JPH07151727A - Composition for enzyme electrode and immobilized enzyme electrode - Google Patents

Abstract

PURPOSE:To make possible the realization of a composition for enzyme electrode a change amount of the conductance of which is not reduced, by using conductive enzyme, and a resin composition including hydrophilic resin and/or hydrophobic resin, as the composition for enzyme electrode. CONSTITUTION:An immobilized enzyme electrode 2 is formed and a single electrode is made, by applying a composition for enzyme electrode on a base material by screen printing. By this, a vary accurate oxygen sensor of easy operation is made. Polyvinyl pyrrolidone is desirable as hydrophilic resin of a component of the composition, and polyvinyl butyral is desirable as hydrophobic resin. Conductive enzyme of a component of the composition is made by keeping a mediator through a covalent bond to a side chain and/or main body of the enzyme. An object selected from glucose oxidase, galactose oxidase, pyruvic acid oxidase, etc., is desirable as the enzyme. The mediator is selected from ferrocene, a ferrocene derivative, a nicotinamide derivative, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme sensor capable of quickly and easily quantifying a specific component contained in a biological sample, and more specifically, an enzyme-immobilized electrode using a conductive enzyme and its electrode. The present invention relates to a composition for an enzyme electrode for manufacturing.

[0002]

2. Description of the Related Art There are known enzyme sensors that measure the abundance of various substances by utilizing the high substrate specificity of enzymes.
For example, a sensor useful for quantitative analysis of glucose has been put into practical use.

An enzyme sensor currently in practical use is, for example, in the case of utilizing an oxidase, hydrogen peroxide produced, hydrogen ions or the amount of consumed oxygen is measured by a hydrogen peroxide electrode,
The substance to be measured is quantified by electrochemically detecting with a hydrogen ion electrode or an oxygen electrode.

In such an enzyme sensor, an enzyme having a high substrate specificity for a substance to be measured is immobilized on a substrate,
The substance to be measured in the sample is brought into contact with the enzyme, and the substance generated by the action of the enzyme is detected and quantified, for example, electrochemically. For example, an oxidase sensor is constructed by immobilizing oxidase in a membrane and mounting the enzyme membrane on a diaphragm oxygen electrode. When the substance to be measured, which is the substrate, is oxidized by the action of the immobilized enzyme, the current value of the oxygen electrode in close contact with the enzyme membrane changes, and the concentration of the substrate can be measured from that value.

In addition, a mediator is also made to exist in a sensor system so that the reaction between the substrate and the enzyme can be easily detected at the electrode. For example, a method has been proposed in which an artificial mediator is applied on the surface of an electrode in a thin film form, and a semitransparent film is further coated on the electrode (EP-786368).
1). In addition, a poorly water-soluble mediator is contained in the electrode (Agric. Biol. Chem., 52 , 1557 (1988)), or a water-soluble mediator is contained in the electrode in advance, and then the electrode surface is highly ionic. Method to prevent elution of mediator in electrolyte by providing a thin film of a composition consisting of molecule and enzyme (Agric. Biol. Chem., 52 , 3187 (1988))
Have been proposed. Furthermore, a method has been proposed in which an enzyme and ferrocene are mixed in paraffin or the like and rubbed into a paste (Talanta, 38 (1) , 107-110 ) .
(1991)).

However, the preparation of electrodes used in conventional enzyme sensors, especially enzyme electrodes in the presence of mediators, is generally complicated. Therefore, in order to obtain stable accuracy, it has been necessary to pay close attention to the quality control of the electrodes during the manufacturing process. Further, it has been pointed out that the conventional enzyme sensor has a problem that the life of the electrode is not sufficient. In addition, if the mediator leaks from the electrode, in-vivo measurement cannot be performed.
Therefore, when a mediator is present, its stabilization has also been an important issue in enzyme sensor development.

Against this background, the inventors of the present invention invented a composition for an enzyme electrode containing a conductive enzyme, a conductive component, and a vehicle (hereinafter referred to as "prior invention"). ,
I filed an application earlier (Japanese Patent Application No. 5-93900). The composition for an enzyme electrode of the invention of the prior application measures the substrate concentration by the change amount of the conductivity of the enzyme composition with the change of the substrate concentration, but when the composition is immobilized on a substrate, There was a problem that the amount of change in conductivity was extremely small or decreased as compared with the case where it was present. Therefore, it may be difficult to measure the substrate concentration at a potential with a small change in conductivity.

[0008]

In order to solve the problems of the invention of the prior application, the present invention provides a composition for an enzyme electrode which does not reduce the amount of change in conductivity even when immobilized on a substrate. The purpose is to provide.

[0009]

Means for Solving the Problems The inventors of the present invention have conducted diligent studies on each component of the composition for enzyme electrodes, and as a result, it is considered that they have conventionally played an extremely important role as components of the composition for enzyme electrodes. By removing the carbon powder, buffer, surfactant, carrier, solvent, etc., which had been used, and using only the enzyme and resin as a composition, it was surprisingly found that the amount of change in conductivity with substrate concentration significantly increased. Completed the invention.

The enzyme electrode composition according to the present invention is an enzyme electrode composition comprising a conductive enzyme and a resin composition containing a hydrophilic resin and / or a hydrophobic resin. Further, the enzyme-immobilized electrode according to the present invention is one in which the enzyme electrode composition is supported on a substrate.

When the composition for an enzyme electrode according to the present invention is supported on an insulating substrate by a technique such as screen printing,
The electrodes can be configured. Therefore, by using the composition for an enzyme electrode according to the present invention, an electrode for an enzyme sensor having stable performance can be produced by a simple method.
The composition according to the present invention is also advantageous in that it is possible to use screen printing, which is a method for easily manufacturing electrodes having various patterns. Further, in the electrode obtained by using the composition for an enzyme electrode according to the present invention, since the mediator is fixed to the enzyme, it is possible to obtain an electrode having stable performance without leakage of the mediator into the test sample. Also excellent in terms.

The present invention will be described in detail below. Composition for Enzyme Electrode The composition for enzyme electrode according to the present invention comprises an enzyme having substrate specificity and conductivity for the substance to be measured.

The term "conductive enzyme" as used herein means an enzyme which changes its conductivity depending on the amount of a substance to be measured which is a substrate present in the system. In the present invention,
The conductive enzyme may be an enzyme having conductivity itself, or an enzyme having conductivity as a result of being modified with various mediators.

Here, the enzyme which has been imparted with conductivity as a result of being modified with a mediator means the following. In general, the active site of an enzyme exists inside a protein that is an enzyme, so it is generally difficult to observe the electron transfer resulting from the action of the enzyme from the outside, for example, through an electrode. In the case of an oxidase, it is also possible to observe the electron transfer through the consumed enzyme or the amount of hydrogen ion or hydrogen peroxide generated,
It is often difficult to stabilize those amounts in the system. Therefore, it is difficult to be an index that accurately reflects the mass of the group present in the system. However, the presence of a mediator that mediates the movement of electrons between the active site and the electrode (for example, by being oxidized or reduced by itself) causes the movement of electrons generated at the active site to be stable and highly accurate at the electrode. Can be observed. Therefore, in the present invention, the enzyme modified with a mediator means an enzyme modified with a mediator having the above-mentioned role and, as a result, imparted with conductivity.

In the present invention, the preferred conductive enzyme is an enzyme modified with this mediator.
Examples of the modified enzyme include (1) a side chain (for example, a sugar chain, an alkyl chain, a peptide chain branched from a main chain, etc.) of an enzyme, to which a mediator is bound via a covalent bond, (2 The present invention includes three embodiments, that is, a mediator bound to the enzyme body via a covalent bond, and (3) a mediator bound to the enzyme body and side chains of the enzyme via a covalent bond. In these aspects,
The mediator and the enzyme may be bound via an appropriate spacer.

In the present invention, the enzyme may be appropriately selected in relation to the substance to be measured, and the mediator is not particularly limited as long as it plays the above role. A specific example of a preferred modifying enzyme is an oxidase modified with a mediator. Here, preferable examples of the oxidase include those selected from the group consisting of glucose oxidase, galactose oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, and choline oxidase. Further, preferred examples of mediators include ferrocene, ferrocene derivatives, p-benzoquinone, potassium ferricyanide, phenazine methosulfate, 2,6-dichlorophenol indophenol, pyrroloquinoline quinone (PQQ),
Examples include flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), and the like. Ferrocene or its derivative is particularly preferable. Preferred ferrocene derivatives include, for example, 1,
1'-dimethylferrocene, ferrocene acetic acid, hydroxyethylferrocene, 1,1'-bis (hydroxymethyl) ferrocene, ferrocene monocarboxylic acid, ferrocene 1,1'-dicarboxylic acid, chloroferrocene, methyltrimethylaminoferrocene and the like can be mentioned. .

Examples of the combination of the enzyme and the mediator include a combination of glucose oxidase (GOD) and ferrocene or a ferrocene derivative. More specifically, GOD is the following reaction:

[0018]

[Chemical 2] And the combination of GOD and ferrocene carries electrons as follows:

[0019]

[Chemical 3] (In the formula, FAD is a flavin adenine dinucleotide.) The mediator ferrocene can be measured even at a low electrode potential as compared with hydrogen peroxide, and many substances that are reduced at a potential similar to hydrogen peroxide (for example, Ascorbic acid, etc.) is not affected.

The introduction of the mediator into the oxidase is
Enzyme body of oxidase (eg, amino group of lysine residue) and / or side chain (eg, sugar chain, alkyl chain,
Peptide chains branched from the main chain).
Furthermore, this introduction may be carried out via a suitable spacer. In particular, the introduction of the mediator into the side chain is preferably carried out via a spacer. As a spacer
Examples of the spacer include isoprene, butadiene, acetylene, derivatives thereof and copolymers thereof, and a preferred spacer is a spacer represented by the following formula (I).

[0021]

[Chemical 4] (Here, n represents an integer of 0 to 8) As a specific example of a preferable modifying enzyme, ferrocenecarboxylic acid is introduced into the amino group of a lysine residue by an acid amide bond in the enzyme body of oxidase, and / or An example is one in which a ferrocenecarboxylic acid is introduced through a spacer represented by the above formula (I) using an aldehyde group formed by modifying the sugar chain of an enzyme. Most preferably,
Examples thereof include those in which ferrocene is introduced into both the enzyme body of the oxidase and the sugar chain. This modified enzyme having a mediator introduced into the enzyme body and sugar chain is novel and has particularly preferable characteristics in the enzyme-immobilized electrode described below, and thus constitutes one embodiment of the present invention.

The amount of the mediator introduced into the enzyme is not particularly limited, but the ratio of the mediator molecule to one molecule of the enzyme is preferably about 1:50, and particularly when the enzyme is oxidase and the mediator is ferrocene, The ratio is preferably about 1: 5 to 20 and more preferably about 1:10 to 15. FIG. 1 is a schematic diagram showing a state in which a mediator is introduced into the main body and / or side chain of an enzyme. In the figure, (A) is a model in which a mediator is covalently bound to the side chain of the enzyme, (B) is a model in which the mediator is covalently bound to the enzyme body and the side chain of the enzyme,
(C) represents a model in which a mediator is covalently bound only to the enzyme body. As the hydrophilic resin, it is preferable to use polyvinyl pyrrolidrin, but it is not particularly limited as long as it is a resin soluble in water and alcohol. Specific examples include sodium polyacrylate, polyacrylic acid, polyvinyl methoxy acetal (acetalized 48%), polyvinyl amine (75% Rh), polyvinyl alcohol, viscose film, cellulose acetate (substitution degree DS = 0.8).
), Polyethylene glycol, polyvinyl methoxy acetal (acetalization 86%), and the like.

Polyvinyl butyral is preferably used as the hydrophobic resin, but is not particularly limited as long as it is a resin which is insoluble in water and soluble in alcohol. Specific examples include cellulose acetate (substitution degree DS = 2.3), polyvinyl acetate, polymethyl methacrylate, polyisobutyl ether and the like. The mixing ratio of the hydrophilic resin and the hydrophobic resin can be arbitrarily set according to the resin used. For example, in the case of using polyvinyl pyrodrine and polyvinyl butyral, the mixing ratio is 15: 1 to 1:
5, especially 5: 1 to 1: 1 is preferable. Also,
If the elution of the modifying enzyme mixed with the resin can be ignored, it is possible to use the hydrophilic resin alone, and, for example, polyvinyl butyral having a low degree of polymerization (DP = less than 1000), It is also possible to use the hydrophobic resin alone. The composition according to the present invention can be produced by mixing these components with an appropriate mixing means such as an agitator, a homomixer and a triple roll.

Production of enzyme-immobilized electrode and its use The enzyme-electrode composition of the present invention can be made into an enzyme-immobilized electrode by supporting it on a substrate.
That is, the composition is applied to a substrate and the vehicle component is evaporated and removed to support the composition on the substrate. The method of applying the composition to the substrate is not particularly limited, but it can be applied to the printing technique. For example, the composition can be applied to the substrate by a technique such as a screen printing method, a roller coating method, a dispenser method or the like. Screen printing is particularly preferable because it is a method that allows various patterns to be printed relatively easily.

The enzyme-immobilized electrode thus obtained can be used as a sensor electrode for measuring the amount of its substrate. The enzyme-immobilized electrode changes its conductivity depending on the amount of the substrate present in the sample through the immobilized enzyme. Therefore, the change in conductivity can be standardized in advance in a system in which the substrate concentration is known, and the substrate concentration of a sample whose substrate concentration is unknown can be measured based on the result. Specifically, the substrate concentration and the change in conductivity of the enzyme-immobilized electrode may be measured by a system using the enzyme-immobilized electrode obtained by the present invention, a counter electrode, and a reference electrode.

FIG. 2 shows a preferred embodiment of the sensor electrode. FIG. 2 (a) shows that the enzyme electrode composition is applied onto the substrate 1 by a screen printing method,
This is a monopolar electrode obtained by forming the enzyme-immobilized electrode 2, and FIG. 2 (b) is a perspective view thereof. In addition, FIG.
Is the same as the composition for enzyme electrodes except that the enzyme-immobilized electrode 2 is formed by applying the composition for enzyme electrodes on the base material 1 by the screen printing method, and the conductive enzyme is not contained. This is a bipolar electrode in which the counter electrode 3 formed by applying the composition of the composition in the same manner is provided on the same base material 1, and FIG. 2D is a perspective view thereof. In the case of the embodiments of FIGS. 2C and 2D, the base material needs to be an insulating substance.

Further, a schematic view of a sensor provided with those sensor electrodes is as shown in FIGS. 3 (A) and 3 (B). The electrode 5 can be separated from the main body 4,
Can be disposable. FIG. 4 shows another preferred embodiment of the sensor electrode. This sensor electrode was prepared by using the enzyme electrode composition 6 of the present invention in wells 7.
In order to use it, the specimen sample may be dropped into the well 7 and the counter electrode and the reference electrode may be installed from above. Such a well-type electrode is superior in that the amount of the specimen sample to be used can be smaller than that of the strip-type electrode as shown in FIG. Further, by providing a plurality of wells as in this embodiment,
It can also be measured continuously. The electrodes in each well may be used only once and may be disposable.

An example of the manufacturing process of this sensor electrode is shown in FIG. First, the substrate 8 is prepared (a), the gold electrode 9 is printed on the surface thereof by screen printing or the like, and baked (b). Then, the enzyme electrode composition 6 is printed on the tip of the gold electrode 9 by screen printing or the like (c). On the other hand, a plate 11 having holes 10 corresponding to the positions of the enzyme electrode composition 6 formed on the substrate is prepared and laminated on the substrate 8 (d). The resin used for the enzyme electrode composition in the sensor electrode may be any of the hydrophilic resin and / or the hydrophobic resin described above, but preferably,
Only hydrophilic resin is used. This is because in such a well-type sensor electrode, there is no problem even if the modifying enzyme is eluted, and the elution causes a faster reaction and better sensitivity.

Production of Conductive Enzyme When the enzyme itself does not have conductivity, it can be made into a conductive enzyme by introducing a mediator.
The introduction of the mediator into the enzyme may be carried out in either the enzyme body or the side chain as described above, and the introduction of the functional group present in the enzyme body or the side chain (the functional group oxidizes or reduces the enzyme). Or a functional group present in the enzyme may be formed by substitution reaction with a functional group present in the enzyme, and a functional group present in the mediator (this functional group is also present in the enzyme by oxidizing or reducing the mediator). Which may be formed by a substitution reaction with a functional group). Further, the introduction of the mediator through the spacer can also be performed by utilizing the functional group of the spacer.

A specific example of the reaction when the enzyme is oxidase is shown below. (1) Introduction of Mediator to Side Chain of Enzyme First, a sugar chain of oxidase is treated with an appropriate oxidizing agent (eg, sodium periodate) in a solvent (eg, phosphate buffer) that does not change the properties of the enzyme. ),
Form an aldehyde group on the sugar chain. Then as a spacer, the following formula (II):

[0031]

[Chemical 5] The dihydrazide represented by is introduced into the aldehyde group. This reactant is reacted with ferrocenecarboxylic acid as a mediator in the presence of a suitable reducing agent (eg sodium cyanoborohydride) or a condensing agent (eg carbodiimide).

(2) Introduction of mediator into enzyme main body and side chain (method A) First, a mediator is introduced into the side chain of the enzyme by the same method as in the above (1). Then denaturing the enzyme (eg urea)
To expose the inside of the protein, and then Y. Degani and A. He
The ferrocenecarboxylic acid is introduced into the main body and side chain of the enzyme by a method similar to the carbodiimide method according to ller (J. Physical Chemistry, 91 , 1285-1289 (1987)).

(3) Introduction of Mediator into Enzyme Main Body and Side Chain (Method B) First, the side chain of the enzyme is oxidized by the same method as in the above (1) to form an aldehyde group on the side chain. Then, dihitrazide represented by the above formula (II) is introduced into the side chain of the enzyme in the presence of the denaturant and the condensing agent. Further, the enzyme modified with a spacer introduced into the side chain is reacted with ferrocenecarboxylic acid.

(4) Introduction of a mediator into the enzyme body It can be carried out according to the above (2). That is, the enzyme is treated with a denaturing agent to expose the inside of the protein, and then ferrocenecarboxylic acid is introduced into the enzyme body by a method similar to the carbodiimide method.

[0035]

EXAMPLES The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. The following abbreviations are used below. GOD: glucose oxidase, HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

Example 1 Introduction of ferrocene into the GOD side chain (No. 1) (a) GOD (Type II, EC1.1.3.4., Asp manufactured by Sigma)
ergillus niger, 100 mg) in 0.1 M pH 6.0 phosphate buffer (1 ml) and mixed with 20 mM sodium periodate previously dissolved in the same buffer as above and mixed at 4 ° C. for 1 hour. I left it. Subsequently, ethylene glycol (100 μl) was added to stop the oxidation reaction. After that, leave it at 4 ℃ for 30 minutes, and then adjust it to pH 6.0, 0.1.
The M phosphate buffer was used as an external solution, and the reaction mixture was dialyzed for 48 hours or more while exchanging the external solution four times.

(B) Adipyl dihydrazide (100 mg
(Dry powder) was dissolved in the above dialyzed solution, and the solution was allowed to stand overnight at room temperature in the dark. Add this solution to pH
Using 6.0 and 0.1 M phosphate buffer as an external solution, the external solution was dialyzed while exchanging the external solution four times to remove unreacted adipyl dihydrazide.

(C) Ferrocene carboxaldehyde (concentration 1 mg / ml, pH 6.0, 0.1 M phosphate buffer 2 m
l) is the GOD hydrazide solution obtained in (b) above (2 ml)
Were mixed with each other and reacted overnight in the dark at 4 ° C. Sodium cyanoborohydride was mixed to a final concentration of 10 mM and reacted for 1 hour. Then pH 6.0, 0.1M
The reaction mixture was dialyzed while the phosphate buffer was used as an external solution and the external solution was exchanged 4 times to obtain GOD in which ferrocene was introduced into the side chain to remove excess ferrocene carboxaldehyde.

Example 2 Introduction of ferrocene into the GOD side chain (part 2) (a) GOD (Type II, EC1.1.3.4., Asp manufactured by Sigma)
(ergillus niger, 500 mg) was dissolved in 0.1 M phosphate buffer (5 ml, pH 6.0) and stirred well at 4 ° C so as not to cause foaming. 20 mM sodium periodate previously dissolved in the same buffer as above was added, and the mixture was allowed to stand at 4 ° C. for 1 hour. Subsequently, ethylene glycol (500 μl) was added to stop the reaction, and the mixture was stirred at 4 ° C. for 30 minutes.
The reaction mixture was dialyzed for 48 hours or longer while using 0.1M phosphate buffer (pH 6.0) as an external solution and changing the external solution 4 times.

(B) Adipyl dihydrazide (500 mg
(Dry powder) was dissolved in the above dialyzed solution and the solution was stirred at 4 ° C. in the dark overnight. This solution was adjusted to pH 6.
The 0, 0.1 M phosphate buffer was used as the external solution, and the external solution was dialyzed for 48 hours or more while exchanging the external solution 4 times to remove unreacted adipyl dihydrazide.

(C) In advance, 1-ethyl-3- (dimethylaminopropyl) carbodiimide (500 mg) and ferrocenecarboxylic acid (450 mg) were added to 0.15M.
Dissolve in HEPES buffer solution (25 ml), pH 7.3
Adjusted to. The ferrocene solution was mixed with the enzyme solution that had been dialyzed, and the mixture was stirred overnight at 4 ° C. in the dark at 2 to 3 times while confirming that the pH was 7.3. This enzyme solution was dialyzed again for 48 hours or longer while using a phosphate buffer as an outer solution and changing the outer solution 4 times or more. Subsequently, unreacted ferrocenecarboxylic acid and other low-molecular components were removed, and ferrocene was introduced into the side chain of GOD.
Got

Example 3 Introduction of ferrocene into GOD body and side chain (1) Y. Degani and A. Heller (J. Physical Chemistry, 91 , 1285-12)
89 (1987)). That is, ferrocene was bound to the GOD body by reacting ferrocenecarboxylic acid with the free amino group of the enzyme itself by the carbodiimide method.

(A) In the same manner as in Example 1 (a), the side chain of GOD was oxidized with sodium periodate. (B) Adipyl dihydrazide was reacted with oxidized GOD in the same manner as in Example 1 (b). (C) Urea (810 mg), water-soluble carbodiimide (1
-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride; 100 mg) and ferrocenecarboxylic acid (80 mg) at pH 7.3, 0.15M HEP
Sonicate in ES buffer (4 ml), pH 7.2
It was set again to 7.3. Ferrocenecarboxylic acid was saturated at this pH. The GOD hydrazide solution (2 ml) obtained in (b) above was added, the pH was checked and adjusted if necessary, and the reaction product was left at 0 ° C. in the dark overnight. Excess reagent is
A 0.1 M phosphate buffer (pH 6.0) was used as an external solution, and the reaction product was dialyzed while exchanging the external solution four times to obtain GOD in which ferrocene was introduced into the main body and side chains.

A conductive enzyme having a spacer of succinyl hydrazide was obtained by the same method except that succinyl hydrazide (n = 2) was used instead of adipyl dihydrazide (n = 4).

Comparative Example 1 An attempt was made to prepare a conductive enzyme by using oxalyl hydrazide (n = 0) and sebacyl hydrazide (n = 8) as spacers instead of adipyl dihydrazide (n = 4) of Example 3. . However, the spacer could not be dissolved in the GOD phosphate buffer, so that the conductive enzyme could not be prepared.

Example 4 Introduction of Ferrocene into GOD Main Body and Side Chain (Part 2) Aldehyde group (—CHO) on oxidized side chain of enzyme,
Both the free carboxyl group (-COOH) of the enzyme body was reacted with adipyl dihydrazide. Then ferrocene aldehyde was reacted with hydrazide.

(A) A portion of the GOD side chain was oxidized with sodium periodate in the same manner as in Example 1 (a).

(B) 0.5M adipyl dihydrazide (adjusted to pH 5: 2 ml), sodium chloride (9 mg), the oxidized GOD solution (2 ml) obtained in the above (a), 1M water-soluble carbodiimide (1 ml) and 3M urea Were mixed and the pH was adjusted to 5. The reaction was allowed to proceed for 4 hours at room temperature in the dark. The pH was adjusted to 5 every 30 minutes. After 4 hours, the reaction solution was adjusted to pH 6.0 and 0.1 M phosphate buffer was used as an external solution, and the reaction solution was dialyzed while exchanging the external solution four times.

(C) The GOD hydrazide solution (2.5 ml) obtained in (b) above was mixed with 0.4 mg / ml ferrocene carboxaldehyde (2.5 ml) and reacted at 4 ° C. in the dark for 4 hours. . Sodium cyanoborohydride was added to a final concentration of 0.1 mM, then pH 6.0,
A 0.1 M phosphate buffer solution was used as an external solution, and the reaction solution was dialyzed while exchanging the external solution four times to obtain GOD in which ferrocene was introduced into the main body and side chains.

Example 5 Introduction of ferrocene into GOD main body and side chain (3) (a) GOD (500 mg) was dissolved in phosphate buffer (5 ml) of 0.1 M and pH 6.0 to prevent foaming. Stir well at 4 ° C. 20 mM sodium periodate previously dissolved in the same buffer as above was added, and the mixture was allowed to stand at 4 ° C. for 1 hour. Subsequently, ethylene glycol (500 μl) was added to stop the reaction, and the mixture was stirred at 4 ° C. for 30 minutes.
The reaction mixture was dialyzed for 48 hours or longer while using 0.1M phosphate buffer (pH 6.0) as an external solution and changing the external solution 4 times.

(B) Adipyl dihydrazide (500 mg
(Dry powder) was dissolved in the above dialyzed solution and the solution was stirred at 4 ° C. in the dark overnight. This solution was adjusted to pH 6.
The 0, 0.1 M phosphate buffer was used as the external solution, and the external solution was dialyzed for 48 hours or more while exchanging the external solution 4 times to remove unreacted adipyl dihydrazide.

(C) Urea (4050 mg), 1-ethyl-3- (dimethylaminopropyl) carbodiimide (5
00 mg) and ferrocenecarboxylic acid (450 mg) were dissolved in 0.15 M HEPES buffer (25 ml), and the pH was
Adjusted to 7.3. This ferrocene solution was mixed with the solution that had been dialyzed, and while checking the pH 2-3 times,
The mixture was stirred in the dark at 0 ° C overnight. For this enzyme solution, use phosphate buffer as the external solution and change the external solution four times or more
Dialysis was performed again for 8 hours or more. Subsequently, unreacted ferrocenecarboxylic acid and other low-molecular components were removed to obtain GOD in which ferrocene was introduced into the main body and side chains.

Example 6 Introduction of ferrocene into GOD body (1) GOD (100 mg) was dissolved in 0.1 M pH 6.0 phosphate buffer (1 ml) and stirred well at 4 ° C. Urea (81
0 mg), water-soluble carbodiimide (1-ethyl-3- (3
-Dimethylaminopropyl) carbodiimide hydrochloride; 100 mg) and ferrocenecarboxylic acid (80 m
g), pH 7.3, 0.15M HEPES buffer (4m
sonicated in l) and re-adjusted to pH 7.2-7.3. Ferrocenecarboxylic acid was saturated at this pH.
The GOD solution was added, the pH was checked and adjusted if necessary, and the reaction was left overnight at 0 ° C. in the dark. As an excess amount of the reagent, a 0.1 M phosphate buffer solution of pH 6.0 was used as an external solution, and the reaction product was dialyzed while exchanging the external solution four times to obtain GOD having ferrocene introduced into the main body.

Example 7 Introduction of Ferrocene into GOD Body (Part 2) GOD (500 mg) was dissolved in 0.1 M phosphate buffer (5 ml) having a pH of 6.0 and stirred well at 4 ° C. Urea (40
50 mg), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (500 mg) and ferrocenecarboxylic acid (450 mg) were added to 0.15 MH 2.
It was dissolved in EPES buffer (25 ml) and the pH was adjusted to 7.3. The above GOD solution was added to this solution, and 2-3
While confirming the pH once, the mixture was left overnight at 4 ° C. in a dark place. As an excess amount of the reagent, a 0.1 M phosphate buffer solution of pH 6.0 was used as an external solution, and the reaction product was dialyzed while exchanging the external solution four times to obtain GOD having ferrocene introduced into the main body.

Example 8 Evaluation of conductive enzyme (1) Gold working electrode (diameter 1 mm), platinum counter electrode, Ag / A
The conductive enzymes prepared in the above examples were evaluated by cyclic voltammetry using a gCl reference electrode. The working electrode was polished with aluminum oxide (0.075 μm) before use. Measured by Hokuto Denko Potentiostat / Galvanostat model HAB-151 and Graphtec W
Performed using an X1200 recorder. As the modified enzyme solution, the one obtained in the example was used without dilution. Catalase at a final concentration of 0.1 mg / ml was added to avoid interference with hydrogen peroxide. First, the cyclic voltammogram of the conductive enzyme was measured in the absence of the substrate. Next, glucose was added to a final concentration of 10 mM, and then cyclic voltammograms were measured under the same conditions. During the measurement, nitrogen was sufficiently bubbled to eliminate the influence of oxygen.

The results for the conducting enzymes of Examples 1, 2, 3 (spacer adipyl dihydrazide), 5, 6 and 7 are shown in FIGS. 6, 7, 8, 9, 10 and 11, respectively.
As shown in. As is clear from these figures, an increase in conductivity was observed when glucose was added as a substrate. In Examples 2, 5 and 7, a gold electrode having a diameter of 5 mm was used, and a glucose solution was added so that the final concentration was 8.25 × 10 ⁻² mM.

Example 9 Evaluation of Conductive Enzyme (2) Two kinds of conductive enzymes prepared in Example 3 (when the spacer is adipyl dihydrazide (n = 4) and succinyl hydrazide (n = 2)) Case). A cyclic voltammogram was measured in the same manner as in Example 11 except that the glucose solution was added so that the final concentration was 1.0 mg / ml. The results are as shown in FIG. The conductive enzyme using adipyl dihydrazide as a spacer showed a higher response than that using succinyl hydrazide.

Example 10 Evaluation of Conductive Enzyme (3) The conductive enzyme of Example 3 (spacer adipyldihydrazide) was used in a final glucose concentration of 0, 0.02, 0.
It was evaluated by measuring cyclic voltammograms at 06 and 0.08 mg / ml. The measurement was performed in the same manner as in Example 11 except that the glucose concentration was changed. The results are as shown in FIG. A positive correlation was found between the glucose concentration and the increase in conductivity in the solution system containing the conductive enzyme.

Example 11 Evaluation of Conductive Enzyme (4) ICP (inductively coupled plasma atomic emission)
The average number of ferrocene molecules introduced per GOD molecule was measured for the conductive enzymes prepared in Examples 2, 5 and 7 by spectroscopy analysis. The results are 1 for each of the conductive enzymes of Examples 2, 5, and 7.
The numbers were 1.8, 14.2 and 9.2. These results correlated very well with the increase in conductivity (FIGS. 7, 9, 11).

Comparative Example 2 A system in which GOD and a mediator were simply mixed was used in Example 1.
It evaluated similarly to 1. That is, the same evaluation apparatus as in Example 11 was used to measure the change in conductivity of the following mixture. GOD (unmodified) 100 mg Ferrocene 1 mg Phosphate buffer (pH 6.0, 0.1 M) 30 ml Catalase 3 mg Glucose 10 mM or 0 mM The results are shown in FIG. As is clear from this figure, it can be said that GOD and ferrocene, which is a mediator, do not substantially contribute to the change in conductivity of GOD when they are simply mixed.

Example 12 Preparation of Sensor Electrode (Part 1) The conductive enzymes obtained in Example 3 and the components shown in the following table were mixed to obtain compositions A, B and C.

Table 1 ─────────────────────────────────── A B C ──────── ──────────────────────────── Conductive enzyme 14.3 1.5 1.8 Carbon powder-36.5 13.9 Buffer ¹⁾ -15.0 29.6 Resin ²⁾ 85.7 20.0 1.2 Surfactant (Kao Span 20) -2.0 10.0 Butyl cellosolve-15.0 27.8 Amyl alcohol-10. 0 15.8 ──────────────────────────────────── 100.0 100.0 100.0

¹⁾ : 30 g of citric acid, 120 g of sodium citrate, and 504 g of amyl alcohol were placed in a ball mill pot and mixed well for 30 hours or more to form a slurry. ²⁾ : Polyvinylpyrrolidone (BASF Kollidon 90) 5
0.4 g, polyvinyl butyral (Sekisui Chemical Bx-1)
After thoroughly stirring 9.0 g and 282.6 g of amyl alcohol with an agitator, the one left to stand overnight or more was used. For the compositions A and B, the current value at each glucose concentration when the potential is +450 mV is shown in the following table.

Table 2 ───────────────────────────────── Glucose concentration (mM) 0 5 10 Composition A ( 220nA 310nA 460nA Composition B (unipolar electrode) 79nA 130nA 176nA ─────────────────────────────── ───

Compared to composition B, composition A has a large change in current due to an increase in glucose concentration, and is suitable for preparing a calibration curve. Compositions A and C were then applied to the substrate and their characteristics evaluated with a cyclic voltammogram. The result of the composition A is shown in FIG. 15, and the result of the composition C is shown in FIG.
6 shows. When the composition A was used, an increase in conductivity was observed with an increase in glucose as observed in the solution system, but when the composition C was used, such an increase in conductivity was observed. Was not done.

Example 13 Preparation of Electrode for Sensor (Part 2) The conductive enzyme obtained in Example 2, a hydrophilic resin (polyvinylpyrrolidone; BASF Kollidon 90) and ethanol (manufactured by Wako) are shown in the following table. The composition for an enzyme electrode was obtained by mixing with the compounding amounts shown in. The conductive enzyme obtained in Example 2 was used as it was, that is, in a solution state.

Table 3 ─────────────────────────── Compounding amount ───────────────── ─────────── Conductive enzyme 900 μl PVP 0.123 g Ethanol 100 μl ───────────────────────────

A glass plate is prepared as a substrate, and is shown in FIG.
Like gold paste (organic paste, NEChem
Cat Ltd.) was screen printed and fired. Then, the obtained composition for enzyme electrode was screen-printed on the tip of the formed gold electrode so that the diameter was 3 mm and the thickness was 10 μm. Then a thickness of 3 with a hole with a diameter of 9 mm
An adhesive was applied to the back of a polyvinyl chloride plate having a size of mm, and the polyvinyl chloride plate was overlaid and adhered to the substrate as shown in FIG.

Using the enzyme electrode sensor thus prepared, the final concentrations of glucose were 0, 0.5 and 1.0 mg.
The cyclic voltammogram at the time of / ml was measured. In addition, platinum was used as the counter electrode and Ag / AgCl was used as the counter electrode counter electrode. The results are shown in the graph of FIG. The current value measured at each glucose concentration at the potential of +700 mV is shown in the graph of FIG.

[0070]

EFFECTS OF THE INVENTION The composition for enzyme electrode of the present invention does not reduce the amount of change in conductivity even when immobilized on a substrate, so that a calibration curve can be prepared easily and the operation is simple and highly accurate. Provide an enzyme sensor.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a state in which a mediator is introduced into the main body and / or side chain of an enzyme.

FIG. 2 shows a preferred embodiment of a sensor electrode according to the present invention. FIG. 2A is a monopolar electrode obtained by forming the enzyme-immobilized electrode 2 on the substrate 1, and FIG. 2B is a perspective view thereof. In addition, FIG. 2C shows a counter electrode formed by forming the enzyme-immobilized electrode 2 and applying a composition having the same composition as the composition for the enzyme electrode except that it does not contain a conductive enzyme in the same manner. 3 is a bipolar electrode provided on the same base material 1, and FIG. 2 (d) is a perspective view thereof.

FIG. 3 is a schematic view of a sensor including a sensor electrode according to the present invention.

FIG. 4 shows another preferred embodiment of the sensor electrode according to the present invention.

5 is a schematic view showing an example of a manufacturing process of the sensor electrode in FIG.

6 is a cyclic voltammogram of GOD in which ferrocene was introduced into the side chain obtained in Example 1. FIG.

7 is a cyclic voltammogram of GOD in which ferrocene was introduced into the side chain obtained in Example 2. FIG.

FIG. 8 is a cyclic voltammogram of GOD in which ferrocene was introduced into the main body and side chain obtained in Example 4.

FIG. 9 is a cyclic voltammogram of GOD in which ferrocene was introduced into the main body and side chain obtained in Example 5.

FIG. 10 is a cyclic voltammogram of GOD in which ferrocene was introduced into the main body obtained in Example 6.

11 is a cyclic voltammogram of GOD in which ferrocene was introduced into the main body obtained in Example 7. FIG.

FIG. 12 is a cyclic voltammogram of the GOD into which the main body and the side chain ferrocene obtained in Example 3 were introduced when the carbon number of the spacer was n = 2 or n = 4.

FIG. 13 is a cyclic voltammogram of GOD in which ferrocene was introduced into the main body and side chains obtained in Example 3, when the glucose concentration was changed.

FIG. 14 is a cyclic voltammogram of a system in which GOD and mediator are simply mixed.

FIG. 15 is a cyclic voltammogram of a composition comprising a conductive enzyme and a resin. The amount of glucose added was 40 μl.

FIG. 16 is a cyclic voltammogram of a composition containing a conductive enzyme, carbon powder, a buffer, a resin, a surfactant, butyl celloslub, and amyl alcohol. The amount of glucose added was 40 μl.

17 is a cyclic voltammogram obtained by the enzyme electrode sensor using the conductive enzyme obtained in Example 2. FIG.

FIG. 18 is a graph of the current value at each glucose concentration when the potential is +700 mV by the enzyme electrode sensor using the conductive enzyme obtained in Example 2.

[Explanation of symbols]

 1 ... Substrate 2, 6 ... Enzyme-immobilized electrode 3 ... Counter electrode 4 ... Main body 5 ... Electrode 7 ... Well 8 ... Substrate 9 ... Gold electrode 10 ... Hole 11 ... Plate

Claims (11)

[Claims] 1. A composition for an enzyme electrode comprising a conductive enzyme and a resin composition containing a hydrophilic resin and / or a hydrophobic resin. 2. The hydrophilic resin is polyvinylpyrrolidrin and the hydrophobic resin is polyvinyl butyral.
The composition for an enzyme electrode according to claim 1. 3. The composition for an enzyme electrode according to claim 1, wherein the conductive enzyme has a mediator supported by an enzyme through a covalent bond. 4. The composition for an enzyme electrode according to claim 3, wherein the enzyme is an oxidase. 5. The enzyme electrode according to claim 4, wherein the oxidase is selected from the group consisting of glucose oxidase, galactose oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase and choline oxidase. Composition. 6. The composition for an enzyme electrode according to claim 3, wherein the mediator is selected from the group consisting of ferrocene, ferrocene derivatives, nicotinamide derivatives, flavin derivatives, quinones and quinone derivatives. 7. The composition for an enzyme electrode according to claim 3, wherein the mediator is introduced into the side chain of the enzyme and / or the enzyme body. 8. The composition for an enzyme electrode according to claim 7, wherein the mediator is introduced into the side chain of the enzyme via a spacer represented by the following formula (I). [Chemical 1] (Here, n represents an integer of 1 to 7.) 9. The enzyme is glucose oxidase, the mediator is ferrocene or a ferrocene derivative, and the mediator of the enzyme is present in the enzyme body and via the spacer represented by the formula (I) defined in claim 8. The enzyme electrode composition according to claim 8, which is introduced into a side chain. 10. An enzyme-immobilized electrode comprising a base material carrying the composition for an enzyme electrode according to claim 1. 11. An enzyme-immobilized electrode having the composition for an enzyme electrode according to any one of claims 1 to 9 inside a well.