JP2005292022A - Manufacturing method for reference electrode - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a manufacturing method for an inexpensive and easily manufactured reference electrode of high performance for electrochemical measurement, and to provide a manufacturing method for an electrochemical sensor provided with the reference electrode. <P>SOLUTION: In this manufacturing method for the reference electrode in an electrode array for the electrochemical measurement, having two or more of electrode pairs including at least the reference electrode and a measuring electrode, the reference electrode is manufactured by applying a silver paste on a reference electrode portion of a conductive pattern, and by treating a surface with hypochlorous acid to form silver chloride. The is also provided the reference electrode manufactured by the method, and an electrochemical measuring device provided with the reference electrode. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a reference electrode used for electrochemical measurement, and a method for producing an electrochemical sensor such as a biosensor. The present invention further relates to a reference electrode obtained by the method of the present invention, and an electrochemical measurement device comprising the reference electrode of the present invention.

  As an element for detecting an electrode, an electrochemical sensor for detecting a current change or a potential change that occurs at an electrode interface due to a substance to be measured has been applied to various fields and has been put into practical use. In particular, in recent years, the demand for biosensors for measuring substances such as proteins, enzymes, and low-molecular complexes existing in living bodies has been increasing because they are small and easy to multiplex. In an electrochemical sensor, electrodes such as a working electrode, a counter electrode, and a reference electrode are usually formed on an insulating substrate. In the case of potential detection, the potential difference between the working electrode and the reference electrode is measured, and in the case of current detection, the potential difference between the working electrode and the reference electrode is made constant by an external circuit, and at that time, between the working electrode and the counter electrode Measure the flowing current. As described above, the reference electrode is a reference electrode for applying a potential or measuring a potential, and is an electrode indispensable as an electrochemical sensor. In the electrochemical measurement, the reference electrode is based on a standard hydrogen electrode. However, since handling is difficult such as using hydrogen gas, a silver-silver chloride electrode is generally used as a reference electrode in an electrochemical sensor or the like.

  As a conventional method of manufacturing a reference electrode, a platinum electrode is used in a plating solution containing dicyano silver (I) acid ions on a reference electrode portion of an electrode pattern formed of gold, platinum, or the like. In some cases, silver plating was applied to a predetermined portion by applying about 0 V, and the surface was silver chloride by applying a voltage of about +1.2 V in 3 M NaCl. However, this method not only complicates the manufacturing process but also has problems such as the treatment of the plating solution, which makes it difficult to reduce the cost and mass production.

As a method for overcoming these drawbacks, a method in which an electrode body is composed of a silver-silver chloride paste material is disclosed (for example, see Patent Document 1). The purpose of this is to reduce the cost by eliminating the degreasing cleaning of silver, the silver chloride treatment, the cleaning / drying step and the like. However, the cost of the silver-silver chloride paste itself is high, and since the electrode itself is configured by printing the silver-silver chloride paste, it is difficult to obtain a fine electrode pattern, and if the pattern is thin, the resistance is high. There was a problem of becoming.
JP 2001-242115 A

  Thus, in a conventional electrochemical sensor, a method for manufacturing a reference electrode with low cost and high reliability has not been established, which has been one of the problems of downsizing and cost reduction of the sensor. In view of the above problems, an object of the present invention is to provide a method for manufacturing an electrochemical sensor including a low-cost, easy-to-manufacture and high-performance reference electrode.

  In order to achieve the above object, the present invention has the following configuration. That is, the present invention is a method of manufacturing a reference electrode in an electrode array for electrochemical measurement having two or more electrode pairs including at least a reference electrode and a measurement electrode, and forms a conductive pattern including the reference electrode on an insulating substrate Manufacturing a reference electrode, comprising: a step of forming a composite containing silver on a reference electrode portion of the conductive pattern; and a step of chlorinating the composite containing silver Provide a method.

  The present invention further includes a detection unit having a hole part into which a sample and a reagent are injected, a channel part through which the sample and the reagent can move, and an electrode array having two or more electrode pairs including at least a reference electrode and a measurement electrode. In the manufacturing method of the electrochemical measuring device comprised by the part, the reference electrode in the said detection part is manufactured by the method of the said invention, The manufacturing method of the electrochemical measuring device characterized by the above-mentioned is provided.

  The present invention is also a reference electrode used in an electrochemical measurement electrode array having two or more electrode pairs including at least a reference electrode and a measurement electrode, wherein the reference electrode is formed of a composite containing silver, Provided is a reference electrode for electrochemical measurement whose surface is silver chloride. The reference electrode of the present invention can be prepared by the above method.

  The present invention further includes a detection unit having a hole part into which a sample and a reagent are injected, a channel part through which the sample and the reagent can move, and an electrode array having two or more electrode pairs including at least a reference electrode and a measurement electrode. An electrochemical measurement device comprising: a reference electrode is formed of a composite containing silver, and the surface thereof is silver chloride. . The present invention further provides an electrochemical sensor that electrochemically measures a substance contained in a sample, comprising the electrochemical measurement device of the present invention. The sensor of the present invention can be suitably used as a biosensor whose measurement target substance is a biological substance.

  According to the present invention, it is possible to provide an electrochemical sensor including a low-cost, easy-to-manufacture and high-performance reference electrode, and a manufacturing method thereof.

Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 is a process diagram showing an embodiment of the present invention.
Step 1 is formation of a conductive pattern on an insulating substrate. As a method for forming a conductive pattern on an insulating substrate, for example, printing of a conductive material on the insulating substrate 1, etching after sputtering or vapor deposition of the conductive material or removal processing by a laser, electrode pattern using a mask Direct sputtering, etc. can be considered. FIG. 2 shows an electrode substrate on which a conductive pattern including electrodes is formed. On the insulating substrate 1, a working electrode 2, a counter electrode 3, and a reference electrode 4 are formed. Each electrode can be selected from noble metals such as gold, platinum and palladium, carbon, and the like, but it is desirable to select gold from the viewpoint of surface state stability. Each electrode is electrically coupled by a conductive pattern 5 to a terminal portion 6 which is a connection portion with an external circuit. From the viewpoint of the manufacturing process, it is desirable that the conductive pattern 5 and the terminal portion 6 are also formed of the same material as the electrode portion. As the insulating substrate 1, a semiconductor such as silicon or germanium, glass such as quartz glass, lead glass, or borosilicate glass, ceramic, or resin can be selected. It is desirable to select a resin material in view of ease of handling and cost. Resin materials include polystyrene (PS), polypropylene (PP), polyimide (PI), polytetrafluoroethylene (PTFE), polyphenylene sulfide (PPS), polyetheretherketone (PEEK), polyethylene terephthalate (PET), poly Methyl methacrylate (PMMA), polyethylene-2,6-naphthalate (PEN) and the like can be selected. Among them, in the case of forming a gold electrode by sputtering, it is preferable to select polyethylene terephthalate from the viewpoint of adhesion with gold.

  The electrode substrate can be used as it is, but as shown in FIG. 3, it may be coated with an insulating film 21 from the viewpoint of defining the area of the electrode portion and insulating / protecting the conductive pattern 5. As the insulating film 21, for example, a resin material thin film such as polyimide can be used.

  Process 2 is formation of the composite containing silver to the reference electrode part formed on the insulating substrate. FIG. 4 is an electrode substrate in which a composite containing silver is formed on the surface of a reference electrode portion in an electrode substrate on which a conductive pattern including electrodes is formed. A composite 31 containing silver is coated on the reference electrode 4. The composite 31 containing silver is preferably a material that contains silver, has conductivity, has good adhesion to the reference electrode 4, and does not react with a measurement sample such as water or an organic solvent. Examples of the composite 31 containing silver include a silver paste in which silver particles are dispersed in a resin material.

  The particle diameter of the silver particles contained in the silver paste is not particularly limited as long as it is about 0.5 to 20 μm which is usually used. In recent years, a silver paste using silver nanoparticles having a particle diameter of 5 to 10 nm has been developed, but the use of such a nanoparticle paste is not particularly limited.

  The silver particle content in the silver paste is preferably about 50 to 95%. The remaining components may be resin materials, solvents, and other conventionally known silver paste components. As a resin material used as the binder component of the silver paste used in the present invention, those usually used as the resin material of the silver paste may be used without any particular limitation. For example, epoxy resin, phenol resin, acrylic resin, polyester , PVC, cellulose and the like are preferably used. The solvent may be selected depending on the resin material, and is generally selected from aromatic hydrocarbons such as toluene and xylene, ketones such as methyl ethyl ketone and methyl isobutyl ketone, alcohols such as normal butanol, ethanol and methanol. When the substrate 1 is a resin material, a silver paste using an acrylic resin that is cured at a relatively low temperature as a binder is particularly preferable.

  In the method of the present invention, printing, discharging, brushing, and the like can be selected as the silver paste application method. The film thickness after drying of the silver paste depends on the coating method and is not particularly limited, but is about 1 μm to 100 μm, particularly preferably 2 to 25 μm, and more preferably about 5 to 15 μm.

  Step 3 is silver chloride of the composite containing silver. The composite 31 containing silver is converted into silver chloride by a chemical reaction. The reagent used for the chemical reaction may be any oxidant containing chlorine, but hypochlorous acid is preferably used from the viewpoint of handling and cost.

  In one embodiment of the present invention, a silver chloride reaction is allowed to proceed from the surface of the silver-containing composite 31 in which an aqueous solution of about 5% sodium hypochlorite and the silver-containing composite 31 are brought into contact. At this time, in order to stabilize hypochlorous acid, it is preferable from the viewpoint of safety to add about 0.5% to 1% sodium hydroxide to make it alkaline. The pH of the aqueous sodium hypochlorite solution is not particularly limited as long as it reduces the chlorine generated from hypochlorous acid and has little influence on the substrate material, etc., but is adjusted to about pH 11-12. obtain.

  The time for bringing the aqueous sodium hypochlorite solution into contact with the surface of the silver paste is not particularly limited as long as the silver chloride reaction on the surface portion of the silver paste proceeds, but the aqueous hypochlorous acid solution is alkaline. For this reason, the influence on the substrate material or the like is taken into consideration, preferably 30 seconds to 30 minutes, and more preferably about 1 minute to 15 minutes.

  FIG. 5 shows an electrochemical measurement device according to an embodiment of the present invention. The housing material 41 used in the electrochemical measurement device according to the present invention may be any material as long as it does not react with the sample solution, such as a semiconductor such as silicon or germanium, a glass such as quartz glass, lead glass, or borosilicate glass, or a ceramic. However, considering the use as a disposable biosensor, it is desirable to select a resin material in view of ease of processing and cost. Resin materials include polystyrene (PS), polypropylene (PP), polyimide (PI), polytetrafluoroethylene (PTFE), polyphenylene sulfide (PPS), polyetheretherketone (PEEK), polyethylene terephthalate (PET), poly Methyl methacrylate (PMMA), polyethylene-2,6-naphthalate (PEN), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS) and the like can be selected. Polymethyl methacrylate is particularly preferable because of its good transparency and easy fine cutting. As a method of forming the cavity 42 and the flow path 43 formed on the housing material 41, when the housing material 41 is a resin material, in addition to cutting, molding by a mold, embossing by thermal transfer, or the like can be used. It is also possible to stick sheets having through holes.

  The housing material 41 including the cavity 42 and the flow path 43 thus formed is bonded to the substrate 43 including the electrodes. At this time, it is preferable that the housing 43 and the substrate 43 including the electrodes are completely adhered or closely adhered to seal the sample inside the cavity 2. For example, an acrylic, epoxy, silicone adhesive, double-sided tape, or the like can be used.

  The housing material 41 is provided with a sample injection hole 44 for injecting a sample into the cavity 42 and the flow path 43. At least one sample injection hole 44 suffices. However, if two or more sample injection holes 44 are provided, the sample injection holes 44 other than those used at the time of sample injection function as air escape paths inside the cavity 42 and the flow path 43, and the sample Is preferable because it can be injected quickly. The inside of the cavity 42 and the flow path 43 is preferably subjected to a known hydrophilic treatment because the sample is introduced more quickly. The injection hole 44 can be sealed by any method after injecting the sample, but can be used in an open state for a short time measurement.

  Hereinafter, the electrode and electrochemical device of the present invention will be described more specifically. The present invention is not limited to the following examples.

  (Example 1) An example in which cyclic voltammetry, which is a basic electrochemical measurement, was performed using an electrode according to an embodiment of the present invention will be described below.

An electrode was prepared using a polyethylene terephthalate sheet having a thickness of 188 μm as an insulating substrate.
First, a 700 Å gold thin film was formed on a polyethylene terephthalate sheet by sputtering. Next, a working electrode, a counter electrode, a reference electrode, a conductive pattern, and a terminal as shown in FIG. 2 were simultaneously formed by etching, and cut into a predetermined size to produce an electrode plate. The electrode area of the working electrode was 2.0 mm ² .

  Next, the conductive paste a (D-550 manufactured by Fujikura Kasei Co., Ltd.) and the conductive paste b (D-723S manufactured by Fujikura Kasei Co., Ltd.) are respectively applied to the reference electrode surface of the electrode plate prepared according to the above. The composite containing silver was formed on the surface of the reference electrode. Table 1 shows the resin materials, drying conditions, and film thickness after drying contained in each conductive paste.

  Thereafter, 5% sodium hypochlorite and 0.5% sodium hydroxide were dropped onto the surface of the composite containing silver, and left for 10 minutes, whereby the surface of the composite containing silver was converted to silver chloride. The electrodes thus prepared were referred to as an electrode A and an electrode B, respectively.

  Further, a silver chloride paste (DB2275 manufactured by Nippon Atson Co., Ltd.) was applied to the surface of the reference electrode of the electrode plate produced according to the above, and dried under the condition of 80 ° C. × 0.5 h. The electrode thus produced was designated as Conventional Example a.

  On the other hand, a silver plating solution (SILVREX JS-2 basic solution, Tanaka Kikinzoku Kogyo Co., Ltd., silver potassium cyanide, Wako Pure Chemical Industries, Ltd.) is dropped only on the surface portion of the reference electrode 4, and platinum is applied to the surface of the silver plating solution. The surface of the reference electrode was silver plated by immersing the wire and applying a voltage of 1.0 V for 10 seconds with the reference electrode side minus and the platinum wire side plus. After sufficiently washing the silver plating solution with pure water, a 3M sodium chloride aqueous solution was dropped only on the surface of the reference electrode on which the silver plating was applied, and a platinum wire was immersed in the surface of the sodium chloride aqueous solution. The surface of the silver plating was converted to silver chloride by applying a voltage of 1.2 V for 10 seconds with the reference electrode side being plus and the platinum wire side being minus. The electrode thus fabricated was designated as Conventional Example b.

  Using the electrode prepared as described above, cyclic voltammetry of 10 mM potassium ferricyanide / 50 mM potassium chloride aqueous solution was measured at a sweep rate of 100 mV / sec. A cyclic voltammogram at that time is shown in FIG. In FIG. 6, the redox peak of ferricyanide ion is measured well, and the redox peak of ferricyanide ion at electrode A and electrode B is almost the same potential as in conventional example a and conventional example b. Is shown. Thus, it was shown that the reference electrode in the electrode A and the electrode B produced by the reference electrode manufacturing method according to this example functions as a good reference electrode.

  (Example 2) An example in which pyrophosphoric acid, which is a small molecule in a living body, was measured using the electrode according to one embodiment will be described below.

  An electrode was prepared using a polyethylene terephthalate sheet having a thickness of 188 μm as the substrate 1.

First, a 700 Å gold thin film was formed on a polyethylene terephthalate sheet by sputtering. Next, as shown in FIG. 2, the working electrode 2, the counter electrode 3, the reference electrode 4, the conductive pattern 5, and the terminal 6 were simultaneously formed by etching and cut into a predetermined size to produce an electrode plate. The electrode area of the working electrode was 7.1 mm ² .

  Next, the conductive paste a used in Example 1 was applied to the surface of the reference electrode of the electrode plate prepared according to the above, dried under the conditions shown in Table 1, and a composite containing silver on the surface of the reference electrode Formed.

  Thereafter, 5% sodium hypochlorite, 0.5% sodium hydroxide, 0.01% sodium alkyl ether sulfate is dropped on the surface of the composite containing silver, and the composite containing silver is allowed to stand for 10 minutes. The body surface was converted to silver chloride. The electrode thus fabricated was designated as F.

  Moreover, the housing material was produced by cutting polymethyl methacrylate.

  The electrode F and the housing material produced as described above were bonded using silicone (KE44 manufactured by Shin-Etsu Silicone Co., Ltd.) to produce an electrochemical measurement device as shown in FIG.

  Using the electrochemical measurement device thus produced, pyrophosphoric acid, which is a small molecule in the living body, was measured.

First, the reaction solution was prepared according to the following procedure. 10 μl of 100 mM nicotinamide adenine dinucleotide (oxidized form) (final concentration 1 mM), 10 μl of 100 mM potassium ferricyanide (final concentration 1 mM), 16 μl of 100 mM magnesium chloride (final concentration 1.6 mM), and 35 μl of 30 mM glyceraldehyde 3-phosphate (final concentration 1 mM) (Final concentration 1 mM) was dissolved in 45 mM Tricine-NaOH buffer and adjusted to pH 8.8 to a total volume of 145 μl. 14.5 μl of the above solution was transferred to another container, 0.5 μl of 200 unit / ml pyrophosphatase (final concentration 1 unit / ml), 800 unit / ml glyceraldehyde-3-phosphate dehydrogenase 4.0 μl (32 unit / ml), 1000 unit 1 μl / ml diaphorase (10 units / ml) and 80 μl of 50 μM pyrophosphoric acid (final concentration 40 μM) were added to make a total volume of 100 μl.
Next, using the electrochemical measurement device as a sample, the reaction solution prepared as described above is used as a sample, and a potential of +600 mV is applied to the working electrode with respect to the reference electrode. It was measured. Similarly, the current values observed when the above 50 μM pyrophosphate was 100 μM (final concentration 80 μM), 150 μM (final concentration 120 μM), 200 μM (final concentration 160 μM), and 0 μM (final concentration 0 μM), respectively. It was measured.
FIG. 7 shows the current value measured 100 seconds after the start of the reaction when the amount of pyrophosphate was changed. In FIG. 7, the vertical axis represents the current value (μA), the horizontal axis represents the pyrophosphate concentration (μM) in the sample, and the current value at each pyrophosphate concentration is indicated by a black circle. As shown in FIG. 7, the amount of added pyrophosphoric acid and the current value 100 seconds after the start of the reaction showed a good correlation. Thus, it was shown that the quantitative measurement of pyrophosphoric acid, which is a small molecule in the living body, can be performed with the electrode manufactured according to this example.

  The method for producing an electrochemical sensor according to the present invention can provide a method for producing an electrochemical sensor having a high-performance reference electrode that is inexpensive and easy to produce. This is useful as a method for producing a biosensor for measuring a substance such as a molecular complex.

It is process drawing which shows one embodiment of this invention. It is a perspective view which shows the structure of the electrode in the process of this invention. It is a perspective view which shows the structure of the electrode in the process of this invention. It is a perspective view which shows the structure of the electrode in the process of this invention. It is a perspective view which shows the structure of the electrochemical device which concerns on one embodiment of this invention. It is a cyclic voltammogram measured using the electrode of the present invention. It is a graph which shows the result measured using the electrochemical device of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Board | substrate 2 Working electrode 3 Counter electrode 4 Reference electrode 5 Conductive pattern 6 Terminal part 21 Insulating film 31 Composite containing silver 41 Housing material 42 Cavity 43 Channel 44 Injection hole

Claims (12)

  A method of manufacturing a reference electrode in an electrode array for electrochemical measurement having two or more electrode pairs including at least a reference electrode and a measurement electrode, the step of forming a conductive pattern including the reference electrode on an insulating substrate, A method for producing a reference electrode, comprising: a step of forming a composite containing silver on a reference electrode portion of a conductive pattern; and a step of chlorinating the composite containing silver.   The method for producing a reference electrode according to claim 1, wherein the composite containing silver is a silver paste containing a resin material and silver particles.   The method for manufacturing a reference electrode according to claim 2, wherein the resin material is selected from an epoxy resin, a phenol resin, an acrylic resin, and polyester.   The method for producing a reference electrode according to claim 1, wherein the step of converting the silver-containing composite to silver chloride is performed by bringing a chlorine-containing oxidizing agent into contact with the surface of the reference electrode.   The method for manufacturing a reference electrode according to claim 4, wherein the oxidizing agent containing chlorine is hypochlorous acid.   A hole portion into which a sample and a reagent are injected, a flow path portion to which the sample and the reagent can move, and a detection portion having an electrode array having at least two electrode pairs including a reference electrode and a measurement electrode. A method for producing an electrochemical measurement device, wherein the reference electrode in the detection unit is produced by the method according to claim 1.   A reference electrode used in an electrode array for electrochemical measurement having at least two electrode pairs including a reference electrode and a measurement electrode, wherein the reference electrode is formed of a composite containing silver, and the surface thereof is silver chloride A reference electrode for electrochemical measurement.   The reference electrode according to claim 7, wherein the composite containing silver is a cured product of a silver paste containing a resin material and silver particles.   The reference electrode according to claim 7, wherein the resin material is selected from epoxy resin, phenol resin, acrylic resin, and polyester.   A hole portion into which a sample and a reagent are injected, a flow path portion to which the sample and the reagent can move, and a detection portion having an electrode array having at least two electrode pairs including a reference electrode and a measurement electrode. An electrochemical measurement device, wherein the reference electrode is the reference electrode according to claim 7.   An electrochemical sensor comprising the electrochemical measurement device according to claim 10. The electrochemical sensor according to claim 11, which is for quantitatively measuring a biological substance.

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