JPH03179248A - Fine pore array electrode for electrochemical analysis and manufacture thereof - Google Patents

Abstract

PURPOSE:To achieve higher sensitivity and selectivity in an electrode wherein a plural ity of numerous fine pore electrodes are arranged by modifying upper electrodes with a high polymer film, a low polymer film, an enzyme film or the like to promote an electrode reaction of an interfering substance or to cause a chemical reaction of the interfering substance in a catalytic manner among the electrodes located at upper, middle and bottom parts. CONSTITUTION:At least more than one working electrodes are arrayed being separated from one another at very small intervals and reaction with an interfering substance is suppressed by increasing a current amplification factor by a redox cycle between the electrodes or shortening a period during which a desired material diffuses between the electrodes. Moreover, some electrodes are modified using a substance which acts as a catalysis for electrochemical reaction of the interfering substance to promote a reaction while an area of the electrodes is made larger so as to check a harmful substance from diffusing near an electrode for detecting the desired material. Low and high polymer thin films and an enzyme thin film modifying the upper electrodes herein used are polyvinyl ferrocene, polyvinyl pyridine. An insulating substrate is used for a substrate and metal and an organic oxidizing/reducing high polymer thin film for a reference electrode.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is applicable to flow cells or liquid chromatography,
The present invention relates to an electrochemical measurement electrode consisting of a microporous Frey electrode used in biosensors, etc., and a method for manufacturing the same.

[Conventional technology]

In addition to the target substance, electrochemical cells can be used to quantify trace components and intracellular substances in body fluids such as blood and urine.

When there is a substance with almost the same redox potential as the target substance, minimize its influence as much as possible.

It is important to quantify the target substance. In a typical electrochemical cell, nine of the three working electrodes (one working electrode, one reference electrode, and one counter electrode) are modified with polymer or low molecular thin films, enzymes, antibodies, etc. to impart selectivity and quantify the target substance. was going on.

However, when the concentration of a low-molecular interfering substance is very high, the diffusion rate in the membrane is high, so the amount that passes through the coating film on the ffi electrode and causes an electrode reaction on the electrode surface cannot be ignored, and the target substance This has been a major problem in quantifying the reaction current. Also.

When a coating film is present on the 11111 constant electrode, the diffusion rate of the target substance itself is reduced, resulting in a problem of reduced sensitivity.

In order to solve this problem, a method has been considered in which two working electrodes are used, one of which electrochemically inactivates the interfering substance, and the remaining working electrode is used to quantify the target substance. However, although this method is effective when the difference in concentration between the interfering substance and the target substance is not very large, if the interfering substance is present in excess compared to the target substance, pre-electrolysis at only one of the above may eliminate all the interfering substances. There was a problem that the substance could not be inactivated and the S/N ratio decreased.

When the target substance is a reversible redox substance, the signal can be amplified using redox cycles between electrodes. In this redox cycle, when one of two adjacent electrodes is fixed above the oxidation potential of the target substance and the other below the reduction potential, the target substance repeats the redox reaction between the two electrodes, resulting in a 1'u flow value. This is a phenomenon that is apparently amplified several dozen times.

However, with one normal-sized electrode, the diffusion distance between the electrodes is long, and even if two electrodes are arranged side by side, hardly any amplification effect can be expected.

On the other hand, in recent years, microelectrodes have been actively researched for the purpose of analyzing minute amounts of solution samples and microscopic areas such as inside living organisms, and attempts have been made to apply them to sensors and the measurement of substances in living cells. It is also known for its high sensitivity and quick response, and is attracting attention as a detection electrode for flow injection and chromatography.

Most microelectrodes are used by enclosing metal wires such as platinum or gold, carbon fibers, etc. in glass capillary tubes.

The response behavior of this microelectrode depends on the electrode shape, and as the electrode size decreases, the response behavior becomes faster, so various electrode shapes and miniaturization are being considered.

However, when the electrode radius is reduced to about 1/J, the detectable current value drops to below the nA order.

Increased noise and decreased sensitivity occur during measurement, making it difficult to measure samples with low concentrations, and electrical shielding is required during measurement. Furthermore, it has been proposed to increase the number of nine working electrodes (array) in order to improve the sensitivity of the electrodes.

In recent years, it has been used as a method to fabricate minute array electrodes.

Applications of lithography techniques have been proposed. In this method, a resist is applied to a substrate, an image mask with an electrode pattern is placed over the substrate, and after exposure to n-light and development, a thin metal film is formed by vapor deposition, etc., and the resist is peeled off to form minute electrodes on the substrate. After creating a metal thin film on an insulating substrate, a resist is applied, an image mask with an electrode pattern is overlaid, exposed and developed, and the remaining resist is used as a mask to create a metal thin film on the exposed parts. A method is known in which an electrode pattern is obtained by etching.

With this method, a large number of microelectrodes having an arbitrary shape and a constant distance between electrodes can be produced on a substrate with good reproducibility.

Furthermore, since multiple electrodes (two or more) can be arranged close to each other, applying different potentials to two close working electrodes causes a redox cycle of redox species, increasing the signal current by more than 40 times. amplification (e.g., J, El
electroanal, Che+a,.

Prelia+1nary note, Vol, 26
7 (1989), p. 291).

In addition, microelectrochemical transistors (e.g., J
, Phys, Chem,, Vol. 89 (198
5), p, 5133), < Electrochemical measurement of low molecules or polymer complexes using an L-shaped platinum electrode (Anal.

Chew, 58 (1986), p. 601).

Furthermore, a positive resist (polymethyl methacrylate: PMMA) was applied onto the conductive substrate and exposed.

By development, a large number of small circular holes are made in the resist to produce a working electrode having a large number of small circular patterns (J, Electrochem, Soc.).

Vol, 133 (1986), p, 752). In this system, PMMA is used for the insulating film in order to obtain a large number of minute circular electrodes, but in a linear polymer film like PMMA, pinholes are likely to occur and the solvent is also limited. However, there were problems with practicality.

[Problem to be solved by the invention]

When a microarray electrode is composed of a pair of close working electrodes, such as a two-comb electrode, a redox cycle of the substance to be measured occurs between the electrodes.

Since the diffusion layers around each band electrode constituting the comb-shaped electrode hardly overlap, the characteristics as a microelectrode are maintained even if the band electrodes are densely arranged.

On the other hand, with micropore array electrodes of the same size, if the electrodes are arranged closely, the diffusion layers of each micropore electrode overlap,9 resulting in the same response behavior as a normal-sized electrode, and the characteristics as a microelectrode are lost. I'll get lost. Therefore, each micropore electrode.

It is necessary to arrange them at a certain distance.

In order to obtain a high current value, a large area of the entire electrode is required.

To solve this problem, if a second working electrode is provided at the top of the microhole, the first electrode at the bottom of the microhole and the second electrode at the top are separated by a step gap corresponding to the depth of the microhole. This is because active species generated at the bottom electrode can be immediately reduced by the top electrode.

Interaction with adjacent microporous electrodes is suppressed, and the characteristics of microelectrodes can be obtained even if the electrodes are placed close to each other. Furthermore, since a large redox cycle occurs between the upper and lower electrodes that are close to each other, amplification of the current value can be expected.

Furthermore, this electrode uses a large-area upper electrode.

If the lower electrode in an array is used as a pre-electrolysis electrode to remove interfering substances, and the lower electrode is used as a detection electrode, the amount of interfering substances will be reduced and the target substance will undergo a redox cycle between the upper and lower electrodes. High selectivity can be expected through amplification.

However, if the interfering substance has a slow electron transfer rate,
When a high potential is applied to the upper electrode in order to rapidly remove interfering substances, problems arise such as part of the target substance being decomposed and shortening the life of the electrode.

There was a drawback that electrochemical measurements could not be performed with good reproducibility.

An object of the present invention is to solve the above-mentioned drawbacks of the prior art, and to provide an electrode for electrochemical analysis in which a large number of microporous electrodes are arranged, in which the bottom and the top or bottom of each microporous electrode are arranged. Electrodes are arranged in the middle and upper parts, and the upper electrode is made of a polymer or low molecular membrane or an enzyme membrane that can promote the electrode reaction of interfering substances or catalytically chemically react interfering substances. An object of the present invention is to provide a micropore array electrode for electrochemical analysis that can perform electrochemical analysis with high sensitivity and good selectivity by modifying the electrode, and a method for manufacturing the same.

[Means to solve the problem]

In order to achieve the above object of the present invention, at least two working electrodes for substance detection are provided, including a surface electrode having a large number of micropores, one surface electrode at the bottom or middle part of the micropores, and a mutually insulating electrode. at least one insulated by micropore walls
In an electrochemical analysis electrode having two or more electrodes, a small molecule that has a property of oxidizing an interfering substance on the surface electrode, or a property of promoting an electrochemical reaction of an interfering substance on the surface electrode. Alternatively, it is a micropore array electrode for electrochemical osteogenesis having a structure modified with a thin film of a polymer compound or a thin film of 1 g element. Materials such as metals, semimetals, and semiconductors can be suitably used as materials for the electrodes. Namely.

Torora's electrodes for electrochemical analysis contain a large amount of interfering substances.
When the electrode reaction rate is slow, it is not possible to remove the interfering substance sufficiently with only one working electrode, and therefore a large amount of the interfering substance reaches the electrode for detecting the target substance. In addition, it is difficult to prevent the target substance that has been electrochemically oxidized or reduced at one electrode from chemically reacting with interfering substances before it diffuses to the detection electrode and is detected. Ta. Furthermore, when the electrode size increases, the current amplification of the target substance due to the redox cycle does not occur, resulting in high sensitivity Δ1.
It was difficult to determine q.

In contrast, the two electrodes of the present invention have a structure in which at least two or more working electrodes are arranged with a small gap between them, so the current amplification rate due to the redox cycle between the electrodes is large, and the target substance can be transferred between the electrodes. Since the diffusion time is also extremely short, reactions with interfering substances can be suppressed. Furthermore, because one electrode is modified with a substance that promotes the electrochemical reaction of the interfering substance by becoming f@@ soot and the electrode area is large, the interfering substance will diffuse into the vicinity of the electrode for detecting the target substance. The present invention has been completed based on the discovery that the above can be suppressed.

2. Next, as a method for producing a micropore array electrode for electrochemical analysis according to the present invention, conductive thin films and insulating thin films made of metals, semimetals, semiconductors, etc. are alternately deposited on a substrate whose surface or entire surface is insulating. After stacking each layer at least once to form a microporous resist pattern, a large number of micropores are made using an etching method until the conductive film surface of the electrode is exposed. The electrode of the present invention can be obtained by modifying with a thin film of a low molecule or polymer or a thin film of an enzyme that oxidizes the interfering substance or promotes the oxidation.

Examples of substrates whose surface or entire surface is insulating include silicon substrates with oxide films + Z] English plates, V-based aluminum 11 substrates,
Examples include glass substrates and plastic substrates. Gold, platinum, silver, palladium, chromium, titanium, stainless steel, etc. can be used as metals for electrodes, and semiconductors for electrodes include p-type and n-type silicon, p- and n-type germanium, cadmium sulfide, and titanium dioxide. .

Zinc oxide, gallium 11 phosphate, gallium arsenide, indium phosphide, cadmium selenium, cadmium tellurium.

Molybdenum disulfide, tungsten selenide, copper dioxide,
Tin oxide, indium a-oxide, indium tin oxide, etc. are used. Moreover, it is preferable to use conductive carbon as the semimetal. Silicon oxide and silicon dioxide are used as insulating thin films.

Examples include silicon nitride, silicone resin, polyimide and its derivatives, epoxy resin, and thermoset polymers.

Examples of low molecules, polymer thin films, and enzyme thin films that modify the upper electrode include polymeric organic materials such as polyvinylferrocene, polyvinylpyridine and their transition metal complexes, vinylpyridine, vinylbipyridine copolymers, and their transition metal complexes. metal complex.

Examples include electropolymerized conductive polymers such as polypyrrole, polythiophene, and polyaniline, charge transfer complexes such as TTF-TCNQ, thin films of phthalocyanine and porphyrin, and polymer/enzyme composite films in which L-ascobate oxidase is dispersed. can.

When fabricating microelectrodes, conductive thin films and insulating thin films are alternately laminated on a substrate, and these fabrications involve vapor deposition,
Sputtering, CVD, coating methods, etc. can be used. A resist is applied onto the substrate, and an image mask having an electrode pattern is overlaid on it, or the pattern is directly exposed using an electron beam lithography system, developed, and the pattern is transferred to the resist on the substrate. Using the prepared resist pattern as a mask, the laminated film of the conductive thin film and the insulating thin film is etched to form microscopic holes to form a large number of microscopic working electrodes.

After that, a resist is applied to the upper insulating thin film, and an image mask with an electrode pattern is placed thereon, or a pattern is directly exposed to light in areas without micropores using an electron beam lithography system, and then developed. After transferring the pattern to a resist on a substrate, a metal, semimetal, or semiconductor thin film is formed by sputtering, vapor deposition, CVD, or coating, and then a reference electrode and a counter electrode are created by a lift-off method in which the resist is peeled off. It is also possible to obtain a microelectrochemical cell in which one working electrode, a reference electrode, and a counter electrode are integrated.

To prepare a reference electrode, after preparing electrodes in addition to the above-mentioned microporous working electrode, a metal serving as an indicator substance,
A thin film of organic redox polymer is produced by plating, electrolytic polymerization, etc.

As the reference electrode material, silver, silver/silver chloride, polyvinylferrocene film, etc. are used.

A method of modifying the upper electrode with a substance that acts as a catalyst for the electrochemical reaction of the interfering substance and promotes the reaction with the interfering substance is to support a vinyl group, an aromatic ring, or a heterocyclic compound such as tetraethylammonium verchlorate. Examples include a method of electrolytically polymerizing by dissolving the material together with an electrolyte in a suitable solvent such as acetonitrile, immersing the electrode cell and a counter electrode such as platinum in the solution, and then applying a voltage to the working electrode. In addition, a redox substance with catalytic action is mixed with an electrolytically polymerizable substance such as pyrrole.

By electrolytically polymerizing this, a composite membrane is produced. [Example] An example of the present invention will be given below with reference to two drawings.
6. The present invention will be explained in more detail.The present invention is not limited to the scope of the following examples.

(Example 1) As shown in Fig. 2(a), a silicon substrate with an oxide film made of a silicon wafer (manufactured by Osaka Titanium Co., Ltd.) with a silicon oxide film of 1t1m thick was sputtered using a sputtering device (manufactured by ANELVA: S). PF-332H) with a metal mask, and applied 50 W of power in argon at a pressure of 1.3 Pa to sputter chromium for 1 hour.
Do this for 0 seconds, then continue to power 70 without breaking the vacuum.
Platinum was sputtered with W for 1 minute to obtain a film thickness of 1100 mm.
A lower electrode 2 made of a laminated film of n chromium and platinum was formed [FIG. 2(b)].

Next, the metal mask was removed and an insulating thin film 3 made of silicon dioxide was deposited by sputtering [Figure 2 (C)]
]. The film forming conditions at this time were such that sputtering was performed for 10 minutes at a power of 50 W and a temperature of 250° C. to obtain a 300 nm thick silicon dioxide thin film. Another metal mask was attached again, and 1100 nm of the surface electrode 4 made of a laminated film of chromium and platinum was deposited [FIG. 2(d)].

Thereafter, photoresist 5 (MP-1400-27, manufactured by Silapray) was applied to the knee thickness on the silicon substrate. This resist-coated silicon substrate was placed in an oven and baked at a temperature of 80° C. for 30 minutes. Thereafter, contact exposure was performed for 20 seconds with a mask aligner (Canon fJJ: PLA301) using a chrome mask. The exposed silicon substrate was placed in a resist developer (MF-319 manufactured by Silapray).

Development was carried out at 20° C. for 60 seconds, washed with water, and dried to transfer the mask pattern to the resist [FIG. 2(e)].

After development, the above substrate is placed in an ion milling device! 1 (manufactured by Commonwealth Corporation: Miratron type 4), and the argon gas pressure was set to 1. lXl0-'Torr, IE amount 123CCM
(7 minutes per standard cubic centimeter), acceleration voltage 550V.

After etching the laminated film 4 of chromium and platinum for 2 minutes under the conditions of current: 250 mA, etching was carried out in a 2-reactive ion etching device (manufactured by ANELVA: DE M-451) with a CF4 gas flow of 25 scM and a pressure of 3. Pa,
The insulating thin film 3 made of silicon dioxide was etched for 10 minutes at a power of 130 W to form a large number of disk electrode patterns 8 having micropores [Fig.
)]. The diameter of each disc electrode is 0.5 cape.

The number was set to 40,000. After that, the substrate is immersed in methyl ethyl ketone and subjected to ultrasonic cleaning. ! An electrode pattern was obtained by peeling off the resist other than the electrode pattern.

Furthermore, this substrate was used along with a platinum wire counter electrode.

Vinyl ferrocene lom moQ/Q! Divinylbenzene 2fflIIloQ/Q, tetrabutylammonium barchlorate 0.0 as supporting electrolyte. Imo
By immersing it in a dimethylpol-11 amide solution containing Q/Q and applying a voltage of -2,5 V to the silver/silver chloride electrode to the top electrode for 3 minutes, both vinylferrocene and divinylbenzene were deposited on the two-part electrode. A poly(vinylferrocene/divinylbenzene) copolymer film 6 consisting of a polymer was formed (
Figure 2 (g)]. The structure of the prepared micropore array electrode is shown in FIG.

This electrode together with the reference and counter electrodes.

1mmoQ/Q ferrocenylmethyltrimethylammonium bromide (water-soluble ferrocene), 10mmo
D/IA containing L-ascorbic acid ρ1 (7) immersed in phosphate buffer solution, modified upper electrode and lower electrode (in the hole)
were each connected to a dual potentiostat via a lead wire, and the potential of the upper electrode was swept from 0 (zero) to 0.6 μm V/see.

Fix the lower electrode at OV and measure the current value.
The two-part electrode was compared to not being modified with vinylferrocene/divinylbenzene copolymer and to not having L-ascorbic acid added.

■, ~ When ascorbic acid is not added, the peak current (120 μ
A), and on the reduction side, the limiting current (2,3 μA) based on the reduction of oxidized water-soluble ferrocene was B 1tIQ. In this case, the upper electrode has a large electrode size, so
It shows the same linear diffusion-limited response behavior as the electrode, but a steady-state current was obtained at the bottom electrode because minute disk electrodes were gathered together.

When 10 times as much L-ascorbic acid as ferrocene exists in the solution, the oxidation of L-ascorbic acid mediated by ferrocene in the aqueous solution occurs at the upper electrode.
l'l and its magnitude increased to 900 μA.

On the other hand, the limiting current accompanying the reduction of ferrocene measured at the lower electrode decreased to 0.72 μA. This indicates that some of the ferrocene oxidized at the upper electrode was reduced to i'ji by -ascorbic acid when it reached the lower electrode.

Next, when measurements are performed using a modified micropore array electrode,
The magnitude of the reduction peak of the water-soluble oxidized ferrocene observed on the reduction side increased from 0.72 μA to 1.1 μA. This is due to the presence of a vinyl ferrocene copolymer on the upper electrode that acts as a catalyst for the oxidation reaction of L-ascorbic acid, which promotes the oxidation of L-ascorbic acid and acts as a reducing agent for V-converted ferrocene. This is thought to be due to a decrease in the concentration of L-ascorbic acid at the electrode IH surface.

C\Example 2) A micropore array electrode was produced in the same manner as in Example 1. The size of the upper 'rli electrode is Jcm x 1cm+ The diameter of the microholes of the do part electrode is 0.5/JTII, and the number is 4000.
It was set to 0. Furthermore, this substrate was used along with a platinum wire counter electrode.

Vinylferrocene: lOm mo Q/Q, divinylbenzene: 2armoQ/Ω, tetrabutylammonium barchlorate O, Imo Q as indicator electrolyte
/ +2 immersed in a dimethylformamide solution containing
A voltage of -2.5 V relative to the silver/silver chloride electrode was applied to the upper electrode for 2 minutes to form a vinylferrocene/divinylbenzene copolymer film on the upper electrode.

This micropore array electrode, together with a reference electrode and a counter electrode, is 0. Im mo flood/flood dopamine, 1 river voQ
Phosphoric acid a of PH4,3 containing L-ascorbic acid of /Q
Immerse in a buffer solution and modify the two-part electrode and the bottom electrode (in the hole)
are connected to the dual potentiostat via lead wires, and the upper electrode is set at 10 mV from O to 0.8
/see, the lower electrode was fixed at OV, the current value was measured, and compared with the case where the upper electrode was not modified with the vinylferrocene/divinylbenzene copolymer.

When measurements are performed using an electrode with an unmodified upper electrode, L-ascorbic acid and dopamine are oxidized at the upper electrode, but the amount of L-ascorbic acid is large, and electrons are exchanged between dopamine and L-ascorbic acid. Because a transfer reaction occurs, the two could not be distinguished. The magnitude of the peak current was 50 μA. On the other hand, a limiting current of 0.20 μA due to the reduction of dopamine was obtained at the lower electrode. the upper electrode.

When modified with vinylferrocene/divinylbenzene copolymer, the current value was 72μA compared to an unmodified electrode.
increased to This is because the oxidation of L-ascorbic acid proceeds catalytically through the vinylferrocene copolymer film on the upper electrode. Furthermore, the limiting current of oxidized dopamine detected on the reduction side increased from 0.20 μA to 0.27 μA. This is because the oxidation rate of L-ascorbic acid oxidized at the electrode interface increases, resulting in L-ascorbic acid oxidizing near the electrode.
This is considered to be because the electron transfer reaction between oxidized dopamine and L-ascorbic acid is suppressed as the ascorbic acid concentration decreases, and the current value observed at the reduction side electrode increases.

(Example 3) A micropore array electrode was produced in the same manner as in Example 1. The size of the upper electrode was 1 cm x 1 cm, and the diameter of the micropores of the lower electrode was 10,000 (1-9). moreover,
This substrate along with a platinum wire counter electrode.

Polypyrrole was added to the upper electrode by immersing it in an aqueous solution containing 0.1 mo Q/Q of pyrrole and 0.1 mo Q/Q of lithium berchlorate as a supporting electrolyte, and applying a voltage of 2 V between the upper electrode and the counter electrode for 20 seconds. A film was formed.

This array electrode was immersed in a PH4,3 phosphate buffer solution containing 0.1 mmo Q/Q epinephrine and 1 mmo Q/fl L-ascorbic acid, and the modified upper electrode and lower electrode (in the hole) were each placed into a dual-potential tube. Connect to the Ostat via the lead wire, and connect the upper electrode from O to 0.
．． Sweep the potential at ton V/see up to 8V.

The lower electrode was fixed at OV and the current value was measured.

A comparison was made with a case where the upper electrode was not modified with a polypyrrole film.

When measurements were performed using an electrode with an unmodified upper electrode, L-ascorbic acid and epinephrine were oxidized at the upper electrode, resulting in a peak current of 45 μA;
High amount of ascorbic acid.

The two could not be distinguished because epinephrine acts as a catalyst for the oxidation of L-ascorbic acid. Also.

This is because L-ascorbic acid has a low electron transfer rate.

It is very gradual from rise to peak.

Furthermore, on the reduction side, a limiting current of <0.18 μA was obtained based on the reduction of oxidized epinephrine. on the other hand.

When the upper electrode was modified with a polypyrrole film, the peak Mization current increased from 45 μA to 75 μA.

This is because L-ascorbic acid is oxidized via the polypyrrole coated on the upper electrode.

This is because the electron transfer rate increases, resulting in a large oxidation current.

Further, the reduction current also increased to 0.23 μA. this is.

"As the amount of monoascorbic acid oxidized at the bipartite electrode increases, the concentration of L-ascorbic acid near the electrode cell decreases, and the reaction in which oxidized epinephrine is reduced by L-ascorbic acid is suppressed. This is thought to be because the amount of epinephrine that diffuses to the lower electrode (reduction side electrode) increases.

(Example 4) A micropore array electrode was produced in the same manner as in Example 1. The size of the upper electrode was 1 cm x 1 cm, the diameter of the micropores of the lower electrode was 0.5 -, and the number of holes was 40,000. Furthermore, this substrate was used along with a platinum wire counter electrode.

It was immersed in an aqueous solution containing 0.1 mo Q/Q of pyrrole, 10 mg of L-ascobate oxidase, and 0.1 mo R,/Q of lithium verchlorate as a supporting electrolyte, and was placed facing the upper electrode. T! When a voltage of 2V was applied between the electrodes for 30 seconds, a polypyrrole film incorporating L-ascobate oxidase was formed on the upper electrode.

This microhole array electrode is 0.1m long! 1100 /
The modified upper electrode and lower electrode (in the hole) were each connected to a dual potentiostat via a lead wire, immersed in a PH4,3 phosphorus M buffer solution containing Q dopamine, 1+nmoQ/Q L-ascorbic acid, The potential of the upper electrode was swept from 0 to 0.8 V at 10 IIV/sec, the lower electrode was fixed at OV, and the current value was measured.
A comparison was made with a case where the upper electrode was not modified with a polypyrrole/L-ascobate oxidase composite membrane.

Al using a micropore array where the top electrode is unmodified
When carrying out the Il determination, L-ascorbic acid and dopamine were oxidized at the upper electrode, but the two could not be distinguished because of the large amount of L-ascorbic acid. At the lower reduction side electrode, 0.19μ due to reduction of dopamine oxidized product
A limiting current of A was obtained.

On the other hand, when the -upper electrode was modified with a polypyrrole/L-ascobate oxidase film, the current value observed on the reduction side increased to 0.21 μA compared to the unmodified electrode. This is because the oxidation of L-ascorbic acid occurs not only on the upper electrode but also on L-ascobate oxidase immobilized on the electrode.
- Ascorbic acid concentration is reduced compared to unmodified electrodes.

This is thought to be because electron transfer between oxidized dopamine and L-ascorbic acid is suppressed, and the current value observed on the reduction side increases.

(Example 5) A micropore array electrode was produced in the same manner as in Example 1. The size of the two-part electrode was 1 cm x 1 cm, the diameter of the micropores of the lower electrode was 0.5-, and the number was 40,000. Furthermore, this substrate was used along with a platinum wire counter electrode.

Vinylferrocene 10m moQ/Q, divinylbenzene 2a+moQ/Q, tetrabutylammonium barchlorate 0.1moQ/Q as indicator electrolyte
The upper part 11i is immersed in a dimethylformamide solution containing
A voltage of -2.5 V was applied to the silver/silver chloride electrode for 3 minutes to form a vinylferrocene/divinylbenzene copolymer film on the upper electrode.

This micropore array electrode is incorporated into a flow cell.

A silver/silver chloride electrode and a stainless steel plate were used as a reference electrode and a counter electrode, respectively. A phosphate buffer solution of pH 4.3 was used as the carrier solution, and 0.7 V was applied to the upper electrode.

After applying a potential of OV to the lower electrode, 0.1 m mo
Inject 5 μl of phosphoric acid Hwt solution containing fl/Q of dopamine and 1n+moQ/Q of L-ascorbic acid.

The current values obtained at the upper and lower electrodes were recorded.

The unmodified bipartite electrode was also measured and compared with the results obtained with the modified electrode.

When measurements were performed using a micropore array electrode with an unmodified upper tff pole, a current of 24 μA was obtained at the upper electrode and 0.11 μA at the lower electrode.

On the other hand, when the upper electrode was modified with vinylferrocene/divinylbenzene copolymer, compared to the unmodified electrode,
The current value increased to 47 μA. This is because the oxidation of (1),-ascorbic acid proceeds catalytically through the vinylferrocene copolymer film on the upper electrode. Furthermore, the limiting current of oxidized dopamine detected on the reduction side also increased to 0.15 μA. This is because the oxidation rate of L-ascorbic acid oxidized at the 'T11 polar interface increases, so the L-ascorbic acid concentration near the electrode decreases, and as a result, there is a gap between 9M dopamine and L-ascorbic acid. As a result, the ff1 observed at the reduction side electrode is suppressed.
This is thought to be because the A value increases.

〔Effect of the invention〕

As explained in detail above, since the micropore array electrode for electrochemical analysis of the present invention is produced using phosphorography technology, it is possible to obtain a large amount of electrodes with arbitrary sizes, shapes, and tit-to-electrode distances. . Additionally, the upper and lower electrodes are extremely close to each other.

Since the upper electrode is modified with a material that promotes the electrochemical oxidation reaction of interfering substances, when detecting reversible redox species on the reduction side, it is possible to suppress the influence of interfering substances and selectively detect the target substance. can be detected. In addition, the target substance causes a redox cycle between two adjacent electrodes, increasing the apparent current value, making the highly sensitive 81 measurement also 6I capability. It can be applied as an extremely effective detector.

[Brief explanation of drawings]

Fig. 1 is a schematic diagram showing the structure of the micropore array electrode for electrochemical analysis exemplified in Example 1 of the present invention, and Fig. 2 shows the manufacturing process of the micropore array electrode exemplified in Example ↓ of the present invention. It is an explanatory diagram. 1...Silicon substrate with oxide film t'...Silicon oxide film 2...Lower part ff made of a laminated film of chromium and platinum! pole 3
...Insulating thin film made of silicon dioxide 4...Surface electrode 5 made of a laminated film of chromium and platinum...Photoresist 6...Poly (vinylferrocene/divinylbenzene)
J (polymer film 7 micropore electrode 8/disk fti pole pattern

Claims (1)

[Claims] 1. Having at least two working electrodes for substance detection;
The working electrode includes a surface electrode having a plurality of micropores arranged therein, and at least one or more electrodes at the bottoms or intermediate portions of the micropores that are insulated from each other by insulating micropore walls. In a micropore array electrode for electrochemical analysis consisting of a micropore electrode, the surface electrode has a property of oxidizing an interfering substance on the surface electrode, or a property of promoting an electrolytic oxidation reaction of an interfering substance on the surface electrode. A micropore array electrode for electrochemical analysis, characterized in that it is modified with a thin film made of a low-molecular or high-molecular compound or an enzyme. 2. A micropore array electrode for electrochemical analysis according to claim 1, wherein the electrode is made of a metal, a semimetal, or a semiconductor. 3. In the micropore array electrode according to claim 1, the modification material that oxidizes the interfering substance on the surface electrode or promotes the oxidation reaction is a low molecule or polymer that has the property of oxidizing L-ascorbic acid. A micropore array electrode for electrochemical analysis characterized by being a thin film made of a compound or an enzyme. 4. Having at least two or more working electrodes for substance detection;
The working electrode includes a surface electrode having a plurality of micropores arranged therein, and at least one or more electrodes at the bottoms or intermediate portions of the micropores that are insulated from each other by insulating micropore walls. In a method for manufacturing a micropore array electrode for electrochemical analysis consisting of a micropore electrode, a conductive thin film made of a metal, semimetal, or semiconductor constituting the electrode and an insulating film are placed on a substrate whose surface or entire surface is insulating. After forming a resist pattern having a plurality of micropores using lithography technology, the laminated films are stacked alternately at least once with conductive thin films, and then the laminated films are etched until the bottom layer of the conductive thin film surface appears. Etched and
A plurality of micropores are formed to form a micropore array electrode, and then the surface electrode has a property of oxidizing L-ascorbic acid, which is an interfering substance, or a property of promoting an electrolytic oxidation reaction on the surface electrode. A method for producing a micropore array electrode for electrochemical analysis, characterized in that the electrode is modified with a thin film made of a low-molecular or high-molecular compound or an enzyme.