JPH061252B2 - Oxidase electrode - Google Patents

Description

TECHNICAL FIELD The present invention relates to an oxidase electrode. More specifically, the concentration of substances that can be substrates for oxidase enzymes such as glucose and galactose present in electrolytes can be measured amperometrically, especially in biological samples such as serum, plasma, urine, etc., or substrates in enzyme reactors. Useful for measuring concentration,
Further, the present invention relates to an oxidase electrode useful as an electrode of an enzyme reactor or a fuel cell.

(B) Conventional technology Recently, redox enzyme-immobilized electrodes have been receiving attention. This electrode behaves as a substitute for a chemical electron acceptor or a donor for an enzyme reaction and is called a so-called biocatalyst electrode. It is an enzyme electrode, a flow system detector, a biochemical fuel cell, an enzyme reactor. It is proposed that it can be used for new applications such as.

Such an electrode is an electrode used in a conventional enzyme sensor, that is, an enzyme-immobilized film is laminated on a sensitive surface such as an enzyme electrode or a platinum electrode, and the amount of a substance such as hydrogen peroxide generated by an enzyme reaction is measured. Unlike the electrode that indirectly measures the substrate that contributes to the enzymatic reaction, this is a method that directly uses the current or voltage related to the enzymatic reaction.

In this regard, the present inventors have previously found that the glucose oxidase-immobilized electrode acts as a biocatalytic electrode for electrooxidation of glucose in the presence of an electron transfer catalyst (reagent) in solution.

(C) Purpose The present invention is intended to provide a new oxidase-immobilized electrode, and in particular, does not require oxygen as an electron acceptor and changes in the oxygen partial pressure in the measured liquid. The main object of the present invention is to provide an oxidase electrode capable of measuring the concentrations of various substrates for an oxidase enzyme without being affected by the above.

(D) Structure Thus, according to the present invention, a paste-like electrode containing an electron-accepting compound in a graphite paste, an oxidase enzyme immobilized on the surface of the paste-like electrode and an oxidase enzyme coating the outer surface thereof. There is provided an oxidase electrode comprising a substrate-sensing part composed of a permeable thin film of a substrate.

The oxidase electrode of the present invention is usually used by setting an electrolysis system in which the paste-like electrode serves as an anode in a state where the substrate sensitive part is in contact with or immersed in the solution to be measured. That is, it can be used as an amperometric electrode, especially as a sensor. The cathode (counter electrode) in this case is not particularly limited, and various electrodes such as a platinum electrode, a silver / silver chloride electrode, a mercury / mercury chloride first electrode and the like can be used.
The measurement is usually carried out by constant voltage electrolysis.

The electrode of the present invention is usually in a form in which a substrate sensitive portion is set at one end of a cylindrical insulator and a lead electrode is appropriately provided. However, it may be in a form other than this (for example, a form directly incorporated in the flow channel) depending on the application, and at least the substrate sensitive part may be configured as described above. In some cases, the counter electrode may be incorporated in the sensor support material.

The most characteristic feature of the present invention is that a paste-like electrode is used as an electrode for immobilizing an oxidase enzyme, thereby eliminating the presence of an electron transfer medium such as oxygen in the liquid to be measured. In order to enable amperometric measurement of various substrates with such a constitution, a graphite paste containing an electron-accepting compound is used as the pasty electrode.

As the graphite paste, a mixture of graphite (graphite) powder and a non-polar binder such as fluid paraffin or undecane is used. The graphite content in this paste is usually
About 60 to 70% by weight is suitable in terms of conductivity and shape retention. Further, as the graphite powder, one having a size of about 1 μm to 50 μm is suitable.

Examples of the electron-accepting compound to be contained in the graphite paste include p-benzoquinone, potassium ferriciyanide, dibromoide salicyl silicate, dichlorophenylindophenol (DCIP), phenazine methosulfate (PMS), and the like. In addition to this, a compound that participates in the reaction as an electron acceptor in the presence of the oxidase enzyme and the substrate and can be stably mixed in the graphite paste can be used. Although the appropriate content of these varies depending on the enzyme used, for example, when p-benzoquinone is used, 0.2 to 30% by weight is suitable. If the content is less than 0.2% by weight in the graphite paste, it is not preferable from the viewpoint of current sensitivity, and if it exceeds 30% by weight, it is not preferable from the viewpoints of shape maintenance of the electrode and economical efficiency. The most preferable content is 15 to 25% by weight.

As the oxidase enzyme immobilized on the paste-like electrode, various ones can be appropriately selected according to the substrate intended for detection or conversion. Typical examples of the enzyme / substrate combination are glucose oxidase / glucose, galactose oxidase / galactose, alcohol oxidase / ethanol, cholesterol oxidase / cholesterol, amino acid oxidase / amino acid, and urate oxidase / uric acid.

Immobilization of the above oxidase enzyme on the paste electrode is
Usually, it is carried out by a solution method, that is, by applying a solution of an oxidase enzyme to the surface of the paste-like electrode, holding it by dropping or the like, and evaporating the solvent. At this time, it is preferable that the surface of the paste electrode is made as smooth as possible. The fixed amount of oxidase used is preferably about 10 to 200 μg / cm ^{2 in} the case of glucose oxidase.

The permeable thin film of the substrate in the present invention serves to retain and fix the oxidase enzyme attached to the electrode surface. As the permeable thin film, a cellulose acetate film, a nitrocellulose film, a K-carrageenan gel film, a polyacrylamide gel film or the like can be used, and in particular, a nitrocellulose film formed by applying a liquid containing collodion and drying it. Is preferably adopted. Although the film thickness is not limited, generally, a thinner film is suitable for shortening the response time, and a thicker film is suitable for expanding the response concentration to a wider concentration range. In general, about 20 to 500 μm is suitable, though it depends on the kind of film material.

(E) Examples Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 (Using glucose oxidase as the oxidase,
Glucose oxidase electrode using p-benzoquinone as electron-accepting compound) (A) Preparation of electrode Reagent Glucose oxidase (GOD) …………………… T
ypeII, manufactured by Sigma (E derived from Aspergillus niger
C1,1,3.4) p-benzoquinone (BQ) ………… Wako Pure Chemical Industries, Ltd. (sublimates and purifies before use) Collodion liquid (5%) ……… Wako Pure Chemical Industries, Ltd. Flow Paraffin: Graphite powder made by Merck Co., Ltd. NO.ACP made by Nippon Graphite Co., Ltd. (10 μm) A predetermined amount of p-benzoquinone was mixed with 3 ml of fluid paraffin, and then 5 g of graphite powder was added. It was The p-benzoquinone-graphite paste thus obtained was
One end of a glass tube having an inner diameter of 3.4 mm was filled. The exposed surface is smoothed with wax paper to 9.0 × 1.
0 ^-2 was set pasty electrode having a surface area of cm ^2.

Next, a GOD aqueous solution having a predetermined concentration was dropped and held on the surface of the paste-like electrode with a syringe to evaporate the solvent. Then, a 20 μl collodion-ethanol (volume ratio 1: 4) mixed solution was cast and dried to form a thin film of nitrocellulose on the electrode surface.

By attaching a lead electrode made of a platinum wire to the thus obtained electrode, and further attaching a nylon net and a Teflon tube for supporting a nitrocellulose membrane, the glucose oxidase electrode of the present invention as shown in FIG. 1 ( GOD-immobilized benzoquinone-graphite paste electrode) (1) was obtained. In the figure, (2) is a p-benzoquinone-graphite paste electrode, and (3) is a fixed GO.
D, (4) is a nitrocellulose thin film, (5) is a platinum wire, (6) is a glass tube, (7) is a nylon net, and (8) is a heat-shrinkable Teflon tube.

The electrode was washed several times with distilled water until it was used as a sensor, and the pH 5.0 acetate was immersed and stored in a buffer overnight.

(B) Cyclic voltammetry The glucose oxidase was subjected to voltammetry by the triode method using an electrode. 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 Denki) Electrolyte desorption Oxygenated 0.1 M acetate buffer with pH 5.0 Temperature: 23 ± 1 ° C. Stirring speed: 500 r. p. m Results 18 μg of GOD was immobilized on a graphite paste electrode containing 0.25% by weight of p-benzoquinone. The electrode of the present invention was immersed in the acetate buffer described above to obtain 50 mV / s.
Cyclic voltammograms were recorded at potential scan rates of. As shown by the solid line in FIG. 2A, −0.07V and + 0.40V for the cathode wave and the anode wave, respectively.
A peak was observed in (vsSCE). Cyclical potential scanning was 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. 2A did not occur.

As described above, the voltammetric peak is based on the electrochemical reduction and reoxidation reaction of p-benzoquinone transferred from the paste to the boundary region between the graphite paste electrode and the nitrocellulose thin film, and the p-benzoquinone molecule is It was found to contribute to the electrochemical reduction reaction.

Note that p-benzoquinone may gradually leak into the electrolyte through the nitrocellulose thin film, but since these are replenished from the graphite paste medium, p-benzoquinone at the interface between the graphite paste and the nitrocellulose thin film is The benzoquinone concentration remains substantially constant.

(C) Electrocatalytic Oxidation of Glucose When glucose was added to the above buffer, the second
It was observed that the anode current of the electrode of the present invention significantly increased as shown by the solid line in FIG. Then, it was found that as the glucose concentration in the liquid to be measured was increased, the steady-state current Is increased at a given potential as shown in FIGS. 3A and 3B. It was found that this current Is increased according to the amount of p-benzoquinone in the range of 0.25 to 10% by weight of the p-benzoquinone in the graphite paste, and reached a limit value when it exceeded 10% by weight. Further current I
s also depends on the amount of GOD on the electrode surface. For example, in the case of a paste electrode containing 20% by weight of p-benzoquinone, values of 2 μA and 32 μA when 1.8 μg and 18 μg of GOD were immobilized, respectively. Was shown.

These results indicate that the immobilized GOD remains active, so that the p-benzoquinone trapped at the interface between the immobilized GOD and the paste electrode acts as an electron carrier. .

18 μg of GOD was added to p-benzoquinone (30% by weight)-
The dependence of Is on the glucose concentration (Cglu) at an applied voltage of 0.5 V is shown in FIGS. 3A and 3B for the electrodes immobilized on the graphite paste. From the extrapolation intersection and slope of the double reciprocal plot of the data in the figure, the maximum current Is ^max and the apparent Michaelis constant are 124 μA and 10 respectively.
It was determined to be 4 mM. From this Is ^max value,
Immobilized GOD molecules and GO dissolved in the solution to be measured
Assuming that the D molecules have the same catalytic activity, the amount of GOD having the electrocatalytic activity immobilized on the electrode is 3 × 10 ^−11.
Mol / cm ² , which is about 3% of the amount of GOD used
Equivalent to.

As shown in FIG. 3A, the above electrode shows a linear response up to a glucose concentration of 15 mM, and its correlation coefficient is 0.9.
It was 999. Since the background current is very small (0.2 ± 0.01 μA), glucose below 1 mM can be measured with a coefficient of variation (n = 5; 5.3%) when Cgul is 1 mM (No. (See FIG. 3, B). The current response is rapid and reaches a steady current within 20 seconds.

The response to 8.3 mM glucose was tested periodically to assess its stability as a sensor and it was found that the initial activity was retained for more than a week. Here, the electrode was stored in a buffer at 5 ° C. or lower when not in use.

18 μg of GOD was added to p-benzoquinone (30% by weight)-
When four sensors immobilized on a graphite paste electrode were manufactured and the reproducibility of electrode preparation was evaluated by the response to 8.3 mM glucose, the average value of Is was 7.9 μm.
In A, the standard deviation was 0.7 μA.

In order to evaluate the effect of oxygen, Is of the air-saturated liquid and Is of the degassed liquid were measured for the glucose solution of 5 to 40 mM, respectively. This result is as shown in FIG. 4, and it was found that the glucose concentration in this concentration range had little effect. This concentration range covers the concentrations normally found in blood samples.

The electrode of the present invention works well in a liquid having a pH of 5-8.

Example 2 A glucose oxidase electrode was produced in the same manner as in Example 1 except that potassium ferriciyanide was used as the electron-accepting compound instead of p-benzoquinone.

When cyclic voltammetry was performed on this electrode in the same manner as in Example 1, it was found that a voltammetric peak was generated as shown in FIG. 5, and potassium ferricyanide contributed to the electrochemical reduction reaction.

Example 3 An electrode having a structure in which a counter electrode is incorporated in a glass tube (1) is used in Example 1. It was prepared according to (A). This structure is shown in FIG. In the figure, (10) shows a silver / silver chloride electrode which is a counter electrode (cathode), which is wound and fixed on the internal support (6 '), and (9) shows acetic acid containing potassium chloride. It shows the electrode internal liquid consisting of buffer, and the others correspond to the above-mentioned numbers.

(F) Effect The oxidase electrode of the present invention shows a large current response to a substrate such as glucose without adding any other electron transfer medium to the liquid to be measured. Therefore,
It can be suitably used as a sensor for various substrates.
In addition, this electrode does not require the presence of oxygen in the liquid to be measured, and has the effect of being substantially unaffected by fluctuations in the oxygen partial pressure when oxygen is present. In addition, the peaking phenomenon of measured values in the high concentration region due to oxygen deficiency, which is observed in conventional glucose measuring electrodes and the like,
According to the electrode of this invention, it can be eliminated.

[Brief description of drawings]

FIG. 1 is a structural explanatory view showing a glucose oxidase electrode of one embodiment of the present invention. FIGS. 2A and 2B are graphs showing cyclic voltammograms, respectively, where A is the one when glucose is not added, B is the one when 41 mM glucose is added, and broken lines c and d are p-benzoquinone-free in the paste. Of the electrode (comparative example), and solid lines a and b show the electrode of the present invention. FIGS. 3A and 3B are graphs showing the relationship between the glucose concentration and the anode current Is when 0.5 V is applied to the electrode of the present invention. Similarly, FIG. 4 is a graph showing the relationship between the glucose concentration and the anode current Is when 0.5 V is applied. The plot ◯ is for the degassed measurement liquid, and the plot ● is for the air saturation measurement liquid. Figure 5 shows
It is a graph which shows the cyclic voltammogram of the glucose oxidase electrode of the other Example of this invention. FIG. 6 is a structural explanatory view showing a glucose oxidase electrode according to still another embodiment of the present invention. (1) ………… Glucose oxidase electrode, (2) ………… p−
Benzoquinone-graphite paste electrode, (3) ………… GOD,
(4) ……… Nitrocellulose thin film, (5) ……… Platinum wire,
(6) ……… Glass tube.

Claims (1)

[Claims] 1. A tubular insulator comprising a substrate-sensitive portion at one end thereof, the substrate-sensitive portion comprising a paste-like electrode obtained by mixing an electron-accepting compound in graphite paste, and a paste-like electrode of the paste-like electrode. An oxidase electrode comprising a surface-immobilized oxidase enzyme and a permeable thin film of a substrate of the oxidase enzyme covering the outer surface thereof.