JP2002071620A - Electrode for electrochemical measurement and manufacturing method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide electrodes for electrochemical measurement wherein only one or a plurality of arbitrary electrodes have a modifying substance or arbitrary electrodes among the plurality of electrodes have a different modifying substance, and to provide a manufacturing method thereof. SOLUTION: These electrodes for electrochemical measurement have the electrodes 2 set in array on a substrate 1 and only relevant portions of the electrodes 2 are selectively modified by the modifying substances.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an electrode for electrochemical measurement and a method for manufacturing the same.

[0002]

2. Description of the Related Art Detection of a specific substance in a solution or a gas by an electrochemical method is widely used because the apparatus is simple and inexpensive. In particular, highly versatile electrodes such as pH electrodes and ion electrodes are indispensable for environmental measurements such as water quality management.

In recent years, with the miniaturization of electrodes (for example, M. Morita, O. Niwa, T. Horiuchi,
chemical Acta, 42, 3177-3183, 1997), making it possible to perform “in-situ” measurements with high temporal resolution in the microscopic range, and the electrodes have also been used for measurements on living organisms. (For example, K. Torimitsu, O.
Niwa, Neuro Report, 8, 1353-1358, 1997). The electrochemical measurement method can reduce the damage to living tissues and cells, so that the same living sample can be used for a long time and can be used repeatedly.

[0004] Further, attempts have been made to perform multi-point measurement of a living body by an electrochemical method using an array-type electrode using a large number of microelectrodes (N. Kasai, Y. Jimbo, O. Niwa, T. et al.
Matsue, K. Torimitsu, Abstract for International M
eeting of ElectrochemicalSociety, 99-2, 1986, 199
9). The measurement method using an array-type electrode is a very excellent technique for simultaneously measuring a change in the concentration of a substance in a plurality of minute regions and obtaining a dynamic change in the distribution of the substance.

[0005]

As a method frequently used for imparting specificity to an electrode in electrochemical detection, there is a method of modifying an electrode with a modifier. The modified product is at least one of an enzyme and an electron transfer mediator for efficiently detecting an enzymatic reaction.

However, conventionally, when one or a plurality of microelectrodes as described above are modified with a modifier, at least one of an enzyme and an electron transfer mediator (hereinafter, referred to as “enzyme or electron transfer mediator”).
It was difficult to modify only those regions where "enzymes / mediators" were desired. That is, the solution containing the enzyme / mediator could not be applied only on the electrode, but was applied on the entire electrode and its peripheral portion.

However, in this method, it is difficult to control the amount of the enzyme / mediator that modifies each microelectrode and to perform uniform modification. Also, it is not possible to modify different enzymes / mediators on any of the electrodes. Also, if the modified electrochemically active substance is located between the electrodes, depending on the conditions,
Mutual interference between the electrodes occurs, for example, a current flows through the substance. Furthermore, since the absolute amount of the enzyme to be modified is large, the substrate to be measured in the measurement solution is consumed by the enzyme, and the concentration of the solution decreases, which may impair the reliability of the measurement itself.

[0008] Therefore, a technique for modifying the enzyme / mediator only on one or a plurality of arbitrary electrodes has come to an indispensable stage.

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems in the prior art, and to provide an electrochemical measurement electrode in which a modification is modified only on one or a plurality of arbitrary electrodes, and a method of manufacturing the same. To provide.

Another object of the present invention is to provide an electrode for electrochemical measurement in which a different modification is modified on any one of a plurality of electrodes, and a method for producing the same.

[0011]

According to the present invention, there is provided an electrode for electrochemical measurement according to the present invention, wherein the electrodes are arranged in an array on a substrate, and only the electrode portion is selectively modified by a modifier. It is characterized by having been done.

Further, the above-mentioned electrode has a plurality of groups modified by different modifications.

[0013] The modified product is an enzyme.

The modified product is an electron transfer mediator.

In the method of manufacturing an electrode for electrochemical measurement according to the present invention, an electric current is applied to a predetermined electrode arranged in an array on a substrate in a predetermined solution to modify only the predetermined electrode portion. It is characterized by being selectively modified by an object.

In addition, a step of passing a current to predetermined electrodes arranged in an array on a substrate in a predetermined solution and selectively modifying only the predetermined electrode portions with a modifier is repeated a predetermined number of times. And forming a plurality of electrode groups modified with different modifiers.

[0017] The present invention is characterized in that a polymer is electrochemically deposited on the electrode to modify the electrode with the modifier.

According to the above configuration, it is possible to provide an electrochemical measurement electrode in which a modification is modified only on one or a plurality of arbitrary electrodes, and a method for producing the same.

Further, it is possible to provide an electrode for electrochemical measurement in which a different modifier is modified on an arbitrary electrode among a plurality of electrodes, and a method for producing the same.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The point of the present invention is to efficiently modify an enzyme / mediator only on an electrode by using a current flowing through the electrode. In other words, by applying an electric potential to the electrode or flowing an electric current, for example, when the polymer is electrochemically deposited on the electrode surface, that is, when the polymer is oxidized or reduced and deposited, the enzyme / mediator is simultaneously placed on the electrode. Fixed on top (Examples 1 to 3 below)
4). A polymer is a molecule having a molecular weight of 10,000 or more,
A compound mainly linked by covalent bonds and having a structure in which certain structural units are regularly repeated. In the present invention, it is also possible to use a polymer to which an enzyme / mediator is bound (for example, polyvinyl bipyridine described below is an enzyme called horseradish peroxidase and osmium bipyridine ([Os (bpy) ₂ C
l] ^{2 + / 3 +} ) is covalently bound.

Alternatively, when a single molecule (a molecule that is not a polymer; a low molecule) is polymerized on the electrode surface to become a polymer, an enzyme / mediator is simultaneously taken in and fixed to the electrode surface as a membrane. In the present invention, a single molecule or a polymer may play a role as a mediator. For example, toluidine blue described below is a single molecule, but is also an electron transfer mediator (Example 5 below).

In the present invention, by modifying the same enzyme / mediator on different electrodes, it is possible to observe changes in the distribution of a specific substance in different regions.
Further, by modifying different enzymes / mediators on different electrodes, it is possible to simultaneously detect different substances and observe changes in concentration. In the former case, it is used for the analysis of multiple substances contained in a uniform solution by combining with the change in the distribution of a specific substance in a biological sample, and in the latter case, combining it with a flow injection method or high-performance liquid chromatography. It is possible.

Further, according to the present invention, it is possible to control the degree of the reaction or polymerization by controlling the amount of the current, and at the same time, to control the amount of the immobilized enzyme / mediator. In addition, since no polymer is generated or deposited or the enzyme / mediator is not modified on the electrode to which no current is applied, the electrode for modifying the enzyme / mediator can be arbitrarily selected by a very simple method. It is possible. Further, after a current is passed through a predetermined group of electrodes in a solution containing an enzyme / mediator to modify the enzyme / mediator on these electrodes, the solution is exchanged to obtain a solution containing another enzyme / mediator in the solution containing another enzyme / mediator. It is also possible to apply another current group arbitrarily selected to modify another enzyme / mediator. In addition, since the electrochemically active substance, which is a modified modifier, exists only on the electrodes, mutual interference of current between the electrodes can be reduced. In addition, since the absolute amount of the modified enzyme can be controlled to be small, the consumption of the substrate to be measured can be minimized, and the concentration of the substrate can be prevented from lowering, and the concentration of the substrate can be accurately adjusted. Can be measured.

According to the present invention, it is possible to modify an enzyme / mediator with high accuracy on only an arbitrary electrode, even for a very minute electrode, and it is extremely effective as a minute sensor capable of detecting a plurality of substances. In addition, it is also possible to simultaneously measure a specific substance at different positions and determine a change in the concentration distribution in two or three dimensions.

[0025]

Embodiments of the present invention will be described below in detail with reference to the drawings. In the drawings described below, those having the same functions are denoted by the same reference numerals, and the repeated description thereof will be omitted.

Example 1 FIG. 1 is a top view illustrating an electrode (working electrode) used for modifying an enzyme / mediator according to this example.

1 is a substrate, 2 is an electrode (working electrode), and 3 is a lead.

In this embodiment, a substrate (for example, a glass substrate)
After forming a flat indium tin oxide thin film on 1, a micro flat electrode array composed of square electrodes 2 each having a side length of 20 μm was prepared and used by lithography. The lead 3 of the electrode 2 was completely covered with an insulating film (not shown) so as not to come into contact with the solution. In this embodiment, the substrate 1 other than the electrode 2 is covered. In addition to the generally used disk electrode and flat plate electrode, other than the flat plate array electrode, a fine needle type electrode and the like can be used. As a material of the electrode 2, a metal such as gold or platinum, a metal oxide, carbon, or the like may be used in addition to indium tin oxide. The reference electrode or counter electrode may be produced on the substrate 1 in the same manner as the working electrode.

FIG. 2 is a diagram showing a schematic configuration of an apparatus used for modifying the enzyme / mediator according to the present embodiment.

4 is a multipotentiostat, 5 is a personal computer, 1 is a substrate, 6 is a measuring cell (integrated cylindrical glass with no bottom and substrate 1 is formed), 7 is a solution, Reference numeral 8 denotes a reference electrode, 9 denotes a counter electrode, 10 denotes an inverted microscope, 11 denotes a CCD (charge coupled device), 12 denotes a shield box, 13 denotes a monitor, and 14 denotes a ground. The electrode (working electrode) 2 and the lead portion 3 are shown in an enlarged manner.

A plurality of electrodes 2 installed in a measuring cell 6 using a multi-potentiostat 4 (manufactured by Hokuto Denko)
Are controlled respectively, and the enzyme /
Modified mediator. The control of the potential and the like of each electrode 2 and the monitoring of the current value and the like were performed by the personal computer 5. A silver / silver chloride electrode was used for the reference electrode 8 and a platinum wire was used for the counter electrode 9. In the present embodiment, the case of using a three-electrode method (working electrode, counter electrode, reference electrode) and using a plurality of working electrodes (electrode 2) is described. Working electrode and reference electrode that also serves as counter electrode)
But you can do it. If necessary, a current amplifier may be used between the electrode 2 and the multipotentiostat 4.

The horseradish peroxidase and [Os
(Bpy)₂Cl]^{2 + / 3 +}(Osmium bipyridi
Phosphoric acid containing polyvinyl bipyridine solution containing
A buffer solution (pH 7.4) is placed in the cell 6 of FIG.
Sweep the potential of 2 to remove enzyme (horseradish
) And electron transfer mediator ([Os (bpy)
₂Cl]^{2 + / 3 +}) Was modified on electrode 2. In addition, high
Polyvinyl bipyridine is a horseradish peruvian
An enzyme called oxidase and [Os (bpy)₂Cl]
^{2 + / 3 +}Is a covalent bond
I agree. In addition to sweeping the potential of the electrode 2,
Apply a potential step to the electrode 2
To control the current flowing through the electrode 2
It is.

Next, using a device similar to the device shown in FIG. 2, the sensing characteristics of the electrode 2 modified with the enzyme / mediator with respect to hydrogen peroxide were examined. The modified solution in the cell 6 was replaced with a measurement solution (pH 7.4, HEPES buffer), the potential of each electrode 2 was fixed at -100 mV, and the current response value at each electrode 2 when hydrogen peroxide was added to the solution (Reduction current value) was measured. The result is shown in FIG. Arrowheads (black downward triangles) in the figure indicate the addition of hydrogen peroxide, and the final concentrations were 80, 200, 320, 460, and respectively.
580 μmol / l.

When hydrogen peroxide was added to the solution, the electrode 2 was subjected to a mediator [Os (bp) due to an enzymatic reaction of a peroxidase derived from horseradish as a modified enzyme on the electrode 2.
y) ₂ Cl] ³⁺ ) Reduction current will be detected. That is, hydrogen peroxide is oxidized by horseradish peroxidase to generate water molecules, and at the same time, the mediator [Os (bpy) ₂ Cl] ²⁺ is oxidized to generate the mediator [Os (bpy) ₂ Cl] ³⁺ . -100
Since mV is a potential at which the mediator [Os (bpy) ₂ Cl] ³⁺ is sufficiently reduced, the concentration of hydrogen peroxide is calculated by measuring a change in the reduction current at the electrode 2. This is the principle of sensing. In FIG. 3, an increase in the reduction current value with respect to hydrogen peroxide was observed. The responses between the electrodes 2 were confirmed to be independent of each other, and the microscope 9 and the monitor 12 also confirmed that the enzyme was uniformly modified only on the electrodes 2. Also, by using different enzymes, it is possible to measure substances other than hydrogen peroxide.

Example 2 In this example, two kinds of enzymes were placed on a plurality of electrodes 2 using a flat plate array electrode having the same material and dimensions as the electrode 2 shown in FIG. 1 and the apparatus shown in FIG. Here is a modified example.

In this embodiment, after a flat indium tin oxide thin film was formed on a substrate (glass substrate) 1, a micro flat electrode array composed of square electrodes 2 each having a side of 20 μm was prepared by lithography and used. The lead 3 of the electrode 2 was completely covered with an insulating film (not shown) so as not to come into contact with the solution. In addition to the generally used disk electrode and flat plate electrode, other than the flat plate array electrode, a fine needle type electrode and the like can be used. As a material of the electrode 2, a metal, a metal oxide, carbon, or the like may be used in addition to indium tin oxide. The reference electrode 8 has a silver / silver chloride electrode and the counter electrode 9
Although a platinum wire was used as the reference electrode, the reference electrode 8 or the counter electrode 9 may be produced on the substrate 1 in the same manner as the working electrode (electrode 2). In this embodiment, the working electrode (electrode 2) is formed by a three-electrode method.
Are shown, but when the current value is extremely small, the detection can also be performed by the two-electrode method. If necessary, a current amplifier may be used.

The method of modifying the electrode 2 with the enzyme / mediator is as follows. A polyvinyl bipyridine solution containing horseradish peroxidase and [Os (bpy) ₂ Cl] ^{2 + / 3 +} and a phosphate buffer solution (pH 7.4) containing glutamate oxidase were placed in the cell 6 of FIG. The potential was swept and the enzyme (horseradish peroxidase and glutamate oxidase) and the electron transfer mediator ([Os (bpy) ₂ Cl]
^{2 + / 3 +} ) on electrode 2.

Next, the modifying solution in the cell 6 was added to the measuring solution (p
H7.4, HEPES buffer), the potential of each electrode 2 was fixed at -100 mV, and the current response value (reduction current value) at each electrode 2 when glutamic acid was added to the solution.
Was measured. FIG. 4 shows the results. The black arrowheads (black downward triangles) in the figure indicate the addition of glutamic acid, indicating the final concentrations of 17, 29, 38, and 47 μmol / l, respectively, and the white arrowheads (white downward triangles) indicate the final concentration of 27 μM.
2 shows the addition of ascorbic acid in Example 1.

By adding glutamic acid, the electrode 2 becomes
Eater [Os (bpy)₂Cl] ³⁺Of the reduction current
Put out. That is, glutamic acid becomes glutamic acid oxida
Oxidized by hydrogenase to produce hydrogen peroxide,
Hydrogen is reduced by horseradish peroxidase
And mediator [Os (bpy)₂Cl]²⁺
Is oxidized. Mediator [Os (bpy)₂Cl]
³⁺By measuring the change in the reduction current of
The concentration of the acid can be calculated. From this result, glutamic acid
That the reduction current increases with the concentration of
I understood. Against ascorbic acid, one of the interfering substances
Response is small, and even in the presence of ascorbic acid
It was confirmed that a change in the concentration of glutamic acid could be detected.
Confirm that the response between each electrode 2 is independent of each other
In the microscope 10 and the monitor 13, only the enzyme is placed on the electrode 2.
It was confirmed that the element was uniformly modified.

Embodiment 3 In this embodiment, an example will be described in which enzymes / mediators are modified on a plurality of electrodes 2 by the method of Embodiment 2 and used for measuring a biological sample.

A plate array electrode having the same material and dimensions as the electrode 2 shown in FIG. 1 and the apparatus shown in FIG. 2 were used. After a flat indium tin oxide thin film was formed on a substrate (glass substrate) 1, a micro flat electrode array composed of square electrodes 2 each having a side length of 20 μm was prepared by lithography and used.
The lead 3 of the electrode 2 was completely covered with an insulating film (not shown) so as not to come into contact with the solution. In addition to the generally used disk electrode and flat plate electrode, other than the flat plate array electrode, a fine needle type electrode and the like can be used. As a material of the electrode 2, a metal, a metal oxide, carbon, or the like may be used in addition to indium tin oxide. Although a silver / silver chloride electrode was used for the reference electrode 8 and a platinum wire was used for the counter electrode 9, the reference electrode 8 or the counter electrode 9 may be formed on the substrate 1 in the same manner as the working electrode (electrode 2). . Further, in this embodiment, the case where a plurality of working electrodes (electrodes 2) are provided by the three-electrode method is described. However, when the current value is extremely small, the detection can be performed by the two-electrode method. If necessary, a current amplifier may be used.

In a cell 6 in FIG. 2, a polyvinyl bipyridine solution containing horseradish peroxidase and [Os (bpy) ₂ Cl] ^{2 + / 3 +} , and a phosphate buffer (pH 7.4) containing glutamate oxidase were placed. By sweeping the potential of the electrode 2, the enzyme horseradish-derived peroxidase and glutamate oxidase, and the electron transfer mediator [Os (bpy) ₂ Cl]
^{2 + / 3 +} was modified on electrode 2.

FIG. 5 is a diagram showing a schematic configuration of an apparatus used for measuring a biological sample.

Reference numeral 15 denotes a hippocampal slice, 16 denotes a nylon mesh, and 17 denotes a Teflon (registered trademark) tube.

A hippocampal slice 15 (400 μm thick) of a Wistar rat on the second day after birth was allowed to stand on an electrode array composed of the electrodes 2, and was brought into close contact with a nylon mesh 16. The measurement was performed using a HEPES buffer solution (pH 7.4).
The change in the current was observed at a potential of the electrode 2 of −100 mV by the multipotentiostat 4. Glutamate is released using mucimol, and a Teflon tube 1 is injected with an injection pump.
From No. 7 was injected into the measurement solution.

As a result, it was observed that even in the same section 15, the glutamic acid concentration changes differently depending on the site, even when musimole was added. That is, the electrode 2 according to the present example enabled real-time multipoint measurement of glutamate, which is a typical neurotransmitter responsible for information transmission between nerve cells in the rat hippocampus. As a result, it is possible to obtain not only the mechanism of memory in the brain but also knowledge about the action of glutamate in vivo.

Example 4 This example shows an example in which glucose oxidase as an enzyme and polyvinyl ferrocene as a polymer are modified.

A plate array electrode having the same material and dimensions as the electrode 2 shown in FIG. 1 and the apparatus shown in FIG. 2 were used. After a flat indium tin oxide thin film was formed on a substrate (glass substrate) 1, a micro flat electrode array composed of square electrodes 2 each having a side length of 20 μm was prepared by lithography and used.
The lead 3 of the electrode 2 was completely covered with an insulating film (not shown) so as not to come into contact with the solution. In addition to the generally used disk electrode and flat plate electrode, other than the flat plate array electrode, a fine needle type electrode and the like can be used. As a material of the electrode 2, a metal, a metal oxide, carbon, or the like may be used in addition to indium tin oxide. Although a silver / silver chloride electrode was used for the reference electrode 8 and a platinum wire was used for the counter electrode 9, the reference electrode 8 or the counter electrode 9 may be formed on the substrate 1 in the same manner as the working electrode (electrode 2). . Further, in this embodiment, the case where a plurality of working electrodes (electrodes 2) are provided by the three-electrode method is described. However, when the current value is extremely small, the detection can be performed by the two-electrode method. If necessary, a current amplifier may be used.

A HEPES buffer solution (pH 7.4) containing polyvinyl ferrocene and glucose oxidase is placed in the cell 6 of FIG. 2 and the potentials of the electrodes 2 are swept, so that polyvinyl ferrocene and glucose are placed on the electrodes 2. The oxidase was modified. Thereafter, the solution in the cell 6 was replaced with a HEPES buffer solution (pH 7.4), and the potential of the electrode 2 was fixed at 500 mV, and the current response value (oxidation current value) of each electrode 2 to the addition of glucose was examined. FIG. 6 shows the result. Arrowheads (black downward triangles) in the figure indicate the addition of glucose, and indicate the final concentrations of 12, 35, 75, and 110 μmol / l, respectively.

As shown in FIG. 6, an increase in the oxidation current was observed with the addition of glucose. Glucose is oxidized by glucose oxidase, and at the same time ferrocene in polyvinyl ferrocene is reduced. The observed current is an oxidation current observed to oxidize the reduced form of ferrocene on each electrode 2. From these results, it was confirmed that polyvinyl ferrocene and glucose oxidase, which are polymers, were modified on each electrode.

Embodiment 5 This embodiment shows an example in which different enzymes are modified on different electrodes 2 in an array.

A plate array electrode having the same material and dimensions as the electrode 2 shown in FIG. 1 and the apparatus shown in FIG. 2 were used. One side 2 made by lithography on a substrate (glass substrate) 1
A microplate electrode array consisting of 0 μm square gold electrodes 2 was used. The lead 3 of the electrode 2 was completely covered with an insulating film (not shown) so as not to come into contact with the solution. In addition to the generally used disk electrode and flat plate electrode, other than the flat plate array electrode, a fine needle type electrode and the like can be used. As a material of the electrode 2, a metal other than gold, a metal oxide, carbon, or the like may be used. Although a silver / silver chloride electrode was used for the reference electrode 8 and a platinum wire was used for the counter electrode 9, the reference electrode 8 or the counter electrode 9 may be formed on the substrate 1 in the same manner as the working electrode (electrode 2). . Further, in this embodiment, the case where a plurality of working electrodes (electrodes 2) are provided by the three-electrode method is described. However, when the current value is extremely small, the detection can be performed by the two-electrode method. If necessary, a current amplifier may be used.

A HEPES buffer solution containing a single molecule toluidine blue and glucose oxidase (pH
7.4) is placed in the cell 6 of FIG. 2, the potentials of the plurality of electrodes 2 are swept, and then the potentials of these electrodes 2 are further reduced by 1.2.
By fixing to V, glucose oxidase was modified on a plurality of electrodes 2 (group A).

Subsequently, the solution in the cell 6 was washed with HEP containing toluidine blue and horseradish-derived peroxidase.
By exchanging with an ES buffer solution (pH 7.4), sweeping the potential of a plurality of electrodes 2 (group B) different from the electrode 2 of group A, and further fixing the potential of these electrodes 2 to 1.2 V A plurality of electrodes 2 (group B) were modified with horseradish peroxidase.

Thereafter, the solution in the cell 6 was exchanged for a HEPES buffer solution (pH 7.4), and the electrode 2 (group A and group B) was replaced.
Was fixed at 500 mV, and the current response value (oxidation current value) of each electrode 2 to the addition of glucose and hydrogen peroxide was examined. FIG. 7 shows the result. The black arrowheads (black downward triangles) in the figure indicate the addition of glucose, indicating the final concentrations of 0.1, 0.4, and 0.8 mmol / l, respectively, and the white arrowheads (white downward triangles) indicate hydrogen peroxide. And the final concentrations of 0.1, 0.2 and 0.5 mmol / l, respectively.

An increase in the oxidation current was observed at the electrode 2 in group A with respect to the addition of glucose, but the current value was hardly changed at the electrode 2 in group B. On the other hand, when hydrogen peroxide was added to the solution, an increase in the oxidation current was observed at the electrode 2 of the group B, but almost no change in the current value was observed at the electrode 2 of the group A. At this time, glucose is oxidized by glucose oxidase, and at the same time, toluidine blue molecule becomes an electron transfer mediator, is reduced by glucose oxidase, and is oxidized on the electrode 2, so that an oxidation current is observed. In addition, hydrogen peroxide is oxidized by horseradish peroxidase, and at the same time, the toluidine blue molecule becomes an electron transfer mediator, is reduced by horseradish peroxidase, and is oxidized on the electrode 2, so that an oxidation current is observed.

From these results, it is found that both the electrodes 2 of the group A and the group B have
By changing the potential of the electrode 2, each enzyme could be modified on the electrode 2 at the same time that toluidine blue was deposited on the electrode 2.

The electrode for electrochemical measurement modified with the enzyme / mediator in each of the above embodiments can effectively utilize the specificity of the enzyme while making use of the excellent characteristics of the microelectrode, and can also perform multipoint measurement. This is an excellent technique. When an enzyme / mediator is modified on a microelectrode, a change in the concentration of a specific substance in an arbitrary microregion can be detected with high sensitivity, both in increasing and decreasing concentrations.

Further, when a biological sample or the like is used as in the third embodiment, a long-time measurement or continuous measurement can be performed because the biological sample is not adversely affected during the measurement. When the measurement is performed by modifying a plurality of electrodes, it is possible to clarify the action and mechanism of the specific substance in a living body by grasping the distribution of the specific substance and a change in the distribution with respect to the stimulus.

Further, when different enzymes / mediators are respectively modified on a plurality of microelectrodes as in Example 5, multi-sensing can be performed by a simple technique of electrodes, for example, flow injection or high-performance liquid chromatography. A plurality of substances can be electrochemically detected simultaneously as a detector.

Further, since the absolute amount of the modified enzyme is small, consumption of the substrate as the target substance is suppressed, and accurate measurement becomes possible.

As described above, in the above embodiment, by modifying the enzyme / mediator only on the electrodes of the existing microelectrode array, high-performance “in-situ” measurement can be performed by a simple method, It is extremely effective for real-time multi-sensing in the environmental field such as water quality analysis, which is receiving attention, and real-time monitoring of the distribution of neurotransmitters in multiple cells and living tissues.

Although the present invention has been described in detail with reference to the embodiments, the present invention is not limited to the above-described embodiments, and it is needless to say that various modifications can be made without departing from the scope of the invention. . For example, in Examples 1 to 4, when the polymer is deposited electrochemically, that is, when it is oxidized or reduced and deposited, the enzyme / mediator, which is a modifier, is simultaneously modified on the electrode. Alternatively, it may be modified using a single molecule (a molecule other than a high molecule; a low molecule). Specifically, it is possible to simultaneously incorporate enzymes / mediators when electrochemically polymerizing a single molecule such as pyrrole or aniline on the electrode surface in addition to toluidine blue. In this case, the single molecule may serve as a so-called binder (crosslinking agent, fixing agent, carrier), or may further act as a mediator. When a single molecule acts as a mediator, modification of the molecule serving as a mediator is unnecessary.

[0064]

As described above, according to the present invention,
It is possible to provide an electrochemical measurement electrode in which a modification is modified only on one or more arbitrary electrodes, and a method for producing the same. Further, it is possible to provide an electrode for electrochemical measurement in which a different modifier is modified on an arbitrary electrode among a plurality of electrodes, and a method for manufacturing the same. In the present invention,
Real-time multi-sensing in the environmental field, such as water quality analysis, which has been attracting public attention in recent years, and a neurotransmitter targeting multiple cells and biological tissues It is extremely effective for real-time monitoring of the distribution of the water and has a wide range of use.

[Brief description of the drawings]

FIG. 1 is a top view of an electrode array to be modified with a modification.

FIG. 2 is a schematic configuration diagram of an apparatus for modifying a modification on an electrode.

[FIG. 3] In Example 1, the enzyme peroxidase derived from horseradish and a mediator [Os (bpy) ₂ Cl]
It is a figure which shows the current response of each electrode when adding hydrogen peroxide to a solution using the electrode array modified by ^{2 + / 3 +} .

FIG. 4 In Example 2, glutamic acid / ascorbic acid was added to a solution using an enzyme-modified horseradish peroxidase and glutamate oxidase and an electrode array modified with a mediator [Os (bpy) ₂ Cl] ^{2 + / 3 +} . FIG. 6 is a diagram showing a current response of each electrode at the time.

FIG. 5 is a schematic configuration diagram of a device for measuring a biological sample using an enzyme / mediator-modified electrode array prepared using the device of FIG. 2 in Example 3.

FIG. 6 is a diagram showing the current response of each electrode when glucose is added to a solution using an electrode array modified with the enzymes polyvinyl ferrocene and glucose oxidase in Example 4.

FIG. 7 shows an electrode array having an electrode modified with glucose oxidase (Group A) and an electrode modified with horseradish peroxidase (Group B) in Example 5.
It is a figure which shows the current response of each electrode when glucose and hydrogen peroxide are added to a solution.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Substrate, 2 ... Electrode, 3 ... Lead part, 4 ... Multipotentiostat, 5 ... Personal computer, 6 ... Measurement cell, 7 ... Solution, 8 ... Reference electrode, 9 ... Counter electrode, 10 ... Inverted microscope, 11 ... CCD, 12 ... Shield box, 13
... monitor, 14 ... ground, 15 ... hippocampal slice, 16 ... nylon mesh, 17 ... Teflon tube.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) G01N 27/46 336G (72) Inventor Yasuhiko Jimbo 2-3-1 Otemachi, Chiyoda-ku, Tokyo Nippon Telegraph and Telephone Telephone Co., Ltd. (72) Inventor Osamu Niwa 2-3-1 Otemachi, Chiyoda-ku, Tokyo Japan Telegraph and Telephone Co., Ltd. (72) Inventor Katsuyoshi Hayashi 2-3-1, Otemachi, Chiyoda-ku, Tokyo Sun Within the Telegraph and Telephone Corporation

Claims (7)

[Claims] 1. An electrode for electrochemical measurement, wherein electrodes are arranged in an array on a substrate, and only the electrode portion is selectively modified with a modification. 2. The electrode for electrochemical measurement according to claim 1, wherein the electrode has a plurality of groups modified with different modifiers. 3. The electrode for electrochemical measurement according to claim 1, wherein the modified product is an enzyme. 4. The electrode for electrochemical measurements according to claim 1, wherein the modified product is an electron transfer mediator. 5. A method according to claim 1, wherein an electric current is applied to predetermined electrodes arranged in an array on the substrate in a predetermined solution, and only the predetermined electrode portions are selectively modified by a modifier. Manufacturing method of electrode for chemical measurement. 6. A process in which a current is applied to predetermined electrodes arranged in an array on a substrate in a predetermined solution to selectively modify only the predetermined electrode portion with a modification is repeated a predetermined number of times. And forming a plurality of electrode groups modified by different modifiers. 7. The method for producing an electrode for electrochemical measurement according to claim 5, wherein the modification is performed by electrochemically depositing a polymer on the electrode.