Patent Publication Number: US-2022221424-A1

Title: Electrochemical detection method for catalytic reaction product, electrochemical detection apparatus 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. It is thereby possible to avoid a problem of evaporation causing a decrease in the volume of the solution and making detection impossible. 
     The state in which the solution is covered with a liquid needs to be maintained stably and appropriately in a progression process of a catalytic reaction and in a process of detecting a catalytic reaction product. For example, when a situation occurs in which a liquid-liquid interface between the solution and the liquid is disturbed or the shape of the solution is changed considerably by application of vibration or shock, the progression of the catalytic reaction or detection processing is thereby affected, resulting in deterioration of detection accuracy or detection errors. 
     Even when a well is used, vibration or shock may cause part of the solution to spill out of the well or the liquid may flow into the well. 
     It is an object of the present invention to provide a technique capable of detecting a catalytic reaction product stably and with high sensitivity. 
     Means to Solve the Problems 
     Technical matters described here will be described not to explicitly or implicitly limit the present invention claimed in the claims or to further express a possibility of admitting such a limitation imposed by persons other than those who benefit from the present invention (e.g., applicant and patentee), but to simply make it easy to understand main points of the present invention. A summary of the present invention from other standpoints can be understood from the claims at the time of filing the present 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 first lump of liquid is held to a liquid-retaining structure that allows the first lump of liquid to permeate and can retain the first lump of liquid. The liquid-retaining structure is located in the vicinity of the working electrode. 
     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. 
     Effects of the Invention 
     According to the present invention, since the liquid-retaining structure stably and appropriately maintains the state in which the first lump of liquid is covered with the second lump of liquid, it is possible to stably detect a catalytic reaction product with high sensitivity. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram for describing an overview of an electrochemical detection apparatus according to an embodiment; 
         FIG. 2A  is a diagram for describing a first example of a retaining structure; 
         FIG. 2B  is a diagram for describing a second example of the retaining structure; 
         FIG. 2C  is a diagram for describing a third example of the retaining structure; 
         FIG. 2D  is a diagram for describing a fourth example of the retaining structure; 
         FIG. 3A  is a diagram for describing a fifth example of the retaining structure; 
         FIG. 3B  is a diagram for describing a sixth example of the retaining structure; 
         FIG. 4  is a diagram for describing the retaining structure to be placed in a well; 
         FIG. 5A  is a plan view of a transducer according to an embodiment; 
         FIG. 5B  is a cross-sectional view of the transducer shown in  FIG. 5A ; 
         FIG. 6  is a perspective view of the transducer shown in  FIG. 5A ; and 
         FIG. 7  is a diagram for describing an array of electrodes in the transducer shown in  FIG. 5A . 
     
    
    
     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  illustrates only one working electrode  40 , two or more working electrodes  40  arranged, for example, in an array are generally installed at a substrate and the substrate is located on 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. 
     Next, the liquid-retaining structure to stably and appropriately maintain the state in which the first lump of liquid  20  is covered with the second lump of liquid  30  will be described. In the above-described example of ELISA, the liquid liquid-retaining structure is formed before step (1). Hereinafter, the liquid liquid-retaining structure will simply be referred to as a “retaining structure.” 
     The retaining structure allows the first lump of liquid  20  to permeate and can also retain the first lump of liquid  20 . The retaining structure is, for example, a porous body having a hydrophilic surface (in this regard, the “surface” is an air-contacting part when the retaining structure is placed in the air, and the inner surface of pores is also included) or dry gel formed by a polymer that forms hydrogels when moistened with water. Although not shown in  FIG. 1 , the retaining structure is placed at a place in the liquid bath  10  where the first lump of liquid  20  is located, that is, in a local space facing the working electrode  40 . 
       FIG. 2  and  FIG. 3  show several examples of the retaining structure. From the standpoint of easy-to-understand illustration, only one working electrode  40  is shown in  FIG. 2  and  FIG. 3  and the working electrode  40  has a circular surface shape in this example. In  FIG. 2  and  FIG. 3 , reference numeral  100  denotes a substrate at which the working electrode  40  is installed. 
     First Example: FIG.  2 A 
     A retaining structure  91  is a porous body. Examples of the material of the porous body include materials made of an organic polymer such as cellulose having a pore diameter of on the order of 0.1 to 10 μm, nitrocellulose, acetylcellulose and polyvinylidene difluoride that has been appropriately subjected to a hydrophilic treatment or include inorganic materials such as silica, silicon and alumina (silicon dioxide). 
     The retaining structure  91 , which is a porous body of the organic polymer, is formed by dissolving the polymer as a raw material in an appropriate solvent, placing the solution  91 ′ on the working electrode  40  using a technique such as spotting, screen printing and inkjet printing, and then drying the solvent. The solvent contains an appropriate cross-linking agent if necessary. 
     For example, the retaining structure  91 , which is a porous body of nitrocellulose, is formed by dropping, onto the working electrode  40 , droplets of the solution  91 ′ obtained by dissolving nitrocellulose in a methyl isobutyl ketone (4-methyl-2-pentanone) solvent using a micropipette, and then removing the solvent by natural drying. 
     The retaining structure  91 , which is a porous body of an inorganic material, is formed using, for example, a technique of forming porous silica on the working electrode  40  by a sol-gel method or a technique of forming porous silicon or porous alumina on the working electrode  40  by an anode oxidation method. 
     Second Example: FIG.  2 B 
     A retaining structure  92  is a porous body. In this example, the above-described porous body is formed in advance as a small block  92 ′ and the small block  92 ′ are pasted onto the working electrode  40  as the retaining structure  92 . 
     Third Example: FIG.  2 C 
     A porous body is formed in the form of a sheet on the substrate  100  where the working electrode  40  is located, using a technique such as spin coating. The porous body is impregnated with resin, which is insoluble in both of the first lump of liquid  20  and the second lump of liquid  30 , to form a resin-impregnated sheet-like porous body  93  in which all the pores are filled with the resin. After that, only the resin on the working electrode  40  is selectively removed by an appropriate solvent. As a result, a retaining structure  94 , which is a porous body, is formed. 
     A sheet-like porous body is formed by, for example, coating the substrate  100 , which the working electrode  40  is located at, by spin coating with a solution resulting from dissolving nitrocellulose in a methyl isobutyl ketone solvent, and then removing the solvent by natural drying. Furthermore, negative photosensitive resin is dropped onto the porous body to impregnate the porous body with the photosensitive resin. By selectively removing only the photosensitive resin on the working electrode  40  by photolithography, the retaining structure  94 , which is a porous body, is formed. 
     Fourth Example: FIG.  2 D 
     The retaining structure  94  is formed by selectively impregnating the region other than the working electrode  40  of the sheet-like porous body  93 ′ described in the third example with resin insoluble in both of the first lump of liquid  20  and the second lump of liquid  30 , using a technique such as spotting, screen printing and inkjet printing. In  FIG. 2D , reference numeral  93  denotes the resin-impregnated sheet-like porous body. 
     Fifth Example: FIG.  3 A 
     The porous body can be formed by agglomerated minute particles. A retaining structure  95 , which is a porous body, is formed by, for example, agglomerating on the working electrode  40  minute particles such as polystyrene minute particles each having a diameter of on the order of 0.1 to 10 μm, silica minute particles, alumina minute particles, magnetic minute particles used for protein separation and refining or the like or agarose minute particles used as carriers for an affinity column. 
     For example, binder molecules for binding minute particles together can be used to agglomerate the minute particles. A retaining structure  95 , which is an agglomerate of minute particles, is formed by suspending the binder molecules and the minute particles in an appropriate solvent, dropping droplets of a suspension  95 ′ onto the working electrode  40 , and then drying the solvent. When molecules, a binding process by which progresses by an operation such as heating or light irradiation, are used as the binder molecules, the operation are executed before or after drying the solvent. 
     Minute particles may be agglomerated on the working electrode  40  to which a DC voltage or AC voltage is applied, using an electrophoretic force or dielectrophoretic force produced thereby. 
     Sixth Example: FIG.  3 B 
     A retaining structure  96  is formed by a polymer that forms hydrogels by absorption of water. Examples of the polymer that forms hydrogels by absorption of water include polyacrylic amide (poly(2-propenamide)), agarose, sodium alginate or collagen. By placing on the working electrode  40  a solution  96 ′ of the polymer, which is a raw material, dissolved in an appropriate solvent, using a technique such as spotting, screen printing or inkjet printing, and then drying the solvent, the retaining structure  96 , which is dry gel, is formed. The solvent may contain an appropriate cross-linking agent if necessary. 
     Collagen hydrogels are obtained by, for example, dropping droplets of the solution  96 ′, which is obtained by dissolving collagen as a polymer and glutalaldehyde (1,5-pentanedial) as a cross-linking agent in a phosphoric acid buffer solution, onto the working electrode  40  using a micropipette, and then causing a cross-linking reaction to progress at a room temperature. The retaining structure  96  is formed by allowing the hydrogels to dry naturally. 
     In a first example shown in  FIG. 2A  and in fifth and sixth examples shown in  FIGS. 3A  and B, when the polymer solution  91 ′ or  96 ′ or a suspension  95 ′ of minute particles is dropped onto the working electrode  40 , the droplets are spread over regions other than a desired region. To avoid this, a well may be formed at the bottom surface  11  of the liquid bath  10 . 
       FIG. 4  shows an example in which a well  105  is formed at the substrate  100  located on the bottom surface  11  of the liquid bath  10 . The working electrode  40  is located in the well  105 . The well  105  is formed by placing hydrophobic resin, which is insoluble in both of the first lump of liquid  20  and the second lump of liquid  30 , on the substrate  100  using a technique such as photolithography or screen printing. Reference numeral  106  in  FIG. 4  denotes a layer of hardened resin. Like the example in  FIG. 2A ,  FIG. 4  shows an example in which a retaining structure  91  is formed by drying the solution  91 ′ of a polymer. The solution  91 ′ is dropped into the well  105 . As a result, the retaining structure  91  is placed in the well  105 . 
     In the following description, the retaining structure  91 ,  92 ,  94 ,  95  and  96  are generically called “retaining structure  90 .” Placing on the working electrode  40  the retaining structure  90 , which allows the first lump of liquid  20  to permeate, can retain the first lump of liquid  20 , and is insoluble in both of the first lump of liquid  20  and the second lump of liquid  30 , strongly retains the first lump of liquid  20  on the working electrode  40 . 
     The retaining structure  90  is provided for each of working electrodes  40 . Alternatively, a combination of the well  105  and the retaining structure  90  is provided for each of working electrodes  40 . 
     After dropping the first lumps of liquid  20  onto the retaining structures  90 , the second lump of liquid  30  is poured into the liquid bath  10 . Progression of the enzyme reaction in ELISA and detection of the enzyme reaction product are performed for each working electrode  40  with the first lumps of liquid  20  retained to the retaining structures  90 . The enzyme reactions corresponding to the working electrodes  40  proceed independently of each other, that is, any two of them do not affect each other. Similarly, detections of the enzyme reaction products corresponding to the working electrodes  40  are performed independently of each other, that is, any two of them do not affect each other. 
     According to the embodiment of the electrochemical detection method, the following effects are produced. 
     1) Since the retaining structure  90  strongly retains the first lump of liquid  20  on the working electrode  40 , even though vibration or shock or the like is applied to the liquid bath  10 , the state in which the first lump of liquid  20  is covered with the second lump of liquid  30  is maintained stably and appropriately. Therefore, high accuracy detection is performed stably. 
     2) Since the retaining structure  90  which is a three-dimensional structure has a larger surface area than a flat surface which is the electrode surface, it is possible to considerably increase the amount of catalyst supported thereby. Therefore, the catalytic reaction progresses efficiently and detection sensitivity improves noticeably. 
     As is clear from the embodiment, the catalytic reaction product is detected electrochemically. Therefore, the retaining structure  90  capable of securing the substance diffusion and the conduction path such as a porous body or gel does not adversely affect the catalytic reaction field. 
     Without being limited to the above-described embodiment, implementation conditions are as follows: 
     a) An object to be detected is the product generated by progress of a catalytic reaction in the first lump of liquid  20 ,
 
b) The concentration of the catalytic reaction product in the first lump of liquid  20  increases as the catalytic reaction progresses,
 
c) The catalytic reaction product can be detected electrochemically, and
 
d) The second lump of liquid  30  in which the catalytic reaction product does not dissolve can be selected.
 
     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. 5 to 7 . 
     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 a 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. 5B  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. 5A, 5B and 6 , 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. 
     The LSI chip  130  in this example has a configuration in which the retaining structure  90  that allows an aqueous solution to permeate and can retain the aqueous solution is provided on each electrode  132  or a configuration in which the electrode  132  is located in each well  105  arranged in an array in the sensor region  131  and the retaining structure  90  is provided in each well  105 . Therefore, the present transducer can strongly retain on each electrode  132  the first lump of liquid where a catalytic reaction takes place. In  FIGS. 5 and 6 , the solution  110  is the second lump of liquid that covers the first lump of liquid and the first lump of liquid is not shown. A counter electrode  160  and a reference electrode  170  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. 
     Addendum 
     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 
           90 ,  91 ,  92 ,  94 ,  95 ,  96  retaining structure 
           91 ′,  96 ′ solution 
           92 ′ small block 
           93  resin-impregnated sheet-like porous body 
           93 ′ sheet-like porous body 
           95 ′ suspension 
           100  substrate 
           105  well 
           106  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