JPS6350748A - Enzyme electrode system - Google Patents

Abstract

PURPOSE:To enhance substrate response, by constituting an enzyme electrode system by providing a first acting electrode, a second acting electrode, an opposed electrode and a differential output detection system. CONSTITUTION:A first acting electrode is constituted by providing an immobilized polymer membrane of hydrogen peroxide generating enzyme composed of an electrolytic polymerization polymer membrane on the response part of a hydrogen peroxide electrode. The second acting electrode is constituted by providing a polymer membrane composed of the electrolytic polymerization polymer membrane having no enzyme immobilized thereon or having deactivated hydrogen peroxide generating enzyme immobilized thereon on the response part of the hydrogen peroxide electrode. Further, an opposed electrode constitutes an electrolytic system along with the first and second acting electrodes. In a differential output detection system, the output difference between the electrolytic current in the first acting electrode and that in the second acting electrode is detected and the electrolytic current substantially corresponding to a hydrogen peroxide part, that is, the concn. of a substrate intended to measure is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to an enzyme electrode system. More specifically, the present invention relates to an enzyme electrolyte system that uses a hydrogen peroxide electrode type enzyme electrode and can selectively and quantitatively analyze the concentration of a specific component in a sample quickly and easily.

(Example) Prior Art In recent years, enzyme electrodes have attracted attention as a means of quickly and accurately quantifying the concentration of a specific component in a sample by making good use of the substrate specificity of enzymes.

Among these enzyme electrodes, in the reaction where the substrate consumes dissolved oxygen and produces hydrogen peroxide in the presence of the enzyme, there is an oxygen electrode method that captures the oxygen reduction mechanism, and a hydrogen peroxide electrode that captures the generated hydrogen peroxide. There is a method.

The oxygen electrode method described above uses an enzyme electrode in which an oxygen permeable membrane is attached to the surface of the oxygen electrode and an enzyme with an oxygen generating system is immobilized on this membrane, and has the advantage that the electrode system and the test solution can be completely separated. There is. Recently, a proposal has been made to use a polymer thin film formed by electrolytic polymerization as an enzyme immobilization membrane having an oxygen generating system [[Oxygen permeability and molecular identification function of electrolytic synthesis enzyme membrane], Electrochemistry Collection of Abstracts from the 53rd Conference of the Society C2o3 (1986)).

On the other hand, in the hydrogen peroxide electrode method, hydrogen peroxide needs to reach the electrode surface, so a membrane that allows the test solution to pass through is used.
Hydrogen peroxide mill 1. An enzyme having a hydrogen peroxide generating system is immobilized on this to create a hydrogen peroxide mill. : Depicted as being covered. Therefore, the test solution comes into contact with the electrode surface, and there is a drawback that interference current is generated due to various reducing substances contained in the test solution. Regarding this point, recently two methods have been developed as a means of removing this interference current.
A photopolymerized polymer membrane using a working electrode (hydrogen peroxide electrode) with an enzyme immobilized on one side and an enzyme with no enzyme immobilized or an inactivated enzyme immobilized on the other side. A method has been proposed for detecting the differential output of these working electrodes by coating them [Takatsu Ichi eta 1, "Differential output type glucose sensor", Institute of Electrical Engineers of Japan Research Group Materials (19
1985)).

(c) Problems to be Solved by the Invention However, the linearity limit of the substrate responsiveness of the enzyme electrode of the oxygen electrode method using the enzyme-immobilized membrane produced by electrolytic polymerization is as narrow as about 100++g/dl; 31 of the samples with substrate concentrations exceeding! There were problems in that a large error occurred in the I constant and a dilution operation was required in advance.

On the other hand, even in the hydrogen peroxide electrode type Ff elementary electrode system using the differential output detection method, the good response was at most about 100111 (1/dl), and there were problems similar to those described above.

The present invention has been made to solve these conventional problems, and particularly to provide an enzyme electrode system with improved substrate responsiveness.

(d) Means to Solve the Problems As a result of intensive research, the present inventor has developed the electropolymerized thin film described above, which is used as a medium for an oxygen-permeable enzyme-processed membrane in an enzyme electrode of an oxygen electrode type. Differential output detection - By using it as a medium for the immobilization membrane of the hydrogen peroxide generating enzyme in the hydrogen peroxide electrode type enzyme electrode system,
The present invention was achieved by discovering the fact that substrate responsiveness is significantly improved.

Thus, according to the present invention, (a first working electrode comprising a hydrogen peroxide generating enzyme immobilized polymer film made of an electropolymerized polymer thin film on the sensitive part of a hydrogen peroxide electrode;
+ a second working electrode comprising, on the sensitive part of the hydrogen peroxide electrode, a polymer membrane made of an electropolymerized polymer thin film on which no enzyme is immobilized or an inactivated hydrogen peroxide generating enzyme is immobilized; (c) a counter electrode that forms an electrolytic system with the first and second working electrodes; +d+ a differential output that detects the output difference between the electrolytic current caused by the first working electrode and the electrolytic current caused by the second working electrode; An enzyme electrode system comprising a detection system is provided.

The electropolymerized polymer thin film of this invention uses various noble metals (platinum, gold, etc.) and carbon electrodes that can be used as hydrogen peroxide electrodes as electrodes for electrolysis, and is immersed in an electrolyte solution of seven polymers for polymerization. It can be formed by performing electrolysis to advance polymerization. As the monomer for the crown, any monomer that is water-soluble and polymerizable in an aqueous solution is suitable, and aromatic compounds having functional groups such as hydroxyl and yamino groups are suitable, such as aniline, 〇-phenylenediamine, Examples include phenol.

However, even water-insoluble monomers such as virol and thiophene, which can be polymerized in a non-aqueous electrolytic solution, are also applicable. In addition, it is suitable to add a suitable supporting salt to make the electrolysis proceed smoothly, and this is especially necessary when polymerization is carried out in a non-aqueous solvent system. Further, the hydrogen peroxide electrode is not limited to a rod-shaped electrode, but may be, for example, a film-shaped electrode placed on an insulating substrate. Another advantage of the present invention is that a uniform thin polymer film can be formed regardless of the shape of the electrode, and that an enzyme electrode of a shape depending on the application can be created and used.

The first working electrode in this invention is a main electrode for detecting an electrolytic current corresponding to the amount of hydrogen peroxide generated by the enzymatic reaction of the hydrogen peroxide generating enzyme.

In this first working electrode, a predetermined hydrogen peroxide generating enzyme is contained in the monomer electrolyte solution when forming a thin polymer film on the sensitive part of the hydrogen peroxide electrode by the above-mentioned electrolytic polymerization method. By doing so, it can be produced easily and efficiently.

The electrolytic polymerization at this time is suitably carried out at a mild temperature such as room temperature, and is preferably carried out at around 0°C so as not to reduce the activity of the enzyme as much as possible. Further, as the electrolytic conditions, either a constant potential method or a potential scanning method may be used. However, in some cases, after the polymer thin film is formed, the hydrogen peroxide-generating enzyme may be immobilized on the surface thereof by a known immobilization method such as the Schiff base method. This method is particularly suitable when using a non-aqueous solvent-based electrolytically polymerized membrane.

The thickness of the immobilized polymer membrane is approximately 0.1 to 0.5 mm thick. If the thickness is too thin, it is difficult to control the thickness and the sensitivity tends to decrease, and if the thickness is too thick, hydrogen peroxide is likely to be inhibited from reaching the electrode surface, and production by electrolytic polymerization itself is difficult, which is undesirable.

The second working electrode in this invention is an auxiliary electrode for detecting the influence of various reducing substances in the test solution on the electrolytic current, and the second working electrode is an auxiliary electrode for detecting the influence of various reducing substances in the test solution on the electrolytic current. An electrode-polymerized thin film in which the enzyme is immobilized, either unimmobilized or inactivated, is formed on the sensitive part of the hydrogen peroxide electrode. Here, the deactivated Vf accumulation is immobilized by forming an immobilized polymer film on the electrode surface in the same manner as the first working electrode, and then at a temperature sufficient to deactivate the enzyme (e.g., 100°C). degree)'c It can be easily carried out by heating.

The hydrogen peroxide generating enzyme in this invention has a reaction system that generates hydrogen peroxide, and for example, glucose oxidase (GOD), which uses glucose as a substrate, is a typical example, and other enzymes/substrates are also used. Combinations include uricase/urinary resistance, lactate oxidase/lactic acid, oxalate oxidase/oxalic acid, amino acid oxidase/amino acids, monoamine oxidase/monoamines, pyruvate oxidase/pyruvic acid, alcohol oxidase/alcohol, galactose oxidase/galactose, etc. It will be done. These enzymes are selected depending on the substance (substrate) intended to be detected.

Note that two or more types of these enzymes may be used.

In particular, by combining enzymes from other species that can produce substrates for the above-mentioned hydrogen peroxide-generating enzymes, the types of detectable substances can be increased.

Examples of this include the combination of invertase/mutarotase/GOD using sucrose as the detection substance, and the combination of glucoamylase (or maltase) using maltose as the detection substance.
Examples include a combination of GOD, a combination of cholesterol esterase/cholesterol oxidase using cholesterol ester as a detection substance, a combination of phospholipase/choline oxidase using phosphatidylcholine as a detection substance, and at least any combination that can ultimately generate hydrogen peroxide. Bye.

Measurements using the enzyme electrode system of the present invention are performed based on electrolytic current in constant potential electrolysis between the counter electrode and each working electrode. Here, the electrolytic voltage is between the first working electrode and the second working electrode.
In order for the member bending electrode or carbon electrode, which is the base of the working electrode, to move together with the hydrogen peroxide electrode, it is set to a potential at which hydrogen peroxide is reduced (usually 0.6 V vs. Ag/Ag01 or higher). . Therefore, usually silver-silver chloride ffrf
It is suitable to monitor the electrolytic potential using a reference electrode such as a calomel electrode or the like, and to control the electrolytic potential at a constant level using a potentiostat or the like. However, the counter electrode itself can also act as a reference electrode, and in this case there is no need to use a special reference electrode.

For the counter electrode, the same material as the material Y that can be used for the hydrogen peroxide electrode can be used. It is preferable to use a electrode with a surface area larger than that of the second working electrode (usually 100 times or more) in order to reduce interelectrode resistance. For example, it is suitable to use a platinum black electrode, which has a significantly larger surface area than a platinum electrode. There is.

Furthermore, it is suitable to use an operational amplifier or the like as a differential power output detection system that detects the difference in output between the electrolytic current produced by the first working electrode and the electrolytic current produced by the second working electrode.

(e) Action At the first working electrode, hydrogen peroxide is generated by the immobilized enzyme in the presence of a substrate, and this permeates through the electropolymerized polymer thin film and undergoes reduction on the surface of the hydrogen peroxide electrode. A reduction current (electrolytic current) flows. On the other hand, at the second working electrode, hydrogen peroxide is not generated by the enzyme;
Various reducing substances contained in the sample reach the surface of the hydrogen peroxide electrode, and a current corresponding to the error flows. Then, in a differential output detection system, the output difference between the electrolytic current at the first working electrode and the electrolytic current at the second working electrode is detected, which substantially affects the generated hydrogen peroxide valve and thus the group intended to be measured.
An electrolytic current corresponding to the concentration of 1 is obtained.

(Embodiment Figure 1 shows an example of the configuration of each electrode in the enzyme electrode system of the present invention, where A is a schematic diagram and B is a bottom view. Each of the electrodes is configured as follows. did.

First, at the tip of a cylindrical insulating substrate 1 (made of vinyl chloride resin), measurement electrodes 2 and 3 made of platinum wires with a diameter of 0.5 mm and reference 1 heptode 5 (Ag/AgC
An electrode system was constructed in which a counter electrode 4 made of platinum was attached to the outer periphery of the tip, and a counter electrode 4 made of platinum was attached to the outer periphery of the tip. This electrode system was immersed in an aqueous solution (PH 7, 0) containing the monomers 0, 1 M aniline, and 2 ma, -' ml glu]-rulesidase (GOD). Measuring electrode 2
A polyaniline-〇〇D immobilized (encompassing) polymer film was formed on the exposed surface of the tip. Next, this tip was immersed in an aqueous solution at 100°C for 5 minutes to deactivate GOD in 1 gI of the polymer, thereby producing a second working electrode. Next, measurement electrode 3
Polyaniline-〇〇D is immobilized (inclusive) on the exposed tip end surface of the measurement electrode 3 in the same manner as above by electrolytic polymerization between the electrode 4 and the counter electrode 4.
A first working electrode was set up by forming polymeric FnWA. In the figure, 6a is a polymer thin film immobilized with deactivated GOD;
b1. Each of them showed a polymer 'aIIl with tGOD immobilized thereon, and each had a thickness of about 0.3-.

The enzyme electrode system of this invention using this electrode structure (
A circuit diagram of the glucose concentration detection device (glucose concentration detection device) is shown in FIG. 3, and a circuit diagram is shown in FIG.

FIG. 5 shows the results of measuring changes in response current to glucose concentration using the enzyme electrode system described above. The test solution was prepared at pH 7, 0, 37° C. and without stirring, and the electrolytic potential was set at +0, 7V VSAg/AgC1.

In this way, a calibration curve with good linearity was obtained up to 500 ma/di in terms of glucose concentration, and at most 1001 g/di.
It was found that the response was significantly improved compared to the conventional enzyme electrode method, in which the linearity limit was approximately dl.

Furthermore, when a similar enzyme electrode system was repeatedly produced and the responsiveness was examined, it was found that the reproducibility was good, and it was also found that enzyme immobilization can be performed with good reproducibility by the electrolytic polymerization method.

Furthermore, when measurements were performed using a test solution containing ascorbic acid and a test solution containing uric acid as interfering substances, these oxidation currents were canceled out by the second working electrode and the cancellation circuit, and the oxidation current was substantially reduced to only glucose. It was also found that it responded.

Further, as another example, FIG. 2 shows a schematic diagram in which one platinum electrode is used as the reference electrode in FIG. 1 and also serves as the counter electrode. Similar measurements were possible with this electrode system, but more stable measurements were possible when the counter electrode, the platinum electrode, was replaced with a platinum black electrode.

(G) Effects of the Invention According to the enzyme electrode system of the present invention, the following effects can be obtained.

■ Substrate responsiveness can be significantly improved compared to conventional oxygen electrode methods and hydrogen peroxide electrode methods.

- Since it uses a hydrogen peroxide electrode method, there is no need to first saturate the test solution with a element, which is required in the enzyme electrode method.

■ Because it detects the differential output of electrolytic current, it is not affected by interfering substances and has high measurement accuracy.

■ Because the enzyme immobilization membrane is produced by electrolytic polymerization, it can be produced extremely thin and with good developability, and the membrane thickness can be easily adjusted, and it is not affected by the size or shape of the electrode. Enzyme can be immobilized on any electrode,
The degree of freedom in designing enzyme electrodes and their systems, especially in miniaturization, will be significantly improved.

■ Because it is immobilized using an electrolytic polymer membrane, the polymer membrane thickness is thinner than that of Haku's polymer membrane, and the measurement sensitivity is also excellent.

[Brief explanation of the drawing]

FIG. 1 shows an example of the configuration of each electric bridge in the enzyme electrode system of the present invention, where Δ in FIG. 1 is a schematic cross-sectional view and FIG. 1B is a bottom view thereof. FIG. 2 similarly shows another example, in which FIG. 2A is a schematic cross-sectional view and FIG. 2B is a bottom view thereof. FIG. 3 is a configuration explanatory diagram illustrating the enzyme electrode system of the present invention, and FIG. 4 is a corresponding circuit diagram. FIG. 5 is a graph diagram illustrating the relationship between substrate concentration and response current according to the enzyme electrode system of the present invention. 1... Insulated substrate, 2, 3... Measurement electrode, 4... Counter electrode, 5... Reference electrode, 5a, 5b... Polymer thin film.

Claims (1)

[Scope of Claims] 1. (a) A first working electrode comprising a hydrogen peroxide generating enzyme immobilized polymer film made of an electropolymerized polymer thin film on the sensitive part of the hydrogen peroxide electrode; b) a second working electrode comprising, on the sensitive part of the hydrogen peroxide electrode, a polymer membrane made of an electropolymerized polymer thin film on which no enzyme is immobilized or on which an inactivated hydrogen peroxide generating enzyme is immobilized; (c) a counter electrode that constitutes an electrolytic system with the first working electrode and the second working electrode; (d) a difference for detecting the output difference between the electrolytic current by the first working electrode and the electrolytic current by the second working electrode; An enzyme electrode system comprising a dynamic force detection system.