JPH0529062B2 - - Google Patents

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 electrode 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 with high precision.

(B) 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 enzyme and produces hydrogen peroxide in the presence of an enzyme, there is an oxygen electrode method that captures the amount of oxygen loss, 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 Abstracts of the 53rd Annual Conference of the Association C 203 (1986)].

On the other hand, in the hydrogen peroxide electrode method, since hydrogen peroxide needs to reach the electrode surface, a membrane through which the test solution can permeate is used, and an enzyme with a hydrogen peroxide generating system is immobilized on this membrane to generate hydrogen peroxide. It is configured by being attached to an electrode. 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, as a means to remove this interfering current, two working electrodes (hydrogen peroxide electrodes) have been used, one of which has an enzyme immobilized with a photopolymerized polymer membrane, and the other with no enzyme immobilized. Alternatively, a method has been proposed in which the differential output of these working electrodes is detected by coating a photopolymerized polymer film with immobilized inactivated enzymes [Ichiro Takatsu et al., "Differential output type glucose sensor"] , Materials of the Institute of Electrical Engineers of Japan Research Group (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 mg/dl. There were problems in that a large error occurred when measuring a sample with a substrate concentration exceeding that of the conventional method, and a dilution operation was required in advance.

On the other hand, even in the enzyme electrode system of the hydrogen peroxide electrode method using the differential output detection method, good response is at most about 100 mg/dl;
I had the same problem as 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 for Solving the Problems As a result of extensive research, the present inventor has discovered that an electropolymerized polymer peritoneal membrane is used as a medium for an oxygen-permeable enzyme-processed membrane in an enzyme electrode of an oxygen electrode type.
The present invention was achieved based on the discovery that substrate responsiveness is significantly improved by using the enzyme as a medium for immobilizing a hydrogen peroxide-generating enzyme in the enzyme electrode system of the differential output detection-hydrogen peroxide electrode method.

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

The electropolymerized polymer thin film in this invention includes various noble metals (platinum, gold, etc.) that can be used as hydrogen peroxide electrodes.
It can be formed by using a carbon electrode as an electrode for electrolysis and performing electrolysis while immersing it in an electrolyte solution of a monomer for polymerization to advance polymerization. The monomer for polymerization may be any monomer as long as it is water-soluble and polymerizable in an aqueous solution, and aromatic compounds having a functional group such as a hydroxyl group or an amino group are suitable, such as aniline,
Examples include o-phenylenediamine and phenol. However, water-insoluble monomers such as pyrrole and thiophene that can be polymerized in a non-aqueous electrolyte 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, and may be, for example, a film-shaped electrode deposited 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 simply 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 forming a thin polymer film, on this surface,
For example, the hydrogen peroxide generating enzyme may be immobilized 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 above immobilized polymer membrane is approximately 0.1 to 0.5 μm
is appropriate. 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. The inactivated enzyme is immobilized here 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 (for example, about 100°C). ) can be easily carried out by heating.

The hydrogen peroxide generating enzyme in this invention has a reaction system that generates hydrogen peroxide,
For example, glucose oxidase (GOD), which uses glucose as a substrate, is typical; other enzyme/substrate combinations include uricase/uric acid, lactate oxidase/lactic acid, oxalate oxidase/oxalic acid, amino acid oxidase/amino acid, Examples include monoamine oxidase/monoamine, pyruvate oxidase/pyruvic acid, alcohol oxidase/alcohol, galactose oxidase/galactose, and the like. 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 of other species that can produce substrates for the above-mentioned hydrogen peroxide-generating enzymes,
The types of substances that can be detected can be increased. Examples of this include an invertase/mutarotase/GOD combination using sucrose as a detection substance, a glucoamylase (or maltase)/GOD combination using maltose as a detection substance, and a cholesterol esterase/cholesterol oxidase combination using cholesterol ester as a detection substance. , a combination of phospholipase/choline oxidase using phosphatidylcholine as a detection substance, etc., and any combination that can at least ultimately generate hydrogen peroxide may be used.

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
The noble metal electrode or carbon electrode that is the base of the first working electrode and the second working electrode works as a hydrogen peroxide electrode, so that the potential at which hydrogen peroxide is reduced (usually
0.6V vs Ag/AgCl). Therefore, it is usually suitable to monitor the electrolytic potential using a reference electrode such as a silver-silver chloride electrode or a calomel electrode, 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.

The counter electrode can be made of the same material as the hydrogen peroxide electrode, but it is recommended to use one with a larger area (usually 100 times or more) than the first and second working electrodes to reduce interelectrode resistance. For example, it is preferable to use a platinum black electrode, which has a significantly larger surface area than a platinum electrode.

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, which 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, but various reducing substances contained in the sample reach the surface of the hydrogen peroxide electrode, and an electrolytic current corresponding to the error flows. Then, in the 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, and corresponds substantially to the generated hydrogen peroxide content and, by extension, to the concentration of the substrate intended to be measured. This results in an electrolytic current that can be obtained.

(f) Example FIG. 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 these electrodes was constructed as follows.

First, at the tip of a cylindrical insulating substrate 1 (made of vinyl chloride resin), measuring electrodes 2 and 3 made of platinum wires with a diameter of 0.5 mm and a reference electrode 5, which will serve as hydrogen peroxide electrodes, are placed on the tip of a cylindrical insulating substrate 1 (made of vinyl chloride resin).
(Ag/AgCl electrode) was embedded and attached so that each sensitive part was exposed, and a counter electrode 4 made of platinum was further attached to the outer periphery of the tip to constitute an electrode system. This electrode system was mixed with the monomer 0.1M aniline and 2mg/
First, measure electrode 2 and counter electrode 4 are immersed in an aqueous solution (PH7.0) containing ml glycol oxidase (GOD).
Constant potential electrolytic polymerization (1.2V
vs Ag/AgCl), a polyaniline-GOD immobilized (encompassing) polymer thin film was formed on the exposed surface of the tip of measurement electrode 2. Next, this tip was immersed in an aqueous solution at 100° C. for 5 minutes to deactivate GOD in the polymer thin film, thereby producing a second working electrode. Next, polyaniline is applied to the exposed end surface of the measuring electrode 3 by electrolytic polymerization between the measuring electrode 3 and the counter electrode 4 in the same manner as above.
The first step is to form a GOD immobilization (encompassing) polymer thin film.
A working electrode was set up. In the figure, 6a has been deactivated.
6b shows a polymer thin film with immobilized GOD, and 6b shows a polymer thin film with GOD immobilized, each having a thickness of approximately
It was 0.3 μm.

A configuration diagram of an enzyme electrode system (glucose concentration detection device) of the present invention using this electrode structure is shown in FIG. 3, and a circuit diagram is shown in FIG. 4.

The results of measuring the change in response current to glucose concentration using the enzyme electrode system described above are shown in the fifth section.
Shown in the figure. The test solution was tested at PH7.0, 37℃, and without stirring, and the electrolytic potential was +0.7V vs.
The setting was Ag/AgCl.

In this way, a calibration curve with good linearity was obtained up to a glucose concentration of approximately 500 mg/dl, and at most 100 mg/dl.
It was found that the response was significantly improved compared to the conventional enzyme electrode method, whose linearity limit was around mg/dl.

In addition, when we repeatedly fabricated a similar enzyme electrode system and examined its response, we found that the reproducibility was good.
It was also found that enzyme immobilization can be performed with good reproducibility by electrolytic polymerization.

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 substantially only glucose was detected. It was also found that it responded to

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. Although similar measurements were possible with this electrode system, more stable measurements were possible by using a platinum black electrode as the counter electrode.

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

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

Since it uses a hydrogen peroxide electrode method, there is no need to first saturate the test solution with enzymes, which is unlike 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.

Since the enzyme immobilization membrane is produced by electrolytic polymerization, it can be made extremely thin and with good reproducibility, and the membrane thickness can be easily adjusted. Moreover, it can be made to any desired size without being affected by the size or shape of the electrode. Enzymes can be immobilized on electrodes, significantly increasing the degree of freedom in designing enzyme electrodes and their systems, especially in miniaturization.

Because it is immobilized by an electrolytic polymer membrane, the polymer membrane thickness is thinner than other polymer membranes, and the measurement sensitivity is also excellent.

[Brief explanation of the drawing]

FIG. 1 shows an example of the configuration of each electrode in the enzyme electrode system of the present invention. FIG. 1A 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. Third
The figure 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... Insulating substrate, 2, 3... Measurement electrode, 4... Counter electrode, 5... Reference electrode, 6a, 6b... Polymer thin film.

[Claims]

1 In the coulometric analysis method in which a sample liquid is supplied into a measurement cell containing an electrolytic solution and the content of the substance to be measured in the sample liquid is determined by electrolyzing the substance to be measured in the sample liquid, multiple samples of the same volume are A non-conductive plate having through-holes and a non-conductive plate having a plurality of through-holes provided so as to communicate with the plurality of through-holes of the plate are arranged so that these through-holes alternate. The electrolyte in the measurement cell is circulated through one set of communicating through holes, and the analysis is performed while the electrolyte is circulated through the through holes.After the coulometric analysis is completed, another set of communicating through holes is connected. After supplying the sample liquid to the hole, rotate the plate with the same volume of through-holes and make the through-holes of the rotated plate communicate with the through-holes of the non-rotated plate to create the same volume that is in communication with the measurement cell. The sample solution is injected into the measurement cell through the through-hole of the cell by circulating the electrolyte within the measurement cell, and at the same time, an amount of electrolyte corresponding to the amount of the injected sample solution is injected from the through-hole of the same volume, which is not in communication with the measurement cell. A sample liquid injection method for continuous coulometric analysis characterized by draining the sample liquid out of the system and repeating this process. 2 Non-conductive with multiple through holes of the same volume