JP2508297B2 - Enzyme electrode - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to an enzyme electrode, and more particularly to an enzyme electrode capable of quickly and simply measuring the concentration of a substrate.

[Prior Art] Many oxidoreductases that require coenzymes can be applied engineeringly, such as alcohol dehydrogenase and lactate dehydrogenase. However, in order to apply these enzymes to enzyme electrodes, a technology for immobilizing a large amount of coenzymes (for example, nicotine adenine dinucleotide; hereinafter referred to as NAD) on the electrode surface while maintaining high activity is required. is there. As an example of a conventional electrode on which a coenzyme is immobilized, there is an electrode in which an enzyme and a coenzyme solution are coated on carbon (Japanese Patent Application No. 56-6961 (Japanese Patent Application Laid-Open No. 57-120853)).

The present inventor previously mixed alcohol dehydrogenase with NAD, which is used as a complement to this enzyme, in an aqueous solution of bovine serum albumin (hereinafter, referred to as BSA), and cross-linked it with glutaraldehyde (hereinafter, referred to as GA). "Japanese Patent Application No. 1-201208 (Japanese Patent Laid-Open No. 3-65644)" in which an enzyme electrode having a film formed on a substrate electrode is manufactured.

[Problems to be Solved by the Invention] However, if a large amount of an enzyme or a coenzyme is to be immobilized on a BSA membrane crosslinked with GA, the physical properties of the membrane are deteriorated, and a good membrane cannot be produced. Therefore, it is impossible to immobilize high-concentration enzyme and coenzyme on a membrane containing both enzyme and coenzyme prepared by the conventional method, resulting in problems in measurement sensitivity and repeatability.

The present invention has been made to solve the above-mentioned conventional problems, and an object thereof is to provide an enzyme electrode having a large amount of coenzyme immobilized together with an enzyme and having excellent measurement sensitivity and repeatability. And

[Means for Solving the Problems] The present invention is directed to an electrode, a membrane containing an enzyme, a membrane containing a coenzyme used in a complementary manner with the enzyme, and a membrane with slow diffusion of the coenzyme (outer layer). Membrane) is sequentially formed on the substrate, which is an enzyme electrode.

The outer membrane used in the present invention is preferably one having a small ratio of the diffusion rate of the coenzyme to the diffusion rate of the substrate, and examples thereof include a bovine serum albumin membrane crosslinked with glutaraldehyde.

[Action] By separating the enzyme-containing film and the coenzyme-containing film,
Each of them can be immobilized at a high concentration on the electrode, and the measurement sensitivity of the obtained enzyme electrode to the substrate and the repeatability are improved. In addition, since the membrane is provided on the electrode in the order of the enzyme membrane and the coenzyme membrane, the enzyme reaction occurs at a position closer to the electrode,
The reduced form of coenzyme produced as a result of the enzymatic reaction can be more captured by the electrode. Therefore, the measurement sensitivity is further improved.

[Embodiment] Next, an embodiment of the present invention will be described in detail with reference to the drawings.

An enzyme electrode according to the present invention was prepared using alcohol dehydrogenase as an enzyme and NAD as a coenzyme.
First, 2 U / μ was formed on an electrode (electrode area 3.52 mm ² ) on which gold was vapor-deposited on a sapphire substrate, which was manufactured by the method described in Japanese Patent Application No. 63-282721 (JP-A-2-128152).
A solution prepared by adding GA to 0.5% of alcohol dehydrogenase and 15% BSA was added dropwise, spin coating was performed at 2000 rpm for 30 seconds, and the mixture was allowed to stand at room temperature for 2 hours to prepare an enzyme membrane.

Next, a solution prepared by adding GA to 0.5% of 100 mM NAD and 15% BSA was added dropwise, spin was applied at 2000 rpm for 30 seconds, and left standing at room temperature for 2 hours to prepare a coenzyme membrane.

Finally, the surface of the electrode on which the above film was formed was covered with an outer film. GA was added to 15% BSA on the electrode where the coenzyme membrane was introduced.
Solution was added dropwise so as to be 5%, 2000 rpm, 30
It was spin-coated for 2 seconds and left at room temperature for 2 hours. The obtained enzyme electrode is designated as A.

On the other hand, as a comparative example, an enzyme electrode prepared in the same manner as in the above example except that the enzyme and the coenzyme were immobilized on the same membrane on the surface of the electrode is designated as B.

FIG. 1 (a) is a sectional view of an enzyme electrode A to which an enzyme and a coenzyme are sequentially immobilized by applying the present invention. Fig. 1 (b)
FIG. 4 is a sectional view of an enzyme electrode B in which a conventional enzyme and coenzyme are immobilized on the same membrane. In the figure, 1 is a substrate, 2 is an electrode, 3 is an enzyme-immobilized membrane, 4 is a coenzyme-immobilized membrane, 5 is a membrane on which both an enzyme and a coenzyme are immobilized, and 6 is an outer membrane that prevents the dissipation of coenzyme. is there.

The current response to ethanol was measured for the A and B electrodes. The structure of the measuring device is shown in FIG. In the figure,
11 is an enzyme electrode, 12 is a counter electrode, 13 is a silver / silver chloride reference electrode, 14
Is a buffer solution containing ethanol, 15 is a potentiostat, and 16 is a recorder. The potential required for the oxidation of the reduced form of NAD produced by the enzyme reaction, 0.75 V vs. Ag / AgCl, was applied to the enzyme electrode, and the change in the current obtained by adding ethanol to the reaction solution was recorded.

FIG. 3 is a diagram showing the current response to 500 mM ethanol obtained with the A and B electrodes. Electrode A having a separate immobilization membrane for the enzyme and coenzyme had a larger current value than Electrode B in which the enzyme and coenzyme were immobilized on the same membrane, and the measurement sensitivity was improved. FIG. 4 is a diagram showing changes in current response to repeated measurement of 500 mM ethanol. In the figure, □ indicates the case of the electrode A, and + indicates the case of the electrode B. Compared with the electrode B, the electrode A had a large amount of both the enzyme and the coenzyme immobilized in an active state, so that the reusability was improved and the measurement could be repeated 10 times or more.

[Effects of the Invention] As described above, the enzyme electrode of the present invention has the following advantages over conventional enzyme electrodes in which an enzyme and a coenzyme are immobilized on the same membrane.
Since both enzymes and coenzymes can be immobilized in large amounts in the active state, measurement sensitivity and repeatability are greatly improved.

[Brief description of drawings]

1 (a) is a sectional view of an enzyme electrode according to the present invention, FIG. 1 (b) is a sectional view of an enzyme electrode according to a conventional example, and FIG. 2 is a configuration diagram of an apparatus for measuring ethanol concentration using the enzyme electrode. ,
FIG. 3 is a characteristic diagram showing the current response of the enzyme electrode to ethanol in one embodiment of the present invention in comparison with the conventional example, and FIG. 4 is the current response to the repeated measurement of the enzyme electrode in one embodiment of the present invention. It is a characteristic view shown in comparison with an example. 1 ... Substrate, 2 ... Electrode 3 ... Enzyme-immobilized membrane, 4 ... Coenzyme-immobilized membrane 5 ... Membrane with both enzyme and coenzyme immobilized 6 ... Outer membrane, 11 ... Enzyme electrode 12 …… Counter electrode, 13 …… Silver / silver chloride reference electrode 14 …… Buffer solution, 15 …… Potentiostat 16 …… Recorder

Claims (1)

(57) [Claims] 1. An electrode, a film containing an enzyme, a film containing a coenzyme that is used complementarily to the enzyme, and a film (outer film) in which the coenzyme diffuses slowly are sequentially formed on a substrate. An enzyme electrode characterized by being formed.