JP2007298325A - Electrode chip and manufacturing method therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately prescribe the area of the electrode body part of a working electrode or the like, without providing an insulating layer in an electrode chip utilizable as a biosensor or the like. <P>SOLUTION: The acting electrode 14 and a counter electrode 15 are formed on an insulating substrate 12. The predetermined region 22 of the insulating substrate 12 is provided with a liquid-holding layer 21 which can hold a sample solution and is brought into direct contact with the insulating substrate 12, the working electrode 14 and the counter electrode 15 that cover them. The parts, brought into contact with the liquid holding layer 21 of the acting electrode 14 and the counter electrode 15, function as electrode body parts 14a and 15a that react electrochemically with the sample solution. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an electrode chip that can be used as an electrochemical sensor including a biosensor and a method for manufacturing the electrode chip.

  Electrode type electrochemical sensors consisting of a chip (electrode chip) provided with an electrode on an insulating substrate and detecting changes in current and potential caused by a substance to be measured at the electrode interface have been applied and put into practical use in various fields. . In particular, in recent years, the demand for applying electrode-type electrochemical sensors to biosensors that measure substances such as proteins, enzymes, and low-molecular complexes existing in living organisms because they are easy to miniaturize and multi-size. Is growing.

  This type of electrochemical sensor is roughly divided into a tripolar type and a bipolar type. The tripolar electrochemical sensor includes a working electrode, a counter electrode, and a reference electrode on an insulating substrate. These working electrode, counter electrode, and reference electrode are insulated except for the part that interacts electrochemically with the liquid to be measured (electrode body part) and the part for electrical continuity with the external circuit (contact terminal part). Covered with layers. In the case of potential measurement, the potential difference between the working electrode and the reference electrode is measured. In the case of current measurement, the current flowing between the working electrode and the counter electrode is measured while the potential difference between the working electrode and the reference electrode is kept constant by an external circuit. On the other hand, a bipolar electrochemical sensor includes only a counter electrode and a working electrode, applies a voltage between the counter electrode and the working electrode, and detects a current flowing at that time to detect a specific component (for example, , Glucose) concentration.

  For example, Patent Document 1 discloses a tripolar electrode chip constituting an electrochemical biosensor. FIG. 10 shows the electrode tip disclosed in Patent Document 1. A counter electrode 2, a working electrode 3, and a reference electrode 4 are provided on the insulating substrate 1 by screen printing a conductive carbon paste. An insulating layer 5 is provided on the insulating substrate 1 and the electrodes 2 to 4, leaving the contact terminal portions and the electrode main body portions 2 ′, 3 ′ and 4 ′. In other words, the opening 5 a is provided in the insulating layer 5, and the electrode body 2 ′ to 4 ′ of the counter electrode 2, the working electrode 3, and the reference electrode 4 are defined by the opening 5 a. In addition, a CMC (carboxymethylcellulose) -GOD (glucose oxidase) layer, which is a layer of a hydrophilic polymer and an oxidoreductase, is provided on the surfaces of the electrode body portions 2 ′ to 4 ′. Patent Document 2 also discloses an electrode chip having a structure in which an electrode main body is defined by an opening formed in an insulating layer covering the electrode.

  However, providing an insulating layer that covers the entire electrode excluding the electrode main body and the contact terminal portion complicates the structure of the electrode tip and complicates the manufacturing process. In particular, when the dimensions of the electrode body are miniaturized, it is necessary to reduce the dimensions of the openings of the insulating layer, and the alignment of the openings of the insulating layer and the individual electrodes is required to be highly accurate. It is required to form the layer with high accuracy. Furthermore, when the insulating layer is formed in a process including heating such as thermal printing, there is a restriction on the selection of the insulating base material with respect to thermal deformation characteristics. For example, a resin is generally selected as a material for an insulating substrate from the viewpoint of cost and disposableness. However, in order to form an insulating layer by thermal printing, a resin that has a high glass transition temperature and is not easily thermally deformed is used as a material for the insulating substrate. There is a need to choose. From the above points, the structure that defines the electrode main body portion of each electrode by the insulating layer is complicated, and the handleability and mass productivity are not good.

Japanese Patent Laid-Open No. 2-62952 Japanese Patent Laid-Open No. 2-310457

  In view of the above-described conventional problems, the present invention provides an electrode chip that can be used as an electrochemical sensor including a biosensor, accurately defines the area of the electrode body without providing an insulating layer, and improves handling and mass productivity. The task is to do.

  According to a first aspect of the present invention, there is provided an electrode chip for electrochemically detecting a reaction in a liquid, wherein the electrode chip is formed on an insulating base, and is formed on the insulating base and in one region on the insulating base. A working electrode partially disposed thereon, a counter electrode formed on the insulating substrate at a distance from the working electrode, and partially disposed in the region on the insulating substrate; A liquid holding layer capable of holding a liquid and covering the region in direct contact with and covering the insulating substrate, the working electrode, and the counter electrode, and being in contact with the liquid holding layer of the working electrode and the counter electrode Provided is an electrode tip in which each part functions as an electrode body part that electrochemically acts on the liquid.

  The liquid holding layer holds the liquid, and only the region of the insulating substrate where the liquid holding layer is disposed is wetted by the liquid. Therefore, the area of the working electrode and the counter electrode that act electrochemically, that is, the area of the electrode body portion of the working electrode and the counter electrode is accurately defined as the contact area of the working electrode and the counter electrode with the liquid holding layer. In other words, the area of the working electrode and the electrode main body of the counter electrode can be accurately defined without forming an insulating layer, and high measurement accuracy and measurement reproducibility can be obtained. In addition, since the electrode tip has a simple structure without an insulating layer, it is easy to handle and easy to manufacture and suitable for mass production.

  Specifically, the area of the electrode body portion of the working electrode is smaller than the area of the electrode body portion of the counter electrode.

  The electrode tip of the present invention is not limited to a two-pole type, and may be a three-pole type. By using the three-pole system, it is possible to measure the natural potential and measure the current with high accuracy. Specifically, the electrode tip further includes a reference electrode formed on the insulating base with a distance from the working electrode and the counter electrode, and a part of the reference electrode disposed in the region on the insulating base. The portion of the reference electrode that contacts the liquid holding layer may function as an electrode main body that electrochemically acts on the liquid.

  By making the electrode body portion of the reference electrode silver-silver chloride, the reference potential is stabilized and measurement with higher accuracy is possible.

  The measurement accuracy can be improved by using gold, platinum, palladium, or carbon that is highly conductive and chemically stable for the working electrode and the electrode main body of the counter electrode.

  Preferably, the liquid holding layer is made of a polymer material that is supported on the surface of the insulating base in a dry state before the liquid is supplied, and dissolves and gels when the liquid is supplied. When the liquid holding layer is gelled and becomes highly viscous, the contact area of the electrode such as the working electrode with the liquid holding layer, that is, the area of the electrode main body of the electrode such as the working electrode can be reliably maintained at a desired width. For example, the gelled liquid holding layer preferably has a viscosity of about 2 to 100 cp (centipoise).

  Preferably, the liquid holding layer contains a molecule having a non-specific adsorption property to the electrode part while allowing a low molecular weight molecule functioning as a redox substance to pass through the electrode part contained in the liquid. High molecular weight molecules have a molecular network structure that does not allow passage. While the electrical or electrochemical interaction between the redox substance contained in the liquid and the electrode main body is ensured, the electrical characteristics of the electrode main body over time have been caused by non-specific adsorption of proteins, etc. Along with this, it can be prevented from decreasing.

  The liquid holding layer is preferably made of a hydrophilic material. Since the wettability of the electrode body is improved, the area of the electrode body can be defined with higher accuracy.

  As a material constituting the liquid holding layer, for example, one type of material selected from starch type, carboxymethyl cellulose type, gelatin type, acrylate type, vinyl alcohol type, vinyl pyrrolidone type, maleic anhydride type or two or more types It is possible to employ a mixture of the following systems. Carboxymethylcellulose (CMC), which is a kind of cellulose-based hydrophilic polymer material and has a nonspecific adsorption inhibiting effect, is particularly suitable as a material constituting the liquid holding layer.

  According to a second aspect of the present invention, an electrode including at least a working electrode and a counter electrode is formed on the insulating substrate so that a part thereof is disposed in one region on the insulating substrate, and the region on the insulating substrate is formed in the region. A method of manufacturing an electrode chip is provided, wherein a liquid holding layer capable of holding a liquid is formed, and the liquid holding layer is covered with the insulating substrate and the electrode in the region.

  Specifically, an opening member having a shape coinciding with the region and having an opening penetrating in the thickness direction is prepared, and the opening member is arranged so that the opening is aligned with the region. A polymer dispersion liquid disposed on an insulating substrate and having a polymer material dispersed in a solvent is injected into the opening, and the solvent is evaporated from the polymer dispersion liquid injected into the opening. The liquid holding layer is formed by drying the polymer dispersion.

  The method for drying the polymer dispersion is not particularly limited, and for example, room temperature drying, vacuum drying, or freeze drying can be employed.

  The electrode tip according to the present invention has a high measurement accuracy and reproducibility because the area of the electrode main body portion that acts electrochemically with the liquid among the working electrode and the counter electrode is accurately defined as the contact area with the liquid holding layer. Is obtained. In addition, since an insulating layer is not provided, it is easy to handle and easy to manufacture and suitable for mass production. Therefore, the electrode chip according to the present invention is useful as various electrochemical measurement chips including biosensors for measuring substances such as proteins, enzymes, and low molecular complexes present in living bodies.

  Next, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In order to facilitate understanding of the structure, the ratio of the dimension in the thickness direction to the dimension in the thickness direction and the dimension in the width direction in the drawings is shown to be larger than actual.

(First embodiment)
1 to 3 show a bipolar electrode tip 11 that can be used as an electrochemical sensor according to a first embodiment of the present invention. The electrode tip 11 has a two-layer structure in which an opening member 13 is joined to an insulating base 12. Specifically, the lower surface 13a of the plate-shaped opening member 13 is joined to the upper surface 12a of the plate-shaped insulating base 12 on which a working electrode 14, a counter electrode 15, and a liquid holding layer 21 to be described later are formed. The opening member 13 is provided with an opening 13b penetrating in the thickness direction.

  In the present embodiment, the insulating base 12 and the opening member 13 are rectangular plates having the same width, and these end faces are aligned with each other on the left side in the drawing. On the other hand, on the right side in the figure, the end surface of the insulating base 12 protrudes from the end surface of the opening member 13. In other words, in this portion, the insulating base 12 is not covered with the opening member 13, and the upper surface 12a is exposed.

  As shown most clearly in FIG. 3, on the upper surface 12a of the insulating base 12, two working electrodes 14 and counter electrodes 15 which are conductor patterns separated from each other are formed with a space therebetween. The working electrode 14 includes an electrode main body portion 14a, a wiring portion 14b extending from the electrode main body portion 14a, and a contact terminal portion 14c for electrical continuity with an external device provided at the other end of the wiring portion 14b. Similarly, the counter electrode 15 includes an electrode body portion 15a, a wiring portion 15b extending from the electrode body portion 15a, and a contact terminal portion 15c provided at the other end of the wiring portion 15b.

  Although the materials and the like will be described in detail later, the liquid holding layer 21 has a function of holding the sample solution. Specifically, when the sample solution is dropped onto the liquid holding layer 21, the sample solution spreads uniformly over the entire liquid holding layer 21, but the sample solution does not leak from the liquid holding layer 21 as long as the amount is appropriate. . The liquid holding layer 21 is provided in a predetermined region 22 on the upper surface 12 a of the insulating substrate 12. In the present embodiment, this region 22 matches the shape of the opening 13b of the opening member 13 in plan view. In other words, the liquid holding layer 21 in the present embodiment is formed in the opening 13 b of the opening member 13. A part of the working electrode 14, that is, the vicinity of the tip on the left side in the drawing, is arranged in a region 22 where the liquid holding layer 21 is formed. Similarly, a part of the counter electrode 15, that is, the vicinity of the tip on the left side in the drawing is arranged in a region 22 where the liquid holding layer 21 is formed. In other words, the liquid holding layer 21 is disposed so as to straddle the working electrode 14 and the counter electrode 15.

  The liquid holding layer 21 is in direct contact with the insulating substrate 12, the working electrode 14, and the counter electrode 15 in the region 22 without passing through other layers such as an insulating layer. 12, the working electrode 14, and the counter electrode 15 are covered with a liquid holding layer 21. Accordingly, the portion of the working electrode 14 that comes into contact with the liquid holding layer 21 functions as a portion that acts electrochemically with the redox substance in the sample solution, that is, the above-described electrode body portion 14a. Similarly, a portion of the counter electrode 15 that contacts the liquid holding layer 21 functions as a portion that electrochemically acts on the redox material in the sample solution, that is, the above-described electrode main body portion 15a. The width of the working electrode 14 is set smaller than that of the counter electrode 15, and the area of the electrode main body portion 14 a of the working electrode 14 is set smaller than the area of the electrode main body portion 15 a of the counter electrode 15. Parts other than the electrode main body part 14a of the working electrode 14 (wiring part 14b and contact terminal part 14c) and parts other than the electrode main body part 15a of the counter electrode 15 (wiring part 15b and contact terminal part 15c) do not contact the sample solution. Therefore, it is not necessary to cover the wiring portions 14b and 15b with an insulating material or the like.

  When the electrochemical measurement by the electrode chip 11 of this embodiment is outlined, a sample solution obtained by adding a reaction reagent to a liquid sample (for example, a biological sample including blood, serum, etc.) is supplied to the liquid holding layer 21 from the opening of the opening member 13. For example, by applying a voltage between the counter electrode 15 and the working electrode 14 and detecting a current flowing between the counter electrode 15 and the working electrode 14 at that time, the concentration of a specific component contained in the measurement liquid, etc. Is measured.

  FIG. 4A shows a state in which the sample solution 23 is supplied to the liquid holding layer 21 of the electrode chip 11 (the opening member 13 is not shown for easy understanding). On the other hand, FIG. 4B shows a configuration in which the liquid holding layer 21 is not provided and the sample is similarly formed in the vicinity of the working electrode 14 and the tip of the counter electrode 15 (in the region corresponding to the electrode main body portions 14a and 15a in the electrode tip 11 of this embodiment). The state where the solution 23 is supplied is shown. As shown in FIG. 4A, in the electrode chip 11 of the present embodiment, the sample solution 23 is held in the liquid holding layer 21, so that the working electrode 14 and the counter electrode 15 are electrically connected via the sample solution 23 which is an electrolyte solution. To do. In addition, the sample solution 23 spreads over the entire liquid holding layer 21, but does not leak outside from the liquid holding layer 21. Therefore, the area of the electrode bodies 14 a and 15 a that act electrochemically out of the working electrode 14 and the counter electrode 15 is as follows. Accurately defined. Therefore, as long as the components in the sample solution 23 do not change, the resistance flowing when the counter electrode 15 and the working electrode 14 are held at a certain potential difference is constant. In other words, measurement with high accuracy and high reproducibility is possible. On the other hand, in the configuration without the liquid holding layer 21 in FIG. 4B, the sample solution 23 spreads irregularly on the insulating substrate 12, so that the contact area between the working electrode 14, the counter electrode 15, and the sample solution 23 changes each time the measurement is repeated. Is not constant. When the contact area between the counter electrode 15 and the sample solution 23 is sufficiently larger than the contact area between the working electrode 14 and the sample solution 23, the current value is proportional to the contact area between the working electrode 14 and the sample solution 23. Therefore, in the configuration without the liquid holding layer 21 of FIG. 4B, the current value varies even if the potential difference is the same as the component in the sample solution 23, and the measurement accuracy and reproducibility are low.

  As described above, in the electrode chip 11 of the present embodiment, the liquid holding layer 21 is provided to accurately define the areas of the electrode main body portions 14a and 15a that electrochemically act on the sample solution among the working electrode 14 and the counter electrode 15. Therefore, high measurement accuracy and reproducibility can be obtained. In addition, since it has a simple structure without an insulating layer, it is easy to handle and easy to manufacture and suitable for mass production.

  As a material of the insulating base 12, a semiconductor such as silicon or germanium, glass such as quartz glass, lead glass, borosilicate glass, ceramic, resin, or the like can be selected. Considering the use of the disposable biosensor, a resin material is suitable as the material of the insulating substrate 12 in terms of processability and cost. As resin materials suitable for the insulating substrate 12, polystyrene (PS), polypropylene (PP), polyimide (PI), polytetrafluoroethylene (PTFE), polyphenylene sulfide (PPS), polyether ether ketone (PEEK), polyethylene Examples include terephthalate (PET), polymethyl methacrylate (PMMA), and polyethylene-2,6-naphthalate (PEN). In particular, when the working electrode 14 and the counter electrode 15 are formed by sputtering of gold, polyethylene terephthalate having high adhesion to gold is suitable as a material for the insulating substrate 12.

  The material of the electrode main body portions 14a and 15a of the working electrode 14 and the counter electrode 15 can be selected from noble metals such as gold, platinum, and palladium, carbon, and the like that are highly conductive and chemically stable. In particular, it is preferable to select gold as the material of the electrode main body portions 14a and 15a from the viewpoint of the stability of the surface state. From the viewpoint of improving the efficiency of the manufacturing process, the other portions of the working electrode 14 and the counter electrode 15, that is, the wiring portions 14b and 15b and the contact terminal portions 14c and 15c may be formed of the same material as the electrode main body portions 14a and 15a. preferable.

  Next, the material of the liquid holding layer 21 will be described in detail. In the present embodiment, the liquid holding layer 21 is formed by supporting a material described later in a dry state. When the sample solution is supplied, the material constituting the liquid holding layer 21 is dissolved and contains moisture. Gels. When the liquid holding layer 21 is gelled and becomes highly viscous, the contact area of the working electrode 14 and the counter electrode 15 with respect to the liquid holding layer 21, that is, the area of the electrode main body portions 14a and 15a can be reliably maintained at a desired size. For example, the gelled liquid holding layer preferably has a viscosity of about 2 to 100 cp (centipoise). However, the liquid holding layer 21 may be gelated from the stage before the sample solution is supplied.

  The gelled liquid holding layer 21 has an electrode coating characteristic, that is, a property of blocking the arrival of the component electrode main body portions 14a and 15a that adsorb nonspecifically to a metal electrode such as protein contained in the sample solution. preferable. The material having such electrode coating characteristics allows low molecular weight molecules that function as redox substances to pass through the electrode body portions 14a and 15a contained in the sample solution, but does not pass through the electrode body portions 14a and 15a. High molecular weight (large molecular structure) molecules such as proteins having specific adsorptivity have a molecular network structure that does not allow passage. Since the liquid holding layer 21 has an electrode coating characteristic, even if a component having non-specific adsorptivity is contained in the sample solution, the working electrode 14 and the counter electrode 15 are caused by the adsorption of such a component. It is possible to prevent the current flowing between them from decreasing with the passage of time after the start of measurement. In other words, current measurement between the working electrode 14 and the counter electrode 15 is not affected by components having nonspecific adsorption, and stable measurement is possible.

  The liquid holding layer 21 preferably has hydrophilicity. By increasing the wettability by imparting hydrophilicity to the liquid holding layer 21, the contact area between the working electrode 14 and the counter electrode 15 and the sample solution in the liquid holding layer 21, that is, the area of the electrode main body portions 14 a and 15 a is higher. The accuracy can be specified, and the accuracy and reproducibility of the measurement can be further improved.

  Specifically, one material selected from, for example, starch, carboxymethyl cellulose, gelatin, acrylate, vinyl alcohol, vinyl pyrrolidone, and maleic anhydride as a material constituting the liquid holding layer 21. Or a mixture of two or more systems. These polymer materials can be easily made into a solution, and a thin film having an appropriate thickness can be formed by dropping a solution of an appropriate concentration and amount and drying. Further, these polymer materials have the aforementioned electrode coating characteristics and hydrophilicity. In particular, carboxymethyl cellulose (CMC), which is a kind of cellulosic hydrophilic polymer material and has a non-specific adsorption suppressing effect, is suitable as a material constituting the liquid holding layer 21.

  The material of the opening member 13 is not particularly limited as long as it has corrosion resistance to the sample solution and the polymer dispersion described later. A semiconductor such as silicon or germanium, glass such as quartz glass, lead glass, or borosilicate glass, ceramic, resin, or the like can be selected as the material of the opening member 13. A resin material is suitable as a material for the opening member 13 in terms of workability and cost. As resin materials suitable for the opening member, polystyrene (PS), polypropylene (PP), polyimide (PI), polytetrafluoroethylene (PTFE), polyphenylene sulfide (PPS), polyether ether ketone (PEEK), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyethylene-2,6-naphthalate (PEN), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS) and the like can be selected. Polydimethyloxane is particularly preferable because it is transparent, easily processed finely, and can be bonded to the insulating substrate 12 with slight adhesiveness.

  In the case of a resin material, the opening 13b formed in the opening member 13 can be formed not only by cutting, but also by molding using a mold, embossing using thermal transfer, or the like. Moreover, you may form the opening member 13 which has the opening 13b by bonding the some sheet | seat which has a through-hole in a lamination | stacking state.

  Next, a method for manufacturing the electrode tip 11 according to this embodiment will be described. In the method of manufacturing the electrode tip 11, the step of forming the working electrode 14 and the counter electrode 15 (FIG. 5A) so that a part thereof is disposed in the region 22 on the insulating substrate 12, and the liquid holding layer 21 in the region 22. To cover the insulating substrate 12, the working electrode 14, and the counter electrode 15 (FIGS. 5B and 5C).

  As a method of forming the working electrode 14 and the counter electrode 15 on the upper surface 12a of the insulating substrate 12, the conductive material is printed, the conductive material is sputtered or deposited after etching or laser removal, or the mask is used for sputtering. There is formation without accompanying.

  As a step of forming the liquid holding layer 21 in the predetermined region 22 on the insulating base 12, first, as shown in FIGS. 5B and 5C, the opening member 13 so that the opening 13 b is aligned with the predetermined region 22. Is disposed on the insulating substrate 12. In the present embodiment, the lower surface 13a of the opening member 13 is joined to the upper surface 12a of the insulating base 12 as described above. Next, a polymer dispersion liquid in which a polymer material suitable for the liquid holding layer 21 is dispersed in a solvent is injected into the opening 13b. Thereafter, the solvent is evaporated from the polymer dispersion injected into the opening to dry the polymer dispersion, thereby forming the liquid holding layer 21.

  Solvents for preparing the polymer dispersion include water, distilled water, water such as pure water and ultrapure water, physiological saline, buffer solutions in which various chemical substances and salts are melted, toluene, xylene, etc. Aromatic hydrocarbons, ketones such as methyl ethyl ketone and methyl isobutyl ketone, alcohols such as normal butanol, ethanol and methanol, petroleum and the like. In particular, it is preferable to use ultrapure water as a solvent and dissolve the polymer solution in a predetermined concentration and use it as a polymer dispersion. The concentration of the polymer material in the polymer dispersion is preferably in the range of 0.01% to 30% by weight, particularly preferably 2.5% by weight.

  The drying method of the polymer dispersion injected into the opening 13b is not particularly limited, and techniques known to those skilled in the art can be applied. For example, room temperature drying, vacuum drying, reduced pressure drying, freeze drying, rotary drying, heat drying and the like can be applied. Vacuum drying is particularly preferable from the viewpoints of adhesion between the liquid holding layer 21 and the insulating substrate 12, ease of manufacture, and rapidity.

  As described above, in the electrode tip 11 of the present embodiment, the area of the electrode main body portions 14 a and 15 a that electrochemically act on the sample solution of the working electrode 14 and the counter electrode 15 is accurate as the contact area with the liquid holding layer 21. Therefore, high measurement accuracy and reproducibility can be obtained. In addition, since an insulating layer is not provided, it is easy to handle and easy to manufacture and suitable for mass production. Therefore, the electrode chip according to the present invention is useful as various electrochemical measurement chips including biosensors for measuring substances such as proteins, enzymes, and low molecular complexes present in living bodies. In the present embodiment, the opening member 13 remains attached to the upper surface 12a of the insulating base 12 after the liquid holding layer 21 is formed. However, the opening member 13 is removed from the insulating base 12 after the liquid holding layer 21 is formed. (See FIG. 4A).

(Second Embodiment)
6 to 8 show an electrode tip 11 according to a second embodiment of the present invention. The electrode tip 11 of the first embodiment is a bipolar type having a working electrode 14 and a counter electrode 15, whereas the electrode tip 11 of the second embodiment has a reference electrode 16 in addition to the working electrode 14 and the counter electrode 3 3. It is a polar type. The reference electrode 16 formed on the insulating substrate 12 at a distance from the working electrode 14 and the counter electrode 15 is an electrode main body portion 16a, which is a covering portion where the liquid holding layer 21 is in direct contact within the region 22, and an electrode main body. The wiring part 16b extended from the part 16a and the contact terminal part 16c provided in the other end of the wiring part 16b are provided. Similar to the electrode main body portions 14 a and 15 a of the working electrode 14 and the counter electrode 15, the area of the electrode main body portion 16 a of the reference electrode 16 is accurately defined as a contact area with the liquid holding layer 21. Further, since the sample solution is held in the liquid holding layer 21, it is not necessary to cover the wiring portion 16b and the contact terminal portion 16c of the reference electrode 16 with an insulating material or the like.

  The material of the reference electrode 16 can be selected from noble metals such as gold, platinum and palladium, carbon, and the like, as with the working electrode 14 and the counter electrode 15, and gold is particularly preferable. The surface of the electrode body portion 16a of the reference electrode portion 16 is silver chloride by a method known to those skilled in the art, such as electroplating or paste coating. In the case of electroplating, the reference electrode 16 formed of gold, platinum, or the like is applied by applying a voltage of about −1.0 V to the platinum electrode in a plating solution containing dicyanic silver (I) acid ions. Then, silver plating is performed on this part, and silver chloride is formed by applying a voltage of about +1.2 V in 3 M NaCl. Further, in the case of paste application, for example, a silver-silver chloride paste material is spotted on the surface. As a result, silver degreasing cleaning, silver chloride treatment, cleaning / drying steps, and the like can be omitted, thereby reducing costs.

  The sample solution is held by the liquid holding layer 21. In the case of potential measurement, the potential difference between the working electrode 14 and the reference electrode 16 is measured in this state. In the case of current measurement (constant potential measurement), the current flowing between the working electrode 14 and the counter electrode 15 is measured while the potential difference between the working electrode 14 and the reference electrode 16 is kept constant by an external circuit. . Since the areas of the electrode main body portions 14a, 15a and 16a of the working electrode 14, the counter electrode 15, and the reference electrode 16, which are contact areas with the sample solution, are accurately defined, highly accurate and highly reproducible measurement is possible. is there.

  Also in the electrode chip 11 of the second embodiment, the opening member 13 may be removed from the insulating base 12 after the liquid holding layer 21 is formed. Since other configurations, manufacturing methods, and operational effects of the second embodiment are the same as those of the first embodiment, the same elements are denoted by the same reference numerals and description thereof is omitted.

(Example)
The electrode tip 11 according to the second embodiment of the present invention was actually manufactured, and constant potential measurement, which is basic electrochemical measurement, was performed.

First, manufacture of the electrode tip 11 will be described. A 2000 mm metal film was formed on 1 mm polyethylene terephthalate (PET), which is an insulating substrate 12, by sputtering, and the working electrode 14, the counter electrode 15, and the reference electrode 16 were formed by photolithography. Further, a silver-silver chloride paste was dropped onto the electrode body 16a of the reference electrode 16, and dried and fixed for 2 days. The opening member 13 was made of polydimethylsiloxane. The thickness of the opening member 13 was 1 mm, and the opening 13b was formed as a circle having a diameter of 3 mm. The opening member 13 was placed on the insulating substrate 12 on which the working electrode 14, the counter electrode 15, and the reference electrode 16 had been formed, and pressure was applied from above to lightly adhere. Area of the working electrode 14, counter electrode 15 and reference electrode 16, in the opening 13b, i.e. the electrode body portion 14a, 15a, the area of 16a, respectively ^{1 mm 2,} 2.1 mm 2, and a 0.8 mm ^2. The polymer dispersion was prepared by dispersing carboxymethyl cellulose (CMC) in ultrapure water so as to have a final concentration of 2.5% by weight. 11 μL of this polymer dispersion was injected. Subsequently, the electrode tip 11 was placed in a desiccator and vacuumed for 15 minutes at room temperature by a turbo pump from the outside to evaporate ultrapure water as a solvent from the polymer dispersion. As a result, the liquid holding layer 21 made of carboxymethyl cellulose was dried and fixed in a film shape on the working electrode 14, the counter electrode 15, and the reference electrode 16 in the region 22. The opening member 13 is pasted on the insulating base 12 on which the working electrode 14, the counter electrode 15, and the reference electrode 16 are formed, and the electrode tip 11 (hereinafter referred to as the electrode tip 11 </ b> A) provided with the liquid holding layer 21. However, two electrode chips of comparative examples (hereinafter referred to as electrode chips 11B) in which the liquid holding layer 21 was not formed were manufactured.

  Using these electrode tips 11A and 11B, the electron transfer of the redox substance, that is, the current value observed at the electrode was measured. As a sample solution, a mixed liquid of potassium ferrocyanide and potassium ferricyanide was used. Specifically, potassium ferrocyanide and potassium ferricyanide were mixed in 50 mM Tris buffer (pH 8.5) at three concentrations from 1 to 3 shown in Table 1 below. The final concentrations of ferrocyanic potassium at concentrations 1 to 3 are 0, 0.5 mM, and 1.0 mM, respectively. For each of the three types of concentrations 1 to 3, 10 μL of the sample solution was dropped onto the liquid holding layer 21 of the electrode tip 11A of the example. Moreover, 10 microliters of sample solutions were inject | poured in the opening 13b of the electrode tip 11 of a comparative example about each of three types of density | concentrations 1-3. After supplying each electrode sample solution, a constant voltage of +400 mV was applied between the reference electrode 16 and the working electrode 14 of each electrode tip 11A, 11B, and the current flowing between the working electrode 14 and the counter electrode 15 was measured for 60 seconds. .

The measurement result about each density | concentration 1-3 of electrode tip 11A, 11B is shown in FIG. In FIG. 9, the vertical axis represents the current value (nA), and the horizontal axis represents the ferrocyanic potassium concentration (concentrations 1 to 3). Moreover, a white circle shows the measurement data of the electrode tip 11A of an Example, and a black circle shows the measurement data of a comparative example. Table 2 below shows the electrode tip 11A, the coefficient of determination ^{R 2} measurements for 11B. Table 2 also shows the coefficient of variation CV of measured values obtained by performing constant voltage measurement 10 times for 0.5 mM (concentration 2) of potassium ferrocyanide for each of the electrode tips 11A and 11B.

As is apparent from FIG. 9 and Table 2, in the electrode tip 11A of the example, the concentration of potassium ferrocyanide and the current value 60 seconds after the start of the reaction showed a good correlation. On the other hand, in the electrode tip 11B of the comparative example, the variation in current was large with respect to the concentration of potassium ferrocyanide, and a good correlation was not shown. Specifically, for the coefficient of determination R ^2, whereas a 0.99 in electrode tip 11A, 0.81 in electrode tip 11B, and the concentration of potassium ferrocyanide than the comparative example better examples The correlation between current values is stronger. The coefficient of variation CV is 21.85% for the electrode tip 11B, and 2.85% for the electrode tip 11A, and the concentration of potassium ferrocyanide in the example is the same as that in the comparative example. In this case, the variation in current value is significantly small.

  The state of the measurement solution observed visually during the constant potential measurement was the same as that shown in FIG. 4A for the electrode tip 11A, and the measurement solution was generally held in the liquid holding layer 21. On the other hand, the electrode tip 11B was the same as the state shown in FIG. 4B described above, and the measurement solution spread irregularly on the electrode. From the above results, it was confirmed that the electrode tip 11A of the example can perform a highly accurate and stable electrochemical reaction.

  In the examples, in consideration of reaction stability, a potassium ferricyanide-potassium ferrocyanide system was used as an electron acceptor. A stable reaction is possible even when P-benzoquinone is used as an electron acceptor. If P-benzoquinone is used, the reaction rate is increased, so that the measurement can be speeded up. In addition, 2,6-dichlorophenolindophenol, methylene blue, phenazine methosulfate, β-naphthoquinone, potassium 4-sulfonate, ferrocene and the like can be used.

1 is a perspective view showing an electrochemical measurement chip (electrode chip) according to a first embodiment of the present invention. The top view which shows the electrochemical measurement chip | tip which concerns on 1st Embodiment of this invention. 1 is an exploded perspective view showing an electrochemical measurement chip according to a first embodiment of the present invention. The typical perspective view which shows the contact area of the liquid sample and electrode in the electrochemical measuring chip which concerns on 1st Embodiment. The typical perspective view which shows the contact area of the liquid sample and electrode in the electrochemical measurement chip | tip which does not have a liquid holding layer. The perspective view which shows formation of the conductive pattern to an insulation base | substrate. The perspective view which shows attachment of the opening member to an insulation base | substrate. The perspective view which shows the insulation base | substrate which attached the opening member. The perspective view which shows the electrochemical measurement chip | tip which concerns on 2nd Embodiment of this invention. The top view which shows the electrochemical measurement chip | tip which concerns on 2nd Embodiment of this invention. The disassembled perspective view which shows the electrochemical measuring chip which concerns on 2nd Embodiment of this invention. The graph which shows the relationship between a ferrocyan potassium concentration and an electric current value. The perspective view which shows an example of the conventional electrochemical measurement chip | tip.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Electrode chip 12 Insulation base | substrate 12a Upper surface 13 Opening member 13a Lower surface 13b Opening 14 Working electrode 14a Electrode main-body part 14b Wiring part 14c Contact terminal part 15 Counter electrode 15a Electrode main-body part 15b Wiring part 15c Contact terminal part 16 Reference electrode 16a Electrode main-body part 16b Wiring portion 16c Contact terminal portion 21 Liquid holding layer 22 Region 23 Sample solution

Claims (12)

An electrode chip for electrochemically detecting a reaction in a liquid,
An insulating substrate;
A working electrode formed on the insulating substrate and partially disposed in a region on the insulating substrate;
A counter electrode formed on the insulating substrate at a distance from the working electrode and partially disposed in the region on the insulating substrate;
A liquid holding layer capable of holding the liquid and covering the region in direct contact with the insulating substrate, the working electrode, and the counter electrode;
An electrode tip in which portions of the working electrode and the counter electrode that are in contact with the liquid holding layer function as electrode main bodies that electrochemically act on the liquid.   The electrode tip according to claim 1, wherein an area of the electrode main body portion of the working electrode is smaller than an area of the electrode main body portion of the counter electrode. A reference electrode formed on the insulating substrate at a distance from the working electrode and the counter electrode, and a part of the reference electrode being disposed in the region on the insulating substrate;
The electrode tip according to claim 1, wherein a portion of the reference electrode that contacts the liquid holding layer functions as an electrode main body portion that electrochemically acts on the liquid.   The electrode tip according to claim 3, wherein the electrode body portion of the reference electrode is made of silver-silver chloride.   The electrode tip according to claim 1, wherein the working electrode and the electrode main body portion of the counter electrode are gold, platinum, palladium, or carbon.   The liquid holding layer is made of a polymer material that is supported on the surface of the insulating substrate in a dry state before the liquid is supplied and that dissolves and gels when the liquid is supplied. The electrode tip according to 1.   The liquid holding layer allows low molecular weight molecules that function as a redox substance to pass through the electrode part contained in the liquid, but has a high molecular weight containing molecules that have non-specific adsorptivity to the electrode part. The electrode tip according to claim 1, which has a molecular network structure that prevents molecules from passing therethrough.   The electrode tip according to claim 1, wherein the liquid holding layer is made of a hydrophilic material.   The electrode tip according to claim 1, wherein the liquid holding layer is carboxymethylcellulose. Forming an electrode including at least a working electrode and a counter electrode on the insulating substrate so that a part thereof is disposed in one region on the insulating substrate;
A method of manufacturing an electrode chip, wherein a liquid holding layer capable of holding a liquid is formed in the region on the insulating substrate, and the liquid holding layer is covered with the insulating substrate and the electrode in direct contact with the region. Preparing an opening member having a shape that matches the region and having an opening penetrating in the thickness direction;
Placing the opening member on the insulating substrate such that the opening is aligned with the region;
A polymer dispersion in which a polymer material is dispersed in a solvent is injected into the opening, and the solvent is evaporated from the polymer dispersion injected into the opening to dry the polymer dispersion. The method of manufacturing an electrode tip according to claim 10, wherein the liquid holding layer is formed.   The method for producing an electrode chip according to claim 11, wherein the polymer dispersion is dried by room temperature drying, vacuum drying, or freeze drying.

Images