JPH0560723A - Manufacture of enzyme electrode - Google Patents

Abstract

PURPOSE:To improve reproducibility and responsiveness by executing electrolytic polymerization in a buffer solution which is kept at specified pH and by controlling the amount of immobilization of enzyme by the pH and the time of impression of potential for the electrolytic polymerization. CONSTITUTION:An electroconductive base (a metal such as platinum, carbon or the like) as an electrode is brought into contact with a buffer solution (phosphoric acid buffer solution or the like) which contains pyrrole and/or a derivative thereof (obtained by substituting various kind of group for hydrogen at the 3- and 4- positions of pyrrole)(the concentration is about 0.01 to 0.5M), oxidoreductase (glucose oxidase or the like) and an electrolyte (potassium chloride of about 0.01 to 1M or the like) and which is kept at pH 5 to 7, and thereby electrolytic polymerization of the pyrrole and/or the derivative thereof is executed at a constant potential of about 1.0 to 2.0V. On the occasion, the amount of immobilization of enzyme can be controlled highly precisely by the pH of the buffer solution and the time of impression of the potential. According to this constitution, an immobilized-enzyme electrode wherein enzyme is immobilized in a prescribed amount in an electroconductive macromolecular film formed on the surface of the electroconductive base and constituted of polypyrrole and/or the derivative thereof can be prepared.

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 as an enzyme sensor for measuring the concentration of a trace amount of biological substrate contained in body fluid components such as blood and urine. ..

[0002]

2. Description of the Related Art Analytical methods utilizing the excellent substrate specificity of enzymes have attracted attention in the fields of clinical analytical chemistry, food manufacturing, environmental chemistry and the like. In particular, in the field of clinical analytical chemistry, enzyme electrodes capable of selectively detecting glucose, urea, uric acid and the like have been conventionally known. These enzyme electrodes are generally composed of an electrode and an enzyme-immobilized membrane, and the substance change due to the enzyme reaction is read by the electrode as a change amount of an electric signal to measure the concentration of a substrate on which the enzyme specifically acts. It is a thing.

An example of an enzyme electrode is one in which the concentration of a biological substrate is measured by monitoring an electrode active substance such as hydrogen peroxide or oxygen produced or consumed by an enzymatic reaction at the electrode. However, the enzyme electrode based on such a principle has the following problems.

Stoichiometric oxygen is required for the reaction of the substrate, but since the amount of dissolved oxygen is insufficient in the measurement system, the sample blood is diluted or oxygen is supplemented by some method. We need to take steps. When electrically monitoring hydrogen peroxide, if a substance that oxidizes at the same potential as hydrogen peroxide, such as a reducing substance such as ascorbic acid, is present in the sample solution, the oxidation current of these interfering substances is added to the measurement current. Is added, which causes a measurement error. Therefore, various means for removing these errors have been required.

As described above, the sensor based on the principle of measuring the concentration of the substance produced or consumed by the enzymatic reaction has a problem of influence of dissolved oxygen and influence of interfering substances. Further, there is a limit to miniaturization because it requires a form in which an enzyme-immobilized membrane is attached to oxygen and hydrogen peroxide electrodes. On the other hand, in order to solve these problems, an enzyme electrode using a conductive polymer such as polypyrrole or polyaniline and an enzyme electrode using an electron mediator have been proposed. Since these enzyme electrodes directly detect the electron transfer associated with the enzyme reaction, they have the advantage that they are not affected by the amount of dissolved oxygen.

An enzyme electrode using an electron mediator encloses an electron mediator such as ferrocene, benzoquinone, ferricyanide ion, and N-methylphenazinium in carbon paste or the like to immobilize the enzyme on the surface of the carbon paste electrode. , Coated with a suitable polymer film. This is to detect the electron transfer accompanying the enzymatic reaction as the electron transfer in the electron mediator.

[0007] Therefore, in addition to the advantage that it is not affected by the amount of dissolved oxygen, the electrode potential at this time is significantly smaller than the potential when monitoring hydrogen peroxide as described above (about 0.7 V relative to Ag / AgCl) ( Since it is about 0.1 to 0.2 V compared to Ag / AgCl, oxidation of interfering substances is unlikely to occur, and as a result, there is an advantage that highly accurate measurement can be performed. However, in the enzyme electrode using this electron mediator, the conductivity is low and the response and response time are insufficient, and since the electron mediator is in the form of being dispersed in the carbon paste, elution of the electron mediator, This includes the problem of a decrease in responsiveness over time due to desorption.

Further, as a method for producing an enzyme electrode using a conductive polymer, as disclosed in JP-A-64-28556, the enzyme is formed on a previously polymerized conductive polymer film by a known method. There are known methods of immobilizing and methods of capturing an enzyme in a polymerized film during electropolymerization of a conductive polymer, as disclosed in JP-A-62-115285. In the enzyme electrode using these conductive polymers, the electrons generated by the enzymatic reaction are transferred through the π-electron conjugated system of the conductive polymer, so the concentration of the target substance can be measured without depending on the amount of dissolved oxygen. ..

However, in the former method of immobilizing the enzyme on the prepolymerized conductive polymer membrane, the polymerization and the immobilization of the enzyme are separate steps, and in addition to being complicated, the enzyme and the high conductivity of the enzyme are high. Electrons may not be efficiently transferred due to insufficient electrical contact with molecules. On the other hand, the latter,
As a method for trapping an enzyme during electropolymerization of a conductive polymer, the method described in JP-A-62-115285 mentioned above is performed by an electropolymerization method in a neutral aqueous solution. However, with this method, the amount of enzyme immobilized can be controlled only by the content of the enzyme in the reaction solution, and it is difficult to control the amount of enzyme immobilized with high accuracy. The sex was not enough.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides a method for producing an enzyme electrode by trapping an enzyme in a polymerized film during electropolymerization of a conductive polymer, and controlling the amount of immobilized enzyme with high accuracy. Another object of the present invention is to provide a method capable of producing an enzyme electrode with good reproducibility and obtaining an enzyme electrode having excellent responsiveness.

[0011]

The present inventors have completed the present invention by obtaining the following knowledge. When an enzyme is allowed to coexist during the electropolymerization of a conductive polymer and the enzyme is immobilized in the conductive polymer film, the capture of the enzyme in the conductive polymer is
In addition to the enzyme existing near the electrode being physically trapped in the polymer chain during polymerization, the enzyme existing as a weak anion in the aqueous solution acts as a kind of dopant and is electrochemically trapped. It was estimated that this was due to Since the trapping of the enzyme based on this latter action is a competitive reaction with the electrolyte anion in the aqueous solution, it is considered preferable to gradually carry out the polymerization reaction in order to trap more enzyme.

On the other hand, the film forming rate of polypyrrole largely depends on the pH in the aqueous solution, and the film forming rate is improved by lowering the pH. Further, the present inventors, during the electropolymerization, the pH in the vicinity of the electrode gradually changes with the polymerization, the film formation rate of pyrrole, and thus the doping competition reaction cannot be kept constant, and the enzyme immobilization amount It has been found that can not be controlled with high precision. Based on these findings, the present inventors have found that, if electrolytic polymerization is carried out in a buffer solution, the pH in the solution and in the vicinity of the electrodes can be kept constant, and therefore the above-mentioned doping competition reaction can be kept constant. The inventors have found that the amount of immobilized enzyme can be easily controlled depending on the time, and have completed the present invention.

In the present invention, a conductive substrate is brought into contact with a solution containing pyrrole and / or a derivative thereof, a redox enzyme, and an electrolyte to carry out electrolytic polymerization.
In a method for producing an enzyme electrode by immobilizing an enzyme on a polymerized film of polypyrrole and / or its derivative formed on the surface of a conductive substrate, the electrolytic polymerization is performed in a buffer solution maintained at pH 5 to 7, A method for producing an enzyme electrode is characterized in that an enzyme electrode having a predetermined responsiveness is produced with good reproducibility by controlling the amount of enzyme immobilized by controlling the pH of a buffer solution and a potential application time of electrolytic polymerization.

[0014]

As a monomer for forming a conductive polymer by electrolytic polymerization, in addition to pyrrole, those in which hydrogen at the 3- and 4-positions of pyrrole is substituted with various groups, and pyrrole derivatives in which hydrogen on nitrogen is substituted are used. One or more can be used. Mixtures of pyrrole and one or more of its derivatives can also be used. The concentration of pyrrole and / or its derivative is not particularly limited, but is preferably about 0.01 to 0.5M. If the monomer concentration is too low, the formation of the polypyrrole film is poor, and if the monomer concentration is too high, it becomes difficult to dissolve it in water.

The oxidoreductase used in the method of the present invention can be appropriately selected in consideration of the target substance and the intended chemical reaction in consideration of the substrate specificity and reaction specificity of the enzyme. The enzyme that can be used is not particularly limited, and examples thereof include glucose oxidase, alcohol dehydrogenase, peroxidase, catalase, lactate dehydrogenase, galactose oxidase and the like. A combination of oxidoreductase and coenzyme is also possible.

As the electrolyte in the method of the present invention, those generally used as a supporting electrolyte for an aqueous solution can be used. For example, NaCl, KCl, Na ₂ SO ₄ , K ₂ SO ₄ , NaClO ₄ , KC can be used.
Examples include lO ₄ , sodium trifluoroacetate, potassium trifluoroacetate, sodium paratoluenesulfonate, potassium paratoluenesulfonate and the like. The concentration of the supporting electrolyte may be such that the aqueous solution can maintain ionic conductivity, and is usually in the range of 0.01 to 1M, preferably 0.05 to 0.5M.

In the method of the present invention, the buffer solution for maintaining a constant pH in the aqueous solution is maintained at a desired pH, and the component compounds of the buffer solution are electrochemically oxidized or reduced during electrolytic polymerization. It may be stable as long as it does not exist. From this point of view, the buffer solution used in the present invention is preferably a phthalate buffer solution, an acetate buffer solution, a phosphate buffer solution, a citrate buffer solution, or the like.

In the present invention, the electrolytic polymerization reaction is carried out while maintaining the pH of the above buffer solution at 5 to 7. At a pH of 5 or less, the polymerization reaction proceeds rapidly and a film is formed in a short time, so the anion doping of the supporting electrolyte becomes dominant in the above-mentioned doping competition reaction, and sufficient enzyme trapping cannot be performed. On the other hand, at pH 7 or higher, the film formation rate is slow, the film itself becomes non-uniform, and the amount of enzyme trapped increases, but the conductivity of the obtained conductive film itself decreases. As a result, the response current obtained as the enzyme electrode is reduced.

As the conductive substrate to be brought into contact with the buffer solution,
Metal electrodes such as platinum, gold, silver, carbon electrodes, ITO (in
(dium-tin-oxide) It is not particularly limited as long as it does not affect the activity of the enzyme such as a glass electrode.

In the method of the present invention, pyrrole and / or
Or a derivative thereof, including a redox enzyme and an electrolyte,
The above-mentioned conductive substrate is brought into contact with a buffer solution kept at pH 5 to 7 as an electrode to carry out electrolytic polymerization of pyrrole and / or its derivative at a constant potential of about 1.0 to 2.0V. At that time, the amount of enzyme immobilization is controlled by the pH of the buffer solution and the potential application time. As a result, an enzyme-immobilized electrode in which a predetermined amount of enzyme is immobilized in a conductive polymer film made of polypyrrole and / or its derivative can be obtained.

Substances that can be measured with the enzyme electrode of the present invention include sugars such as glucose, trace biological substances in blood and urine such as lactic acid and alcohol, sugars and alcohols in food processing processes. By using the enzyme electrode of the present invention, it is possible to selectively and highly accurately analyze the above substances repeatedly over a long period of time. Also,
Not only the measurement of a substance but also a bioreactor or the like can be used.

[0022]

【Example】

[0023]

Example 1 In the coexistence of 0.1 M pyrrole and 35 mg glucose oxidase (manufactured by Sigma, type VII, 211.2 U / mg),
10 ml of 1/15 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride as an electrolyte was added to a platinum wire (diameter: 0.5 mm,
(Length: 7 mm) as working electrode, platinum foil as counter electrode, and Ag / AgCl as reference electrode at a constant potential of 1.4 V (vs. Ag / AgCl) for 50 minutes to carry out electrolytic polymerization to obtain an enzyme-immobilized electrode. .. As shown in Table 1, the same operation was performed when the potential application time was 100 minutes and 120 minutes.

The obtained enzyme-immobilized electrode was immersed in an 8M urea aqueous solution for 12 hours or more in the dark. Next, the amount of immobilized enzyme was calculated from the fluorescence intensity at 520 nm of the obtained flavin adenine dinucleotide elimination solution. The amount of immobilized enzyme at each potential application time is shown in Table 1.

Using the enzyme-immobilized electrode obtained at a potential application time of 50 minutes as a working electrode, a platinum plate as a counter electrode, and Ag / AgCl as a reference electrode, a potential of 0.5 V (compared with Ag / AgCl) was applied. ,
Substrate responsiveness was measured by dropwise addition of a glucose standard solution (0.1 M) in a 1/15 M phosphate buffer with stirring under nitrogen saturation or air saturation. The result is shown in FIG. As is apparent from FIG. 1, the response current with respect to the glucose concentration maintains the linearity of the normal blood glucose level measurement under both nitrogen saturation and air saturation.

[0026]

Example 2 An enzyme electrode was prepared in the same manner as in Example 1 except that a 1 / 15M phosphate buffer solution having a pH of 6 was used. Table 1 shows the results of the amount of immobilized enzyme on this electrode determined in the same manner as in Example 1.

[0027]

Example 3 An enzyme electrode was prepared in the same manner as in Example 1 except that a 1 / 15M phosphate buffer solution having a pH of 5 was used. Table 1 shows the results of obtaining the amount of immobilized enzyme on this electrode in the same manner as in Example 1.

[0028]

[Comparative Example 1] An enzyme electrode was prepared in the same manner as in Example 1 except that the pH of the electrolytic polymerization solution was 7.5. According to this method, the film formation rate of the polypyrrole film was slow and the film was not uniform.

[0029]

[Comparative Example 2] An enzyme electrode was prepared in the same manner as in Example 1 except that the pH of the electrolytic polymerization solution was 4.5. When the glucose responsiveness was measured using the electrode obtained by this method in the same manner as in Example 1, almost no response was obtained.

[0030]

[Table 1]

[0031]

According to the method for producing an enzyme electrode of the present invention, the electropolymerization of a conductive polymer is carried out in a buffer solution, so that the amount of immobilized enzyme can be controlled with high accuracy by adjusting the pH of the solution and the potential application time. It becomes possible with. Therefore, the reproducibility in the production of the enzyme electrode is improved, and the enzyme electrode capable of measuring the target substance with high accuracy can be produced. Further, the enzyme electrode obtained by the method of the present invention adopts a method of directly detecting the electron transfer accompanying the enzymatic reaction, and therefore is not affected by the amount of dissolved oxygen and is not affected by the electrochemical interfering substance. It is an enzyme electrode with excellent responsiveness.

[Brief description of drawings]

FIG. 1 is a graph showing the substrate responsiveness of the enzyme electrode prepared in Example 1.

Claims (1)

[Claims] 1. A polypyrrole formed on the surface of a conductive substrate by bringing the conductive substrate into contact with a solution containing pyrrole and / or a derivative thereof, a redox enzyme, and an electrolyte to carry out electrolytic polymerization. In the method for producing an enzyme electrode by immobilizing an enzyme on a polymerized film of a derivative thereof and / or a derivative thereof, the electrolytic polymerization is carried out in a buffer solution maintained at pH 5 to 7, and the pH of the buffer solution and a potential application time of the electrolytic polymerization are applied. A method for producing an enzyme electrode, which comprises producing an enzyme electrode having a predetermined responsiveness with good reproducibility by controlling the amount of enzyme immobilized by the method.