Patent Publication Number: US-2022229010-A1

Title: Electrochemical detection method for catalytic reaction product, and transducer

Description:
TECHNICAL FIELD 
     The present invention relates to a technique for electrochemically detecting a product generated by progress of a catalytic reaction in a solution and dissolved in the solution. 
     BACKGROUND ART 
     Detection sensitivity of a catalytic reaction product generated by catalytic reaction such as enzyme reaction and dissolved in a solution depends on the concentration of the product in the solution. In order to improve the concentration of the product in the solution, for example, longer catalytic reaction time is preferable or a smaller volume of the solution is preferable. 
     When the volume of the solution is extremely small, evaporation leads to a decrease in the volume of the solution, making detection impossible. Such a problem arises noticeably in the case of a long catalytic reaction time. 
     Patent literature 1 and Non-patent literature 1 disclose a configuration that can prevent a solution from evaporating. Patent literature 1 discloses, as a technique related to ELISA (Enzyme-Linked ImmunoSorbent Assay), a configuration in which droplets of a hydrophilic solvent, which is an enzyme reaction field, are placed in a storage part (well) and the storage part is sealed with a hydrophobic solvent. 
     Regarding an ELISA-related technique, Non-patent literature 1 discloses a configuration in which a pattern of hydrophilic region is formed by forming a hydrophobic region on a hydrophilic surface, and droplets located on the pattern of hydrophilic region (that is, the enzyme reaction field) are covered with oil. 
     PRIOR ART LITERATURE 
     Patent Literature 
     
         
         Patent literature 1: International Publication No. WO2012/121310 
       
    
     Non-Patent Literature 
     
         
         Non-patent literature 1: S. Sakakihara et al., “A Single-molecule enzymatic assay in a directly accessible femtoliter droplet array”, Lab Chip, 2010, 10, 3355-3362 
       
    
     SUMMARY OF THE INVENTION 
     Problems to be Solved by the Invention 
     As described above, evaporation of the solution can be prevented by covering a solution, which is a catalytic reaction field, with a liquid which is different from the solution. Thus, it is possible to achieve detection with high sensitivity. 
     The techniques disclosed in Patent literature 1 and Non-patent literature 1 are spectroscopic detection techniques using a fluorescent substrate and require a large-scale measuring apparatus. Moreover, the fluorescent substrate is expensive. Therefore, these techniques require a large cost. 
     An object of the present invention is to provide a technique that can inexpensively and easily detect a catalytic reaction product with high sensitivity. 
     Means to Solve the Problems 
     The following technical matters are described simply to facilitate the understanding of the main points of the present invention, not to limit the invention claimed in the claims explicitly or implicitly and not to express the possibility of accepting such a limitation that is imposed by a person other than those who will benefit from the present invention (for example, the applicant and the right holder). The general outline of the present invention described from other perspectives can be understood from, for example, the claims of this application as originally filed at the time of application. 
     The technique disclosed here is an electrochemical detection technique to which the technique that a first lump of liquid, where a catalytic reaction progresses, is covered with a second lump of liquid is applied. 
     The technique uses a liquid bath that contains a working electrode, a counter electrode, a first lump of liquid and a second lump of liquid. 
     The first lump of liquid has conductivity. The working electrode is located in the first lump of liquid. 
     The second lump of liquid has conductivity. The first lump of liquid and the second lump of liquid form a liquid-liquid interface and the catalytic reaction product is insoluble in the second lump of liquid. The counter electrode is located in the second lump of liquid. 
     The current is detected which flows through the working electrode by an oxidation reduction reaction between the catalytic reaction product and the working electrode. 
     Effects of the Invention 
     According to the present invention, it is possible to detect a catalytic reaction product inexpensively and easily with high sensitivity. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic diagram of a detection apparatus; 
         FIG. 2A  is a diagram illustrating a first example of a structure that holds a first lump of liquid; 
         FIG. 2B  is a diagram illustrating a second example of the structure that holds the first lump of liquid; 
         FIG. 2C  is a diagram illustrating a third example of the structure that holds the first lump of liquid; 
         FIG. 2D  is a diagram illustrating a fourth example of the structure that holds the first lump of liquid; 
         FIG. 2E  is a diagram illustrating a fifth example of the structure that holds the first lump of liquid; 
         FIG. 3A  is a diagram illustrating a sixth example of the structure that holds the first lump of liquid; 
         FIG. 3B  is a diagram illustrating a seventh example of the structure that holds the first lump of liquid; 
         FIG. 3C  is a diagram illustrating an eighth example of the structure that holds the first lump of liquid; 
         FIG. 4  is a diagram for describing an example of the structure that holds the first lump of liquid.  FIG. 4( a )  is a schematic diagram of a well,  FIG. 4( b )  is a schematic diagram illustrating a state in which the first lump of liquid is introduced into the well, and  FIG. 4( c )  is a schematic diagram illustrating a state in which the second lump of liquid covers the first lump of liquid; 
         FIG. 5  is a diagram illustrating another example of the structure that holds the first lump of liquid; 
         FIG. 6A  is a plan view illustrating an embodiment of a transducer; 
         FIG. 6B  is a cross-sectional view illustrating the embodiment of the transducer; 
         FIG. 7  is a perspective view of the transducer shown in  FIG. 6A ; and 
         FIG. 8  is a diagram for describing an array of electrodes in the transducer shown in  FIG. 6A . 
     
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
     The present embodiment will be described with reference to the accompanying drawings. 
     According to the embodiment, electrochemically detected is a product generated by progress of a catalytic reaction in a first lump of liquid (that is, a lump of solution, which is a catalytic reaction field) and dissolved in the first lump of liquid.  FIG. 1  schematically shows a configuration example of a detection apparatus  1  of the present embodiment. 
     The detection apparatus  1  includes a liquid bath  10 , a working electrode  40 , a counter electrode  50 , a reference electrode  60  and a potentiostat  80 . The liquid bath  10  contains a first lump of liquid  20  and a second lump of liquid  30 . The first lump of liquid  20  and the second lump of liquid  30  form a liquid-liquid interface (that is, an interface between two liquids). As shown in  FIG. 1 , the first lump of liquid  20  is placed on a bottom surface  11  of the liquid bath  10  and is covered with the second lump of liquid  30 . 
     The working electrode  40  is located at the bottom surface  11  of the liquid bath  10  and covered with the first lump of liquid  20 . That is, the working electrode  40  is in contact with the first lump of liquid  20 , whereas it is not in contact with the second lump of liquid  30 . The counter electrode  50  and the reference electrode  60  are placed in the second lump of liquid  30  and are electrically connected to the working electrode  40  via the liquid-liquid interface between the first lump of liquid  20  and the second lump of liquid  30 . In  FIG. 1 , reference numeral  70  denotes a salt bridge. 
     The working electrode  40 , the counter electrode  50  and the reference electrode  60  are connected to the potentiostat  80  in this example. The potentiostat  80  functions as a constant-voltage power-supply apparatus and includes a variable power supply  81 , a voltmeter  82  and an ammeter  83 . 
     The catalytic reaction product is confined in the first lump of liquid  20  and is not dissolved in the second lump of liquid  30  (that is, the product does not move from the first lump of liquid  20  to the second lump of liquid  30 ). An oxidation reduction reaction between the catalytic reaction product and the working electrode  40  causes a current to flow through the working electrode  40 . By detecting this current, the catalytic reaction product is detected or a quantitative analysis is performed. 
     Although  FIG. 1  shows only one working electrode  40 , two or more working electrodes  40  arranged, for example, in an array are generally installed at the bottom surface  11  of the liquid bath  10 . When the detection apparatus  1  includes two or more working electrodes  40 , the detection apparatus  1  includes two or more first lumps of liquid  20 . Each of the two or more working electrodes  40  is covered with the corresponding one of the two or more first lumps of liquid  20 . The first lumps of liquid  20  are independent of each other and the first lumps of liquid  20 , which are different from each other, are separated by the second lump of liquid  30 . The second lump of liquid  30  is a single liquid lump. The second lump of liquid  30  and any one of the two or more first lumps of liquid  20  form a liquid-liquid interface. Each of the two or more first lumps of liquid  20  is covered with the second lump of liquid  30 . 
     Hereinafter, an electrochemical detection method according to the present embodiment applied to ELISA will be described. 
     According to ELISA, an antigen-antibody complex is detected or quantitative analysis is performed, for example, by labeling an antigen or antibody (that is, immunoglobulin) contained in a sample with an enzyme and detecting a product obtained by a reaction between the enzyme and a substrate. For example, the following operation is performed in a combination of a sandwich ELISA (sandwich ELISA protocol) and an electrochemical detection method. However, operation such as cleaning, incubation (leaving a product at a constant temperature) is not specified. 
     (1) Binding of capture antibody to a solid phase (the solid phase includes a surface of the working electrode and a surface of a solid substance in the vicinity of the working electrode) 
     (2) Blocking treatment of the solid phase 
     (3) Addition of antigen (protein to be detected) 
     (4) Addition of primary antibody 
     (5) Addition of enzyme-labeled secondary antibody 
     (6) Addition of substrate-containing first lump of liquid (by enzyme reaction, enzyme reaction product is accumulated in the vicinity of the working electrode) 
     (7) Electrochemical detection of enzyme reaction product using the working electrode 
     In the embodiment, an operation of covering the entire first lump of liquid  20  with the second lump of liquid  30  is added as shown in  FIG. 1 . 
     The second lump of liquid  30  is insoluble in the conductive first lump of liquid  20 , and is a conductive liquid. In ELISA, the first lump of liquid  20  is generally an aqueous solution having a buffering ability, and thus the second lump of liquid  30  is, for example, an organic solvent that is insoluble in water and can dissolve a support electrolyte that is for conductivity. 
     The organic solvent is preferably a liquid that can be easily handled as a solvent for electrochemical detection, in other words, preferably, the organic solvent is a liquid at ordinary temperature and has low reactivity against water and electrode materials (such as, gold, platinum) within a detection potential range. For example, nitrobenzene, 1,2-dichlorobenzene, 1-nitro-2-(n-octyloxy) benzene, 1,2-dichloroethane, 1,4-dichlorobutane, 1,6-dichlorohexane, 1-octanol or 1,9-decadiene is suitable for the organic solvent. 
     As the support electrolyte that is soluble in these organic solvents and can impart conductivity to the organic solvents, a support electrolyte used for electrochemical detection in a common non-aqueous solution may be adopted. For example, the support electrolyte is preferably a salt containing, as its anion, any one of chloride ion, bromide ion, iodide ion, sulfate ion, nitrate ion, hyperchloric acid ion, tetrafluoroboric acid ion, hexafluorophosphoric acid ion and sulfonic acid ion, and, as its cation, any one of lithium ion, sodium ion, potassium ion, rubidium ion, cesium ion, ammonium ion and tetraalkyl ammonium ion having alkyl groups of arbitrary chain lengths. 
     A combination of the labeling enzyme and the substrate is a combination having electrochemical activity and capable of generating a product soluble in the first lump of liquid  20  and not soluble in the second lump of liquid  30 . When the first lump of liquid  20  is an aqueous solution and the second lump of liquid  30  is the aforementioned organic solvent, a combination of, for example, alkaline phosphatase and phosphoric acid 4-aminophenyl ester or a combination of, for example, horseradish peroxidase and potassium ferricyanide is suitable for a combination of the labeling enzyme and the substrate. 
     In the embodiment, as shown in  FIG. 1 , for high sensitivity detection, an extremely small amount of first lump of liquid  20  is held in contact with the working electrode  40 , and further, a state is established in which the working electrode  40 , the counter electrode  50  and the reference electrode  60  are electrically connected to one another via the first lump of liquid  20  and the second lump of liquid  30  that covers the first lump of liquid  20 . However, when the second lump of liquid  30  is introduced into the liquid bath  10  without taking any special measures after, for example, dropping the first lump of liquid  20  on the working electrode  40  located at the bottom surface  11  of the liquid bath  10 , the first lump of liquid  20  is swept away from a top surface of the working electrode  40  due to hydrodynamic action of the second lump of liquid  30 . Alternatively, when the density of the first lump of liquid  20  is smaller than the density of the second lump of liquid  30 , the first lump of liquid  20  is separated from the working electrode  40  due to buoyancy action. Therefore, the first lump of liquid  20  needs to be held to the top surface of the working electrode  40  strongly enough to exceed the hydrodynamic action and the buoyancy action. 
     In the embodiment, for instance, the following holding structure is adopted to hold the first lump of liquid  20 . 
     (a) Structure in which the surfaces of all the working electrodes  40  are subjected to a hydrophilic treatment 
     (b) Structure in which annular portions of the bottom surface  11  of the liquid bath  10 , each of which surrounding the surface of a corresponding one of the working electrodes  40 , are subjected to a hydrophilic treatment 
     (c) Structure in which the surfaces of all the working electrodes  40  and all the annular portions are subjected to a hydrophilic treatment 
     (d) Structure in which all or part of the bottom surface  11  of the liquid bath  10  except the surfaces of all the working electrodes  40  is subjected to a hydrophobic treatment (this structure includes a structure in which a region of the bottom surface  11  of the liquid bath  10  in contact with the second lump of liquid  30  is subjected to a hydrophobic treatment) 
     (e) Structure in which the surfaces of all the working electrodes  40  are subjected to a hydrophilic treatment and all or part of the bottom surface  11  of the liquid bath  10  except the surfaces of all the working electrodes  40  is subjected to a hydrophobic treatment 
     (f) Structure in which annular portions of the bottom surface  11  of the liquid bath  10 , each of which surrounding the surface of a corresponding one of the working electrodes  40 , are subjected to a hydrophilic treatment and all or part of the bottom surface  11  of the liquid bath  10  except the surfaces of all the working electrodes  40  and all the annular portions is subjected to a hydrophobic treatment 
     (g) Structure in which the surfaces of all the working electrodes  40  and all the annular portions are subjected to a hydrophilic treatment and all or part of the bottom surface  11  of the liquid bath  10  except the surfaces of all the working electrodes  40  and all the annular portions is subjected to a hydrophobic treatment 
       FIGS. 2A, 2B, 2C, 2D, 2E, 3A, 3B and 3C  show examples of the above-described holding structure of the first lump of liquid  20 . In the drawings, reference numeral  91  denotes hydrophilic-treated region, reference numeral  92  denotes hydrophobic-treated region. From the standpoint of easy-to-understand illustration, only one working electrode  40  is taken as an example in  FIGS. 2A, 2B, 2C, 2D, 2E, 3A, 3B and 3C , and the working electrode  40  located at the bottom surface  11  of the liquid bath  10  has a circular surface shape in this example. 
       FIG. 2A  shows example (g) in which the surface of the working electrode  40  and the annular portion of the bottom surface  11  of the liquid bath  10 , which surrounds the surface of the working electrode  40 , are subjected to a hydrophilic treatment and the region of the bottom surface  11  of the liquid bath  10  except the surface of the working electrode  40  and the annular portion is subjected to a hydrophobic treatment. 
       FIG. 2B  shows example (f) in which the surface of the working electrode  40  is not subjected to a hydrophilic treatment, but only the annular portion of the bottom surface  11  of the liquid bath  10 , which surrounds the surface of the working electrode  40 , is subjected to a hydrophilic treatment, and further the region of the bottom surface  11  of the liquid bath  10  except the surface of the working electrode  40  and the annular portion is subjected to a hydrophobic treatment. 
       FIG. 2C  shows example (c) in which the surface of the working electrode  40  and the annular portion of the bottom surface  11  of the liquid bath  10 , which surrounds the surface of the working electrode  40 , are subjected to a hydrophilic treatment. 
       FIG. 2D  shows example (b) in which the surface of the working electrode  40  is not subjected to a hydrophilic treatment, but only the annular portion of the bottom surface  11  of the liquid bath  10 , which surrounds the surface of the working electrode  40 , is subjected to a hydrophilic treatment. 
       FIG. 2E  shows example (d) in which no hydrophilization is performed and the region of the bottom surface  11  of the liquid bath  10  except the surface of the working electrode  40  and the annular portion is subjected to a hydrophobic treatment. 
       FIG. 3A  shows example (e) in which the surface of the working electrode  40  is subjected to a hydrophilic treatment and the region of the bottom surface  11  of the liquid bath  10  other than the surface of the working electrode  40 , that is, the region in contact with the second lump of liquid  30  is subjected to a hydrophobic treatment. 
       FIG. 3B  shows example (a) in which the surface of the working electrode  40  is subjected to a hydrophilic treatment. 
       FIG. 3C  shows example (d) in which no hydrophilization is performed and the region of the bottom surface  11  of the liquid bath  10  other than the surface of the working electrode  40 , that is, the region in contact with the second lump of liquid  30  is subjected to a hydrophobic treatment. 
     Generally, the one first lump of liquid  20  on the bottom surface  11  of the liquid bath  10  is larger in size than the working electrode  40 . Thus, the holding structures for the first lump of liquid  20  as shown in  FIGS. 2A to 2E  are adopted. However, the holding structures for the first lump of liquid  20  as shown in  FIGS. 3A to 3C  may also be adopted in light of the size of the working electrode  40  and the lump size of the first lump of liquid  20 . 
     From the standpoint of strongly holding the first lump of liquid  20  to the surface of the working electrode  40 , the structures in (e), (f) and (g) above as shown in  FIG. 2A  or  FIG. 3A  are preferable. However, it would be difficult to completely deny the possibility that detection sensitivity may degrade due to decrease of electron moving speed at the solid-liquid interface between the surface of working electrode  40  and the first lump of liquid  20 , which is caused by hydrophilization of the working electrode  40 . The holding structures for the first lump of liquid  20  shown in  FIGS. 2B, 2D, 2E  or  FIG. 3C  are preferable in light of avoidance of the sensitivity degradation. 
     Hydrophilization of the surface of the working electrode  40  can be performed by chemical treatment on the surface of the working electrode  40  using a hydrophilic agent having a hydrophilic group. When the material of the working electrode  40  is gold or platinum, examples of the hydrophilic agent include 2-mercaptoethanesulfonic acid, 2-amino-1-ethanethiol, and 3-mercaptopropionic acid. These hydrophilic agents have in a molecule a functional group that can be selectively combined with gold or platinum, and thus by only applying a solution containing these hydrophilic agents to the bottom surface  11  of the liquid bath  10 , it is possible to selectively apply chemical treatment to only the surface of the working electrode  40 . 
     When the hydrophilic agent is, for instance, 2-amino-1-ethanethiol or 3-mercaptopropionic acid, it is also possible to use the active amino group or the carboxyl group introduced onto the working electrode  40  as anchor molecules in the process of binding treatment of a capture antibody onto the working electrode  40 , which will be performed later. 
     Next, hydrophilization of the annular portion which is part of the bottom surface  11  of the liquid bath  10  and surrounds the surface of the working electrode  40  will be described. The working electrode  40  is generally formed on, for instance, a substrate and this substrate is located on or at the bottom surface  11  of the liquid bath  10 . Thus, the annular portion surrounding the surface of the working electrode  40  is, for example, part of a surface of a substrate. 
     When material of the substrate is, for instance, glass, quartz, aluminum oxide or silicon, or when a silicon nitride film is formed on the surface of the silicon, hydrophilization of the surface of the substrate can be achieved by surface treatment with, for instance, silane coupling agent including in a molecule a hydrophilic group such as a hydroxyl group, an amino group, a carboxyl group, which is performed after temporarily activating the surface through, for instance, ashing process or UV (ultraviolet) ozone process. 
     When a substrate in which the working electrode  40  is formed is located at the bottom surface  11  of the liquid bath  10 , the second lump of liquid  30  is located on the substrate. 
     Hydrophobization of the surface of the substrate can be performed by photolithography using hydrophobic photosensitive resin. The method allows only a predetermined region to be selectively subjected to a hydrophobic treatment. Alternatively, only a portion that is not subjected to a hydrophobic treatment is masked with appropriate photosensitive resin, the whole substrate surface is treated with a hydrophobic agent, and then the photosensitive resin used for masking is dissolved and removed using a solvent, resulting in selective hydrophobization of the surface of the substrate. When material of the substrate is the aforementioned material, organic silane compound having a hydrophobic group such as di(trimethylsilyl)amine, chlorodimethyl(octadecyl)silane can be used as the hydrophobic agent. 
     Next, the holding structure for the first lump of liquid  20  using a well will be described with reference to  FIG. 4 . 
       FIG. 4 ( 1 ) shows the bottom surface  11  of the liquid bath  10  with a well  100  formed thereat. The working electrode  40  is located at the bottom of the well  100 . The well  100  can be formed by photolithography using, for example, photosensitive resin. In the drawing, reference numeral  101  denotes a layer of hardened resin. 
       FIG. 4 ( 2 ) shows the well  100  with the first lump of liquid  20  introduced thereinto by dropping.  FIG. 4 ( 3 ) further shows the liquid bath  10  with the second lump of liquid  30  introduced thereinto. The aforementioned first lump of liquid  20  can be used as the herein-described first lump of liquid  20  and the aforementioned second lump of liquid  30  can be used as the herein-described second lump of liquid  30 . The first lump of liquid  20  can be held onto the working electrode  40  by the well  100 . 
     When two or more working electrodes  40  arranged in an array are located at the bottom surface  11  of the liquid bath  10 , two or more wells  100  arranged in an array in a one-to-one correspondence with the two or more working electrodes  40  are formed at the bottom surface  11  of the liquid bath  10  and each of the working electrodes  40  is located at the bottom of a corresponding one of the wells  100 . Each of the two or more wells  100  stores the first lump of liquid  20 . 
       FIG. 5  shows an example in which the same hydrophilization as the aforementioned hydrophilization is applied to the surface of the working electrode  40 , the bottom surface of the liquid bath  10  located at the bottom of the well  100 , or to be more specific, the surface of the substrate surrounding the surface of the working electrode  40  in order to more satisfactorily hold the first lump of liquid  20  in the well  100 . The inner bottom surface of the well  100  may also be subjected to the herein-described hydrophilic treatment. Note that a hydrophilic-treated region  91  may be limited to only the surface of the working electrode  40  or only the annular portion surrounding the surface of the working electrode  40  at the inner bottom surface of the well  100 . 
     The electrochemical detection method of the present embodiment produces the following effects. 
     1) Because the first lump of liquid  20  is covered with the second lump of liquid  30 , it is possible to prevent evaporation of the first lump of liquid  20 . Thus, an object to be detected can be detected with high sensitivity. 
     2) Compared to a method requiring a relatively large-scale measuring apparatus and using quite an expensive substrate such as spectroscopic measurement, it is possible to inexpensively detect an object to be detected with a compact apparatus configuration. 
     3) When N is a predetermined integer equal to or greater than 2, in multi-point detection processing (that is, comprehensive multi-point detection processing) that performs N detection processes simultaneously using N working electrodes  40 , crosstalk can be prevented because different first lumps of liquid  20  are separated by the second lump of liquid  30 . Therefore, it is possible to detect an object to be detected with high sensitivity. 
     4) Although a small amount of the first lump of liquid  20  is used to improve the concentration of the catalytic reaction product, it is not necessary to use a micro counter electrode  50  and/or a micro reference electrode  60  because the counter electrode  50  and the reference electrode  60  are arranged in the second lump of liquid  30 . Thus, it is possible to use the counter electrode  50  and the reference electrode  60  used in the prior art. 
     5) In multi-point detection, one counter electrode  50  and one reference electrode  60  may be prepared in the liquid bath  10  for the working electrodes  40 . Therefore, it is possible to detect an object to be detected with a compact apparatus configuration with high sensibility. 
     The electrochemical detection method of the present embodiment is applicable to a detection scheme in which: the product of a catalytic reaction progressing in the first lump of liquid  20  is the object to be detected; the concentration of the catalytic reaction product in the first lump of liquid  20  increases as the catalytic reaction progresses; the catalytic reaction product can be electrochemically detected; and the second lump of liquid  30  that dissolves the catalytic reaction product can be chosen. 
     ELISA uses an enzyme as a catalyst, but the catalyst is not limited to an enzyme. Examples of the catalyst may include metal catalyst, ribozyme, cells containing enzymes on the surface thereof or inside, organelle, minute particles that artificially adsorb or are artificially bound to these elements, and vesicle. 
     The sandwich ELISA forms a composite of capture antibody, antigen, primary antibody and enzyme-labeled secondary antibody, and thereby causes the catalyst to be indirectly bound to the solid phase, but the catalyst binding method is not limited to this. For example, the catalyst may be indirectly bound to the solid phase by hybridizing a probe DNA preliminarily bound to the solid phase surface with a single-strand DNA complementary with the probe DNA and labeled with the catalyst. 
     Alternatively, the catalyst may be indirectly bound to the solid phase by making antigen, peptide or sugar chain, these being preliminarily bound to the surface of the solid phase, interact with an antibody or a lectin that can specifically bind to those molecules and are labeled with the catalyst. 
     Such a binding method is already well known in expression analysis of using a DNA chip or a protein chip. 
     Alternatively, when measuring activity of the catalyst itself, the catalyst may be directly bound to the surface of the solid phase. 
     Next, an embodiment of a transducer suitable for the aforementioned electrochemical detection of the catalytic reaction product will be described with reference to  FIGS. 6 to 8 . 
     The transducer of the present embodiment has a structure in which a liquid bath  120  that can contain a solution  110  is mounted on an LSI chip (large scale integrated chip)  130 . The liquid bath  120  has hole  121  at the center thereof and the LSI chip  130  is placed at lower end of the hole  121  and covers the hole  121 . 
     The LSI chip  130  and the liquid bath  120  are fixed to a substrate  140  and a wiring pattern  141  for connecting the LSI chip  130  to an external apparatus that controls the transducer is formed on the substrate  140 . Reference numeral  150  in  FIG. 6B  denotes a bonding wire for connecting the LSI chip  130  to the wiring pattern  141 . 
     A sensor region  131  is formed on the top surface of the LSI chip  130 . The sensor region  131  is located in the hole  121  at the bottom surface of the liquid bath  120 . 
     Although details are omitted in  FIGS. 6A, 6B and 7 , 400 electrodes  132  of φ40 μm that function as working electrodes are formed in the sensor region  131  in this example. The 400 electrodes  132  constitute a 20×20 array, and are arranged at intervals of 250 μm.  FIG. 8  shows part of the sensor region  131  in which the electrodes  132  are formed. The material of the electrodes  132  is gold in this example and a silicon nitride film is formed on the top surface of the LSI chip  130  including at least the sensor region  131  except the electrodes  132 . The LSI chip  130  has a function to detect currents generated by oxidation reduction reaction between each electrode  132  and an object to be detected, and amplify the respective detected currents. 
     In the LSI chip  130 , the above-described holding structures (see  FIGS. 2A, 2B, 2C, 2D, 2E, 3A, 3B, 3C, 4 and 5 ) are applied to each electrode  132  and the sensor region  131 . For example, the hydrophilization shown in  FIG. 2  and  FIG. 3  is applied to the surface of each electrode  132  and an annular surface surrounding the surface of each electrode  132 , or the well shown in  FIG. 4  and  FIG. 5  is formed. The transducer of the embodiment can strongly hold the first lumps of liquid in which a catalytic reaction progresses to the electrodes  132  by using the holding structures. In  FIGS. 6A, 6B and 7 , the solution  110  is the second lump of liquid that covers the first lumps of liquid, and the first lumps of liquid are not shown. 
     A counter electrode  160  and a reference electrode  170  shown in  FIGS. 6A and 6B  are not necessarily essential components of the transducer. 
     The counter electrode  160  and the reference electrode  170  are introduced into the second lump of liquid before implementing the method of the present embodiment. 
     &lt;Addendum&gt; 
     Although the present invention has been described with reference to the illustrative embodiment, those skilled in the art will understand that various changes can be made without departing from the scope of the present invention and the elements thereof can be replaced by equivalents. Moreover, many modifications can be made to adapt a specific system, device or components thereof to the teachings of the present invention without departing from the intrinsic scope of the present invention. Therefore, the present invention is not limited to a specific embodiment disclosed to implement the present invention, but includes all embodiments included in the appended scope of claims. 
     Furthermore, the terms like “first,” “second” or the like are used not to indicate order or importance, but to distinguish the elements. The terms used in the present specification is intended to describe the embodiment and is in no way intended to limit the present invention. The term “include” and inflections thereof, when used in the present specification and/or the appended scope of claims, clarify the presence of the mentioned features, steps, operations, elements and/or components, but do not exclude the presence or addition of one or a plurality of other features, steps, operations, elements, components and/or the group thereof. The term “and/or” includes, if present, one or a plurality of all sorts of combinations of related and listed elements. In the scope of claims and the specification, “connection,” “combination,” “joining,” “coupling” or synonyms thereof and all inflections thereof do not necessarily deny the presence of one or more “interconnected” or “combined” or “coupled” intermediate elements unless otherwise specified. 
     All terms used in the present specification (including technical terms and scientific terms) have the same meanings generally understood by those skilled in the art to which the present invention belongs unless otherwise specified. Furthermore, terms such as those defined in commonly used dictionaries should be interpreted as having meanings consistent with meanings in context of the related arts and the present disclosure, and should not be interpreted ideally or excessively formally unless explicitly defined. 
     It will be understood that the present invention has disclosed many techniques and steps in the description thereof. Those techniques and steps have their respective individual advantages and can also be used in combination with one or more or, in some cases, all of other disclosed techniques. Therefore, to avoid complications, the present specification refrains from describing all possible combinations of the individual techniques or steps. However, the specification and claims should be read with an understanding that such combinations are totally included in the present invention and the scope of claims. 
     In the following claims, corresponding structures, materials, actions and equivalents of all functional elements combined with parts or steps, if present, are intended to include structures, materials or actions to execute functions in combination with other claimed elements. 
     Although the embodiment of the present invention has been described so far, the present invention is not limited to the embodiment. Various changes and modifications are allowed without departing from the spirit of the present invention. The selected and described embodiment is intended to describe principles of the present invention and practical applications thereof. The present invention is used as various embodiments along with various changes or modifications, and the various changes or modifications are determined according to expected use. All such changes and modifications are intended to be included in the scope of the present invention defined by the appended claims, and are intended to be granted the same protection when interpreted according to a range given impartially, legally and fairly. 
     DESCRIPTION OF REFERENCE NUMERALS 
     
         
           10  liquid bath 
           11  bottom surface 
           20  first lump of liquid 
           30  second lump of liquid 
           40  working electrode 
           50  counter electrode 
           60  reference electrode 
           70  salt bridge 
           80  potentiostat 
           81  variable power supply 
           82  voltmeter 
           83  ammeter 
           91  hydrophilic-treated region 
           92  hydrophobic-treated region 
           100  well 
           101  resin layer 
           110  solution 
           120  liquid bath 
           121  hole 
           130  LSI chip 
           131  sensor region 
           132  electrode 
           140  substrate 
           141  wiring pattern 
           150  bonding wire 
           160  counter electrode 
           170  reference electrode