JP2636637B2 - Manufacturing method of enzyme electrode - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an enzyme electrode, and more particularly, to a method for producing an enzyme electrode suitable for use in measuring the concentration of a trace amount of a biological substrate contained in body fluid components such as blood and urine. About.

[0002]

2. Description of the Related Art Analytical methods utilizing the excellent substrate specificity of enzymes have received attention in the fields of clinical analytical chemistry, food production, environmental chemistry, and the like. Particularly, in the field of clinical analytical chemistry, an enzyme electrode capable of selectively detecting glucose, urea, uric acid, and the like has been conventionally known.

[0003] These enzyme electrodes are generally composed of an electrode and an enzyme-immobilized membrane. By reading a change in a substance due to an enzyme reaction as a change in an electric signal by the electrode, the concentration of a substrate on which the enzyme specifically acts is determined. Is measured. For example, in a glucose sensor, by monitoring an electrode-active substance such as hydrogen peroxide or oxygen produced or consumed by an enzymatic reaction as shown in the following equation,
Measure biosubstrate concentration.

[0004]

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However, the enzyme electrode based on such a principle has the following problems. As is clear from the above formula, for the substrate to react,
Although stoichiometric oxygen is required, in actual measurement, for example, when measuring the blood glucose concentration of a diabetic patient with a glucose sensor, the amount of dissolved oxygen in the body fluid is insufficient. Therefore, measures such as diluting the sample blood and supplying oxygen by some method are taken.

In the case of electrically monitoring hydrogen peroxide, if a substance which is oxidized at the same potential as that of hydrogen peroxide, for example, a reducing substance such as ascorbic acid, is present in the sample solution, these interfering substances are added to the measured current. The oxidation current of the sample is reflected, causing a measurement error. Therefore, in order to eliminate these errors, the electrode on which the enzyme is not immobilized is corrected as a compensation electrode, or oxygen, hydrogen peroxide molecules and the measurement substrate are transmitted, but electrode active buffer substances such as ascorbic acid are transmitted. It was necessary to attach a permselective membrane that was not allowed to do so.

As described above, a sensor based on the principle of measuring the concentration of a substance produced or consumed in an enzymatic reaction has problems such as the influence of dissolved oxygen and the influence of interfering substances. In addition, since it is necessary to mount the enzyme-immobilized membrane on an oxygen or hydrogen peroxide electrode, there is a limit to miniaturization.

On the other hand, in order to solve these problems, an enzyme electrode using a conductive polymer and an enzyme electrode using an electron mediator have been proposed. The former, polypyrrole, during the electropolymerization of conductive polymers such as polyaniline,
The enzyme is coexisted in the monomer solution, and the enzyme is trapped in the polymerized film at the time of polymerization, or the enzyme-immobilized film is provided by a known method on a prepolymerized conductive polymer film to form a conductive enzyme-immobilized film. What you get. In addition, an enzyme electrode using an electron mediator encloses an electron mediator such as ferrocenes, benzoquinone, ferricyanide ion, N-methylphenazinium in a carbon paste, etc., and immobilizes the enzyme on the carbon paste electrode surface, It is covered with a suitable polymer film.

[0009] However, an enzyme electrode that directly detects electron transfer accompanying an enzymatic reaction using a conductive polymer has the advantage that it is not affected by dissolved oxygen, but has a low response.
There are problems such as a long response time. Furthermore, when taking a method such as capturing an enzyme in a polymerized film during electrolytic polymerization,
It is difficult to control the amount of the immobilized enzyme, and when it is used as an enzyme electrode, it is inevitable that the substrate responsiveness decreases with time due to the desorption of the enzyme. In addition, the conventional enzyme electrode using an electron mediator has low conductivity and is inadequate in terms of responsiveness and response time.In addition, since the electron mediator is in a form dispersed in carbon paste, elution of the electron mediator, There is a problem that the responsiveness decreases with time due to the desorption.

Therefore, the present inventor has previously proposed an enzyme electrode in which a conductive layer containing an organic charge transfer complex crystal is provided on the surface of a conductive substrate in order to solve these drawbacks of the conventional enzyme electrode. (Japanese Patent Application No. 2-244840 (Japanese Unexamined Patent Application Publication No.
No. 22849))). This enzyme electrode has the advantage of not being affected by dissolved oxygen and being less affected by interfering substances by adopting a method of directly detecting electron transfer accompanying the enzymatic reaction. This has the advantage that an accurate response can be given. In addition, the present inventors have further improved this enzyme electrode, and have proposed that by combining an organic charge transfer complex and an electron mediator, electron transfer accompanying the enzyme reaction can be efficiently performed to further improve responsiveness. (Japanese Patent Application No. 3-7908 (JP-A-4-240558), Japanese Patent Application No. 3-86884 (JP-A-4-319658)).

By the way, as a specific application of a biosensor using an enzyme electrode, for example, in consideration of home blood glucose measurement of a diabetic patient, which has been increasing in recent years, cost reduction and easy operability of the biosensor are required. Becomes Conventional enzyme electrodes take a batch or flow type, which requires a large-scale apparatus and high cost.
Not only is it unsuitable for home use, but also because the reproducibility of individual electrodes at the time of enzyme electrode production is insufficient, calibration is required for each measurement, resulting in a problem that the operation becomes complicated.

[0012]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for producing an enzyme electrode at low cost and excellent in reproducibility.

[0013]

Means for Solving the Problems The present inventors have developed an enzyme electrode using an organic charge transfer complex as an electrode material.
The present inventors have found that the oxidized electron mediator can be arranged with high reproducibility in the vicinity of the enzyme by utilizing the printing and coating technique using the polymer thick film technique, and completed the present invention.

The gist of the present invention is that an enzyme electrode comprising (A) a step of forming an organic charge transfer complex on a conductive substrate, and (B) a step of forming a layer containing an enzyme and an electron mediator on the charge transfer complex. A method for producing an enzyme electrode, wherein part or all of the step (B) is performed by a printing method in the production method. Preferred embodiments of the method of the present invention include the following cases.

The step (B) comprises: (1) a step of applying an enzyme on the charge transfer complex; (2) a step of applying a composition containing a reduced mediator thereon; and (3) a step of applying the reduced mediator. A part of or the whole is changed to an oxidized form. The step (B) includes (1) a step of applying an enzyme on the charge transfer complex, and (2) a step of applying a composition containing at least an oxidized mediator thereon. The step (B) includes a step of applying a composition containing at least an enzyme and an oxidized mediator on the charge transfer complex.

The present inventors have also proposed a miniaturized biosensor in which a counter electrode is arranged near an enzyme electrode using an organic charge transfer complex as an electrode material. This biosensor does not require dilution and stirring of a sample, and can accurately measure a small amount of a sample. If the manufacturing method of the present invention is applied to this miniaturized biosensor, a biosensor which can be measured directly by dripping even a small amount of sample without the need for dilution and stirring of the sample can be manufactured with good reproducibility and low cost.

[0017]

In the enzyme electrode in which an organic charge transfer complex and an electron mediator are combined, it is considered that most of the detected response current can be quantified according to the following reaction scheme. Here, the case of an oxidase using tetracyanoquinodimethane (hereinafter, referred to as TCNQ) as an organic electron acceptor is shown.

[0018]

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Enz (ox): Enzyme (oxidized form) Enz (red): 〃 (Reduced form) Med (red): Reduced mediator Med (ox): Oxidized form It comprises (A) a step of forming an organic charge transfer complex on a conductive substrate, and (B) a step of applying an enzyme and a mediator. In order to form an organic charge transfer complex on a conductive substrate, it is preferable to form the organic charge transfer complex directly on the surface of the conductive substrate. For example, by the method described below, the complex crystal can be easily obtained by growing the complex crystal in the thickness direction, and can have good conductivity in the thickness direction.

Examples of the conductive substrate include a metal such as copper, silver, platinum, and gold, a carbon electrode, a substrate having a conductive layer made of such a conductive material provided on the surface by means of vapor deposition or the like, Substrates made from a paste containing a powder of a conductive material can be used. Here, the organic charge transfer complex (hereinafter, referred to as an organic CT complex) is a compound formed from an organic electron acceptor and an electron donor by a charge transfer reaction between the two.

The organic electron acceptor used for forming the organic CT complex is not particularly limited, but is preferably a compound having a cyanomethylene functional group, and particularly preferably a compound having a dicyanomethylene functional group and a quinone or naphthoquinone skeleton. It is. Among them, especially 7,7 ', 8,8'-
Tetracyanoquinodimethane (TCNQ) has a strong CT complex forming ability and the obtained organic CT complex has high electrical conductivity, which is advantageous in response time and responsiveness. It is also preferable because it is relatively easily available commercially.

The electron donor used for forming the organic CT complex is not particularly limited as long as it can form a CT complex having conductivity with the organic electron acceptor used. Any of the above can be used. Specifically, as the inorganic material, copper, silver, cobalt, nickel,
Iron, manganese and the like, and organic materials include tetracenes such as tetrathiafulvalene and tetraselenofulvalene, and derivatives thereof, and known electron donors such as 2,2′-bispyridinium and N-methylphenazinium. Can be used.

For growing the organic CT complex crystal, known methods in a liquid phase and a gas phase can be used. Organic in liquid phase
Methods for growing CT complex crystals include, for example, the following methods. First, a substrate provided with an electron donor layer on its surface or a part or all of a substrate such as a copper plate which also functions as an electron donor is brought into contact with a solution containing an organic electron acceptor. As a result, the organic CT complex electron acceptor in the solution undergoes a CT complexation reaction with the electron donor constituting the surface of the substrate, and the complex grows.

As the solvent used for preparing the solution containing the organic electron acceptor, a polar aprotic solvent such as acetonitrile, dioxane, N, N-dimethylformamide,
Dimethyl sulfoxide, hexamethylphosphoramide,
Methyl ethyl ketone and the like are preferred. The concentration of the organic electron acceptor in this solution is usually based on 100 parts by weight of the solvent.
A suitable range is from 0.01 parts by weight to saturated concentration, preferably from 0.1 parts by weight to saturated concentration.

The formation of the organic CT complex is usually carried out at a temperature of 10 to 30 ° C. However, depending on the combination of the organic electron acceptor used and the electron donor on the surface of the substrate, the CT complex formation reaction proceeds rapidly, and It may be difficult to obtain a uniform target layer. In such a case, if necessary, the temperature of the solution, the substrate, and the atmosphere may be lowered, or the concentration of the solution may be lowered. Conversely, when the complexing reaction is slow and it takes a long time for the organic CT complex crystal to grow to a required thickness, heating can be performed if necessary. The contact time of the organic electron acceptor-containing solution greatly depends on the combination of the organic electron acceptor and the electron donor to be used and the desired thickness of the conductive layer, but is generally about 10 seconds to 1 hour.

In general, a vacuum deposition method can be used as the vapor phase growth method. First, a complex crystal provided with an electron donor layer on the substrate surface or a copper plate which also functions as an electron donor is placed under reduced pressure (1 × 10 ⁻³ to 1 × 10 ⁻⁷ Torr), and the complex crystal is formed. Set the part to be grown at an appropriate temperature (100 to 300
(° C). Next, the electron acceptor is gradually heated and vaporized. As a result, a complex grows by a complexation reaction between the electron acceptor molecule reaching the substrate surface and the electron donor on the substrate surface. At this time, the thickness of the conductive layer can be easily controlled by the temperature of the substrate, the evaporation rate of the electron acceptor, and the like.

By the way, as is clear from the above reaction scheme, an electrochemically neutral electron acceptor (TCNQ ⁰ ) is required in the electron transfer process accompanying the enzymatic reaction. Therefore, in the step of forming an organic charge transfer complex on a conductive substrate, by allowing a neutral electron acceptor to exist,
Electron transfer can be performed more efficiently. For this purpose, when manufacturing in a liquid phase, application and drying of a solution containing a neutral electron acceptor can be easily repeated,
A neutral electron acceptor can be forced to be present.
In the case of manufacturing in a gas phase, it is possible to make a neutral electron acceptor exist by forming a conductive charge transfer complex and then depositing an electron acceptor without heating. .

The organic CT complex prepared by the liquid phase method or the gas phase method as described above generally becomes fine needle-like crystals,
It grows perpendicular to the substrate surface. The thickness of the conductive layer made of this organic CT complex is not particularly limited,
Usually, it is in the range of 0.01 to 50 μm, preferably 0.1 to 10 μm.
m.

As described above, since the conductive layer is made of fine needle-like crystals grown in the thickness direction, the surface of the conductive layer has a structure having fine irregularities. Therefore, when immobilizing enzymes and electron mediators described below, the immobilization of the enzymes and electron mediators is facilitated by capturing the enzymes and electron mediators in these minute recesses. In addition, because of the fine needle-like crystals, the actual surface area of the electrode portion can be increased, and as a result, the amount of immobilization of the enzyme and the electron mediator is increased, and the current density obtained as the output of the enzyme electrode is increased. It becomes possible.

If the thickness of the conductive layer is less than the above range, a sufficient surface area cannot be obtained, resulting in a small output current value. On the other hand, if the thickness exceeds the above range and is too thick, the resistance value of the conductive layer itself increases. Therefore, when used as an enzyme electrode, a voltage drop occurs on the electrode surface when a voltage is applied. Also, this organic
Since the mechanical strength of the CT complex itself is not large, if it is too thick, structural defects are likely to occur.

Next, a method for forming a layer containing an enzyme and an electron mediator will be described. A first preferred embodiment is
The method comprises (1) a step of applying an enzyme, (2) a step of applying a composition containing a reduced mediator, and (3) a step of changing a part or all of the reduced mediator to an oxidized form.

The enzyme can be present on the organic charge transfer complex using a known covalent bonding method, ion bonding method, adsorption method, entrapment method, cross-linking method, etc., but an aqueous solution containing the enzyme can be used. The method of coating on the organic charge transfer complex is simple and suitable. Specifically, for example, a predetermined amount of the enzyme solution may be simply dropped with a pipette or the like, but from the viewpoint of reproducibility,
By using a known printing technique such as a screen printing method, an offset printing method, and an ink jet method, it is possible to apply a fixed amount to a fixed area, which is more preferable. In the case of the printing method, the viscosity can be adjusted to a range required for printing by dissolving or dispersing the enzyme in water or an organic solvent, and adding a water-soluble or water-insoluble polymer as needed.

The enzyme to be used can be appropriately selected in consideration of the substrate specificity and reaction specificity of the enzyme according to the target substance and the intended chemical reaction. Enzymes that can be used are not particularly limited, for example, glucose oxidase, alcohol dehydrogenase, peroxidase,
Catalase, lactate dehydrogenase, galactose oxidase, penicillinase and the like can be mentioned. A combination of an oxidoreductase and a coenzyme is also possible.

Next, a mediator is placed near the enzyme by forming a layer containing a reduced mediator. The electron mediator used can efficiently perform the electron transfer accompanying the enzyme reaction, that is,
Any material that smoothly transfers electrons from the enzyme to the organic CT complex may be used. 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, the electron mediator itself is reduced, and electrons are supplied to the electrode by an electrode reaction on the conductive layer surface. ,
It has the property of returning to the oxidized form. Examples of such an electron mediator include ferrocene, 1,1′-dimethylferrocene, ferrocene carboxylic acid, ferrocene derivatives such as ferrocene carboxaldehyde, hydroquinone, chloranil, quinones such as bromanil, ferricyan ion, octacyanotungstate ion, Metal complex ions such as octacyanomolybdate ions are suitable.

The application of these electron mediators is usually performed in the form of a solution. In the case of a ferrocene derivative, for example,
In the reduced state, it is easily soluble in many organic solvents, but in the oxidized state, it is often hardly soluble in the organic solvent. Therefore, when an attempt is made to immobilize using a water-insoluble polymer, the solution is in a molecular dispersed state in a reduced state and a particle dispersed state in an oxidized state. To smoothly transfer electrons from the enzyme, it is important to arrange a mediator closer to the oxidation-reduction site of the enzyme. From such a viewpoint, it is desirable to apply the electron mediator in a molecularly dispersed state.

The reduction mediator is applied by a method in which a composition is prepared by dissolving the above-mentioned reduction electron mediator and a water-insoluble polymer in a suitable solvent, followed by printing and coating. Here, the amount of the reduced mediator to be used is usually in the range of 20 to 300 parts by weight, preferably 50 to 200 parts by weight, per 100 parts by weight of the water-insoluble polymer. If it is less than 20 parts by weight, the mediator amount will be insufficient,
Electron transfer cannot be performed sufficiently. Also, 300
If the amount is more than part by weight, the amount of the polymer is insufficient, so that not only is it difficult to obtain a uniform film in the coating process, but also the elution of oxygen or a mediator existing inside is caused.

The above composition containing the reduced mediator is printed on a required portion by a known coating method. The coating method is not particularly limited, but a screen printing method is preferable from the viewpoint of the reproducibility of the obtained enzyme electrode. After printing, coating and drying, the organic solvent is distilled off to obtain a layer containing a reduced mediator. The thickness of the layer containing the reduced mediator is usually 0.01 to 50 μm,
It is preferably about 0.1 to 5 μm. If it is less than 0.01 μm, it is difficult to make the necessary amount of the mediator exist, and it is also difficult to apply it uniformly. On the contrary, 50 μm
If it exceeds, the thickness of the mediator layer increases, which hinders the diffusion of the substrate during the measurement, and as a result, the sensitivity is reduced or the linearity of the response is poor.

The water-insoluble polymer used for coating the enzyme or the reducing mediator can be easily and uniformly formed into a film, and uniformly disperses and fixes the enzyme and the electron mediator, and serves as an enzyme electrode. When used, any material can be used without limitation as long as it does not dissolve and swell in the sample solution and does not cause a decrease in output due to elution of enzymes and electron mediators. Furthermore, if a pinhole is formed in the conductive layer, there is a possibility that the substrate or the electron donor may be eluted into the working solution when used as an enzyme electrode. Elution is prevented by covering.

Examples of such a water-insoluble polymer include thermoplastic polymers such as polyvinyl butyral, polyester, polyamide and polyester amide.
Species or two or more can be used. Depending on the method of immobilizing the enzyme and the electron mediator, the polymer can be appropriately selected in consideration of the diffusivity of the substrate and the like.For example, the polymer vinyl butyral has hydrophilicity, water absorbency while being insoluble in water, and It is suitable because it has very micro pores.

The step of changing a part or all of the reduced mediator to an oxidized form is a step of bringing a neutral electron acceptor into contact with the mediator layer to precipitate an oxidized mediator in the mediator layer. The electron acceptor used has the property of promptly causing a redox reaction with the reducing mediator used, receiving electrons, transforming the mediator to an oxidized type, and diffusing and penetrating into the reduced mediator layer. There is no particular limitation as long as it has the ability to make it work. From such a viewpoint, an inorganic electron acceptor such as a halogen such as iodine or bromine can be used in addition to the electron acceptor exemplified in the step of forming the organic charge transfer complex on the conductive substrate.

Usually, these electron acceptors are dissolved in an appropriate solvent, and coated and printed on the reduced mediator layer. The solvent used at this time has a dissolving power for the electron acceptor, has a low solubility for the polymer constituting the reduced mediator layer, and does not significantly disturb the structure of the polymer layer. I just need. As the electron acceptor and its solvent, for example, when polyvinyl butyral is used as a polymer and a ferrocene derivative is used as a mediator, TCNQ is used as an electron acceptor, and a nitrile-based solvent such as acetonitrile has the above characteristics as the solvent. It is.

The solution containing the electron acceptor is usually 0.01% by weight.
The concentration is adjusted to a saturation concentration, preferably 0.05 to a saturation concentration.
If the concentration is below this range, the reduced mediator cannot be sufficiently converted to the oxidized form.If the concentration exceeds this range, an unnecessarily neutral electron acceptor will be present on the enzyme electrode surface, and May hinder diffusion.
As a method for applying the solution containing the electron acceptor, a method such as screen printing, offset printing, and an ink jet method can be adopted in addition to a method of simply dropping with a pipette or the like. In addition, depending on the printing method, a polymer compound can be added to adjust the viscosity to a required value. The application of the electron acceptor can usually be performed at room temperature, but if the reaction with the reduced mediator is extremely slow, it may be heated if necessary, or conversely, if the reaction is very fast, it may be cooled, Its speed can be adjusted.

In a second preferred embodiment, an enzyme is applied on the organic CT complex, and then a layer containing at least an oxidized mediator is formed. The method includes the second step described above,
The third step is one step. That is, a composition containing a mediator previously oxidized is used. In general, as the number of manufacturing processes increases, the variation in characteristics among manufacturing lots and within lots increases. Therefore, to improve reproducibility at the time of manufacturing, reducing the number of steps is one improvement measure. From this point of view, this method is advantageous.

In this method, the method of applying the enzyme is as follows.
This is the same as the method of the first preferred embodiment described above. The layer containing the oxidized mediator in this method is usually
Contains high molecular components. As the oxidized mediator, the above-mentioned reduced electron mediator is previously oxidized by an oxidation-reduction reaction with an electron acceptor. Further, as the polymer component, a water-insoluble polymer such as the above-mentioned polyvinyl butyral can be used, and when disposable, a water-soluble polymer can be used.

These oxidized mediators and polymer components can be dissolved or dispersed in a suitable solvent to prepare a coating composition. The solvent used at this time is not particularly limited as long as it can dissolve the polymer component. The content of the oxidized mediator in the composition is usually 20 to 300 parts by weight, preferably 50 to 200 parts by weight, based on 100 parts by weight of the polymer component. If the amount is less than 20 parts by weight, the amount of the mediator is insufficient and electrons cannot be transferred sufficiently.On the other hand, if the amount exceeds 300 parts by weight, the amount of the polymer component is insufficient and a uniform film is formed in the coating process. Is difficult to obtain, and may cause the elution of enzymes and mediators present inside. The amount of the solvent used can be appropriately adjusted in order to secure the viscosity required for the coating method.

In addition, a neutral electron acceptor (TCNQ ⁰ ) required in the above-mentioned reaction scheme is added to the above composition in an amount of 5 to 50.
By containing about 10 wt% and applying simultaneously, it is possible to more effectively capture the electron transfer accompanying the enzyme reaction. Further, an additive such as a dispersant may be added. The above composition is prepared by an appropriate kneading method such as a milk bee, a three-roll mill, a kneader, a mixer, etc., to obtain a uniformly dispersed composition.

As a coating method, a known method can be applied as described above, but a screen printing method is preferable from the viewpoint of reproducibility of production and mass productivity. After coating in this manner, the solvent is distilled off by heating, drying, or the like to obtain a layer containing an oxidized mediator. The thickness of the layer containing the oxidized mediator is usually about 0.01 to 50 μm, preferably about 0.1 to 5 μm. If it is less than 0.01 μm, it is difficult to make the necessary amount of the mediator exist, and it is also difficult to apply it uniformly. Conversely, if it exceeds 50 μm, it will hinder the diffusion of the substrate.

In a third preferred embodiment, the first step and the second step in the second embodiment are combined into one step. That is, a composition containing an enzyme and a pre-oxidized mediator is applied on an organic CT. Thus, reducing the number of steps is advantageous for manufacturing stability.

The composition used here can be easily obtained by adding an enzyme to the composition containing an oxidized mediator in the above-mentioned second method. The amount of the enzyme to be added is the composition 10 containing the oxidized mediator described above.
1 to 50 parts by weight, preferably 5 to 0 parts by weight
Parts by weight to 30 parts by weight. If the amount is less than 1 part by weight, the amount of the enzyme present in the layer is small and a sufficient substrate response cannot be obtained.
If the amount is more than 50 parts by weight, the mediator amount is relatively insufficient and the linearity of the response is reduced. The composition used here can form a layer containing an enzyme and an oxidized mediator using the same blending method and coating method as described in the second method.

According to the method of the present invention as described above, an enzyme electrode can be manufactured with good reproducibility and at low cost. Since this enzyme electrode has small variations during manufacture, it is not necessary to calibrate the individual electrodes at the time of measurement, and the enzyme electrode can be easily used for home blood glucose measurement and the like at home.

The enzyme electrode obtained by the method of the present invention comprises:
It has a structure in which an enzyme and an oxidized mediator are brought into direct contact with an organic CT complex, and is structurally simpler than conventional hydrogen peroxide electrodes, oxygen electrodes, etc., and can be miniaturized. Also,
The conductive layer made of organic CT complex crystals not only facilitates electron transfer to and from the enzyme, but also has a remarkably higher electrical conductivity than the ferrocenes used as electron mediators. large. This is because these organic
It is considered that the number of conductive paths in the film increases even with the same content because the CT complex constitutes a developed needle-like crystal, which effectively contributes to electron transfer. In addition, since the electron mediator is immobilized on the surface of the conductive layer, smooth electron mobility is ensured even in areas where electron transfer does not occur structurally despite the contact between the organic CT complex and the enzyme. Thus, the responsiveness can be improved. Also organic
When a CT complex crystal is grown directly on a conductive substrate, a fine irregular surface of needle-like crystals can be obtained, so that the amount of enzymes and electron mediators that directly contact the conductive layer can be increased, and the response of the enzyme electrode can be increased. Properties can be further enhanced.

The enzyme electrode obtained by the method of the present invention is used as a measurement electrode and a counter electrode is provided in the vicinity thereof, for example, to form a sensor as shown in FIG. It is also easy to miniaturize.
In such a sensor, measurement can be performed by covering the measurement electrode and the counter electrode, or furthermore, the compensation electrode with a sample, so that even a small amount of sample can be easily measured by, for example, directly dropping the sensor on the sensor. Here, the compensation electrode is an electrode provided to prevent the influence of electrochemical side reactions and adsorption other than the target enzyme reaction and having the same resistance as the measurement electrode.

Note that the measurement using the biosensor as described above can be performed without using a reference electrode. In such a case, it is desirable that the area of the counter electrode is at least twice, preferably at least 10 times, the area of the measurement electrode. This is because the potential difference applied at the time of measurement is mainly applied to the measurement electrode, so that quantification can be performed with high accuracy.

When a potential is applied to the biosensor to measure a response current due to an enzymatic reaction, it is preferable to apply a pulse potential. When a steady state current is applied, the surface state of the electrode changes due to an electrochemical reaction other than the intended purpose.
Measurement errors are likely to occur. If a pulse potential is applied,
It is preferable because such deterioration of the electrode can be minimized and the stabilization time is short. In the measurement using a biosensor provided with a compensation electrode, a predetermined potential is continuously or simultaneously applied between the measurement electrode and the counter electrode, and between the compensation electrode and the counter electrode, whereby a current flowing between both electrodes is applied. Measure the value,
The difference in the current value between the two can be quantified as the response current due to the enzyme reaction.

A biosensor using an enzyme electrode according to the method of the present invention can measure sugars such as glucose, lactic acid, alcohol and other trace biological substances in blood and urine, and sugars and alcohols in a food processing process.

In a conventional biosensor, when measuring, dilution,
It was necessary to stir, but with the miniaturized biosensor as described above, the sample can be measured as it is without diluting and stirring, and the above substances are selectively analyzed with high precision and over a long period of time. It is possible.

[0057]

【Example】

[0058]

[Example 1] Copper-clad glass epoxy substrate (R made by Matsushita Electric Works)
-1701) was etched to form a measurement electrode having the shape shown in FIG.
(1.5 mm in diameter) and a counter electrode were formed, and the entire surface was electrolytic silver plated to form an electrode. Next, portions other than the electrode portion were screen-printed with an epoxy resin paint and molded. The counter electrode was anodic-polarized in 0.1 M hydrochloric acid at a current density of 0.4 mA / cm ^-2 for 2 minutes to deposit silver chloride on the surface.

1.0 g of 7,7 ′, 8,8′-tetracyanoquinodimethane (reagent, manufactured by Kishida Chemical Co., Ltd .; hereinafter abbreviated as TCNQ) was added to 10 mg of acetonitrile (reagent, for spectrum), and a saturated solution of TCNQ was added. Prepared. 2 μl of the TCNQ saturated solution was measured, dropped at the measurement electrode portion of the electrode at room temperature, and air-dried. This operation was repeated a total of five times to obtain an organic CT complex thin film having dark purple fine needle-like crystals over the entire surface of the measurement electrode.

Glucose oxidase (Aspergillus ni
ger, Sigma, Type VII) (40 mg) was dissolved in 1 ml of 100 mM phosphate buffer (pH 7.0), 4 μl was measured, and the measurement electrode was air-dried.

1.0 g of polyvinyl butyral resin (trade name: ESREC B, BX-L, manufactured by Sekisui Chemical Co., Ltd.) and 1,1'-dimethylferrocene [reagent, Tokyo Chemical Industry Co., Ltd.]
Was dissolved in 2.5 g of ethyl carbitol to give a composition containing a reduced mediator. The composition was printed on the measurement electrode using a screen printer, and dried at 80 ° C. for 10 minutes to form a layer containing a reduced mediator having a thickness of about 3 μm.

Next, 1 μl of the above-mentioned TCNQ solution was measured and taken out.
After coating on the measurement electrode and air-drying, it was washed with pure water to obtain an enzyme electrode. Human serum of each glucose concentration shown in FIG.
20 μl was dropped on the electrode system, and the entire surface of the measurement electrode and the counter electrode was covered with a sample solution. After leaving it at room temperature for 1 minute, a pulse potential of 0.25 V was applied to the counter electrode, and the current value was measured 2 seconds after the application of the potential. Was measured.

FIG. 2 shows the average value of the ten electrodes and the range of variation including the maximum and minimum values of the results obtained by measuring the total of ten electrodes produced in the same manner. In this way, a good linear relationship is obtained up to a glucose concentration of about 30 mM, and there is little variation among the ten electrodes.

[0064]

Example 2 A conductive organic charge transfer complex thin film was obtained in the same manner as in Example 1, and an enzyme solution was applied in the same manner. TCNQ 5.0g
Was suspended in 300 ml of acetone at room temperature, an acetone solution of dimethylferrocene equivalent to TCNQ was gradually added dropwise, and the mixture was further stirred for 2 hours, filtered and dried to obtain a dimethylferrocene-TCNQ complex.

1.0 g of polyvinyl butyral resin, 0.7 g of the above dimethylferrocene-TCNQ complex and 0.3 g of TCNQ were weighed, and 2.5 g of ethyl carbitol was added. Produced. This was printed on the measuring electrode by a screen printing method and dried in the same manner as in Example 1 to a thickness of about 4 μm.
A layer containing the oxidized mediator was prepared as an enzyme electrode.

Next, FIG. 2 shows the measurement results for ten electrodes in the same manner as in Example 1. As described above, not only good linearity was obtained up to a glucose concentration of about 30 mM, but also the number of steps was reduced compared to Example 1.
The variability between the ten electrodes was reduced.

[0067]

EXAMPLE 3 1.0 g of glucose oxidase was mixed with 4.5 g of the composition containing an oxidized mediator prepared in Example 2, and kneaded with a three-roll mill to obtain a composition containing an enzyme and an oxidized mediator.

The composition obtained in this manner was applied to the measuring electrode by a screen printing method on a conductive organic charge transfer complex thin film prepared in the same manner as in Example 1, and dried. Then, a layer having a thickness of about 5 μm containing the enzyme and the oxidized mediator was formed to form an enzyme electrode.

Next, FIG. 2 shows the results of measurement on ten electrodes in the same manner as in Example 1. As described above, not only good linearity was obtained up to a glucose concentration of about 30 mM, but also the variation between the ten electrodes was significantly reduced due to the further reduction in the number of steps as compared with Examples 1 and 2. .

[0070]

According to the method of the present invention, an enzyme electrode having a good response to a biological sample such as serum up to a high substrate concentration can be produced with good reproducibility. Since the obtained enzyme electrode has a small variation at the time of manufacture, it can be used by a simple operation that does not require calibration of individual electrodes at the time of measurement. In addition, low-cost production and mass production are possible.

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of a biosensor manufactured in an example.

FIG. 2 is a diagram showing a relationship between a glucose concentration measured in Examples 1 to 3 and a response current.

[Explanation of symbols]

 1: measurement electrode 2: counter electrode 3: epoxy resin 4: measurement electrode terminal 5: counter electrode terminal 6: glass epoxy substrate

Claims (4)

(57) [Claims] 1. An enzyme electrode manufacturing method comprising: (A) forming an organic charge transfer complex on a conductive substrate; and (B) forming a layer containing an enzyme and an electron mediator on the charge transfer complex. And (B) performing a part or all of the steps by a printing method. 2. The step (B) comprises: (1) a step of applying an enzyme on the charge transfer complex; (2) a step of applying a composition containing a reduced mediator thereon; and (3) a step of applying the enzyme. The method for producing an enzyme electrode according to claim 1, comprising a step of changing a part or all of the type mediator to an oxidized type. 3. The method according to claim 2, wherein the step (B) includes (1) a step of coating an enzyme on the charge transfer complex, and (2) a step of coating a composition containing at least an oxidized mediator thereon. Item 10. A method for producing an enzyme electrode according to Item 1. 4. The method for producing an enzyme electrode according to claim 1, wherein the step (B) includes a step of applying a composition containing at least an enzyme and an oxidized mediator on the charge transfer complex.