JPH0612352B2 - Enzyme functional electrode - Google Patents

Description

Detailed Description of the Invention (a) Field of Industrial Application Enzyme functional electrode. Ie. Glucose present in the electrolyte. Lactic acid and substances that can be substrates for oxidase enzymes such as galactose. alcohol. The present invention relates to an electrode that amperonatically measures the concentration of a substance that can be a substrate for a dehydrogenase enzyme such as glycerol.

(B) Conventional technology Recently. Redox enzyme-immobilized electrodes have come to the fore. This electrode behaves as a substitute for a chemical electron carrier for the enzymatic reaction. Enzyme electrode. Flow system detector. Biochemical fuel cell. It has been proposed that it can be used in new applications such as enzyme reactors.

And. Among these electrodes, as an electrode for measuring the concentration of substances that can be substrates for oxidase enzymes. Laminate the enzyme immobilization membrane on the sensitive surface such as enzyme electrode or platinum electrode. There is a system in which the amount of a substance generated in an enzymatic reaction, such as hydrogen peroxide, is measured to indirectly quantify a substrate that contributes to the enzymatic reaction.

Furthermore. As an electrode for measuring the concentration of substances that can be substrates for dehydrogenase enzyme. NAD (nicotinamide adenine dinucleotide) immobilized with an enzyme. Specifically, a NAD-polymer compound in which NAD is chemically bonded to a polymer compound such as agarose or dextran is trapped on an inner surface of a suitable substrate permeable membrane together with an enzyme and immobilized on a platinum or graphite electrode. Directly chemically bond NAD to the substrate permeable membrane. Immobilized on a platinum or graphite electrode that simultaneously traps the enzyme in this membrane. The substrate permeable membrane was chemically treated to make it difficult for NAD to permeate. There is one in which an enzyme is trapped on the inner surface of this membrane and fixed to a platinum or graphite electrode.

Also. The applicant of the present invention is not limited to the above electrodes. August 31, 1984. A pasty electrode containing an electron-accepting compound in graphite paste. A permeable thin film of an oxidase enzyme or dehydrogenase enzyme immobilized on the surface of the paste-like electrode and a substrate of the oxidase enzyme or dehydrogenase enzyme covering the outer surface thereof. An application has been made for an oxidase electrode or a dehydrogenase electrode which comprises a substrate-sensing part composed of

(C) Problems to be solved by the invention Of the above-mentioned electrodes. Those using oxygen electrodes or platinum electrodes have the problem that they are affected by fluctuations in the oxygen partial pressure in the measured liquid. Also. What is NAD immobilized with enzyme? There was a problem such as leakage of NAD.

Furthermore. In the invention of the applicant's earlier application. Electrodes for immobilizing enzymes are limited to graphite paste electrodes.

(D) Means for Solving Problems This invention is. Unaffected by fluctuations in oxygen partial pressure in the measured liquid. Also. The electrode members were made for the purpose of not being limited to graphite paste. The structure is. In an enzyme functional electrode that transfers substrate changes in the immobilized enzyme layer to the electrode via an electron carrier. It is characterized in that a storage layer of a semi-molten electron carrier is provided in contact with the immobilized enzyme layer so that the electron carrier is gradually dissolved and supplied to the immobilized enzyme layer.

As a storage layer for electron carriers. For example, the one in which the electron carrier is impregnated in the gap of the porous electrode. Paste the electron carrier onto the metal mini-grid electrode with gaps. Some are in gel form and contacted.

(E) Operation In the electrode of the present invention, the electron carrier gradually melts from the storage layer of the electron carrier and is supplied to the immobilized enzyme layer to mediate electron transfer between the immobilized enzyme layer and the electrode. .

(F) Example An example of the present invention will be described below.

Example 1 (Electron carrier-impregnated porous electrode) First, an enzyme functional electrode using a porous electrode in which an electron carrier is impregnated in a gap of a porous electrode as a storage layer of the electron carrier will be described.

(A) Production of Electrode The following is an example of producing an electrode for measuring glucose.

Reagent Glucose oxidase (GOD) ………… Type II,
Made by Sigma (EC1, derived from Asbergillus niger
1,3,4) p-Benzoquinone (BQ) …… Wako Pure Chemical Industries, Ltd. (sublimates and purifies before use) Collodion liquid (5%) …… Wako Pure Chemicals Fluid Paraffin ……………… Merck electrode material Porous graphite plate ... RVC 2x1-100, INF
Chemotronics Co., Ltd. First, the diameter from the porous graphite plate with a cork polar
A 3.4 mm cylinder was cut out and a lead wire was connected to one end of the cylinder using a conductive adhesive. Epoxy adhesive is applied to the entire adhesive surface to prevent solvent penetration, and at the same time the inner diameter is 3.4 mm.
Glued to a glass tube. From above, a heat-shrinkable tube was used to insulate the side surface of the electrode and fix the electrode. A storage layer (a quinone-impregnated porous electrode) was prepared by impregnating the quinone-containing material into the holes of the porous electrode thus prepared. The quinone-containing material is 40 mg of P-benzoquinone and is a liquid paraffin.
It was adjusted by mixing 0.3 ml with an agate mortar.

Then, 10 μl of GOD (1 mg / ml) was placed on the surface of the storage layer (quinone-impregnated porous electrode) to evaporate the water. Then, the surface was covered with a nylon net, and a 20 μl collodion-ethanol (volume ratio 1: 4) mixed solution was cast and dried to form a nitrocellulose thin film on the electrode surface. The nylon net was fixed to the electrode with parafilm, and grease was applied to the parts of the nylon net other than the electrode surface.

Electrodes for glucose measurement obtained as described above (1)
Is shown in FIG. In the figure, (2) is a storage layer and (3) is an immobilized GOD.
Layer, (4) nitrocellulose thin film, (5) lead wire (platinum wire), (6) glass tube, (7) nylon net, (8) heat shrink tube, (9) conductive adhesive Agent (10) is an epoxy resin, respectively.

The electrode (1) was washed several times with distilled water until it was used, and immersed and stored in an acetate buffer of pH 5.0 overnight.

(B) Cyclic voltammetry Voltammetry was performed on the glucose oxidase electrode by the triode method. The measurement conditions are as follows.

Equipment Potentiostat (Fusosha) Signal generator (HB-104; Hokuto Denko) Counter electrode (platinum plate) Reference electrode (saturated calomel electrode) Recorder (XY recorder; Yokogawa Electric Corp.) Electrolyte Deoxidized 0.1M acetate buffer of pH 5.0 Temperature ………… 25 ± 1 ℃ Stirring speed …… 500r ・ p ・ m Results The electrode of the present invention was immersed in the above acetate buffer, and 50
Cyclic voltan monograms were recorded at a potential scanning rate of mV / s. As shown by the solid line in FIG. 2, -0.07V and + 0.40V for cathode and anode waves, respectively.
A peak was observed in (vs SCE). Cyclic potential scanning was further continued for several hours, but no decrease in current was observed. The same evaluation was performed on the same electrode containing no p-benzoquinone, but no voltammetric peak as shown by the broken line in FIG. 2 was generated.

Thus, the voltammetric peak is based on the electrochemical reduction and reoxidation reaction of p-benzoquinone transferred from the storage layer to the boundary region between the quinone-impregnated porous electrode and the nitrocellulose thin film, and the p- It was found that benzoquinone acts as an electron carrier.

(C) Electrocatalytic oxidation of glucose The potential is sufficiently positive (E
= + 0.5 V vs. SCE), glucose was added to the buffer at successively increasing concentrations (O → 100 mM), and the increase in current from the base was measured. The results are shown in FIG.

From these results, it was found that the electrode of the present invention can cope with minute changes in glucose concentration and is useful as an electrode for measuring glucose.

In the above description, only the mixture of p-benzoquinone and liquid paraffin was described as the quinone-containing material used in the storage layer, but of course other than this, those shown in the table below However, it was confirmed that similar results could be obtained.

The electron carrier used in the storage layer is not limited to P-benzoquinone, but may be potassium ferricyanide, dibromoide salicylic acid, dichlorophenyl indophenol (DCIP) phenazine methosulfate (PMS) NAD, or the like.

Furthermore, although the above-mentioned electrode was an electrode for measuring glucose, it can be used as an electrode for measuring various substrates by changing the enzyme to be immobilized.

The enzyme / substrate combination includes alcohol dehydrogenase / ethanol, glucose dehydrogenase /
Examples include glucose, glutamate dehydrogenase / glutamic acid, lactate dehydrogenase / lactic acid, glycerol dehydrogenase / glycerol, galactose oxidase / galactose, alcohol oxidase / ethanol, cholesterol oxidase / cholesterol, amino acid oxidase / amino acid, and urate oxidase / uric acid.

Example 2 (Electron Carrier Contact Minigrid Electrode) About an enzyme functional electrode using a metal minigrid electrode having a gap as a storage layer of the electron carrier, in which the electron carrier is brought into contact with a paste or gel explain.

(A) Production of Electrode The following is an example of producing an electrode for measuring glucose.

The same reagents as in Example 1 were used.

Electrode material Gold mini-grid electrode: 500 lpi; manufactured by Buckbee Meers Co., Ltd. First, 0.3 ml of liquid paraffin was added to 500 mg graphite powder and mixed in a mortar, and further 40 mg of P-benzoquinone was added and uniformly mixed. This was filled in a glass tube having an inner diameter of 3.4 mm and the surface was smoothed on wax paper to prepare a storage layer. Then, GOD solution (1 mg
/ Ml) was evaporated and the solvent was evaporated, and then a gold mini grid electrode was placed and fixed with a nylon net. The nylon net was fixed with Parafilm. Furthermore, from above the nylon net, onto the gold mini grid electrode, GOD solution (1 mg / ml)
Was added to 10 μl and the solvent was evaporated. Then, 20 μl of collodion-ethanol solution (1: 4) was placed on it and dried, and then a mini-grid electrode and a lead wire were connected with a conductive adhesive to obtain an electrode (11) in FIG.

In the figure, the same parts as those in FIG. 1 are designated by the same reference numerals. (1
2) is a storage layer, (13) is a gold mini-grid electrode, and (14) is a collodion film.

The storage until using this electrode (11) was the same as in Example 1.

(B) Cyclic voltammetry When cyclic voltammetry was performed under the same measurement conditions as in Example 1, the results shown in FIG. 5 were obtained. As shown in FIG. 5, also in the electrode according to this example, it was found that P-benzoquinone migrated from the storage layer and acted as an electron carrier.

(C) Electrocatalytic Oxidation of Glucose In the same manner as in Example 1, glucose was added to the buffer at successively increasing concentrations, and the increase in current from the base was measured.

The results are shown in FIG. From this result, it was found that the above-mentioned electrode can cope with minute changes in glucose concentration and is useful as an electrode for glucose measurement.

In the above description, P-benzoquinone is used as the storage layer.
This is explained only by using the pasty one.
Of course, other than, for example, P-benzoquinone Geruo
Similar results were obtained with quinone gel prepared with
It was

Further, the electrode material is not limited to the gold mini grid electrode, and any metal mini grid electrode such as a platinum mini grid electrode can be used.

Furthermore, the electron carrier used for the storage layer is not limited to P-benzoquinone, and it can be prepared for electrodes other than glucose measurement as in Example 1.

The configuration of the entire electrode is not limited to those of the first and second embodiments, and for example, an electron carrier may be provided between a bundle of carbon fibers, glassy carbon grains (manufactured by Tokai Electrode), and beads of 60 to 100 mesh. If a storage layer is prepared and the enzyme is immobilized on this surface, an electrode that exhibits the same action can be formed.

(G) Effect The enzyme-functional electrode of the present invention has the effect of showing a large current response to substrates such as glucose and ethanol without adding any other electron carrier to the solution to be measured, and the electrode material is optional. You can choose one.

[Brief description of drawings]

FIG. 1 shows an embodiment of a porous electrode impregnated with an electron carrier,
The figure shows cyclic voltammetry performed in the buffer using the electrodes shown in Fig. 1, Fig. 3 shows the calibration curve prepared by measuring the glucose response using the electrodes shown in Fig. 1, and Fig. 4 shows the electron An example of a carrier-contacting mini-grid electrode, Fig. 5 shows cyclic voltammetry in a buffer using the electrode of Fig. 4, and Fig. 6 measures glucose response using the electrode of Fig. 4. This is a calibration curve created by 2, 12 ... Storage layer, 3 ... Immobilized GOD layer 5 ... Lead wire, 6 ... Glass tube 13 ... Mini grid electrode

Claims (1)

[Claims] 1. In an enzyme functional electrode for transmitting a change in a substrate in an immobilized enzyme layer to an electrode via an electron carrier, the electron carrier is semi-melted so as to be gradually melted and supplied to the immobilized enzyme layer. An enzyme functional electrode, characterized in that a storage layer of a state electron carrier is provided in contact with an immobilized enzyme layer.