JPH01221653A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To improve the responsiveness in measuring the high concn. of alcohol by using a silicone oily material as a binder in graphite paste which constitutes a pasty electrode of an enzyme electrode. CONSTITUTION:The graphite paste is prepd. by uniformly mixing and kneading graphite powder, silicon grease or silicone oil as the binder and p-benzoquinone as an electron acceptor. This graphite paste is packed in one end of a glass tube 1 to form the pasty electrode 2. The exposed surface of the outside thereof is polished to a uniform surface. A soln. prepd. by dissolving alcohol oxidase in a phosphoric acid buffer soln. is dropped onto this surface and is air-dried to form an enzyme layer 3. Such surface is covered by a dialysis membrane 4 and is fixed by a rubber O-ring 5. A platinum wire 6 is inserted from the other end of the glass tube 1 and is fixed to the pasty electrode 2.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to an enzyme electrode. More specifically, the present invention relates to an enzyme electrode for amperometrically measuring the concentration of various substances in solution that can serve as substrates for enzymes such as oxidase and dehydrogenase.

(b) Conventional technology pH has traditionally been used to measure chemical substances that can serve as substrates for enzymes.
A so-called enzyme electrode in which an enzyme is immobilized is used as the sensitive part of an electrode such as an oxygen electrode, and the pH generated by the enzyme reaction is
Methods are known for quantifying substrate concentration based on changes in oxygen consumption, etc.

However, in enzyme electrodes that measure based on pH changes, it is impossible in principle to avoid being affected by pH changes in the sample itself, and the substrate specificity of enzymes is limited under extremely limited conditions (constant pH). It can only be used in In addition, in a sample that has a buffering effect, even if a chemical substance that causes a pH change is generated, it will not lead to a change in the pH of the system, and measurement itself is impossible.

On the other hand, enzyme electrodes that measure based on oxygen consumption eliminate the above disadvantages, but when the substrate concentration increases, the dissolved oxygen in the sample is consumed and the detection output reaches a plateau. It has the disadvantage that it cannot be applied to measurements and is strongly affected by fluctuations in the partial pressure of dissolved oxygen in the sample.

Therefore, the enzyme il! is measured based on such oxygen consumption. In order to eliminate the drawbacks of electrodes, graphite powder and paraffin,
An oxidase electrode using a paste-like electrode in which an electron transfer medium such as an electron-accepting compound is contained in a graphite paste made of a binder such as undecane has been proposed (
(Japanese Patent Application Laid-Open No. 61-61049). According to such an oxidase electrode, since oxygen is not required for the enzymatic reaction, it is possible to measure the substrate amperometrically based on the enzymatic reaction while preventing the output from peaking due to lack of oxygen and the adverse effects of dissolved oxygen fluctuations. .

(c) Problems to be Solved by the Invention However, when measuring a substrate exhibiting organic solvent properties such as alcohol using the above oxidase electrode, the response may decrease as the substrate concentration increases. There were cases where it became unstable and caused problems in high concentration measurements. Furthermore, similar troubles occurred during measurement with an enzyme electrode in which dehydrogenase was immobilized on the paste-like electrode instead of oxidase.

This invention was made under such circumstances, and aims to provide an enzyme electrode that can accurately and stably measure high concentrations, especially when using substrates exhibiting organic solvent properties such as alcohol. It is something.

(d) Means for Solving the Problems From the above viewpoint, the present inventors conducted intensive research and found that by using a silicone oil as a binder in the graphite paste that constitutes the paste electrode of the enzyme electrode, ,
We have discovered that the responsiveness in measuring high alcohol concentrations is significantly improved and have arrived at this invention.

Thus, according to the present invention, there is provided a pasty electrode comprising an electron acceptor contained in a graphite paste, a permeable membrane of an enzyme substrate covering the externally exposed surface of the pasty electrode, and a permeable membrane containing an enzyme substrate that covers the externally exposed surface of the pasty electrode. Provided is an enzyme electrode comprising a substrate sensitive portion comprising an enzyme layer fixed between the membrane and the graphite paste, wherein the graphite paste is comprised of a mixture of graphite powder and silicone oil. be done.

The silicone-based oil used in this invention means a hydrophobic liquid or semi-liquid material mainly composed of polyorganosiloxane, and includes, for example, silicone oil and silicone grease. By mixing this silicone oil and graphite powder, a graphite paste material constituting the paste electrode of the present invention can be obtained. In this case, the amount of graphite powder is preferably about 50 to 80% by weight from the viewpoint of maintaining the shape of the sensitive surface. Further, the particle size of the graphite powder is preferably about 1 to 50 μm.

The graphite paste used in the present invention can be obtained by incorporating an electron acceptor into the graphite paste. Here, the electron acceptor refers to a compound that acts as an electron acceptor for an enzymatic reaction in the presence of an immobilized enzyme and a substrate,
For example, when targeting oxidase, p-benzoquinone, potassium ferricyanide, dichlorophenylindophenyl, phenazine methosulfate, etc. may be mentioned. Further, when targeting dehydrogenase, NAD, dichlorophenylindophenol, etc. may be mentioned.

It is suitable to incorporate these electron acceptors into the graphite paste by mixing them at the time of preparing the paste.

Although the content of the electron acceptor is not particularly limited, it is usually suitable to be about 0.1 to 30% by weight.

In addition, as a permeable membrane for the enzyme substrate of this invention, Cell 9
Examples include film-like membranes such as acetate membrane, nitrocellulose membrane, K-carrageenan gel membrane, polyacrylamide gel membrane, and transparent tFi membrane.

Further, the enzyme may be immobilized between the permeable membrane and the paste electrode by physical immobilization or chemical immobilization. For example, it may be immobilized on a paste-like electrode or on the above-mentioned permeable membrane.

(e) Effect The decrease in response or destabilization that conventionally occurs when using a sample that contains a large amount of organic solvent substrates such as alcohol is caused by the fact that these substrates partially bind the graphite paste binder. This is thought to be due to the phenomenon of elution and release of the electron acceptor contained therein. Since the binder of the graphite paste in this invention consists of silicone oil,
It is thought that such elution phenomena are significantly suppressed, and as a result, adverse effects on responsiveness are prevented even when the substrate concentration is high.

(f) Example 400 wg of graphite powder (10 μm) and silicone grease (High Vacuum Gre) as a binder.
ase ; 'manufactured by Dow Corning Co., Ltd.) 240μ or silicone oil (WP-30: manufactured by Wako Pure Chemical Industries, Ltd.) 240μQ
and p-benzoquinone 200x9 as an electron acceptor were uniformly mixed and kneaded to prepare two types of graphite pastes.

This graphite paste was filled into one end of a glass tube with an inner diameter of 5 x m, and the externally exposed surface was polished to a uniform surface.
n origin: Boehringer Mannheim) 5 u to pH
20 μe of a solution dissolved in 100 μe of 7.5 O, IN phosphate buffer was added dropwise and air-dried. Next, this surface was covered with a dialysis membrane (manufactured by ViskLng), which was fixed with a rubber O-ring, and a platinum wire was inserted and fixed into the graphite paste layer from the other end of the glass tube.
An enzyme electrode of the present invention as shown in the figure was obtained.

In the figure, (1) is a glass tube, (2) is a paste electrode made of graphite paste, (3) is a retained and fixed enzyme layer, (
4) is a dialysis membrane, (5) is an O-ring, and (6) is a platinum wire.

In order to evaluate this enzyme electrode, an electrolyte (pH 7.5, I
A predetermined amount of ethanol is added to this electrolyte, and constant voltage electrolysis is performed at 30°C with gentle stirring at an applied voltage of 0.45V. , and investigated its current responsiveness.

The results are shown in FIG. In addition, in the figure, plot -
〇- is the one using silicone oil, Plot-Δ- is the one using silicone grease, Plot-・-
This is about an enzyme electrode of a comparative example prepared in the same manner except that liquid paraffin was used instead of the silicone oil.

In this way, the conventional enzyme using liquid paraffin as a binder 1! At the poles, there was a significant drift in the measured values for ethanol of 6 wt% or more (not shown in the figure).
High concentration measurements were not possible. In contrast, the enzyme electrode of the present invention is capable of measuring up to at least 20 wt%, and has been found to have significantly improved responsiveness to high-concentration substrates.

(G) Effects of the Invention According to the enzyme electrode of the present invention, when measuring an organic solvent-based substance, it is possible to perform high-degree measurement, and it is possible to directly measure a highly concentrated sample without diluting it.

Also, the lifespan of the sensor will be improved.

Therefore, it is extremely useful for applications such as biosensors for controlling and monitoring bioreactors and fermentation processes.

[Brief explanation of the drawing]

FIG. 1 is a configuration explanatory diagram showing an enzyme electrode according to an embodiment of the present invention, and FIG. 2 is a graph diagram similarly illustrating its responsiveness together with a comparative example. ! ...Glass tube, 2...Pest-shaped electrode, 3...Enzyme layer, 4...Transparent V
IT film, 5...O-ring, 6...Platinum wire.

Claims (1)

[Scope of Claims] 1. A paste-like electrode containing an electron acceptor in graphite paste, a permeable membrane of an enzyme substrate covering the externally exposed surface of this paste-like electrode, and a permeable membrane containing an enzyme substrate that 1. An enzyme electrode comprising: a substrate-sensitive portion comprising an enzyme layer fixed between a transparent membrane; and wherein the graphite paste is comprised of a mixture of graphite powder and a silicone oil.