent as in Example 1, a self-assembled monolayer (SAM film) was produced in the same manner as in Example 1 on the nucleic acid analysis device as the non-specific adsorption prevention film  107 . The PEG silane film is also formed on the metallic bonding pads, however, since the binding force thereof is extremely weaker to a metal than the bonding to a quartz glass, only the PEG silane film on the bonding pads is selectively peeled off by washing with a 0.1% SDS solution. Subsequently, the nucleic acid analysis device was immersed in a 1% octyl glucoside aqueous solution and reacted at 60° C. for 30 minutes. The device was washed with ultra pure water and baked at 90° C. for 10 minutes using an electric furnace. The microparticle was then reacted to the above treated nucleic acid analysis device at room temperature for 15 minutes or more. The microparticle used was a quantum dot (Qdot (registered trademark) 705 StreptAvidin Conjugated, Invitrogen) having a CdSe core diameter of 15 nm and conjugated with streptavidin. A polystyrene NeutrAvidin-conjugated bead (FluoSpheres (registered trademark) F8773, Invitrogen) having a diameter of 40 nm can also be used. Further, when the nucleic acid analysis device was observed using a scanning electron microscope (SEM), it was revealed that the microparticles were immobilized in an immobilization rate of about 90% or more. An example of the advantage for using the quantum dot as the microparticle is that the FRET effect can be used. In other words, using the quantum dot as the energy donor by light irradiation, the fluorescent dye labeling the nucleic acid molecule to be detected captured when analyzing the nucleic acid is actuated as an acceptor for the above energy, thereby enabling the detection by the fluorescence of the detection target molecule. Since the energy transfer is the phenomenon which only occurs in the vicinity of about less than or equal to 10 nm, it is effective in the case where the nucleic acid molecule to be detected is labeled with the same fluorescent dye and a specific molecule is detected in a high concentration of the sample solution. In this instance, the source of excitation light only needs to excite the semiconductor microparticles, and the light source may be only one kind, hence preferable. 
         [0061]    Also, as in  FIG. 2 , a method in which a single molecule of DNA polymerase  204 , which is a nucleic acid polymerase, is immobilized on a microparticle  203  (enzyme immobilization method) can be used.  FIG. 2   a  shows the state in which a bonding pad  202  is formed on a flat support substrate  201 , and a microparticle  203  on which the DNA polymerase  204  is immobilized is bound on the bonding pad by a hydrophobic interaction via a hydrophobic film formed from linear molecules  205  having an end functional group  206 , and  FIG. 2   b  shows the state in which the microparticles  203  on which the DNA polymerase  204  is immobilized are regularly formed on the bonding pads  202  on the flat support substrate  201 . In this method, as the one-to-one immobilization of the nucleic acid molecule and microparticle described in Example 1, a microparticle attached with a single molecule of DNA polymerase is produced by the probability reaction. The microparticle surface is conjugated with an epoxy group, a tosyl group, an amino group, a carbonyl group to immobilize a DNA polymerase thereon. It is particularly preferable to react the microparticle with an epoxy group which does not deactivate the DNA polymerase and enables a moderate binding reaction to proceed. Next, the produced enzyme-attached microparticles are immobilized on the bonding pads  202 . Subsequently, when fluorescence-labeled 4 kinds of bases (A, T, C, G)  209  are incorporated in the reaction solution, the DNA polymerase captures a nucleic acid molecule  208  to be a template and keeps adding nucleotides one after another complementary to the template, thereby proceeding the DNA elongation reaction. During this process, the microparticles are irradiated with an excitation light and caused the fluorescence of the incorporated nucleotides to light up, which are observed using a CCD camera. Each kind of bases is labeled with a different colored fluorescent dye and the DNA sequence can be determined real time by image processing which identifies the colors. The enzyme immobilization method has an advantage that the position of the bright spots does not blur even in a case of long read since the intake for the enzyme, that is, the fluorescence-labeled bases, is immobilized on the bonding pad  202 . With no blurred position, the signals between the adjacent bonding pads are prevented from being intermixed and hence the bonding pads can be arrayed in a higher density manner, thereby increasing the number of parallels. Further, unlike the method in which the template of nucleic acid is immobilized as in Example 1, when the enzyme once immobilized is deactivated, the sequence reaction is not carried out at that reaction site. However, by using in combination with a removable alkyl glucoside linker, the enzyme-attached microparticle can be exchanged together with the linker and the all the enzymes can be refreshed every sequence reaction cycle. 
       EXAMPLE 3  
       [0062]    An example of the method for producing a nucleic acid analysis device is described with reference to  FIG. 3 . A film is produced by sputtering a material composing the bonding pad, e.g., gold, titanium, nickel or aluminum, on a flat support substrate  301  ( FIG. 3   a ) ( FIG. 3   b ). The film produced is to be a metallic thin film (metal deposition film)  308 . When a quartz glass substrate or a sapphire substrate is used as the flat support substrate  301  and gold, aluminum or nickel is used as the material for the bonding pad, it is preferable to form a titanium or chromium thin film to reinforce the bonding between the substrate material and the bonding pad material. Additionally, the bonding pad as thin as possible is more preferable. This is because when the flat support substrate  301  is thick, the area on the side portion increases, and even when the diameter of microparticle is less than or equal to the diameter of the bonding pad, a plurality of microparticles may increasingly be likely to be immobilized. For this reason, the metallic thin film  308  is preferably produced as thin as possible at the time of film forming. A pattern is formed using a resist  309  on the metallic thin film  308  ( FIG. 3   c ). Next, the metallic thin film  308  other than the resist pattern is removed by etching ( FIG. 3   d ). The resist  309  is further removed to complete the bonding pad  302 . After peeling off the resist, linear molecules  305  selectively adsorbed only by the bonding pad  302  are incorporated ( FIG. 3   e ) to further form a non-specific adsorption prevention film  307  ( FIG. 3   f ). 
       EXAMPLE 4  
       [0063]    An example of the method for producing a nucleic acid analysis device is described with reference to  FIG. 4 . An example is shown in which the same materials are used as in Example 3, provided that, for example, a flat covering film  410  composed of a material such as glass is formed on a metallic thin film (metal deposition film)  408  and the flat covering film  410  is processed to carry out patterning. The film is produced on the flat support substrate  401  using the material which composes the bonding pads. In the case of the present example, the material composing the bonding pad must have visible light transmission properties. For example, titanium or aluminum is sputtered to produce a film. This film is to be the thin film  408  ( FIG. 4A  a,  FIG. 4B  a). A glass substrate or a sapphire substrate is used as the flat support substrate  401 . Additionally, the bonding thin film layer  408  as thin as possible is more preferable. Further, the bonding thin film layer  408  is preferably annealed at 600° C. or more. This is to enhance the transmission rate of the excitation light which irradiates from the bottom side of the substrate. For this reason, the metallic thin film  408  is preferably produced as thin as possible at the time of film forming and annealed. 
         [0064]    In  FIG. 4A , a pattern is formed on this metallic thin film  408  using a resist  409  ( FIG. 4A  b). The flat covering film  410  is formed thereon ( FIG. 4A  c). The resist pattern is removed by etching together with the flat covering film  410  on the resist  409  to complete the patterning of the metal and the flat covering film  410  ( FIG. 4A  d). In this instance, the region on which the flat covering film  410  does not sit on the metallic thin film  408  of  FIG. 4A  d serves as the bonding pad. Further, linear molecules  405  are adsorbed to the bonding pad portion and a non-specific adsorption prevention film  407  is further formed on the flat covering film  410  ( FIG. 4A  e). 
         [0065]    In  FIG. 4B , the flat covering film  410  is formed on the bonding thin film layer  408  ( FIG. 4B  b). The pattern of bonding pads is formed thereon using the resist  409  ( FIG. 4B  c). At this time, by using a positive resist, only the resist of the portion irradiated in the bonding pad patter is removed by etching, and the portion is etched to the outermost surface of the metal. Finally, the remained resist  409  of the resist pattern is removed by etching to complete the patterning of the flat covering film  410  and the metal ( FIG. 4B  d). In this instance, the region on which the flat covering film  410  does not sit on the metallic thin film  408  of  FIG. 4B  d serves as the bonding pad. Further, linear molecules  405  are adsorbed to the bonding pad portion and a non-specific adsorption prevention film  407  is further formed on the flat covering film  410  ( FIG. 4B  e). 
       EXAMPLE 5  
       [0066]    In the present example, an example of the preferable structure for a nucleic acid analyzer which uses the nucleic acid analysis device is described with reference to  FIG. 5 . The nucleic acid analyzer of the present example comprises at least the nucleic acid analysis device, a means for supplying nucleotides having a fluorescent dye, a nucleic acid polymerase, and a nucleic acid sample to the nucleic acid analysis device, a means for irradiating the nucleic acid analysis device with light, and a luminescence detector for measuring the fluorescence of a fluorescent dye incorporated into a nucleic acid chain through the nucleic acid elongation reaction induced by the coexistence of the nucleotide, the nucleic acid polymerase, and the nucleic acid sample on the nucleic acid analysis device. 
         [0067]    More specifically, the above nucleic acid analysis device  505  is set in a reaction chamber consisting of a cover plate  501 , a detection window  502 , and an inlet  503  and an outlet  504 , which are openings for exchanging the solution. PDMS (Polydimethyl siloxane) is used as the material for the cover plate  501  and the detection window  502 . Further, the detection window  502  has a thickness of 0.17 mm. Of the laser lights  509  and  510  oscillated from YAG laser light source  507  (wavelength 532 nm, output 20 mW) and YAG laser light source  508  (wavelength 355 nm, output 20 mW), only the laser light  509  is circular polarized using a lambda/4 plate  511  and the above two laser lights are adjusted so as to be on the same axis using a dichroic mirror  512  (reflects 410 nm or less) and condensed with a lens  513  and irradiate the device  505  at a critical angle or more via a prism  514 . The fluorescent dyes in the nucleic acid chain are excited with the laser light, and a part of the fluorescence is emitted via the detection window  502 . The fluorescence emitted from the detection window  502  is converted to a parallel luminous flux by an objective lens  515  (×60, NA 1.35, working distance 0.15 mm), and the background light and excitation light are blocked off by an optical filter  516 , whereby the luminous flux is imaged on a two-dimensional CCD camera  518  by an image forming lens  517 . The nucleic acid analysis device is caused to move, and the same measurement can be repeatedly carried out targeting the nucleic acid immobilized on the microparticle on each of the bonding pads. 
         [0068]    In the case of the sequential reaction system, a usable nucleotide is that in which 3′-O-allyl group is introduced as a protective group at 3′OH position of the ribose and a fluorescent dye is bound via an allyl group to the 5th position of pyrimidine or the 7th position of purine. Since the allyl group is cut when contact the light irradiation or palladium, both the quenching of dyes and the control of elongation reaction can be achieved at the same times. The unreacted nucleotides do not need to be removed by washing even in the sequential reaction. Further, the real time measurement of the elongation reaction is viable since the washing step is not required. In this case, there is no need to introduce the 3′-O-allyl group as a protective group at 3′OH position of the ribose in the above nucleotide, and the nucleotide, which is bound to a dye via a functional group cuttable by the light irradiation, may be used. 
         [0069]    In the case where a semiconductor microparticle is used as the energy transfer medium to the fluorescent dye, the above example of the nucleic acid analyzer is applicable. For example, when Qdot (registered trademark) 565 StreptAvidin conjugated (Invitrogen) is used as the semiconductor microparticle, the YAG laser light source  507  (wavelength 532 nm, output 20 mW) can cause a sufficient excitation. This excitation energy emits fluorescence when transferred to Alexa 633 (Invitrogen), which is not excited by light of 532 nm. In other words, the dyes attached to the unreacted nucleotides are not excited but light up for the first time when captured by the DNA probe and come close to the semiconductor microparticle where the energy transfer occurs, and hence the captured nucleotide can be identified by the fluorescence measurement. When the material of the microparticle for immobilizing the probe molecule is an organic polymer, the excitation is not caused by the light irradiation using an exterior light source. Accordingly, the fluorescent dyes do not light up by the transfer of the excitation energy, and also the unreacted nucleotides light up, likely causing noises. However, when the microparticle such as semiconductor microparticle which causes the energy transfer, is bound to a nucleic acid polymerase, only the incorporated nucleotides are caused to light up. Alternatively, when gold, silver, platinum, aluminum, or the like, is bound to a nucleic acid polymerase, the fluorescence of the incorporated nucleotides can be enhanced. Alternatively, when gold, silver, platinum, aluminum, or the like is used as the material of the bonding pad for immobilizing the microparticle, the fluorescence around the bonding pad is enhanced, thereby increasing the SIN ratio. 
         [0070]    In the nucleic acid analyzer of the present invention, an evanescent field is used as the means for irradiating the nucleic acid device with light. More specifically, the evanescent light is irradiated from the means for irradiating light, that is, a laser light oscillated from a laser light source irradiates the device  505  via the prism  514  at a critical angle or more. The fluorescent dye in the nucleic acid chain is excited by the laser light, and a part of the fluorescence stays unreflected but is emitted in the form of evanescent light via the detection window  502  and measured by a fluorescence microscope. 
         [0071]    As described above, by assembling a nucleic acid analyzer using the nucleic acid analysis device of the present example, the washing step is obviated, the time required for analysis is cut short, the device and analyzer can be simplified, and the real time nucleotide elongation reaction can also be measured in addition to the sequential reaction system, whereby a significant amount of time is saved in comparison with the conventional art. 
         [0072]    The nucleic acid analyzer comprising the nucleic acid analysis device of the present invention can carry out a wide variety of nucleic acid analyses such as determining a DNA sequence and performing hybridization. Particularly, the DNA sequence can be determined (DNA sequence) using the nucleic acid analyzer of the present invention. The method for carrying out the DNA sequence is not limited and is achieved by the method which employs the fluorometric detection. 
       INDUSTRIAL APPLICABILITY 
       [0073]    In the nucleic acid analysis using the nucleic acid analysis device, the dephasing never occurs, and hence the read length is extended as well as many kinds of DNA fragments to be analyzed can be quickly immobilized in a large amount and analyzed, thereby achieving an extremely high throughput. 
         [0074]    All publications, patents and patent applications cited herein shall be incorporated per se by reference in the specification. 
       REFERENCE SIGNS LIST 
       [0000]    
       
           101 ,  201 ,  301 ,  401  Flat support substrate 
           102 ,  202 ,  302 ,  402  Bonding pad 
           103 ,  203  Microparticle 
           104  Probe molecule 
           105 ,  205 ,  305 ,  405  Linear molecule 
           106 ,  206  End functional group of the linear molecule 
           107 ,  207 ,  307 ,  407  Non-specific adsorption prevention film 
           204  Nucleic acid polymerase 
           208  Template nucleic acid fragment 
           209  Nucleic acid substrate 
           308 ,  408  Metal deposition film 
           309 ,  409  Electron beam resist 
           410  Flat covering film 
           501  Cover plate 
           502  Detection window 
           503  Inlet 
           504  Outlet 
           505  Device 
           506  Flow channel 
           507 ,  508  YAG laser light source 
           509 ,  510  Laser light 
           511  lambda/4 plate 
           512  Dichroic mirror 
           513  Lens 
           514  Prism 
           515  Objective lens 
           516  Optical filter 
           517  Image forming lens 
           518  Two-dimensional CCD camera