JP2590004B2 - Comb-shaped modified microelectrode cell and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a comb-shaped modified microelectrode cell having a pair of intermeshing comb-shaped working electrodes and a method for producing the same.

[Conventional technology]

Ions contained in water, organic solvents, living organisms, etc.
As a method of qualitative or quantitative analysis of molecules, an electrochemical measurement method is known. In the electrochemical measurement, usually, one working electrode for detecting a substance, its counter electrode, and one reference electrode including a reference substance serving as a reference for defining an electric potential are measured by using a potentiostat or other measuring device. Connect to

[Problems to be solved by the invention]

However, this measurement system can directly detect ions in a solution and molecules in which a reaction site can come into direct contact with an electrode, but it is difficult to detect molecules whose reaction sites are covered, such as proteins and enzymes. Further, depending on the substance, the oxidation-reduction potential may be high, exceeding the normal measurement limit. In addition, when two or more ions or molecules having the same oxidation-reduction potential are mixed, these cannot be distinguished and identified.

In order to solve these drawbacks, there is known a method of modifying (coating) the electrode surface with a functional material. That is, in order to detect and quantify extremely low concentrations of ions and molecules and to detect specific molecules, the electrodes are modified with a catalyst having molecular selectivity such as an enzyme. For example, when an electrode modified with glucose oxidase is used, the enzyme reacts only with glucose, so that only glucose in the sample can be quantified by electrochemically quantifying the generated hydrogen peroxide at the electrode. it can. However, according to this method, since the electrode surface is coated with an enzyme film, the generated hydrogen peroxide hardly reaches the electrode surface, and the sensitivity is low.
There are problems such as slow response speed.

In the case of a sample whose reaction site cannot directly contact the electrode, such as an enzyme, or a sample with a high oxidation-reduction potential, a low-molecular redox active substance called a mediator is mixed or chemically bonded and immobilized on the electrode. In this method, a redox of a sample is once performed by a mediator, and the mediator reacts with an electrode to detect a target substance. However, also in this case, since the mediator is in the solid film, there are problems such as low transmission speed, low sensitivity, and low response speed.

Furthermore, when the electrode is modified with an electron transfer system or energy transfer system enzyme such as cytochrome C, and energy conversion or electrochemical photosynthesis is attempted, the reaction takes many steps, and the electrode potential varies according to each step. Must be set, but it is difficult to realize this in a system having only one working electrode.

On the other hand, a microelectrode is used as a means for performing an electrochemical measurement of a microregion. Many applications of microelectrodes to biometric electrodes and biosensors have been proposed. However, most of these are metal wires, carbon fibers,
It is made by enclosing metal chloride etc. In this case, it is difficult to manufacture the same electrode shape, and the obtained electrochemical characteristics also differ depending on the electrode shape, such as ring-disk electrodes In addition, a measurement cell in which the shape of the electrode and the distance between the electrodes are important factors cannot be configured. In addition, only qualitative data can be obtained by measurement using ordinary three electrodes, and when quantitative data is required, it is necessary to test the electrodes in advance, which requires a large amount of measurement time. . In addition, when it is not possible to carry out an assay for reasons such as contamination of electrodes by measurement, it is very difficult to obtain quantitative data.

On the other hand, in recent years, application of a lithography technique has been proposed as a method of manufacturing a microelectrode. In this method, a resist is applied to a substrate, an image mask having an electrode pattern is superimposed, exposed, and developed.
Micro electrodes are obtained on the substrate. According to this method, a large number of microelectrodes having an arbitrary shape and a constant interelectrode distance can be produced on a substrate with high reproducibility. Electrode pairs that can perform the same measurements as ring and disk electrodes, electrochemical devices,
It can be applied to base electrodes of sensors. By applying this fine electrode fabrication method, a micro electrochemical transistor (for example, J. Phys. Chem. 89, 5133 (198
5)), electrochemical measurement of low-molecular or high-molecular complex using a comb-shaped platinum electrode (Anal. Chem., 58, 601 (1986))
And so on.

However, in order to construct an electrochemical element such as a cell for electrochemical measurement or a sensor with these fine electrodes, a reference electrode and a counter electrode are separately required in addition to these fine electrodes, and the size of the entire measurement cell increases. In addition, it is impossible to measure an electrochemical reaction in a minute area. Further, since the distance between the fine electrode serving as the working electrode and the reference electrode or the counter electrode provided outside increases, there is a problem that it is difficult to obtain a sharp response in the measurement of a high-resistance system such as a solid electrolyte.

[Means for solving the problem]

The present invention has been made in order to solve such a problem, and a pair of meshing working electrodes, a reference electrode, and a counter electrode are formed on the same substrate, and one of the comb working electrodes is catalyzed. It is modified with a redox substance having a certain property.

Further, an electrode portion, a lead portion, and a connection pad portion are formed on an insulating substrate by a predetermined method to form a pair of meshed comb-shaped working electrodes, a reference electrode, and a counter electrode. The lead portion is covered with an insulating film while leaving only the connection pad portion, and the reference electrode is covered with a redox material, and one of the comb-shaped working electrodes is modified with a redox material having a catalytic action. Things.

[Action]

Therefore, according to the present invention, it is possible to perform an electrochemical measurement superior to the conventional modified electrode without using an external electrode.

〔Example〕

Hereinafter, a comb-shaped modified microelectrode cell according to the present invention and a method for manufacturing the same will be described in detail.

First, before describing specific embodiments of the present invention,
The outline is described. That is, the present invention relates to a microelectrode cell for electrochemical measurement provided with a pair of intermeshing comb working electrodes, and a pair of close comb working electrodes, a counter electrode, and a reference electrode formed of a metal or a semiconductor or a metalloid. Are integrated on a substrate, and one of the comb-shaped working electrodes is covered with a redox substance having a catalytic action. In addition, as a method of manufacturing the microelectrode cell for electrochemical measurement, a metal or semiconductor or semimetal electrode, lead, and connection pad are formed on a substrate having an insulating surface or whole by a lift-off method or an etching method. To form a pair of comb-shaped working electrodes, a reference electrode, and a counter electrode. Then, the lead part is covered with an insulating film except for the electrode part and the connection pad part of these electrodes, and the reference electrode is made of a redox substance. It is characterized in that one of the comb working electrodes is covered with a redox substance having a catalytic action.

Examples of the substrate having an insulating surface or the whole include a silicon substrate with an oxide film, a quartz plate, an aluminum oxide substrate, a glass substrate, a plastic substrate, and the like.

Examples of the metal for the electrode include gold, platinum, silver, chromium, titanium, and stainless steel. Semiconductors for electrodes include p and n-type silicon, p and n-type germanium, cadmium sulfide, titanium dioxide, zinc oxide, gallium phosphide, gallium arsenide, indium phosphide, cadmium selenium, cadmium telluride, molybdenum disulfide,
Examples thereof include tungsten selenide, copper dioxide, tin oxide, indium oxide, and indium tin oxide. Conductive carbon can be mentioned as a semimetal. Examples of the reference substance on the reference electrode include silver, silver chloride, and polyvinyl ferrocene. Examples of the insulating film include silicon oxide, silicon dioxide, silicon nitride, silicone resin, polyimide and derivatives thereof, epoxy resin, and thermosetting polymer.

Also, when fabricating microelectrodes, a resist is applied on the substrate, an image mask having an electrode pattern is overlaid thereon, or the pattern is directly exposed using an electron beam or the like and developed to develop the pattern on the substrate. After transferring to the upper resist, a metal, semiconductor, or semimetal thin film is formed by sputtering, vapor deposition, CVD, coating method, and then lift-off method to obtain a fine electrochemical cell consisting of four electrodes on the substrate from which the resist is stripped. Form a metal, semiconductor, or metalloid thin film on the substrate by sputtering, vapor deposition, CVD, coating method, apply resist on it, overlay image mask with electrode pattern, or use electron beam etc. After exposing the pattern directly, developing and transferring the pattern to the resist,
By using this as a mask, an underlying metal, semiconductor, or metalloid is etched to obtain a microelectrochemical cell including four electrodes on a substrate.

When fabricating the reference electrode, two
A metal serving as a support and an organic redox polymer are formed on one of the electrodes by plating and electrolytic polymerization to form the electrode.

As a method of covering either one of the comb-shaped working electrodes with a redox substance having a catalytic action, a functional group that causes electrolytic polymerization such as a vinyl group is introduced into this substance, and this is mixed with a supporting electrolyte such as tetraethyl perchlorate in acetonitrile. After dissolving in a suitable solvent such as, and immersing the comb-shaped working electrode and a counter electrode such as platinum, by applying a voltage to the comb-shaped working electrode, there is a method of electrolytic polymerization on the comb-shaped working electrode. . Another example is a method in which a redox substance having a catalytic action is mixed with an electrolytic polymerizable substance such as pyrrole, and this is electrolytically polymerized to be deposited on a comb-shaped electrode as a composite polymer film.

Hereinafter, specific examples thereof will be listed. It should be noted that the present invention is not limited to only the embodiments described below.

Example 1 A photoresist (AZ1400-2 manufactured by Shipley) was placed on a silicon wafer having an oxide film of 1 μm (manufactured by Osaka Titanium).
7) was applied to a thickness of 1 μm. The resist-coated silicon wafer was placed in an oven and baked at 80 ° C. for 30 minutes. Thereafter, contact exposure was performed for 20 seconds with a mask aligner (PLF-501 manufactured by Canon Inc.) using a chrome mask. The exposed silicon wafer was developed in a resist developing solution (AZ Developer, manufactured by Shipley) at 20 ° C. for 120 seconds, washed with water and dried to transfer the mask pattern to the resist.

The substrate with the resist pattern is attached to a predetermined position in a sputtering apparatus (made by Anelva: SPF-332H).
And platinum were sequentially deposited by sputtering. Pressure 10 ^-2 Tor
r, in an argon atmosphere, chromium: 10 seconds and platinum: 1 minute were sputtered to a total thickness of 100 nm. Thereafter, the substrate was immersed in methyl ethyl ketone and subjected to ultrasonic treatment, and the resist except for the portion where the electrode was formed was removed to obtain an electrode pattern. After that, the current density is 1m only on the reference electrode part of the substrate.
A, electricity was applied for 10 seconds, silver plating was performed, and silver was deposited on the reference electrode.

After plating, spin on glass (OCD Type manufactured by Tokyo Ohka)
Using -7), spin-coating is applied to the substrate, and then heat-cured at 450 ° C., a resist is applied again on the substrate, baked at 80 ° C. for 30 minutes, and then masked. Exposure and development were performed, and the resist was covered except for the comb-shaped working electrode portion, the reference electrode tip portion, and the counter electrode portion. Next, using the resist as a mask, spin on glass
Etching was performed with CF ₄ gas (DEM-451 manufactured by Anelva) to expose the comb-shaped working electrode portion, the reference electrode tip portion, and the counter electrode portion.

Next, a lead wire is connected to only one of the comb working electrodes, immersed in an aqueous solution of glucose oxidase and pyrrole, and electrolytically polymerized, thereby depositing a pyrrole polymer film containing glucose oxidase on the comb working electrode. Was.

FIG. 2 shows a schematic view of the cell for electrochemical measurement thus produced. In the figure, 1-1 and 2-1
Reference numeral 3 denotes a reference electrode, 4 denotes a counter electrode, 5 denotes an insulating film (spin-on-glass film), 6 denotes a silicon substrate with an oxide film, and 7 denotes an electrode pad. In the figure, 8
The portion indicated by is a pair of intermeshing comb-shaped working electrode portions, and a partially enlarged view thereof is shown in FIG. In this example,
A pyrrole polymer film 9 containing glucose oxidase is deposited on the electrode part 1-2 of the comb-shaped working electrode 1. Further, in this example, the pitch of the pair of meshing working electrodes was 5 μm, the gap was 2 μm, and the length of the comb was 2 mm.

Phosphate buffer in which glucose was dissolved at various concentrations (0.
1 mol / l, pH 6.8) is dropped on the comb working electrode portion 8, and the unmodified comb working electrode 2, the reference electrode 3,
The pad 7 of the counter electrode 4 was connected to a potentiostat via a lead wire. Next, the comb-shaped working electrode 2
When a potential of 0.6 V was applied, the oxidation potential of hydrogen peroxide generated by the action of glucose oxidase on the opposing modified comb-shaped working electrode 1 was observed. The current was stabilized within 1 second after the application of the voltage, and the current amount was 10 mA per 1 mmol / l of glucose. On the other hand, when a potential was applied thereto using the comb-shaped working electrode 1 modified with an enzyme, the amount of current was 500 nA per 1 mmol / l of glucose. As shown above, the comb-shaped electrode 1 modified with the enzyme
The sensitivity was improved by applying an electric potential to the unmodified comb-shaped electrode 2 arranged in a meshing manner.

In addition, the measurement was sufficiently performed with a sample solution of 1 ml or less, and even in a glucose solution containing a large amount of impurities such as fructose, glucose could be selectively detected without being affected by the impurities.

Example 2: Photoresist (AZ1400-2 manufactured by Shipley) on a silicon wafer with a 1 μm oxide film (manufactured by Osaka Titanium)
7) was applied to a thickness of 1 μm. The resist-coated silicon wafer was placed in an oven and baked at 80 ° C. for 30 minutes. Thereafter, contact exposure was performed for 20 seconds using a mask aligner (manufactured by Canon Inc.) using a chrome mask. The exposed silicon wafer is treated with a resist developer (Zipley, AZ
Developer), develop at 20 ° C for 120 seconds, wash with water,
After drying, the mask pattern was transferred to the resist.

The substrate with the resist pattern was mounted at a predetermined position in a vacuum deposition apparatus (manufactured by JEOL Ltd.), and chromium and gold were sequentially deposited by a resistance wire heating deposition method. Chromium is deposited for 5 seconds and gold for 3 minutes under a pressure of 10 ^-6 Torr, for a total of 100
Deposition was performed to a thickness of about 200 nm. Thereafter, the substrate was immersed in methyl ethyl ketone and subjected to ultrasonic treatment, and the resist except for the portion where the electrode was formed was removed to obtain an electrode pattern. The length of the interlocking working electrode was 2 mm, the pitch was 8 μm, and the gap was 5 μm. Thereafter, silver plating was performed only on the reference electrode under the same conditions as in Example 1 to deposit silver on the reference electrode.

After plating, spin on glass (OCD Type manufactured by Tokyo Ohka)
Using -7), spin-coating is applied to the substrate, and then heat-cured at 450 ° C., a resist is applied again on the substrate, baked at 80 ° C. for 30 minutes, and then masked. Exposure and development were performed, and the resist was covered except for the comb-shaped working electrode portion, the reference electrode tip portion, and the counter electrode portion. Next, using the resist as a mask, spin on glass
Etching was performed with CF ₄ gas (DEM-451 manufactured by Anelva) to expose the comb-shaped working electrode portion, the reference electrode tip portion, and the counter electrode portion.

Next, 0.6 g of the non-enzymatic comb working electrode
When a potential of volts was applied, a current was observed according to the concentration of glucose. The amount of current is glucose 1 mmol / l
12 mA per unit. On the other hand, when used by applying a potential to the electrode modified with the enzyme, the amount of current, glucose 1mmol /
It was 600 nA per liter. As described above, the sensitivity was improved by applying an electric potential to the unmodified comb-shaped electrode arranged in engagement with the comb-shaped electrode modified with the enzyme.

Example 3: An electron beam resist (Φ-MA) was formed on a quartz substrate having a thickness of 0.5 mm.
C, manufactured by Daikin Industries, Ltd.) to a thickness of 0.5 μm. Place the resist-coated quartz substrate in an oven at 180 ° C, 60 ° C.
Bake under the conditions of minutes. After that, it was put into an electron beam exposure system (JEOL: JSM-840), and the electron beam acceleration voltage: 5 kV, exposure amount:
Under the condition of 5 μC / cm ² , only the interdigitated comb-shaped portions were exposed. After the electron beam exposure, the substrate with the resist pattern developed and washed with the dedicated developer was subjected to chromium and platinum sputtering in the same manner as in Example 1, and then the resist was peeled off. Photoresist (Shipley Co., Ltd.)
AZ1400-27) was applied to a thickness of 1 μm, baked at 80 ° C for 30 minutes, aligned with the photomask and brought into close contact with the substrate with resist, and the patterns of the lead, reference electrode, counter electrode and pad were prepared in the examples. After exposure under the same conditions as in 2, development, cleaning, sputter deposition of chromium and platinum, and stripping of the resist were performed to form an electrode cell pattern. The size of the interlocking working electrode manufactured was pitch: 3.5 μm, gap: 0.5 μm, and comb length: 1 mm.

The substrate on which the electrode cell pattern was formed was subjected to silver plating on the reference electrode, a four-electrode, and a spion glass insulating film other than the pad portion in the same manner as in Example 1 to form an electrode cell for microelectrochemical measurement. I got Next, a lead wire is connected to only one of the comb working electrodes, immersed in an aqueous solution of glucose oxidase and pyrrole, and electrolytically polymerized to deposit a pyrrole polymer film containing glucose oxidase on the comb working electrode. Was.

Next, this comb-shaped working electrode was connected to a phosphate buffer (0.1 mmol /
l, pH 6.8) as a carrier, a 1 ml glucose sample having a concentration of 10 mmol / l was injected at a flow rate of 0.8 ml / min, and the output current was recorded. As a result, the comb working electrode responds immediately, peaking at 100 msec,
To return to the original baseline.

Example 4 After a comb working electrode was produced in the same manner as in Example 1, one of the comb working electrodes was connected to a potentiostat device via a lead wire. The comb working electrode was immersed in an acetonitrile solution in which cobalt tetra (aminophenyl) porphyrin and tetraethylammonium perchlorate were dissolved at concentrations of 1 mmol / l and 0.1 mol / l, respectively, and subjected to electrolytic polymerization at a potential of 1.1 V for 1 minute.

Next, a cell in which one of the comb-shaped working electrodes obtained by the above-mentioned method was modified with cobalt tetra (aminophenyl) porphyrin was immersed in a 50 mmol / l aqueous sulfuric acid solution, and oxygen gas was blown therein. When the potential of the modified electrode was set to -0.2 V and the potential of the other electrode was set to 0.6 V, the modified electrode reduced oxygen by the catalysis of reduced porphyrin, and the other electrode reduced it. A current due to the oxidation of hydrogen peroxide, a product of oxygen, was observed. The ratio (capture rate) of the current of the modified electrode to the current of the other electrode was 60%. Furthermore, when the potential of the modified electrode was set to 0.2 V, no current flowed in either electrode.

When a cell not modified with porphyrin was used, the electrode potential had to be reduced to -0.7 V in order to reduce oxygen.

Examples 5 to 8: Comb length: 2 mm, pitch:
4 μm, gap: 1 μm (Example 5), pitch: 6 μm, gap: 3 μm (Example 6), pitch: 7 μm, gap: 4 μm
Comb length: 1 m in the same manner as in Example 3 of (Example 7)
An electrode cell for microelectrochemical measurement including a pair of intermeshing microcomb-type working electrodes having an m, a pitch of 1.5 μm, and a gap of 0.5 μm (Example 8) was produced. A porphyrin film was attached to these electrodes and the electrodes before modification with pyrrole prepared in Examples 2 and 3 by the method of Example 4.

The relationship between the trapping rate of hydrogen peroxide and the size of the micro comb-shaped working electrode measured in the same manner as in Example 4 using these electrode cells was combined with the results measured using the electrodes of Examples 2 to 4. It is shown in the table below.

In each cell, the generation of hydrogen peroxide is controlled by changing the potential of the modified electrode We were able to.

As described above, the microcell for electrochemical measurement according to the present embodiment is manufactured by using the lithography technique. Therefore, a pair of combs having an arbitrary size, shape, and distance between electrodes can be obtained. A large number of measurement cells having a shaped working electrode, a reference electrode, and a counter electrode can be obtained at low cost.

Further, unlike the conventional modified electrode, the electrode for detecting the target substance is not coated, so that the sensitivity is high and the response speed is high.

Furthermore, by selecting a material that modifies the electrode, it is possible to respond to only a specific substance or to change the response potential so that detection is performed at a potential different from the response potential of the interfering substance. In addition, the electrochemical reaction can be switched by changing the potential of the modified electrode.

In addition, it can be used for measurement of a minute area in a minute amount of a sample, a solid, or a high-viscosity solution, and has an extremely remarkable effect as an electrochemical measurement or a sensor element.

〔The invention's effect〕

As described above, according to the comb-shaped modified microelectrode cell and the method of manufacturing the same according to the present invention, a pair of meshed working electrodes, a reference electrode and a counter electrode are formed on the same substrate, and any one of the working electrodes is formed. Since one of them is modified with a redox substance having a catalytic action, an electrode portion, a lead portion, and a connection pad portion are formed on an insulating substrate by a predetermined method, and a pair of intermeshing comb-shaped working electrodes are formed. ,
A reference electrode and a counter electrode are formed, and the lead portion is covered with an insulating film except for the electrode portion and the connection pad portion of these electrodes, and the reference electrode is covered with an oxidation-reduction substance, and either of the comb-shaped electrodes is formed. Since one is modified with a redox substance having a catalytic action, it is possible to perform better electrochemical measurements than conventional modified electrodes without using an external electrode, and it is possible to use a small amount of sample, solid or high viscosity. It is suitable for use in the measurement of minute areas in a solution, and its utility value is extremely high.

[Brief description of the drawings]

FIG. 1 is an enlarged view of the comb-shaped working electrode portion in FIG. 2, and FIG. 2 is a schematic diagram showing an embodiment of a comb-shaped modified microelectrode cell according to the present invention. 1,2 ... comb-shaped working electrode, 1-1,2-1 ... lead part, 1
−2, 2-2: Comb-shaped working electrode section, 3: Reference electrode, 4
... counter electrode, 5 ... insulating film, 6 ... silicon substrate with oxide film, 7 ... electrode pad, 8 ... comb electrode part, 9 ... pyrrole polymer film including glucose oxidase.

Claims (2)

(57) [Claims] 1. A comb-shaped working electrode, a reference electrode, and a counter electrode, which are engaged with each other, are provided on the same substrate, and one of the comb-shaped working electrodes is modified with a catalytic redox substance. And a comb-shaped modified microelectrode cell. 2. An electrode portion on an insulating substrate by a predetermined method,
A lead portion and a connection pad are formed to form a pair of meshed working electrodes, a reference electrode, and a counter electrode, and the lead portion is covered with an insulating film except for the electrode portion and the connection pad portion of these electrodes. And a method of manufacturing a comb-shaped modified microelectrode cell, wherein the reference electrode is covered with an oxidation-reduction substance, and one of the comb-shaped working electrodes is modified with an oxidation-reduction substance having a catalytic action.