JPS5853863B2 - Enzyme electrode system - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improvement in an enzyme electrode system used in a biochemical sensor or an enzyme battery that detects and measures biological substances by using an enzyme, which is a biological catalyst, as an electrochemical catalyst.

More specifically, an enzyme that catalyzes the hydrogen transfer reaction can be added to various dehydrogenase electrode systems that require coenzymes such as NAD (cotinamide adenine dinucleotide) or NADP (cotinamide adenine dinucleotide phosphate). Depending on the biologically relevant substance (substrate) of the enzyme electrode
This improves the response characteristics.

Conventionally, enzyme electrodes containing coenzymes have been used for dehydrogenases, coenzymes,
It is composed of a redox compound and a current collector, and its electrochemical reaction is shown in Figure 1.

That is, the coenzyme and redox compound are conjugated to the dehydrogenation reaction of the substrate AH2, such as an organic acid such as malic acid, lactic acid, or glutamic acid, and a current flows between the current collector and the counter electrode.

However, conventional enzyme electrodes do not have excellent response characteristics and are insufficient for practical use.

As a result of various studies on this type of enzyme electrode, the present inventors found that the rate-limiting coupling reaction between a coenzyme and a redox compound.

In view of the above, enzymes that catalyze hydrogen (electron) transfer reactions, such as ribamide dehydrogenase (diaphorase), reduced NADP dehydrogenase (bright yellow enzyme), reduced NAD(P) dehydrogenase (DT diaphorase), and reduced NAD dehydrogenase By adding (cytochrome C reductase), we succeeded in greatly improving the response characteristics of the enzyme electrode to the substrate.

The present invention will be explained in detail below.

Example l Figure 2 shows a measurement system using an enzyme electrode.

1 is an enzyme electrode system, 2 is a saturated calomel electrode (SC) as a reference electrode.
E), 3 is a platinum counter electrode, 4 is a salt bridge, and 5 is a separator.

6 is the current collector of the enzyme electrode thread, which is made of platinum, and the electrolyte γ contains 5 units of malate dehydrogenase and 1X10 oxidized coenzyme NAD in a phosphate buffer with a pH of 7.0.
'mol/l, methylene blue as an oxidized redox compound is dissolved at 1 x 10-3 mol/l.

The electrolytic solution 8 on the counter electrode side is a phosphate buffer solution with a pH of 7.0.

In the above thread, an equivalent amount of O0 units of diaphorase is added to the electrolytic solution 7, and the working electrode current collector 6 is set at +0.4V (vs.
, 5CE), and the current flowing between the current collector and the counter electrode was measured.

As a result, as shown in FIG. 3A, a steady current value of about 2 μA is obtained by adding malic acid.

On the other hand, the response characteristics of the conventional system in which diaphorase was not added to the electrolytic solution 7 were as shown in curve B.

As described above, according to the electrode system of the present invention, an extremely excellent response can be obtained at a steady current value, which is about IO times that of the conventional system.

Example 2 Using the same three-electrode configuration as in Example 1, P H7
, MgCA, an activator of isocitrate dehydrogenase, in 0 phosphate buffer! Isocitrate dehydrogenase, oxidized coenzyme NADP, and potassium ferricyanide, a redox compound, are added to the electrolytic solution on the working electrode side in which io'' mol/l of 2 is dissolved.

When the working electrode was anodically polarized at +〇, 4V (vs-SCE) and ink citric acid was added, a steady current value of about 0.1 μA was obtained.

Furthermore, when 0.5 unit of NADP peroxidase was added as a hydrogen transfer reaction catalytic enzyme, the steady-state NK value increased about 8 times.

Example 3 As the same three-electrode configuration and polarization conditions as in Example 1, L
-Alanine dehydrogenase, oxidized NAD.

A redox compound, flavin mononucu V otide, is added to the electrolyte on the working electrode side.

When L-alanine was added, a steady current value of about 0.2 μA was obtained.

Furthermore, when NAD peroxidase was added, the steady-state current value increased approximately io times.

Examples of hydrogen transfer reaction catalytic enzymes that have the effect of increasing the response current as described above include reduced NADP tehydrogenase, reduced NAD(P) dehydrogenase, reduced NAD dehydrogenase, and ferredoxin-NADP reductase.

The latter two of these are enzymes whose electron acceptors are natural redox compounds (proteins) such as cytochrome C1 ferredoxin.

The enzyme electrode system according to the present invention allows current to flow in accordance with the substrate concentration of dehydrogenase, so it can be used as a substrate concentration sensor.

Furthermore, when the dehydrogenase concentration is changed in a system containing a large excess of VC as a substrate, a current flows depending on the enzyme concentration.
It is also possible to measure the activity of dehydrogenase.

In addition, when combined with a suitable counter electrode such as an oxygen electrode, a fuel cell using the substrate as fuel can be constructed.

As described above, according to the present invention, the sensitivity of the enzyme electrode system as a sensor, the output as a battery, the reaction efficiency, etc. are significantly improved.

[Brief explanation of drawings]

Figure 1 is a schematic diagram showing the mechanism of an electrochemical reaction using a conventional enzyme electrode, Figure 2 is a schematic diagram showing the configuration of an electrochemical measurement system including an enzyme electrode, and Figure 3 is a diagram showing the structure of an electrochemical measurement system including an enzyme electrode. A comparison of response characteristics when added is shown.

Claims (1)

[Scope of Claims] 1. A dehydrogenase, its coenzyme, a redox compound, and a current collector, and the dehydrogenase, coenzyme, and redox compound are present in an electrolytic solution in which the current collector is immersed. . An enzyme electrode system further comprising an enzyme that catalyzes a hydrogen transfer reaction between the coenzyme and the redox compound and is present in the electrolyte. 2. A patent claim in which the hydrogen transfer reaction catalytic enzyme is selected from the group consisting of diboamide dehydrogenase, reduced NADP dehydrogenase, reduced NAD(P) dehydrogenase, reduced NAD dehydrogenase, fentoxin-NADP reductase, NAD peroxidase, and NADP peroxidase. The enzyme electrode system according to item 1.