JPH04240558A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To obtain a large response current without being affected by dissolved oxygen or electrochemical obstructing matter by immobilizing enzyme and an electron mediator within a conductive film and/or on the surface thereof. CONSTITUTION:As a means for immobilizing an electron mediator, a method preliminarily dispersing the electron mediator in an insulating polymer solution in a stage applying said solution to a substrate to imobilize the same, a method applying an electron mediator-containing polymer solution to a conductive film containing an org. charge transfer complex and a method simultaneously immobilizing both of the electron mediator and enzyme using a polymer solution containing both of them can be designated. Further, by growing the org. charge transfer complex within the polymer layer by a means immersing the polymer layer in an electron acceptor solution, the conductivity of the whole of the film is enhanced and a large response current is obtained.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme electrode, and is particularly suitable as an enzyme sensor for measuring the concentration of minute amounts of biological substrates contained in body fluid components such as blood and urine.

0002

BACKGROUND OF THE INVENTION Analytical methods that utilize the excellent substrate specificity of enzymes are attracting attention in fields such as clinical analytical chemistry, food manufacturing, and environmental chemistry. In particular, in the field of clinical analytical chemistry, enzyme electrodes that can selectively detect glucose, urea, uric acid, etc. have been known.

[0003] These enzyme electrodes generally consist of an electrode and an enzyme-immobilized membrane, and the concentration of the substrate on which the enzyme specifically acts can be determined by reading the substance change caused by the enzyme reaction as the amount of change in electrical signal using the electrode. It is used to measure. Specifically, for example, there is a method that attempts to measure the biological substrate concentration by monitoring electrode-active substances such as hydrogen peroxide and oxygen produced or consumed according to the following formula using an electrode.

[0004] Taking the determination of glucose as an example, glucose oxidase is generally used as the enzyme, and the glucose concentration is measured by electrically monitoring the generated hydrogen peroxide. Here, the method for monitoring hydrogen peroxide is as follows:
This is carried out by measuring the oxidation current obtained by oxidizing under a constant voltage using a platinum electrode, a carbon electrode, or the like.

However, the enzyme electrode based on this principle has the following problems.

(2) As is clear from the above formula, a stoichiometric amount of oxygen is required for the substrate to react. However, taking a glucose sensor as an example, the blood glucose concentration of a diabetic patient is over 15mM, and even for a healthy person it is about 7mM, whereas the amount of dissolved oxygen is 1mM even in water and less than that in body fluids. It is. Therefore, when measuring the blood glucose concentration of diabetic patients, linearity is not possible in the measured concentration range.

[0007] For this reason, measures have been taken to dilute the sample blood or to supply oxygen by some method.

■ When monitoring hydrogen peroxide electrically, if there are substances in the sample solution that are oxidized at the same potential as hydrogen peroxide, such as reducing substances such as ascorbic acid, these may interfere with the measurement current. The oxidation current of the substance is added, causing measurement errors. Therefore, in order to eliminate these errors, we correct the problem by using an electrode that does not have an immobilized enzyme as a compensation electrode, or allow oxygen, hydrogen peroxide molecules, and the measurement substrate to pass through, but do not allow electrode-active buffer substances such as ascorbic acid to pass through. It was necessary to install a selectively permeable membrane.

[0009] As described above, sensors based on the principle of measuring the concentration of substances produced or consumed in enzymatic reactions have problems such as the influence of dissolved oxygen and the influence of interfering substances. Furthermore, since it is necessary to attach the enzyme-immobilized membrane to the oxygen and hydrogen peroxide electrodes, there is a limit to miniaturization.

On the other hand, in order to solve these problems, enzyme electrodes using conductive polymers and enzyme electrodes using electron mediators have been proposed. The former is used during electrolytic polymerization of conductive polymers such as polypyrrole and polyaniline.
A conductive enzyme-immobilized membrane can be created by allowing the enzyme to coexist in a monomer solution and capturing the enzyme in the polymer membrane during polymerization, or by providing the enzyme-immobilized membrane on a pre-polymerized conductive polymer membrane using a known method. It's something you get. In this enzyme electrode, for example, taking glucose as an example, electron transfer of the enzyme is performed by transferring electrons generated by the reaction via the π-electron conjugated system of the conductive polymer. With this method, glucose concentration can be measured without being affected by dissolved oxygen.

[0011] Enzyme electrodes using electron mediators confine electron mediators such as ferrocenes, benzoquinone, ferricyanide ions, N-methylphenazinium, etc. in carbon paste and immobilize enzymes on the surface of the carbon paste electrode. , coated with a suitable polymer film.

[0012] This glucose sensor measures concentration using the following mechanism. That is, when glucose is oxidized, the enzyme becomes a reduced form, and by electron transfer to a mediator, which is an electron acceptor, the enzyme returns to an oxidized form. Glucol concentration is measured by the current generated when this reduced mediator returns to its oxidized form through an electrode reaction.

Therefore, in addition to the advantage of not being affected by the amount of dissolved oxygen, the electrode potential at this time is about the same as the potential used for monitoring hydrogen peroxide (Ag/AgCl ratio of about 0.7
V), which is significantly smaller than that of Ag/AgCl (approximately 0.1 to 0.2 V compared to Ag/AgCl), oxidation of interfering substances is less likely to occur, and as a result, it has the advantage of being able to measure with high precision.

However, enzyme electrodes that use conductive polymers to directly detect electron transfer accompanying enzyme reactions have problems such as low responsiveness and long response times. Furthermore, when using methods such as capturing enzymes in a polymeric membrane during electropolymerization, it is difficult to control the amount of immobilized enzyme, and when used as an enzyme electrode, the substrate Decrease in responsiveness is unavoidable.

[0015] In addition, the conventional enzyme electrodes using electron mediators have low conductivity and are insufficient in terms of response and response time. In addition, since the electron mediators are dispersed in carbon paste, This includes problems such as a decrease in responsiveness over time due to elution and desorption.

[0016] Therefore, in order to solve the drawbacks of these conventional enzyme electrodes, the present inventors first provided a conductive film containing organic charge transfer complex crystals on the surface of a conductive substrate, and We have proposed an enzyme electrode in which an enzyme is immobilized inside and/or on its surface. This enzyme electrode uses a method that directly detects the electron transfer associated with enzyme reactions, so it has the advantage of not being affected by dissolved oxygen or interfering substances, and has excellent stability over time and high performance over long periods of time. It has the advantage of being able to provide accurate responses. However, further improvement is desired in terms of responsiveness, that is, the magnitude of the response current value.

[0017]

SUMMARY OF THE INVENTION The present invention comprises an electrically conductive substrate and an electrically conductive film containing an organic charge transfer complex provided on the surface of the electrically conductive film. The purpose of the present invention is to obtain an enzyme electrode in which an enzyme is immobilized on the surface of the enzyme electrode, which has further improved responsiveness. That is, the object is to provide an enzyme electrode that is not affected by the amount of dissolved oxygen or electrochemical interfering substances, has excellent stability over time, and can provide a large response current.

[0018]

[Means for Solving the Problems] The present inventors have further developed an enzyme electrode produced by utilizing the fact that a conductive material containing an organic charge transfer complex has excellent electrical conductivity and acts as a stable electrode material. have discovered that by immobilizing an electron mediator on an electrode material, electron transfer accompanying an enzyme reaction can be carried out more efficiently and responsiveness can be improved, and the present invention has been completed.

The present invention has an electrically conductive substrate and an electrically conductive film containing an organic charge transfer complex crystal provided on the surface of the electrically conductive film, and enzymes and electron mediators are present in the electrically conductive film and/or on the surface thereof. The subject matter is an enzyme electrode characterized by having immobilized thereon.

[0020]

[Operation] The electrode used in the enzyme electrode of the present invention is composed of a conductive substrate and a conductive coating containing organic charge transfer complex crystals provided on the surface of the substrate.

[0021] Examples of the conductive substrate include metals such as copper, silver, platinum, and gold, carbon electrodes, substrates on the surface of which a conductive layer made of these conductive materials is provided by means such as vapor deposition, or conductive materials such as these. A substrate made from a paste containing powder of a synthetic material, etc. can be used.

The conductive film containing organic charge transfer complex crystals is a film in which organic charge transfer complex crystals are contained in an insulating polymer film so that the film becomes electrically conductive. For example, a film in which organic charge transfer complex crystals are grown to penetrate an insulating polymer film in the thickness direction exhibits conductivity and is currently preferred.

[0023] The organic charge transfer complex (hereinafter referred to as organic CT complex) is a compound formed from an organic electron acceptor and an electron donor due to a charge transfer reaction between the two.

The organic electron acceptor used to form this organic CT complex is not particularly limited, but compounds having a cyanomethylene functional group are preferred, and compounds having a dicyanomethylene functional group and a quinone or naphthoquinone skeleton are particularly preferred. It is. Among these, especially 7, 7'
,8,8'-tetracyanoquinodimethane (TCNQ
) has a strong CT complex forming ability and the obtained organic CT complex has high electrical conductivity, so it is advantageous in terms of response time and responsiveness. It is also suitable industrially because it is relatively easy to obtain.

[0025] As the electron donor used for forming the organic CT complex, the organic electron acceptor used and carbon having conductivity are used.
There are no particular restrictions on the material as long as it can form a T complex, and it may be either organic or inorganic. Specifically, inorganic materials include copper, silver, cobalt, nickel, iron, manganese, etc., and organic materials include tetracenes such as tetrathiafulvalene and tetraselenofulvalene, and derivatives thereof, or 2,2' - Known electron donors such as bispyridinium and N-methylphenazinium can be used.

The insulating polymer containing the organic CT complex crystal is one that can grow the crystal without hindering the CT complex formation reaction between the organic electron acceptor and the electron donor, and has appropriate electrical insulation properties. However, it is desirable to use a film-forming polymer that does not significantly dissolve or swell in the solvent of the organic electron acceptor solution used in forming the CT complex. Examples of such polymers include thermoplastic polymers such as polyvinyl butyral, polyester, polyamide, and polyesteramide, and one or more of these can be used.

[0027] As the above-mentioned insulating polymer, an appropriate polymer can be selected depending on the enzyme immobilization method described below and taking into consideration the diffusivity of the substrate. For example, polyvinyl butyral,
Although it is water-insoluble, it has hydrophilic and water-absorbing properties, and has extremely microscopic pores, making it advantageous for immobilizing enzymes.

The method for growing organic CT complex crystals in the film thickness direction within the above insulating polymer film is as follows:
For example, the following methods are possible.

First, an electron donor layer is provided on the surface of the substrate, or
Alternatively, a substrate such as a copper plate that also functions as an electron donor is used, and a polymer film is formed on the electron donor. The substrate in this case does not need to be electrically conductive, as will be described later. The coating can be formed by a general coating method. For example, a polymer solution prepared by dissolving a polymer in a suitable solvent, or a molten polymer can be coated as it is using a roll coater, a knife coater, or the like.

Considering the reaction rate during the CT complexation reaction between the electron donor and the organic electron acceptor and the electrical conductivity of the obtained film containing the organic CT complex microcrystals, the film thickness of the polymer film is usually 0. It is within the range of .01 to 50 μm, preferably 0.1 to 10 μm. If the polymer film is too thick, the rate of penetration of the organic electron acceptor into the polymer film during the subsequent CT complexation reaction will decrease, and the organic CT complex crystals will grow until they penetrate the polymer film. It will take a long time. Furthermore, if the thickness of the polymer film is too thin, pinholes may occur, and as a result, when used as an electrode material, problems such as elution of the base material may occur.

The thickness of the polymer film can be adjusted to any desired thickness by changing the amount of polymer solution used and the polymer concentration.

After the surface of the substrate having the electron donor layer on the surface is coated with the polymer in this manner, the entire surface is dried if necessary.

Next, part or all of the surface of this polymer coating is brought into contact with a solution containing an organic electron acceptor. As a result, the organic electron acceptor in the solution diffuses and permeates into the polymer film, causing a CT complexation reaction with the electron donor that constitutes the surface of the substrate, and the organic CT complex crystals form the polymer film. It gradually grows from the substrate side and eventually penetrates the polymer film, yielding the desired organic CT complex-containing film.

Suitable solvents used for preparing the organic electron acceptor-containing solution include polar aprotic solvents such as acetonitrile, dioxane, N,N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoramide, and methyl ethyl ketone. It is.

The concentration of the organic electron acceptor in this solution is generally 0.01 parts by weight to saturation concentration, preferably 0.1 parts by weight to saturation concentration, based on 100 parts by weight of the solvent.

[0036] Formation of the organic CT complex is usually carried out for 10 to 30
Although it is carried out at a temperature of .degree. C., depending on the combination of the organic electron acceptor used and the electron donor on the surface of the substrate, the CT complexation reaction may proceed rapidly, making it difficult to obtain a dense and uniform target film. In such a case, the temperature of the solution, the substrate, and the atmosphere may be lowered or the concentration of the solution may be lowered as necessary. Conversely, if the complexation reaction is slow and it takes a long time for the organic CT complex crystal to grow until it penetrates the film, heating can be performed as necessary.

[0037] The contact time of the organic electron acceptor-containing solution largely depends on the combination of the organic electron acceptor and electron donor used and the thickness of the desired polymer film, but is generally about 10 seconds to 1 hour. .

[0038] In this way, a conductive film is formed on the substrate in which organic CT complex crystals are grown so as to penetrate through the insulating polymer film layer in its thickness direction. The conductive film is cleaned and dried as necessary.

If the substrate used is conductive, the substrate on which the conductive coating is formed can be used as is, or the conductive coating can be peeled off as a film from the substrate and then attached to another conductive substrate. It is also possible to use it as an electrode material. If the substrate used is not conductive, the coating is peeled off and attached to a conductive substrate to form an electrode material.

The enzyme electrode of the present invention consists of a combination of an electrode made of a conductive substrate and a conductive film as described above, and an enzyme and an electron mediator immobilized thereon.

[0041] In the enzyme electrode of the present invention, the enzyme to be immobilized in or on the surface of the conductive film may be selected as appropriate depending on the target substance and the desired chemical reaction, taking into account the substrate specificity and reaction specificity of the enzyme. You can choose. Enzymes that can be used include, but are not particularly limited to, glucose oxidase, alcohol dehydrogenase, peroxidase, catalase, lactate dehydrogenase, galactose oxidase, penicillinase, and the like. Furthermore, a combination of an oxidoreductase and a coenzyme is also possible.

In the enzyme electrode of the present invention, the electron mediator fixed in or on the surface of the conductive film can efficiently transfer electrons accompanying the enzyme reaction, that is, it can smoothly transfer electrons from the enzyme to the organic CT complex. It suffices as long as it is made to be carried out by For example, in the case of a reaction in which a substrate is oxidized by an oxidase, electrons are easily received from the reduced enzyme, and the electron mediator itself becomes a reduced form and donates electrons to the electrode through an electrode reaction on the surface of the conductive film. However, it has the property of returning to its oxidized form. Such electron mediators include ferrocene, 1,1
'- Ferrocene derivatives such as dimethylferrocene, ferrocenecarboxylic acid, ferrocenecarboxaldehyde,
Quinones such as hydroquinone, chloranil, and bromanil, metal complex ions such as ferricyanide ion, octacyanotungstate ion, and octacyanomolybdate ion are suitable.

The method for immobilizing the enzyme and electron mediator is not particularly limited, and known methods can be used. These may be immobilized in separate steps, but they can also be immobilized by forming an immobilization film containing both on the conductive coating of the electrode.

[0044] In order to immobilize the enzyme on the conductive coating, the enzyme can be directly immobilized on the conductive coating using a known immobilization method.
An enzyme-immobilized membrane obtained by a known immobilization method may be bonded to a conductive film. Examples of immobilization methods include carrier bonding, covalent bonding, ionic bonding, adsorption, and crosslinking.

Methods for immobilizing the electron mediator on the conductive film include a method of directly immobilizing the electron mediator on the conductive film, or a method of providing a film containing the electron mediator on the conductive film. Examples of immobilization methods include a method of dispersing into a film, a covalent bond method, an adsorption method, a crosslinking method, and the like.

[0046] An organic CT complex is grown by coating an insulating polymer solution on a substrate that is also an electron donor to form a film, and then bringing this into contact with an electron acceptor-containing solution to grow an organic CT complex crystal. When providing a conductive film containing an electron mediator, it is convenient to disperse the electron mediator in the solution at the stage of applying the insulating polymer solution before the formation of the organic CT complex, thereby fixing the electron mediator. Also, organic C
There is a method of forming a film in which an electron mediator is immobilized by applying a polymer solution containing an electron mediator on a conductive film containing a T complex. In this case, if a polymer solution containing both the electron mediator and the enzyme is used, both can be immobilized at the same time. If an organic CT complex is further grown on this polymer layer by immersion in an electron acceptor solution,
This is advantageous in that it increases the conductivity of the entire membrane and provides a large response current.

When forming an enzyme and/or electron mediator immobilized film, responsiveness is improved by ensuring smooth electron transfer from the enzyme to the organic CT complex, from the enzyme to the electron mediator, or from the electron mediator to the organic CT complex. Therefore, the thinner the enzyme-immobilized membrane is, the better. Also, it does not necessarily have to be a uniform film, and enzymes and organic CT
The complex, the enzyme and the electron mediator, or the electron mediator and the organic CT complex may be brought into direct contact.

The enzyme electrode of the present invention thus obtained has a structure in which the enzyme and the electron mediator are immobilized on the conductive film provided on the conductive substrate so as to be in contact with each other,
It is structurally simpler than conventional hydrogen peroxide electrodes, oxygen electrodes, etc., and can be made smaller.

[0049] The conductive film containing the organic CT complex crystal used in the enzyme electrode of the present invention not only facilitates electron transfer between the enzyme and the enzyme, but also is compatible with ferrocenes, etc., which have been conventionally used as electron mediators. In comparison, the electrical conductivity of the crystalline polymer-dispersed film is significantly higher. this is,
It is thought that this is because these organic CT complexes constitute developed needle-like crystals, so that even with the same content, the number of conductive paths in the film increases, contributing effectively to electron transfer.

Further, by growing these complex crystals in the film thickness direction, electron transfer can be carried out more effectively.

[0051] In the present invention, furthermore, in the conductive coating and/or
Or, because an electron mediator is immobilized on the surface, even in parts where electron transfer is structurally difficult even though the organic CT complex and enzyme are in contact with each other.
It becomes possible to ensure smooth electron mobility and improve responsiveness.

As described above, the enzyme electrode of the present invention is superior in responsiveness, response time, and stability over time compared to conventional electrodes that utilize only electron mediators or conductive polymer electrodes.

Substances that can be measured with the enzyme electrode of the present invention include sugars such as glucose, trace biological substances such as lactic acid and alcohol in blood and urine, and sugars and alcohols in food processing processes. If the enzyme electrode of the present invention is used,
It is possible to selectively analyze the above substances with high precision and repeatedly over a long period of time. Moreover, it can be used not only for measuring substances but also for bioreactors and the like.

[0054]

【Example】

[0055]

[Example 1] One side of a 0.5 mm thick copper plate was molded with epoxy resin except for the electrode part (3 mm x 3 mm) and the terminal part, and then treated with 5% by weight sulfuric acid and then washed with pure water. and dried. Polyvinyl butyral resin [Product name: S-LEC B, BX-L, Sekisui Chemical Co., Ltd.
An ethyl alcohol solution containing 5% by weight of 1,1'-dimethylferrocene and 2% by weight of 1,1'-dimethylferrocene was prepared, and 30 μl of this solution was dropped onto the electrode part of the copper plate and dried to obtain a polymer-coated copper electrode. (Polymer film thickness: 5 μm).

7,7',8,8'-tetracyanoquinodimethane (reagent, manufactured by Kishida Chemical Co., Ltd., hereinafter abbreviated as TCNQ)
1.0g acetonitrile (for reagent, spectrum)
A saturated solution of TCNQ was prepared.

The electrode portion of the polymer-coated copper electrode was immersed in this TCNQ saturated solution and left for 10 minutes. Ten minutes after immersion, precipitation of acicular microcrystals was observed on the polymer surface.

[0058] The electrode coated with the organic CT complex microcrystal-containing polymer thus obtained was treated with glucose oxidase.
(from Aspergillus niger, Sig
(manufactured by MA, Type VII) was immersed in a 10 mg/ml aqueous solution and left at 4° C. for 24 hours to obtain an enzyme-immobilized electrode.

The above electrode was used as a working electrode and attached to a measurement cell of a batch type measuring device, and a 100mM phosphate buffer solution (pH
7.0). A 1 cm2 platinum plate was used as a counter electrode, a silver/silver chloride electrode was used as a reference electrode, and a voltage of 0.25 V (Ag/Ag
A potential of Cl (vs.) was applied to measure the response current for each glucose concentration shown in Table 1. The results are shown in Table 1. In this way, a good linear relationship was obtained over the measured concentration range.

Next, using the same measuring device and the same solution, after nitrogen bubbling was performed for 30 minutes, the response current for each glucose concentration shown in Table 1 was measured. The results are shown in Table 1. This result shows that the response current of the present enzyme electrode is hardly affected by the dissolved oxygen concentration.

Furthermore, the buffer was replaced with 100mM phosphate buffer containing 0.1M ascorbic acid (pH
7.0), measurements were performed in the same manner. As is clear from the results shown in Table 1, the response current is hardly affected by ascorbic acid.

[0062] This sensor was also incubated in phosphate buffer for 30 minutes.
Even after being left at room temperature, it maintained approximately 80% of its initial responsiveness and had excellent storage stability.

[0063]

Example 2 Using the same copper plate as used in Example 1, 30 μl of a 3% by weight ethyl alcohol solution of polyvinyl butyral was dropped onto the electrode portion and dried to obtain a polymer-coated copper electrode.

Using this electrode, TCN
An electrode coated with an organic CT complex-containing polymer was obtained by immersion in a Q-saturated solution.

[0065] Take 10 μl of a solution in which 5 mg of glucose oxidase is dispersed in 1 ml of an acetone solution containing 2% by weight of hydroquinone and 1% of polybutyral resin, drop it onto the above electrode, dry it, and immediately apply TCN.
It was immersed in a Q-saturated solution for 5 minutes and dried to obtain an enzyme-immobilized electrode.

Table 1 shows the results of measurements made using this electrode in the same manner as in Example 1.

[0067]

[Example 3] A silver plate of the same shape as in Example 1 was used instead of the copper plate, and the polymer solution was added to a 3% by weight methylene chloride solution of copolyester resin PES-110L (manufactured by Toagosei Kagaku Kogyo Co., Ltd.). , 1,'-dimethylferrocene dissolved in 2% by weight was immersed in a TCNQ saturated solution in the same manner as in Example 1 to obtain organic carbon.
An electrode coated with a T complex-containing polymer was obtained.

1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide meso-P-toluenesulfonic acid 0.127 g, glucose oxidase 10
mg was dissolved in 2 ml of 0.1 M acetate buffer, the above electrode was immersed in this solution, and reacted at room temperature for 3 hours.
An enzyme-immobilized electrode was obtained.

Table 1 shows the results of measurements made using this electrode in the same manner as in Example 1.

[0070]

[Example 4] Using the electrode coated with the organic CT complex-containing polymer obtained in Example 2, 5 mg of glucose oxidase was dissolved in 1 ml of pure water, and then photocrosslinkable polyvinyl alcohol [manufactured by Toyo Gosei Co., Ltd.] was used. , SPP-H-1
3] 10 μl of a uniformly dissolved solution containing 0.5 g and 30 mg of potassium octacyanomolybdate was dropped onto the above electrode, air-dried, and heated under a 60 W fluorescent lamp for 2 hours.
After being irradiated with light for a period of time, it was immersed in a TCNQ saturated solution for 5 minutes and dried to obtain an enzyme-immobilized electrode.

Table 1 shows the results of measurements made in the same manner as in Example 1 using this electrode.

[0072]

[Comparative Example] Table 1 shows the results of measurements carried out in the same manner as in Example 1 using an enzyme electrode prepared using a polymer solution containing no 1,1'-dimethylferrocene.

From Table 1, it is clear that the enzyme electrode of the present invention provides a larger response current and has superior responsiveness than the enzyme electrode of the comparative example that does not contain an electron mediator.

[0074]

[Table 1]

[0075]

[Effects of the Invention] The enzyme electrode of the present invention uses a method that directly detects electron transfer accompanying enzyme reactions, so it is not affected by the amount of dissolved oxygen or by electrochemical interfering substances. . Moreover, it is an enzyme electrode with excellent responsiveness and response life. Therefore, it is possible to perform accurate measurements over a long period of time as an enzyme sensor.

Claims (3)

[Claims] [Claim 1] A conductive substrate comprising an electrically conductive substrate and an electrically conductive coating containing an organic charge transfer complex crystal provided on the surface of the substrate,
An enzyme electrode characterized in that an enzyme and an electron mediator are immobilized in this conductive film and/or on its surface. 2. The enzyme electrode according to claim 1, wherein the enzyme is an oxidoreductase. 3. A conductive film containing organic charge transfer complex crystals is formed by growing crystals of the organic charge transfer complex within an insulating polymer film so as to penetrate through the thickness of the film. The enzyme electrode according to claim 1.