JP2001103994A - Glutamic acid sensor and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a glutamic acid sensor capable of measuring the concentration of a very small amount of glutamic acid being a neurotransmitter contained in a sample solution and to provide a method for producing the glutamic acid sensor. SOLUTION: This glutamic acid sensor has an action electrode 5 modified with a membrane containing a glutamate oxidase treated with a glutaminase inhibitor at a part of a flow channel flowing the sample solution and a cell 3 for preelectrolysis at the upstream part of the action electrode 5. The shape of the flow channel is a thin layer shape along the electrode face of the electrode at a part to be brought into contact with the electrode face of the electrode for the preelectrolysis in the cell 3 for preelectrolysis. The electrode face is modified with a material to promote an electrode reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a glutamate sensor, and more particularly to a biosensor for medical or physiological research, or a medical biosensor for continuous quantitative analysis of a small amount of a sample from a living body. Regarding what is valid.

[0002]

2. Description of the Related Art In researches on neuroscience and molecular biology, many attempts have been made to measure a physiologically active substance contained in a minute region in a living body in real time.
Among them, electrochemical analysis methods are (1) suitable for measurement of very small amounts, (2) relatively high sensitivity, and (3) by modifying electrodes with selective membranes or enzymes. It has features such as being able to perform highly selective measurement and (4) being able to manufacture a simple and low-cost sensor.

[0003] In addition, an online sensor is used in combination with a microdialysis method to dialyze low molecular weight components in the brain and blood vessels.
Attention has been paid to a technique for continuously measuring components contained therein. In this method, conventionally, the dialysate was measured using an analytical instrument such as high-performance liquid chromatography, but since the target substance can be measured continuously,
Particularly, there is a feature that a change of a molecule in a living body can be monitored in real time. In addition, since various coexisting molecules exist in the living body, when electrochemical methods are used for sensors to eliminate the effects, the electrode surface is modified with a highly specific enzyme to selectively target the target substance. A general method is to react and detect a reaction product with an electrode. Up to now, online measurement of glucose, lactic acid, glutamic acid and the like has been reported using such a method.

When measuring the concentration of glucose, lactic acid, choline or the like in a living body by an electrochemical method using such an enzymatic reaction and an electrode reaction, are there any coexisting molecules that hinder the enzymatic reaction? Alternatively, since the concentration is lower than the concentration of the target substance, a highly selective enzyme reaction can be realized, and the concentration of the target substance can be accurately measured.

However, when measuring the concentration of a low concentration of a neurotransmitter molecule such as glutamic acid, the selectivity of the enzyme may become a problem. For example, even when the reaction rate ratio between the target molecule and the coexisting molecule of the enzyme is 10: 1, when the concentration of the coexisting molecule in the living body is 10 times or more larger than the target molecule, the signal from the coexisting molecule is more than the signal from the target molecule. Becomes larger. Examples of such coexisting molecules include electrochemically active L-ascorbic acid and uric acid. Since these molecules are easily oxidized at a low potential, they are directly oxidized on an electrode for measuring the concentration of the target molecule (hereinafter, referred to as a working electrode), causing a problem of causing a measurement error.

For example, hydrogen peroxide, which is a reaction product of an oxidase such as glucose, lactic acid, glutamic acid, and choline, is usually easily oxidized on a platinum electrode at a potential of about 0.5 V with respect to a silver / silver chloride electrode. However, since L-ascorbic acid and uric acid are also easily oxidized at the same potential, in a measurement method for determining the amount of the target molecule from the amount of hydrogen peroxide,
The concentration of the target molecule cannot be measured accurately. Particularly, in the case of glutamic acid or the like in which the concentration in the living body is μM (M represents molar concentration: mol / liter), the change in the concentration is masked by the oxidation current of L-ascorbic acid (brain concentration: several hundred μM). Detection is difficult.

Therefore, in order to remove the influence of coexisting molecules, a method of inserting a pre-electrolysis electrode upstream of the working electrode in the flow path of the sample solution to oxidize and remove coexisting molecules, or using L-ascorbic acid or uric acid Is anionic,
There is known a method in which the surface of a working electrode is modified with an anionic polymer film such as a Nafion film and the amount of coexisting molecules diffused to the electrode surface is reduced by electrostatic repulsion.

FIG. 6 shows a conventional example of an electrochemical sensor using an electrode for pre-electrolysis.

In FIG. 6, a sample solution is pushed out of a syringe 2 of a syringe pump 1 and enters a pre-electrolysis cell 3. The role of the pre-electrolysis cell 3 is to oxidize the electrode when a large amount of L-ascorbic acid is contained in the sample so as not to affect the measurement of the concentration of the target molecule. . An example in which a platinum tube is used as a pre-electrolysis electrode of the pre-electrolysis cell 3 has been reported.

After the electrode oxidation reaction, the sample solution is supplied to the flow cell 4
to go into. In the flow cell 4, a working electrode 5, a counter electrode 6, and a reference electrode 7 are arranged. The application of a voltage to each electrode and the measurement of the electrode current are performed by a potentiostat 8, and the result of the measurement is recorded by a recorder 9. The sample solution after the electrode reaction is discharged out of the flow cell 4 as a waste liquid. In the flow cell 4, the gasket 10
Has a role of maintaining a constant distance between the working electrode 5 and the counter electrode 6 and securing a flow path of the sample solution.

FIG. 7 shows a method for reducing the amount of coexisting molecules diffusing to the electrode surface due to electrostatic repulsion. In the method shown in FIG. 7A, the surface of the working electrode is modified with an anionic polymer film to prevent the anionic coexisting molecules from diffusing to the electrode surface by electrostatic repulsion. If the target molecule is also anionic (such as glutamic acid), the method shown in FIG. 7A will also move the target molecule away from the working electrode. As shown in FIG. 7 (b), an enzyme film is provided on the anionic polymer film so that the enzyme reaction of the target molecule takes place there.

[0012]

As described above, in order to accurately measure the concentration of a neurotransmitter such as glutamate having a low concentration in a living body, it is necessary to completely suppress the diffusion of a coexisting substance to the sensor electrode surface. . Conventionally as an electrode for pre-electrolysis,
It has been reported that a platinum tube is inserted between the microdialysis probe and the sensor. However, the electrode reaction speed of coexisting substances such as L-ascorbic acid is slow, and the diameter of the platinum tube is required to flow the solution smoothly. Needs to be several tens μm or more, and it takes time to oxidize all coexisting molecules flowing near the center of the tube.

When the coexisting substance is anionic (such as L-ascorbic acid), the surface of the working electrode is modified with an anionic polymer as shown in FIG. Although the influence of the substance can be reduced, this method has a problem that the response of the working electrode may be reduced or the enzyme reaction product may not be efficiently collected by the electrode.

Further, when the target substance is glutamic acid, a reaction between glutamate oxidase and aspartic acid or glutamine has been reported (Obrenovitch, et al.
l., J. Neurosci. Methods, 60: 1-9 (1995)), and especially in vivo (in the cerebrospinal fluid), the concentration of glutamine is more than 10 times higher than that of glutamic acid. There is a problem that it may hinder the concentration measurement.

An object of the present invention is to solve the above problems,
An object of the present invention is to provide a glutamate sensor capable of measuring the concentration of a small amount of glutamate, which is a neurotransmitter contained in a sample solution, without being affected by coexisting substances, and a method for producing the same.

[0016]

In order to solve the above-mentioned problems, according to the present invention, a flow path through which a sample solution flows is formed in a thin layer along the electrode surface at a portion in contact with the electrode surface of the pre-electrode electrode. By reducing the time required for the coexisting substance to reach the electrode surface by adopting the shape of, and by modifying the electrode surface with a material that promotes an electrode reaction,
The co-existing substance, L-ascorbic acid, is efficiently electrode-oxidized so as not to affect the measurement of glutamic acid concentration.

Further, in the present invention, glutamine oxidase is treated with a glutaminase inhibitor at the time of manufacturing a sensor, so that glutamine, which is a coexisting substance, does not affect the measurement of glutamic acid concentration.

[0018]

Embodiments of the present invention will be described below in detail with reference to the drawings. The present invention is not limited only to the following examples.

Embodiment 1 FIG. 1 shows the configuration of a glutamate sensor, an enlarged view of a pre-electrolysis cell, and an enlarged view of a working electrode in this embodiment. The configuration of the present sensor is the same as the configuration of the conventional example shown in FIG. 6 except for the configuration of the pre-electrolysis cell and the configuration of the working electrode.

In FIG. 1, a sample solution is pushed out of a syringe 2 of a syringe pump 1 and enters a pre-electrolysis cell 3. The role of the pre-electrolysis cell 3 is to oxidize the electrode when a large amount of L-ascorbic acid is contained in the sample so as not to affect the measurement of the concentration of the target molecule. . The sample solution after the electrode oxidation reaction is flow cell 4
to go into. In the flow cell 4, a working electrode 5, a counter electrode 6, and a reference electrode 7 are arranged. The application of a voltage to each electrode and the measurement of the electrode current are performed by a potentiostat 8, and the result of the measurement is recorded by a recorder 9. The sample solution after the electrode reaction is discharged out of the flow cell 4 as a waste liquid. In the flow cell 4, the gasket 10
Has a role of maintaining a constant distance between the working electrode 5 and the counter electrode 6 and securing a flow path of the sample solution.

In this embodiment, in order to efficiently remove L-ascorbic acid which is an interfering substance when measuring glutamic acid in a living body, as shown in FIG. You are using As the electrode for pre-electrolysis in this thin-layer cell, as shown in the enlarged view of the cell for pre-electrolysis in FIG. 1, a gold electrode modified with platinum black was used.
As the modification means, an electrodeposition reaction of platinum black was used. Since platinum black has a large surface area when viewed microscopically, it is effective for remarkably accelerating the electrode reaction and efficiently removing L-ascorbic acid.

FIG. 2 shows a main part of a method for manufacturing a thin-layer cell. In the figure, the process proceeds from a to j. First, silicon was sputtered on a glass wafer (a) to a thickness of 100 nm by a magnetron sputtering apparatus (manufactured by Nippon Seed Co., Ltd.), and then a positive photoresist was applied to a thickness of 10 μm (c). The electrode pattern was exposed to this using a mask aligner manufactured by Canon. The exposure time is
15 seconds. After the exposure, the wafer was developed with an alkaline developer for 30 seconds, and then washed with water and dried (d). further,
The silicon at the electrode portion was removed by a mixed plasma of chlorine and oxygen (e). Thereafter, the wafer was subjected to wet etching in a hydrofluoric acid solution for a depth of 6 μm for 15 minutes. The remaining resist and silicon were removed by an oxygen plasma and a mixed plasma of oxygen and chlorine, respectively, using a reactive ion etching apparatus (DEM-451, manufactured by Anelva) (f). Thereafter, a positive photoresist is applied (g), and the electrode portion is again exposed and developed, washed with water,
Dried (h). This wafer was placed in a magnetron sputtering apparatus (same as above), and titanium (100 nm) and gold (400
nm) in this order (i). Thereafter, unnecessary portions were removed (lift-off) while applying ultrasonic waves in a methyl ethyl ketone solution to prepare an electrode for pre-electrolysis (j).

Using a dicing saw on this wafer, a width of 400 μm is set so that 1 mm from the end becomes a depth of 400 μm.
It was cut to form a groove for capillary connection. Platinum black was formed on the gold electrode thus manufactured. Platinum black consists of 2 g of chloroplatinic acid hexahydrate (Kanto Chemical) and 0.07 g of lead acetate
(Manufactured by Kanto Chemical Co., Ltd.) in 100 g of pure water was used as an electrolytic solution, and a potential of -1.8 V with respect to a silver reference electrode was applied to a gold electrode.

[0024] Two wafers prepared as described above were bonded together so that the electrode surfaces faced each other to obtain a thin layer cell for pre-electrolysis. Here, since the flow path of the sample solution is a thin layer between two electrodes with an interval of 10 μm,
L-ascorbic acid in the sample solution reaches the electrode surface in a very short time. Moreover, since the electrode modified with platinum black efficiently oxidizes L-ascorbic acid, L-ascorbic acid in the sample solution is oxidized and consumed with high efficiency for the above two reasons.

On the other hand, as a working electrode for detecting glutamic acid, a platinum electrode (diameter 3 mm, bioanalytical system (BA
S), glutamate oxidase was dissolved in a mixed solution of bovine serum albumin (2%) and glutaraldehyde (0.2%), 2 μl of the solution was applied, and then dried. Glutamate in the sample solution is oxidized by glutamate oxidase, and hydrogen peroxide generated quantitatively at that time is electrode-oxidized, and the concentration of glutamic acid is determined by measuring an oxidation current flowing at that time.

After the pre-electrolysis cell 3 and the flow cell 4 prepared as described above are connected by a capillary, in order to enhance the selectivity of glutamate oxidase, 6-diazo-5-oxo-L-norleucine (6- diazo-5-oxo-L-norleucine,
(Hereinafter abbreviated as DON) to treat glutamate oxidase. 5 mM (M represents molarity: mol / liter) of DON was injected into the sensor for 5 minutes, and then 10
The inside of the sensor was washed by injecting a phosphate buffer solution for minutes.
Finally, the working electrode 5 of the sensor and the electrode for pre-electrolysis are each 4
Channel potentiostat (manufactured by Physiotech) 8
And the counter electrode (stainless steel block) 6 and the silver / silver chloride reference electrode 7 were connected to the counter electrode terminal and the reference electrode terminal, respectively.
A syringe was connected to the sensor manufactured as described above, and a sample was injected at a flow rate of 2 μl / min using a syringe pump.
The applied potential was set to 500 mV with respect to the reference electrode for both the pre-electrolysis electrode and the glutamic acid detection working electrode.

First, glutamic acid at a concentration of 10 μM was added.
When a mixed solution with 100 μM L-ascorbic acid was injected, a response current of 54 nA was obtained. Next, when a potential was applied only to the working electrode for detection without applying a potential to the electrode for pre-electrolysis, a response current of 380 nA was obtained. On the other hand, when the same measurement was performed using a flat gold electrode as the electrode for pre-electrolysis, a response current of 85 nA was obtained. As described above, when the platinum black electrode is used as the pre-electrode, the response current is smaller than when no potential is applied to the pre-electrode or when a flat gold electrode is used as the pre-electrode. The reason is that the surface area of the platinum black electrode is large,
This is because L-ascorbic acid was removed with high efficiency.
Using the sensor according to the present invention, when a solution in which L-ascorbic acid was added at various concentrations to a glutamic acid solution having a concentration of 10 μM was measured, the response current was almost constant until the L-ascorbic acid concentration was 300 μM. (55 nA).

Next, glutamic acid 10 μM and glutamine
A 100 μM mixed solution was injected, and glutamic acid was measured. On the other hand, for comparison, the glutamate oxidase DO
A similar experiment was performed for a sensor that did not perform the process using N.

As a result, almost the same response current was obtained as compared with the case where only glutamic acid was measured and the case where a mixed solution of glutamine was measured. That is, the reason that only glutamic acid was selectively detected was that the treatment of glutamic acid oxidase with DON suppressed the reaction of glutamine in the enzyme. When the glutamate oxidase treated with DON before the production of the sensor was immobilized on the electrode, the same effect as the above-mentioned sensor was observed. On the other hand, glutamate oxidase is DO
When a mixed solution of glutamic acid and glutamine was measured without N treatment, the response current increased to 105 nA due to the reaction between glutamic acid oxidase and glutamine.

As described above, by treating glutamic acid oxidase with DON, the effect of glutamine can be suppressed, and only glutamic acid can be detected with high selectivity.

The sensor according to the present embodiment can selectively detect only glutamic acid without being affected by L-ascorbic acid and glutamine which are often present in a biological sample. Therefore, a very small amount of a neurotransmitter in a biological sample such as glutamic acid can be detected with high sensitivity and selectivity, and application to neuroscience and medical research can be expected.

(Example 2) The method of manufacturing a thin layer cell for pre-electrolysis in this example is the same as that of Example 1 except for the method of manufacturing an electrode for pre-electrolysis and a working electrode. In the present embodiment, the efficiency of the electrode reaction is increased in the pre-electrode and the glutamic acid is selectively formed in the working electrode by modifying the pre-electrode and the working electrode with a polymer film having a redox property. Only can be detected. The polymer film having redox properties used in this example is an osmium-polyvinylpyridine complex film, and this film is
It has the property of causing electron transfer from L-ascorbic acid in the oxidized state, and enhances the efficiency of the electrode reaction in the pre-electrolysis electrode. In addition, horseradish peroxidase (HRP), which reduces hydrogen peroxide, is included in this membrane, and the working electrode is modified with the membrane to suppress the reaction of L-ascorbic acid on the working electrode while reducing the glutamic acid concentration. Measurement was made possible.

FIG. 3 shows the structure of the electrode for pre-electrolysis and the working electrode for detection in this embodiment. The electrode for pre-electrolysis is obtained by modifying a gold electrode with an osmium-polyvinylpyridine complex film (in FIG. 3, indicated as an Os-gel film). Here, the flow path of the sample solution is a thin layer sandwiched between two electrodes with a spacing of 10 μm, so that L-ascorbic acid in the sample solution is extremely short-lived. Reach the plane. In addition, osmium in the Os-gel film is oxidized by the gold electrode, and the film in such an oxidized state causes electron transfer from L-ascorbic acid to osmium, and efficiently oxidizes L-ascorbic acid.
For the above two reasons, L-ascorbic acid in the sample solution is oxidized and consumed with high efficiency.

On the other hand, on the working electrode, an osmium-polyvinylpyridine complex film containing horseradish peroxidase (HRP) for reducing hydrogen peroxide in the lower layer (indicated as Os-gel-HRP film in FIG. 3) was modified. Thereafter, glutamate oxidase was immobilized on the upper layer. In this example, glutamate oxidase was treated with DON in the same manner as in Example 1. Glutamate in the sample solution is oxidized by glutamate oxidase, and the hydrogen peroxide generated quantitatively at that time is electrode-reduced through the Os-gel-HRP membrane, and the concentration of glutamic acid is measured by measuring the reduction current flowing at that time. Is required.

At the time of measurement, the potentials applied to the pre-electrode electrode and the working electrode were set to 400 with respect to the silver / silver chloride reference electrode, respectively.
mV and 0 mV. By using a redox osmium-polyvinylpyridine complex film on the working electrode, the potential applied to the working electrode can be set low, so that the interfering substance L-ascorbic acid (the concentration of The reaction on the working electrode (which is significantly reduced by the above) can also be suppressed, and as a result, the effect of L-ascorbic acid can be removed with extremely high efficiency.

A dialysate probe (microdialysis probe) and a syringe were connected to the sensor, and a sample was introduced into the sensor by a syringe pump. The sample introduction speed is
2 μl / min.

When the same measurement as in Example 1 was performed using this sensor, the response current showed a constant value even when the L-ascorbic acid concentration in the sample solution was increased to 300 μM. In other words, the pre-electrode electrode and the working electrode are modified with a polymer having a redox property, whereby the potential applied to the working electrode can be kept low. Therefore, the effect of L-ascorbic acid is suppressed, and high sensitivity is achieved. Glutamic acid was detected with good selectivity. Similarly, even when the glutamine concentration in the sample was increased, the value of the response current was almost constant, and the effect of glutamine as well as L-ascorbic acid could be similarly suppressed.

In vivo measurement was performed using the sensor in this embodiment. FIG. 4 shows the measurement system. A microdialysis probe was inserted into the brain of the rat, and glutamic acid was measured. The microdialysis probe was connected to a syringe, and a sample containing glutamic acid released by administering a 160 mM potassium chloride solution into the brain was injected into the sensor while pushing the syringe at a speed of 2 μm / min.

At this time, it was revealed that a response current of 180 nA was obtained in real time, and that it could be used sufficiently for measurement of a trace amount of neurotransmitter. As a comparison, when the same measurement was performed without applying a potential to the electrode for pre-electrolysis, only a response current of 15 nA was obtained. The reason why a small value was obtained as compared with the case where pre-electrolysis was performed was that L-ascorbic acid reduced osmium in the osmium-polyvinylpyridine complex film containing HRP, and the electrode was reduced accordingly. This is because the amount of the electrode reduction reaction of osmium in the above was reduced.

As described above, by using the glutamate sensor according to the present invention, the concentration of glutamate released in the brain can be detected with high sensitivity and selectivity, which is useful for elucidation of brain functions such as learning and memory. it can. In this example, L-ascorbic acid at a concentration of several hundred μM in a biological sample and glutamic acid several times more than that of glutamine whose concentration in the brain could be detected with high reproducibility without being affected by the pre-electrolysis electrode This is because L-ascorbic acid was removed and the reaction of L-ascorbic acid on the working electrode was suppressed.

(Embodiment 3) In this embodiment, a sensor in which electrodes are modified using a conductive polymer film will be described. The conductive polymer film used was a polypyrrole film. This film is also a polymer film having a redox property, and in the oxidized state (that is, a state maintained at a positive potential), electrons are transferred from L-ascorbic acid to the film, thereby efficiently oxidizing L-ascorbic acid.

A polypyrrole film was formed on the gold electrode manufactured as in Examples 1 and 2. The polypyrrole film was formed by applying a potential of 650 mV to a silver reference electrode from a solution containing 0.2 M of pyrrole and 0.2 M of sodium chloride using an electrolytic polymerization method. Two electrode substrates thus modified with a polypyrrole film were bonded to form a thin layer cell for pre-electrolysis.

On the other hand, glassy carbon is used as a working electrode for detecting glutamic acid, and a polypyrrole film is formed on this electrode by electrolytic polymerization from a solution containing pyrrole, horseradish peroxidase (HRP), and sodium chloride as a lower layer. Glutamate oxidase was immobilized on the upper layer. The method for producing the polypyrrole film is as described above. Glutamic acid in the sample solution is oxidized by glutamate oxidase, and hydrogen peroxide generated quantitatively at that time is electrode-reduced through a polypyrrole film, and the concentration of glutamic acid is determined by measuring a reduction current flowing at that time.

After the preparation of the sensor, the glutamate oxidase was treated with DON in the same manner as in Example 1.

The sensor prepared as described above was connected to a potentiostat, and a potential of 500 mV and -300 mV with respect to a silver / silver chloride reference electrode was applied to each of the pre-electrode and the working electrode to measure glutamic acid. Was done. Glutamic acid 10 μM and various concentrations of L-ascorbic acid and glutamine mixed solutions were measured.
There was not much change around 50 nA, and almost the same value as in Example 1 was obtained. Therefore, it was shown that even when another mediator was used, it was sufficiently effective as an electrode for pre-electrolysis.

(Embodiment 4) FIG. 5 shows the structure of an integrated sensor according to the present invention. In the figure, reference numeral 1 denotes a syringe pump, 2 denotes a syringe, 8 denotes a potentiostat, 9 denotes a recorder, 11 denotes an osmium-polyvinylpyridine complex film containing HRP in a lower layer, and a glutamate oxidase-containing film in an upper layer. A working electrode, 12 is a reference electrode, 13 is a counter electrode, 14 is an electrode for pre-electrolysis, 15 is a glass capillary for sampling, 16 is a glass substrate on which electrodes are formed, and 17 is a thin-layer channel 18 having a rectangular cross section. Glass substrate. As the figure shows, all electrodes are integrated inside one laminar channel 18.

In FIG. 5, a sample continuously taken from the capillary 15 is sucked into the syringe 2 through the thin-layer cell. L-ascorbic acid in the sample solution is oxidized by the electrochemical reaction on the electrode for pre-electrolysis 14 and is no longer an interfering substance, and glutamic acid is oxidized on the working electrode 11 by glutamic acid oxidase and is quantitatively generated at that time. Hydrogen peroxide is electrode-reduced through an osmium-polyvinylpyridine complex film containing HRP, and the concentration of glutamic acid is determined by measuring the reduction current flowing at that time.

The sensor is manufactured by laminating a glass substrate 17 having a thin-layer flow path 18 having a rectangular cross section and a glass substrate 16 having various thin-film electrodes.

The main parts of the manufacturing process of the sensor are described below.

A glass substrate having a thin layer flow path having a rectangular cross section was manufactured as follows. That is, the thickness of polyimide (made by Toray) on Pyrex glass wafer (3 inches)
Spin coating was performed so as to be 20 μm. Next, a photoresist for silicon-based dry etching (manufactured by NTT-AT) is applied with a spin coater (manufactured by Mikasa) at a rate of 2000 / min.
Spin coating was performed by spinning. Then, a photomask on which a flow path pattern is drawn is superimposed on the wafer, and a mask aligner (PLA) is formed.
-501, manufactured by Canon Inc.). After exposure, development was performed in an alkaline developer, washed with water and dried to form a resist pattern. Since parts other than the flow path of the wafer after development are covered with a silicon-based resist pattern, the substrate with the resist is put into a reactive ion etching apparatus (DEM-451, manufactured by Anelva), and the silicon-based resist pattern is used as a mask. The portion of the polyimide film not covered with the resist was removed by oxygen plasma. Etching conditions are pressure 5Pa, power
At 100 W and an oxygen flow rate of 100 SCCM, the time was 110 minutes.
After forming the polyimide pattern, the substrate is put into another reactive ion etching apparatus (DEM-451, manufactured by Anelva),
The glass was etched with C ₂ F ₆ gas plasma using polyimide as a mask. Conditions are pressure 2Pa, power 5
00W, flow rate 100SCCM, time 120 minutes, depth 22
μm. After forming the glass flow path, the remaining polyimide film was removed by oxygen plasma using a reactive ion etching apparatus to obtain a glass flow path. Then, use a dicing saw (manufactured by Disco) about 7 mm from both ends of the flow path so that 1 mm from the end becomes 400 μm deep.
It was cut by 00 μm to form a groove for capillary connection.

On the other hand, a substrate having a thin film electrode was manufactured as follows. That is, first, a 3-inch Pyrex wafer is used, and a carbon thin film (film thickness: 100
nm). In the CVD method, a method in which a quartz substrate is heated to 1000 ° C. in a glass tube, phthalocyanine is used as a starting material, sublimated at 400 ° C., and thermally decomposed on a wafer is used. A silicon-based dry etching (RIE) photoresist (NTT) is formed on a wafer having a carbon film formed thereon.
-AT) by spinner (Mikasa) 4000
Spin coating was performed by spinning. Thereafter, a photomask on which the electrode pattern is drawn is superimposed on the wafer, and a mask aligner (PLA) is formed.
-501, manufactured by Canon Inc.) to expose a pattern of four electrodes. After exposure, development was performed in an alkaline developer, washed with water and dried to form a resist pattern. Since only the electrode pattern is covered with the resist pattern on the wafer after development, the substrate with the resist is put into a reactive ion etching apparatus (DEM-451, manufactured by Anelva), and oxygen plasma is applied using the resist pattern as a mask. The portion of the carbon film not covered with the resist was removed. Etching conditions are: power 70 W, pressure 2 Pa, oxygen flow rate 100 SC
CM. Thereafter, the resist was stripped in acetone to obtain an electrode pattern. The electrodes were used as a pre-electrolysis electrode, a working electrode, a reference electrode, and a counter electrode from the side to which the sampling capillary was connected. As in Example 2, an osmium-polyvinylpyridine complex film was applied to the electrode for pre-electrolysis, an osmium-polyvinylpyridine complex film containing HRP was applied to the lower layer of the working electrode, and a glutamate oxidase-containing polymer was applied to the upper layer. A film was formed. Further, silver was plated as a reference substance on the reference electrode substrate.

After that, the capillary 15 and the syringe-side capillary, whose tips are extended by heat, are connected to the substrate on which the thin-layer flow path having the rectangular cross section is formed, and the substrate on which the thin-film electrode is formed are both connected. After the channels were pressed so as to face each other and aligned, a photocurable adhesive was infiltrated from around. After the adhesive was infiltrated, light was irradiated using a high-pressure mercury lamp to bond the substrates. After this,
Glutamate oxidase was treated with DON. The electrode 14 for pre-electrolysis of the sensor, the working electrode 11 for detecting glutamic acid, and the reference and counter electrodes (12 and 13) were connected to terminals of a potentiostat LC4C (manufactured by BAS).

The measurement was carried out by using the syringe pump 1 to suck the solution from the sampling capillary 15 while applying 400 mV and -50 mV to the pre-electrolysis electrode and the working electrode, respectively.
Was applied. Aspirate the solution at a flow rate of 1 μl / min and aspirate a 1 μM glutamic acid solution as a sample.
A reduction current of 2.4 nA was observed. On the other hand, 100 μM L-
When only ascorbic acid was measured, no response current was observed. From this, it became clear that the sensor according to the present invention can detect only glutamic acid without being affected by L-ascorbic acid at all. In addition, the responsiveness could be further improved by increasing the flow rate.

In-vivo measurement was performed in the same manner as in Example 2 using the sensor according to this example. At this time, a microdialysis probe was used for sampling. When 160 mM potassium chloride was administered, a solution containing the released glutamic acid was injected into the sensor at a rate of 1 μl / min, and the same measurement was performed. The obtained response current was 85 nA.
Met. Thus, the response was further improved, and the release of glutamate could be tracked in real time.
This is because the electrode for pre-electrolysis and other electrodes, particularly the working electrode for detection, are provided and integrated in the same thin layer portion of the sample solution flow path, so that the electrode for pre-electrolysis and the working electrode for detection are interposed. This is because the ineffective volume of the intervening capillary was reduced.

[0055]

As described above, in the glutamic acid sensor for measuring the concentration in a living body according to the present invention, the electrode for pre-electrolysis is modified with platinum black to increase the microscopic electrode area to increase the electrode reaction speed. Alternatively, a method is used in which the electrode for pre-electrolysis is modified with a redox polymer film, and a coexisting substance such as L-ascorbic acid is oxidized through redox of the polymer. By using such an electrode for pre-electrolysis, almost 100% of coexisting substances such as L-ascorbic acid and uric acid contained in the dialysate can be removed online, and glutamate, a trace amount of neurotransmitter contained in the dialysis, can be removed. Can be measured without the influence of coexisting substances.

On the other hand, for the working electrode for measuring glutamate concentration, the electrode is modified only with a glutamate oxidase-containing membrane or with a composite membrane of a peroxidase-containing redox polymer and a glutamate oxidase-containing membrane. Electrode oxidation of hydrogen peroxide generated by enzymatic reaction,
Alternatively, the electrode is reduced via a peroxidase-containing redox polymer, and the concentration of glutamic acid is determined by measuring the oxidation current or reduction current flowing at that time. In either case, before or after modification of glutamate oxidase, the glutamate oxidase is contacted with a solution containing a glutaminase inhibitor, so that the concentration of glutamate is one such as in cerebrospinal fluid.
The enzyme reaction of glutamine which is at least 0 times higher can be suppressed, and only glutamic acid can be selectively detected.

As described above, the use of the glutamate sensor according to the present invention makes it possible to measure the concentration of glutamate in the brain with high sensitivity and in real time while suppressing the influence of interfering substances. And significant application effects to medical research.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of a sensor for measuring glutamic acid, an enlarged view of a cell for pre-electrolysis, and an enlarged view of a working electrode in Example 1.

FIG. 2 is a diagram showing a method for producing a thin-layer cell having a pre-electrolysis electrode according to the present invention.

FIG. 3 is a view illustrating an electrode for pre-electrolysis and a working electrode for detecting glutamic acid according to the present invention.

FIG. 4 is a diagram illustrating in vivo measurement using the glutamate sensor according to the present invention.

FIG. 5 is a diagram illustrating an integrated thin-layer cell for measuring glutamic acid concentration according to the present invention.

FIG. 6 is a diagram showing a conventional example of an electrochemical sensor using a pre-electrolysis electrode.

FIG. 7 is a diagram illustrating a method for reducing the amount of coexisting molecules diffusing to the electrode surface due to electrostatic repulsion.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Syringe pump, 2 ... Syringe, 3 ... Pre-electrolysis cell, 4 ... Flow cell, 5 ... Working electrode, 6 ... Counter electrode, 7
... reference electrode, 8 ... potentiostat, 9 ... recorder,
10 gasket, 11 working electrode, 12 reference electrode,
13: Counter electrode, 14: Electrode for pre-electrolysis, 15: Glass capillary for sampling, 16: Glass substrate on which electrodes are formed, 17: Glass substrate on which thin-layer flow path is formed, 1
8 ... Thin layer flow path.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tsutomu Horiuchi 2-3-1, Otemachi, Chiyoda-ku, Tokyo Within Nippon Telegraph and Telephone Corporation (72) Keiichi Torimitsu 2-chome, Otemachi, Chiyoda-ku, Tokyo No. 1 Nippon Telegraph and Telephone Corporation (72) Inventor Ryoji Kurita 2-1-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo NTT Advanced Technology Co., Ltd. F-term (reference) 4B063 QA01 QA05 QA19 QQ02 QQ80 QR03 QR10 QR50 QR84 QR90 QS20 QS36 QS39 QX04

Claims (8)

[Claims] A working electrode modified with a membrane containing glutamic acid oxidase is provided in a part of a flow path through which a sample solution flows, and a pre-electrolysis electrode is provided in a part of the flow path upstream of a position of the working electrode. A glutamic acid sensor having, wherein the shape of the flow path is a thin layer along the electrode surface at a portion in contact with the electrode surface of the pre-electrode, and the electrode surface is modified with a material that promotes an electrode reaction. A glutamate sensor. 2. The glutamic acid sensor according to claim 1, wherein the material for promoting the electrode reaction is platinum black. 3. The glutamic acid sensor according to claim 1, wherein the material that promotes the electrode reaction is a film that causes electron transfer from L-ascorbic acid in an oxidized state. 4. The glutamic acid sensor according to claim 3, wherein the film that causes electron transfer from L-ascorbic acid in the oxidized state is an osmium-polyvinylpyridine complex film. 5. The glutamic acid sensor according to claim 3, wherein the film that causes electron transfer from L-ascorbic acid in the oxidized state is a polypyrrole film. 6. A flow path through which a sample solution flows, comprising a working electrode modified with a membrane containing glutamic acid oxidase, and a pre-electrode electrode provided upstream of the position of the working electrode in the flow path. A glutamate sensor comprising: a glutamate sensor, wherein the glutamate oxidase is treated with a glutaminase inhibitor. 7. The glutamic acid sensor according to claim 1, wherein the pre-electrolysis electrode and the working electrode are provided in the same thin layer portion of the flow channel. 8. A flow path through which a sample solution flows, a working electrode modified with a membrane containing glutamic acid oxidase, and a pre-electrolysis electrode in a part of the flow path upstream of the position of the working electrode. In the method for producing a glutamate sensor having, before or after the step of modifying the working electrode with the membrane containing the glutamate oxidase, a step of treating the glutamate oxidase with a glutaminase inhibitor aqueous solution, A method for manufacturing a glutamate sensor.