JPH0296649A - Dehydrogenase electrode - Google Patents

Abstract

PURPOSE:To enable measurement of nicotinamide adenine nucleotide or nicotinamide adenine dinucleotide phosphate with high sensitivity by modifying a carbon electrode with charge-transfer complexes and dehydrogenase. CONSTITUTION:A carbon electrode is modified with charge-transfer complexes and dehydrogenase. As for the carbon electrode, a paste-form electrode obtained by mixing the powder of carbon such as carbon black or carbon graphite with a nonpolar binder such as paraffin, is used. A platinum electrode, a silver/silver chloride electrode or the like is used as an opposite electrode. As for the charge- transfer complexes, moreover, Wurster salt or tetracyanoquinodimethane is used. As for the dehydrogenase, glucose-6-phosphate dehydrogenase, lactic dehydrogenase or the like is used. The carbon electrode can be modified simply by kneading the charge-transfer complexes and the dehydrogenase in the carbon- paste-form electrode.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention is directed to the use of nicotinamide adenine dinucleotide (NADH or NAD) or nicotinamide adenine dinucleotide phosphate (NADPH or NA) in a sample.
The present invention relates to a dehydrogenase electrode suitably used for measuring the concentration of DP).

(Prior art) NADH or NAD or NADPH or NAD
Quantification of P provides an important means for biochemical analysis methods such as clinical chemistry analysis 2 food analysis. especially,
Quantification of these substances has become an indispensable means in clinical chemical analysis, for example.

The activity values of taleatine kinase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, etc. in biological samples such as blood are measured by quantifying these.

In such analysis, an appropriate substance is applied to the substance to be measured to bring it into a form that is easy to quantify.Recently, enzymes have been attracting attention as suitable substances, and many are being used. Enzymes are now playing a leading role in analysis items.

The form that is easy to quantify is H10□ or NAD
(H) or NADP(H) is adopted.

When oxidase is used as an enzyme, H2O is stationary, and when dehydrogenase is used.

NAD (H) or NADP (H) will be quantified. Comparing the two, the latter is theoretically better because the former has problems with stoichiometry, is more susceptible to the amount of dissolved oxygen, and is more susceptible to reducing substances, etc. It is said to be excellent. In the latter case, N
Since ADH or NADPH has a characteristic absorption spectrum in the ultraviolet region of 340 nm, it is quantified by measuring the increase or decrease in absorbance at 340 nm.

Since it requires an expensive spectrophotometer, NAD is more convenient.
There are high hopes for the development of a biosensor that can quantify (H) or NADP (H). For example, attempts have been made to develop an electrode (1) that measures the current when NADH is directly oxidized on the electrode.

Also, from this, the ratio of signal (S) to noise (N).

That is, for the purpose of improving the S/N ratio, NADH is not directly oxidized on the electrode, but an enzyme is combined with an appropriate mediator to perform a redox reaction, and an electrode (1) that indirectly obtains a current is used.
2) [Review of Polarography (Revi
ew of Polarography) Vo13
1, 31 (1985)).

On the other hand, the present inventors have proposed a dehydrogenase electrode (3) in which NAD (H), diaphorase, vitamins, and a dehydrogenase surround are immobilized [Review of Polarography of
Polarography) Vol 33,
20 (19B?) and Japanese Unexamined Patent Publication No. 61-61050].

Reference - The present inventors have already developed a dehydrogenase electrode (4) in which diaphorase and the king of diaphorase mediators, vitamin and glucose 6-phosphate dehydrogenase, are immobilized on an electrode that converts the transfer of electrons into electrical signals.
) was proposed (Journal of the Japanese Society of Agricultural Chemistry, Vol. 62, No. 3, 59)
5, 1988).

(Problem to be solved by the invention) Since the electrode in (1) above requires a large electrode applied voltage (approximately 0.7 V), an electrolyte with an electrolytic voltage value lower than the electrode applied voltage coexists in the liquid to be measured. When this occurs, an electrolytic current due to this coexisting substance, that is, an unstable dark current, flows.
The S/N ratio becomes extremely poor, and it cannot be practically used for measuring NAD (H) in a liquid to be measured containing various electrolytes. In addition, the electrode of (2) above has problems with electrode stability, resulting in poor reproducibility of measured values, and furthermore, the mediator is in a free state, so it has not been realized as a practical electrode.

The reality is that none of the above electrodes can quantify NAD (H) or NADP (H) with high sensitivity (up to the order of several nM).

On the other hand, the electrode in (3) above uses enzyme substrates (ethanol,
(lactic acid, glycerol, etc.) down to a few mM.
This level of sensitivity is not sufficient, and furthermore, this electrode is not intended to quantify NAD (H) or NADP (H).

Furthermore, the electrode (3) above is an ultra-sensitive NAD (11)
Alternatively, this method realizes the measurement of NADP (H), but because it uses an electrode with immobilized diaphorase and vitamin K, it is expensive and the reaction system tends to be somewhat complicated, so it is not necessarily optimal for all measurement systems. That doesn't mean there is.

(Means for Solving the Problems) The present inventors have conducted intensive research to solve these problems, and have discovered that a dehydrogenase electrode, which is a carbon electrode modified with a charge transfer complex and dehydrogenase, The inventors have discovered that it is possible to quantify NAD (H) or NADP (H) with high sensitivity, in two steps, and with high precision, and have completed the present invention.

That is, the present invention provides an NA characterized in that a carbon electrode is modified with a charge transfer complex and a dehydrogenase.
The subject matter is a dehydrogenase electrode that is sensitive to D (H) or NADP (H).

Examples of the carbon electrode used in the present invention include a paste-like electrode obtained by mixing carbon powder such as carbon black or carbon graphite with a non-polar binder such as liquid paraffin, and if this electrode is used as an anode, good.

Further, as the cathode (counter electrode), one of various electrodes 1 such as a platinum electrode, a silver/silver chloride electrode, a silver/silver oxide electrode, a mercury/mercurous chloride electrode, etc. can be used.

Examples of charge transfer complexes used in the present invention include Wurster salts or salts having semiquinoids, thiotetracene, chloranil, tetracyanoethylene, tetracyanoquinodimethane, trinitrobenzene, or derivatives thereof as electron acceptors; Among these, salts of tetracyanoquinodimethane or its derivatives are particularly preferred.

Further, as electron donors in these salts, sulfate ions, nitrate ions, halogen ions, etc. are used, including vinylpyridine and vinylimidazole.

Polymeric type salts such as aminostyrene can also be used as electron donors.

The dehydrogenase used in the present invention includes:

Examples include glucose 6-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, glucose dehydrogenase, and glutamate dehydrogenase. Various types of these are used, including those derived from microorganisms and those derived from animals. Among them, those produced by microorganisms having an optimum growth temperature of 50°C to 85°C are preferred. As such a microorganism.

Examples include microorganisms of the genus Bacillus such as Bacillus stearothermophilus, Bacillus thermoproteolyteix, and Bacillus acidocaldarius, Thermoactinomyces, Thermus, and Thermomicrobium. Among these, a particularly preferred microorganism is Bacillus stearothermophilus, and a specific example thereof is ATCC? 933,7954.10194,1
2980, NCA1.503. UK563 strain (FERMP-7275, FERMP-7275, September 1982)
(Deposited on May 29th) etc.

Also, as a substrate for the above dehydrogenase.

For example, glucose 6-phosphate in the case of glucose 6-phosphate dehydrogenase, lactic acid in the case of lactate dehydrogenase, malate in the case of malate dehydrogenase, alcohol in the case of alcohol dehydrogenase, and alcohol in the case of glycerol dehydrogenase. In some cases, glycerol.

In the case of glucose dehydrogenase, glucose is
In the case of glutamate dehydrogenase, glutamic acid is used, and it is a feature of the present invention that such an inexpensive substance can be used effectively.

To modify a carbon electrode with a charge transfer complex and dehydrogenase in the present invention, 1. For example, the above-mentioned carbon paste can be used as an electrode, and the modification can be easily carried out by kneading the charge transfer complex and dehydrogenase into this paste-like electrode. In the case of a charge transfer complex, it can also be modified while being enclosed in a water-insoluble carrier.

In the case of dehydrogenase, it is made into a solution and applied or dropped onto the surface of a paste-like electrode.

The solvent is retained by evaporation, and dehydrogenase, such as a semipermeable membrane, does not permeate the surface, but
Preferably, the dehydrogenase substrate is coated with a permeable thin film.

By coating with such a thin film, dehydrogenase is stably maintained. One example of such a semipermeable membrane is a cellulose acetate membrane.

Cellulose-based membranes such as nitrocellulose membranes, various synthetic polymer membranes, and natural polymer membranes are used.

Moreover, dehydrogenase can also be used by being immobilized on the above-mentioned thin film. This immobilization method is described, for example, in Chibata, ed. [Immobilized Biocatalysts J Kodansha (1986), 12-
Conventionally known covalent bonding methods and adsorption methods as described on page 67 can be employed, and any method such as a crosslinking method or an entrapment method can also be employed.

To measure NAD (H) or NADP (H) using the dehydrogenase electrode of the present invention, for example, N-methylphenadium-7.7.8
．． A dehydrogenase electrode on which 8-tetracyanoquinodimethane and glucose 6-phosphate dehydrogenase are immobilized may be immersed in a buffer containing glucose 6-phosphate, and the change in electrode current caused by the addition of the measurement sample may be measured.

The immobilized product of glucose 6-phosphate dehydrogenase was
-Methylphenadium-? , 7.8.8 Tetracyanoquinodimethane kneaded carbon paste directly coated on the electrode sensitive surface is immersed in a buffer solution containing glucose 6-phosphate, and the change in electrode current caused by the addition of the measurement sample is measured. Bye. Also.

If it is desired to retain glucose 6-phosphate on the electrode surface, an anionic membrane can be used to coat and retain glucose 6-phosphate dehydrogenase on the electrode sensitive surface.

As the solution for measurement, for example, a solution of 0.01 to 0.5M Trjs-HC (! buffer (pH 5 to 10, preferably 7 to 9) may be used.The measurement temperature is 0 to 60
°C, preferably 20 to 40 °C is used.

(Function) The dehydrogenase electrode of the present invention uses the electrode to detect the amount of electron transfer generated by the secondary reaction, thereby detecting NAD (H) or NADP (H) originally present in the sample.
) in the sample as well as the amount of NAD ( ) in the sample.
H), NADP (H) The substance to be measured other than (H) is finally converted to NAD (H) by the action of dehydrogenase using an appropriate means.
) or NADp (H) Wk, and can be measured by measuring the amount of NAD (H) or NADP (H) in the sample.

NAD(P)H+ Mox −−1−−−−−−◆
NAD(P)""MraaM,,, +(
el) M,, + e-(el) (where DH is dehydrogenase, S is the substrate of dehydrogenase, P is the oxide of S + Mr*a is the reduced charge transfer complex + M ox is the oxidized charge transfer Complex.

el represents an electrode, and e- represents an electron. ) (Example) Next, two embodiments of the present invention will be specifically explained using examples.

Example 1 <Preparation> Carbon paste was obtained by mixing 5 g of graphite powder with 3 ml of liquid paraffin. This carbon paste
As shown in Figure 1, a glass tube treated with TA water using dimethyldichlorosilane (outer diameter 511.inner diameter 3.4*+m)
packed at one end. The geometric surface area of the electrode at this time is 0.0
It was 9 cd.

On the surface of this electrode, N-methylphenadium-7,7,8
, 8 tetracyanoquinodimethane 18 mg, polyvinyl chloride 2 mg and tetrahydrahydrofuran 0.6
M! ! 5 μl of the mixed suspension was added dropwise, and the solvent was evaporated. Next, glucose 6-phosphate dehydrogenase [derived from Bacillus stearothermophilus (manufactured by Unitika), 10 mg/m7!] was applied to the surface of this electrode. An enzyme solution was prepared and used. ] 5μ! After dripping and evaporating the solvent, a semipermeable membrane 4 [nitrocellulose membrane (
(manufactured by Union Carbide)].

Furthermore, the tip of the electrode was covered with a nylon net 5 to provide physical strength. This electrode is shown in FIG.

This electrode should be kept in a 0.1 M phosphate buffer (pH
7,0) and stored at 4°C.

Using the above electrodes, a polarographic analyzer (manufactured by Yanagimoto Seisakusho), and an X-Y (t) recorder (manufactured by Yokogawa Hokushin Electric), a 3-electrode system was assembled to measure the 5 basic solutions. as 0.
1 M Tris-HCIl! yt buffer solution (pH 8,5
) and stirred with a magnetic rotor (800 rpm or more). The electrode potential was measured using chloride/chloride as a reference.

The temperature at this time was adjusted to 25°C.

<With NADH1> N-methylphenadium-7 prepared as above
．． 7.8.8 Tetracyanoquinodimethane and glucose 6-phosphate dehydrogenase-modified carbon paste electrodes were mixed with 0.1mM Trjs-HCj21jl solution (pH 8,
5) and apply voltage 1 m in the positive direction from 0.25V.
Sweep at a sweep speed of V/S and measure the cyclic portamogram to find the song &'jla (residual current) in Figure 2.
I got it. NADH was added to this solution to a concentration of 1 mM, and the voltage was swept in the same manner as above to obtain the current-voltage curve shown in FIG. 2, that is, a portamogram.

Further, 4.6mM glucose 6-phosphate was added to this solution, and the voltage was swept in the same manner as above.

A portamogram of the song NfAC in FIG. 2 was obtained.

As you can see from these portamograms.

It was found that a large current can be obtained with an NADH solution containing glucose 6-phosphate, allowing highly sensitive detection.

Furthermore, 10mM glucose 6-phosphate was added to the buffer solution (pH
8,5) and keep the electrode potential at 0.7■.

Steady oxidation current by changing the concentration of NADH! 1. The measurement results (after correcting the residual current) are shown in FIG.

FIG.
7■ A graph showing the relationship between the steady current tut (vertical axis) during application and the NADHi 1 degree (horizontal axis) in the liquid to be measured,
As shown in FIG. 3, it was found that the measurement had linearity over a wide range of NADH and could be measured with high sensitivity.

(Effects of the Invention) The dehydrogenase electrode of the present invention has sensitivity to NAD (H) or NADP (H) because the carbon electrode is modified with a heavy double transfer complex and dehydrogenase. Alternatively, it can show a large sensitive current to NADP(H). Therefore, the dehydrogenase electrode of the present invention can have a large S/N ratio with respect to electrolytic current due to coexisting substances, that is, dark current.

Although simple and inexpensive, NAD (H) or N
It has excellent performance in that it can measure ADP (H) concentration with high sensitivity.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the structure of an electrode showing one embodiment of the present invention, FIG. 2 is a graph showing a cyclic portamogram of the electrode of FIG. 1, and FIG.
- It is a graph showing the relationship between the steady current Itt when 0.7 V is applied in a buffer solution containing phosphoric acid and the degree of N A D HtlA in a liquid to be measured. ■...Glass tube 2--carbon paste electrode 3...N-methylphenadium-7.7.8. F
l Tetracyanoquinodimethane and glucose 6-phosphate dehydrogenase layer 4 - Semipermeable membrane 5 - Nylon net

Claims (1)

[Claims] (1) A dehydrogenase electrode sensitive to nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, which is made by modifying a carbon electrode with a charge transfer complex and dehydrogenase.