JP3527185B2 - Electrochemical measurement method of living tissue - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to electrification of living tissue.
Study method .

[0002]

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

Furthermore, in recent years, along with the miniaturization of electrodes (for example, M. Morita, O. Niwa, T. Horiuchi, Electro
(Chemical Acta, 42, 3177-3183, 1997), it became possible to perform “in-situ” measurement with high time resolution in the extremely small range, so that the electrodes can also be used for measurements on living bodies. (For example, K. Torimitsu, O.
Niwa, Neuro Report, 8, 1353-1358, 1997). Since the electrochemical measurement method can reduce damage to living tissues and cells, the same biological sample can be used for a long time and can be used repeatedly.

Furthermore, attempts have been made to measure biological multipoints by an electrochemical method using an array type electrode using a large number of microelectrodes (N. Kasai, Y. Jimbo, O. Niwa, T.
Matsue, K. Torimitsu, Abstract for International M
eeting of Electrochemical Society, 99-2, 1986, 199
9). The measurement method using an array-type electrode is an extremely excellent method for measuring changes in the concentration of a substance in a plurality of minute regions at the same time and obtaining a dynamic change in the distribution of the substance.

[0005]

As a method frequently used to give specificity to an electrode in electrochemical detection, there is a method of modifying the 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 more microelectrodes as described above are modified with a modifier, "at least one of an enzyme and an electron transfer mediator" (hereinafter,
It was difficult to modify only the regions where "enzymes / mediators") were desired. That is, the solution containing the enzyme / mediator cannot be applied only to the electrode, but is applied to the entire electrode and its peripheral portion.

[0007] However, with this method, it is difficult to control the amount of 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. In addition, when the modified electrochemically active substance is located between the electrodes, depending on the conditions,
Mutual interference occurs between electrodes, such as current flowing through the substance. Further, since the absolute amount of the enzyme to be modified is large, the enzyme consumes the substrate to be measured in the measurement solution and the concentration of the solution becomes low, which may impair the reliability of the measurement itself.

Therefore, the technique of modifying the enzyme / mediator only on one or more arbitrary electrodes is at an indispensable stage.

The object of the present invention is to solve the above-mentioned problems in the prior art, for example, in the rat hippocampus.
Is a typical neurotransmitter responsible for information transmission between nerve cells of
Enables real-time multipoint measurement of certain glutamic acid
Of multiple neurotransmitters targeting multiple cells and biological tissues
Living tissue that enables real-time monitoring of distribution
The object of the present invention is to provide an electrochemical measurement method of.

[0010]

In order to solve the above-mentioned problems, the living body of the present invention
In the method of electrochemical measurement of tissue , electrodes are arranged in an array on a substrate, and only the electrode portions selectively undergo enzyme or electron transfer.
Using an array-shaped working electrode modified with a modified product containing at least one of the dynamic mediators ,
Place the working electrode in close contact with the biological tissue section and immerse it in the measurement solution.
Arrange the counter electrode and the reference electrode in the measurement solution and
It is characterized by measuring the current change of each electrode of the working electrode of . Also, use a brain slice as the living tissue slice.
Is characterized by. In addition, a hippocampal slice is used as the biological tissue slice.
Is used. Also, as the measurement solution
It is characterized by using a HEPES buffer solution. Also,
The modified product contains at least glutamate oxidase.
Using an array-shaped working electrode modified with
It is characterized by Also, at least the above-mentioned modified product
Is also modified with modified products including peroxidase
It is characterized by using an array of working electrodes. Also on
Includes at least osmium bipyridine as a modification
Use working electrodes in array form modified with modifiers
It is characterized by

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The point of the present invention is to efficiently modify an enzyme / mediator only on an electrode by using an electric current flowing through the electrode. That is, when a polymer, for example, is electrochemically deposited on the electrode surface electrochemically, that is, when it is oxidized or reduced, the enzyme / mediator is simultaneously applied to the electrode by an extremely simple method of applying a potential to the electrode or passing a current. fixed to the upper. A polymer is a compound having a molecular weight of 10,000 or more, which is mainly a covalent bond and has a structure in which a certain structural unit is regularly repeated. In the present invention, it is also possible to use a polymer having an enzyme / mediator bound thereto (for example, polyvinyl bipyridine described below includes an enzyme called horseradish peroxidase and osmium bipyridine ([Os (bpy) ₂ Cl] ^{2 + / 3 +} ) is an electron transfer mediator covalently bound).

Alternatively, when a single molecule (a molecule that is not a polymer; a low molecule) is polymerized on the surface of the electrode to form a polymer, the enzyme / mediator is simultaneously taken in and immobilized on the surface of the electrode as a film. In the present invention, a single molecule or a polymer may serve as a mediator. For example below toluidine blue is a single molecule, Ru mower an electron transfer mediator.

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.
In addition, by modifying different electrodes with different enzymes / mediators, it is possible to simultaneously detect different substances and observe changes in concentration. In the case of the former, it is used to change the distribution of a specific substance in a biological sample, and in the case of the latter, it is used in combination with flow injection method or high performance liquid chromatography to analyze multiple substances contained in a homogeneous solution. It is possible.

Further, in the present invention, it is possible to control the degree of reaction or polymerization by controlling the amount of electric current and at the same time control the amount of immobilized enzyme / mediator. In addition, since the generation / precipitation of the polymer and the modification of the enzyme / mediator are not performed on the electrode that does not pass the current, the electrode that modifies the enzyme / mediator can be arbitrarily selected by a very simple method. It is possible. Furthermore, after applying an electric current to a predetermined electrode group in a solution containing a certain enzyme / mediator to modify the enzyme / mediator on these electrodes, the solution is exchanged in a solution containing another enzyme / mediator. It is also possible to apply an electric current to another arbitrarily selected electric current group to modify another enzyme / mediator. In addition, since the electrochemically active substance that is the modified substance exists only on the electrodes, mutual interference of current between the electrodes can be reduced. Furthermore, since the absolute amount of modified enzyme can be controlled so as to be small, it is possible to minimize the consumption of the substrate to be measured, suppress the decrease in the concentration of the substrate, and accurately measure the concentration of the substrate. Can be measured.

In the present invention, it is possible to precisely modify the enzyme / mediator only on an arbitrary electrode even for an extremely minute electrode, and it is extremely effective as a minute sensor capable of detecting a plurality of substances. Alternatively, it is also possible to measure a specific substance at different positions at the same time and obtain a change in concentration distribution in two or three dimensions.

[0025]

Embodiments of the present invention will be described in detail below with reference to the drawings. In the drawings described below, components having the same function are designated by the same reference numeral, and repeated description thereof will be omitted.

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

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

In this reference example, a substrate (for example, a glass substrate)
After forming a flat plate indium tin oxide thin film on 1, a fine flat plate electrode array composed of square electrodes 2 having a side of 20 μm was prepared by lithography and used. The lead portion 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 reference example, the substrate 1 other than the electrodes 2 is covered. In addition to the flat plate array electrode, generally used disc electrodes, flat plate electrodes, and microneedle type electrodes can be used. In addition to indium tin oxide, a metal such as gold or platinum, a metal oxide, or carbon may be used as the material of the electrode 2. The reference electrode or the counter electrode may be formed 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 this reference example.

Reference numeral 4 is a multipotentiostat, 5 is a personal computer, 1 is a substrate, 6 is a measuring cell (a cylindrical glass without a bottom and the substrate 1 are integrally formed), 7 is a solution, Reference numeral 8 is a reference electrode, 9 is a counter electrode, 10 is an inverted microscope, 11 is a CCD (charge coupled device), 12 is a shield box, 13 is a monitor, and 14 is a ground. The electrode (working electrode) 2 and the lead portion 3 are shown enlarged.

A plurality of electrodes 2 installed in a measuring cell 6 using a multipotentiostat 4 (manufactured by Hokuto Denko).
Of the enzyme /
Modified mediator. The control of the potential 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 reference example, the three-electrode method (working electrode, counter electrode, reference electrode) is used, and the case where there are a plurality of working electrodes (electrode 2) is shown. However, when the current value is extremely small, the detection is performed by the two-electrode method ( (Working electrode and reference electrode that also serves as the counter electrode)
But you can do it too. If necessary, a current amplifier may be used between the electrode 2 and the multipotentiostat 4.

Peroxidase from horseradish and [Os
(Bpy)_TwoCl]^{2 + / 3 +}(Osmium bipyridi
Phosphoric acid containing polyvinylbipyridine solution containing
A buffer solution (pH 7.4) was placed in the cell 6 shown in FIG.
The electric potential of 2 is swept and the enzyme (peroxida derived from horseradish)
And an electron transfer mediator ([Os (bpy)
_TwoCl]^{2 + / 3 +}) Was modified on electrode 2. In addition, high
The molecule, polyvinylbipyridine, is
The enzyme called oxidase, [Os (bpy)_TwoCl]
^{2 + / 3 +}Electron transfer mediator
I am fit. Besides sweeping the potential of electrode 2,
Apply a potential step to electrode 2
It is also effective to control the current flowing through the electrode 2
Is.

Next, using a device similar to the device shown in FIG. 2, the sensing characteristics of hydrogen peroxide of the electrode 2 modified with the enzyme / mediator were examined. The modified solution in the cell 6 was replaced with a measurement solution (pH 7.4, HEPES buffer solution), 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 are 80, 200, 320, 460, respectively.
It shows 580 μmol / l.

When hydrogen peroxide is added to the solution, the electrode 2 is treated with a mediator [Os (bp) by the enzymatic reaction of the modified enzyme, horseradish peroxidase, on the electrode 2.
y) ₂ Cl] ³⁺ ) The reduction current will be detected. That is, hydrogen peroxide is oxidized by horseradish-derived 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 the change in the reduction current at the electrode 2. Is the principle of sensing. In FIG. 3, an increase in reduction current value was observed with respect to hydrogen peroxide. It was confirmed that the responses between the electrodes 2 were independent of each other, and it was also confirmed that the enzyme was uniformly modified only on the electrodes 2 in the microscope 9 and the monitor 12. Also, substances other than hydrogen peroxide can be measured by using different enzymes.

Reference Example 2 In this Reference Example, a flat plate array electrode having the same material and size as those of the electrode 2 shown in FIG. 1 and the apparatus shown in FIG. Here is a modified example.

In this reference example, a flat indium tin oxide thin film was formed on a substrate (glass substrate) 1 and then a fine flat plate electrode array consisting of square electrodes 2 having a side of 20 μm was prepared by lithography and used. The lead portion 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 flat plate array electrode, generally used disc electrodes, flat plate electrodes, and microneedle type electrodes can be used. In addition to indium tin oxide, a metal, a metal oxide, carbon, or the like may be used as the material of the electrode 2. The reference electrode 8 is a silver / silver chloride electrode, and the counter electrode 9
Although a platinum wire was used for the reference electrode 8, 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). In addition, in this reference example, the working electrode (electrode 2) is formed by the three-electrode method.
In the above, there are a plurality of cases, 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 polyvinylbipyridine solution containing horseradish-derived 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 is swept to generate enzymes (horseradish peroxidase and glutamate oxidase) and electron transfer mediators ([Os (bpy) ₂ Cl]).
^{2 + / 3 +} ) was modified on electrode 2.

Next, the modified solution in the cell 6 is measured with a measuring solution (p
H7.4, HEPES buffer solution), the electric 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. The result is shown in FIG. The black arrowheads (black downward triangles) in the figure indicate the addition of glutamic acid, showing final concentrations of 17, 29, 38, and 47 μmol / l, and the white arrowheads (white downward triangles) indicate the final concentration of 27 μM.
3 shows the addition of ascorbic acid.

By adding glutamic acid, the electrode 2 is
Eta [Os (bpy)_TwoCl] ³⁺The reduction current of
Put out. In other words, glutamic acid is converted to glutamate oxida.
Oxidation produces hydrogen peroxide and its peroxidation
Hydrogen was reduced by horseradish peroxidase.
At the same time, the mediator [Os (bpy)_TwoCl]²⁺
Is oxidized. Mediator [Os (bpy)_TwoCl]
³⁺Glutamic acid by measuring the change in reduction current of
The concentration of acid can be calculated. From this result, glutamate
That the reduction current increases with increasing concentration
I understood. For 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 lutamic acid could be detected.
Confirm that the response between each electrode 2 is independent of each other
However, even with the microscope 10 and the monitor 13, the fermentation is performed only on the electrode 2.
It was confirmed that the element was uniformly modified.

Example In this example, an enzyme / mediator is modified on a plurality of electrodes 2 by the method of Reference Example 2 and used for measuring a biological sample.

A flat plate array electrode having the same material and size as those of the electrode 2 shown in FIG. 1 and the apparatus of FIG. 2 were used. After forming a flat plate indium tin oxide thin film on a substrate (glass substrate) 1, a fine flat plate electrode array composed of square electrodes 2 having a side of 20 μm was prepared by lithography and used.
The lead portion 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 flat plate array electrode, generally used disc electrodes, flat plate electrodes, and microneedle type electrodes can be used. In addition to indium tin oxide, a metal, a metal oxide, carbon, or the like may be used as the material of the electrode 2. 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 the present embodiment, the case where there are a plurality of working electrodes (electrodes 2) is shown by the three-electrode method, 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.

A polyvinylbipyridine 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 cell 6 of FIG. , The enzymes horseradish peroxidase and glutamate oxidase by sweeping the potential of the electrode 2, and the electron transfer mediator [Os (bpy) ₂ Cl].
^{2 + / 3 +} were modified on electrode 2.

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

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

A hippocampal slice 15 (400 μm thick) of a 2 day-old Wistar rat was placed on the electrode array consisting of the electrodes 2 and adhered thereto using a nylon mesh 16. Measurement is HEPES buffer solution (pH 7.4)
And the change in current was observed at a potential of the electrode 2 of −100 mV by the multipotentiostat 4. For stimulation to release glutamate, Muscimol was used, and Teflon tube 1 was used by injection pump.
It was injected into the measurement solution from 7.

As a result, it was observed that, with respect to the addition of musimol, the concentration of glutamic acid varied depending on the site even within the same section 15. That is, the electrode 2 according to the present example enables real-time multipoint measurement of glutamic acid, which is a typical neurotransmitter responsible for information transmission between nerve cells in rat hippocampus. This makes it possible to obtain not only the mechanism of memory in the brain but also the knowledge of the action of glutamate in vivo.

Reference Example 3 This Reference Example shows an example in which glucose oxidase which is an enzyme and polyvinyl ferrocene which is a polymer are modified.

A flat plate array electrode having the same material and size as those of the electrode 2 shown in FIG. 1 and the apparatus of FIG. 2 were used. After forming a flat plate indium tin oxide thin film on a substrate (glass substrate) 1, a fine flat plate electrode array composed of square electrodes 2 having a side of 20 μm was prepared by lithography and used.
The lead portion 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 flat plate array electrode, generally used disc electrodes, flat plate electrodes, and microneedle type electrodes can be used. In addition to indium tin oxide, a metal, a metal oxide, carbon, or the like may be used as the material of the electrode 2. 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 reference example, the case where the working electrode (electrode 2) is plural by the three-electrode method is 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.

A HEPES buffer solution (pH 7.4) containing polyvinyl ferrocene and glucose oxidase was placed in the cell 6 of FIG. 2 and the potentials of the plurality of electrodes 2 were swept to obtain polyvinyl ferrocene and glucose on the plurality of electrodes 2. The oxidase was modified. Then, the solution in the cell 6 was replaced with a HEPES buffer solution (pH 7.4), the potential of the electrode 2 was fixed at 500 mV, and the current response value (oxidation current value) of each electrode 2 with respect to the addition of glucose was examined. The result is shown in FIG. Arrowheads (black downward triangles) in the figure indicate addition of glucose, and show 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 polyvinylferrocene is reduced. The observed current is the oxidation current observed to oxidize the reductant ferrocene on each electrode 2. From these results, it was confirmed that each electrode was modified with the polymers polyvinylferrocene and glucose oxidase.

Reference Example 4 This reference example shows an example in which different electrodes 2 in the array were modified with different enzymes.

A flat plate array electrode having the same material and size as the electrode 2 shown in FIG. 1 and the apparatus shown in FIG. 2 were used. One side 2 produced by lithography on a substrate (glass substrate) 1
A microplate electrode array consisting of 0 μm square gold electrodes 2 was used. The lead portion 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 flat plate array electrode, generally used disc electrodes, flat plate electrodes, and microneedle type electrodes can be used. As the 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 reference example, the case where the working electrode (electrode 2) is plural by the three-electrode method is 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.

HEPES buffer solution (pH containing single molecule toluidine blue and glucose oxidase)
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 adjusted to 1.2.
By fixing to V, glucose oxidase was modified on the plurality of electrodes 2 (group A).

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

After that, the solution in the cell 6 was replaced with a HEPES buffer solution (pH 7.4), and the electrode 2 (group A and group B) was replaced.
The potential of 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. The result is shown in FIG. 7. In the figure, black arrowheads (black downward triangles) indicate addition of glucose, final concentrations of 0.1, 0.4, and 0.8 mmol / l, respectively, and white arrowheads (white downward triangles) indicate hydrogen peroxide. Of the final concentration of 0.1, 0.2 and 0.5 mmol / l, respectively.

An increase in oxidation current was observed at the electrode 2 of the group A with respect to the addition of glucose, but almost no change was observed in the current value at the electrode 2 of the group B. On the other hand, when hydrogen peroxide was added to the solution, an increase in 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, the toluidine blue molecule serves as an electron transfer mediator, is reduced by glucose oxidase, and is oxidized on the electrode 2. Therefore, an oxidation current is observed. Further, hydrogen peroxide is oxidized by horseradish-derived peroxidase, and at the same time, the toluidine blue molecule serves as an electron transfer mediator, is reduced by horseradish-derived peroxidase, and is oxidized on the electrode 2. Therefore, an oxidation current is observed.

From these results, the electrodes 2 of the groups A and B are
By changing the potential of the electrode 2, toluidine blue was deposited on the electrode 2 and at the same time, each enzyme could be modified on the electrode 2.

[0058] Electrochemical measuring electrodes modified by the enzyme / mediator in the above you施例while taking advantage of excellent properties possessed by the microelectrodes can effectively utilize the specificity with the enzyme, even further multi-point measurement It's a great way to make it possible. When the enzyme / mediator is modified on the microelectrode, a change in the concentration of the specific substance in an arbitrary microregion can be detected with high sensitivity both when the concentration increases and when the concentration decreases.

When a biological sample or the like is used as in the embodiment, the biological sample is less likely to be adversely affected at the time of measurement, so that long-time measurement or continuous measurement is possible. When measuring by modifying multiple electrodes, by grasping the distribution of specific substances and changes in distribution due to stimulation,
It is possible to elucidate the action and mechanism of the substance in the living body.

Further, when different enzymes / mediators are modified on a plurality of microelectrodes as in Reference Example 4 , multisensing by a simple method of electrodes becomes possible. For example, in flow injection or high performance liquid chromatography, It is possible to electrochemically detect a plurality of substances simultaneously as a detector.

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

As described above, in the above-mentioned 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, and socially recently. It is extremely effective for real-time multi-sensing in environmental fields such as water quality analysis, and for real-time monitoring of the distribution of neurotransmitters in multiple cells and biological tissues.

Although the present invention has been specifically described based on the embodiments, the present invention is not limited to the above embodiments, and it is needless to say that various modifications can be made without departing from the scope of the invention. . For example, in the example, when the polymer is deposited electrochemically, that is, when it is oxidized or reduced, the modifier enzyme / mediator is simultaneously modified on the electrode, but as in Reference Example 4 , It may be modified with a single molecule (a molecule that is not a polymer; a low molecule). Specifically, in addition to toluidine blue, it is possible to incorporate the enzyme / mediator at the same time when a single molecule such as pyrrole or aniline is electrochemically polymerized on the electrode surface. In this case, the single molecule is a so-called binder (crosslinking agent,
It may function as an immobilizing agent or carrier and may also act as a mediator. When a single molecule acts as a mediator, modification of the molecule serving as a mediator is unnecessary.

[0064]

According to the present invention as described above, according to the present invention, examples
For example, it is a typical one responsible for information transmission between nerve cells in rat hippocampus
Real-time multipoint of glutamate, a novel neurotransmitter
Measurement is possible, enabling real-time monitoring of the distribution of neurotransmitters that targets multiple cells or living tissue
Becomes

[Brief description of drawings]

FIG. 1 is a top view of an electrode array modified with a modifier.

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

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

FIG. 4 In Reference Example 2, the enzymes horseradish-derived peroxidase and glutamate oxidase and the electrode array modified with the mediator [Os (bpy) ₂ Cl] ^{2 + / 3 +} were used, and glutamic acid / ascorbic acid was added to the solution. It is a figure which shows the current response of each electrode at the time.

[5] Oite Example, using an enzyme / mediator modified electrode array fabricated using the apparatus of FIG. 2 is a schematic configuration diagram of a device for performing biological sample measurement.

FIG. 6 is a diagram showing a current response of each electrode when glucose is added to a solution using an electrode array modified with polyvinyl ferrocene as an enzyme and glucose oxidase in Reference Example 3 .

[Fig. 7] Fig. 7 shows an electrode array having the electrode modified with glucose oxidase (Group A) and the electrode modified with horseradish-derived peroxidase (Group B) in Reference Example 4 ,
It is a figure which shows the current response of each electrode at the time of adding glucose and hydrogen peroxide in 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 ... grounding, 15 ... hippocampal slice, 16 ... nylon mesh, 17 ... Teflon tube.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI G01N 27/30 353R 27/46 336G (72) Inventor Osamu Niwa 2-3-1, Otemachi, Chiyoda-ku, Tokyo Nippon Telegraph and Telephone Stock In-company (72) Inventor Katsuyoshi Hayashi 2-3-1 Otemachi, Chiyoda-ku, Tokyo Inside Nippon Telegraph and Telephone Corporation (56) Reference JP-A-10-177004 (JP, A) (58) Fields investigated ( Int.Cl. ⁷ , DB name) G01N 27/333 G01N 27/327 G01N 27/416 G01N 27/30

Claims (7)

(57) [Claims] 1. A method for electrochemically measuring a biological tissue , wherein electrodes are arranged in an array on a substrate, and only the electrode portions are selectively made of at least an enzyme or an electron transfer mediator.
Array-like effect modified by a modifier containing one
Using a pole, adhere the above-mentioned working electrode array to a tissue section
And soak it in the measurement solution.
By arranging the electrodes, the current change of each electrode of the working electrode in the array above
Electrochemical measurement direction of the living tissue, characterized by measuring the
Law . 2. The electrochemical measurement of a living tissue according to claim 1, wherein the this <br/> using brain slices as the tissue section
Way . 3. The electrochemical meter body tissue according to claim 1 Symbol mounting, characterized in <br/> using hippocampal slices as the tissue section
Measuring method . 4. A HEPES buffer solution as the measurement solution
The living tissue according to claim 1, which is used.
Electrochemical measurement method . 5. At least glutamic acid as the modified product
Array-shaped that has been modified by a modified product containing oxidase
5. The working electrode according to claim 1 , wherein a working electrode is used.
Electrochemical measurement method for biological tissues . 6. At least peroxida as the modified product.
Array-like action modified by a modified product containing a protease
A living body according to any one of claims 1 to 4, wherein a pole is used.
Electrochemical measurement method of tissue . 7. At least osmium vinyl chloride as the modified product.
Array-shaped modified with pyridine-containing modifiers
5. The working electrode according to claim 1 , wherein a working electrode is used.
Electrochemical measurement method for biological tissues .