JPS6161050A - Dehydrogenase electrode - Google Patents

Abstract

PURPOSE:To measure the concn. of a substrate without adding an electron receptor compd. to liquid to be measured by incorporating the electron receptor compd. into graphite paste, immobilizing dehydrogenase enzyme thereto and coating the same with a thin permeable film. CONSTITUTION:Graphite and liquid paraffin are mixed to make a paste and the electron receptor compd. is incorporated therein. Nicotinamide-adenine- dinucleotide or the like is used as the electron receptor compd. Such pasty electrode 2 is put into a glass tube. The dehydrogenase enzyme 3 or the like is immobilized to the electrode 2. The permeable film of the substrate of the dehydrogenase, for example, cellulose acetate film 4 is coated thereon to protect the same. A platinum electrode 5 is connected thereto. Various substrates are measured in the liquid to be measured by the resulted electrode. Since the electron receptor is incorporated into the paste electrode, the electrode exhibits the large current response to the substrate such as ethanol and glucose and the measurement of the substrate with good accuracy is made possible.

Description

[Detailed description of the invention] (b) Industrial application fields This invention relates to dehydrogenase electrodes.

More specifically, it is possible to amperometrically measure the concentration of substances such as lactic acid, alcohol, glycerol, and glucose present in electrolytes that can serve as substrates for dehydrogenase enzymes, and in particular in biological samples such as serum, plasma, and urine. Related to dehydrogenase electrodes useful for measuring substrate concentration in enzyme reactors and also useful as electrodes for enzyme reactors or fuel cells.Prior artRecently, oxidoreductase immobilized electrodes have attracted attention. It has become. This electrode acts as a substitute for a chemical electron acceptor or donor for an enzyme reaction and is called a biocatalytic electrode. It has been proposed that it can be used in new applications such as reactors.

In particular, a proposal has been made to use an immobilized dehydrogenase enzyme, to set up a redox system by combining this with nicotinamide adenine dinucleotide (NAD) as an electron-accepting compound, and to use this for electrode reactions. is being exposed. In order to perform measurements without adding NAD to the sample solution for each measurement, attempts have been made to immobilize NAD together with enzymes.As a typical example, (NAr chemically bonded to a polymer compound) - A method of trapping a polymer compound along with an enzyme on the inner surface of a suitable substrate-permeable membrane and fixing it to a platinum or graphite electrode. A method of simultaneously trapping enzymes within the membrane and immobilizing them on platinum or graphite electrodes. ■ Chemically treating a substrate-permeable membrane to make it difficult for NAD to permeate; A method of fixing it to a graphite electrode is known.

However, the above methods (1) and (2) have a problem in that the enzymatic reaction activity of NAD itself is greatly reduced due to the polymerization of NAD. Furthermore, the method (2) has the problem that leakage of NAD cannot be completely prevented and the enzyme reaction activity decreases over time.

(c) Purpose This invention was made to solve the above-mentioned conventional problems! 0. The object of the present invention is to provide a dehydrogenase electrode that can maintain the enzyme reaction activity of an enzyme and an electron-accepting compound such as NAD for a long period of time without adding an electron-accepting compound such as NAD to a liquid to be measured.

B) Structure According to the present invention, there is provided a paste-like electrode in which an electron-accepting compound is contained in a graphite paste, a dehydrogenase enzyme identified on the surface of the paste-like electrode, and a substrate for the dehydrogenase enzyme whose nuclide is formed on the outer surface of the paste-like electrode. A dehydrogenase enzyme pole is provided, comprising a substrate sensing portion composed of a permeable thin film and a substrate sensing portion.

The dehydrogenase electrode of the present invention is usually used by setting up an electrolytic system in which the substrate-sensitive part is in contact with or immersed in a liquid to be measured and a paste electrode is used as an anode. 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/mercurous chloride electrode, etc. can be used.

Note that measurement as a sensor is usually performed by constant voltage electrolysis.

The reversing polarizer of the present invention usually has a substrate sensing section set at one end of a cylindrical insulator, and an extraction electrode is provided as appropriate. However, depending on the application, other forms (for example, directly incorporated into the flow path) may be used.
It is sufficient that at least the substrate sensitive portion is configured as described above. Note that, depending on the case, the counter electrode may be incorporated into the sensor support material.

The most characteristic feature of this invention is that a paste-like electrode containing an electron-accepting compound such as NAD is used as an electrode for immobilizing the dehydrogenase enzyme, and the electron-accepting compound in the dehydrogenase layer is thereby immobilized. The key point is that the enzyme reaction and electrode reaction are linked while keeping the concentration constant. These electron-accepting compounds consumed in the enzymatic reaction are regenerated in the electrode reaction, so that no net consumption occurs in the reaction. In this way, the intended substrate can be measured without adding these electron-accepting compounds to the liquid to be measured.

The graphite paste used in this invention is a mixture of graphite powder and a nonpolar binder such as liquid paraffin or undecane.

The graphite content in this paste is usually about 60 to 70% by weight from the viewpoint of electrical conductivity and shape retention.

Moreover, as graphite powder, one having a diameter of about 1 μm to 50 μm is suitable.

Typical examples of the electron-accepting compound contained in the graphite paste include NAD and dichlorophenolindophenol (DCIP), which also participate in the reaction as an electron acceptor in the presence of a dehydrogenase enzyme and a substrate. Compounds that are stably miscible in the graphite paste can be used. The appropriate content of these varies depending on the enzyme used, but for example, when using NAD, 1
~10% by weight is suitable. Content is 1 in graphite paste
If it is less than 10% by weight, it is unfavorable in terms of current sensitivity, and if it exceeds 10% by weight, it is unfavorable in terms of electrode shape retention and economical efficiency. The most preferred content is 3-5% by weight.

Various dehydrogenase electrodes can be appropriately selected to be immobilized on the paste-like electrode, depending on the substrate to be detected or converted.

The enzyme/substrate combinations include alcohol dehydrogenase/ethanol, glucose dehydrogenase/
Representative examples include glucose, glutamate dehydrogenase/glutamic acid, lactate dehydrogenase/lactic acid, and glycerol dehydrogenase/glycerol.

The dehydrogenase enzyme is usually immobilized on the pasty electrode by a solution method, that is, by applying a solution of the dehydrogenase enzyme onto the surface of the pasty electrode, dripping it, etc., and retaining the solution, and then evaporating the solvent. At this time, it is preferable to make the surface of the paste electrode as smooth as possible. The fixed amount of dehydrogenase used is, for example, 10 to 20 in the case of alcohol dehydrogenase.
Approximately 0 μf/- is preferable.

The permeable thin film of the substrate in this invention is useful for retaining and immobilizing the dehydrogenase enzyme attached to the electrode surface. As this permeable membrane, cellulose acetate-Nitrocellulose membrane, K-carrageenan gel membrane, polyacrylamide gel membrane, etc. can be used, and in particular, nitrocellulose formed by applying a collodion-containing liquid and drying it. Preferably, a membrane is employed. There is no limit to the film thickness, but in general, a thinner film is recommended to shorten the response time, and a thicker film is required to widen the response concentration over a wider concentration range. Depending on the type of membrane material, usually about 20 to 500 μm is suitable.

(e) Examples Hereinafter, this invention will be explained in detail with reference to Examples.

Example 1 (Alcohol dehydrogenase electrode) (4) Electrode production reagent 0 Alcohol dehydrogenase (ADH)...
...... Sigma IJEC, ], 1.1.
10 Nicotinamide Adenine Dinucleotide (NAD)
・・・・・・・・・・・・O collodion liquid manufactured by Sigma Co., Ltd. (5 units) ・・・・・・・・・・・・・・・O liquid paraffin manufactured by Wako Tsumugi Co., Ltd.・・・・・・・・・・・・・・・
Merck O graphite powder・・・・・・・・・・・・
...AACP manufactured by Nippon Graphite Co., Ltd. (10 μtn) A predetermined amount of NAD was mixed with 3 m of liquid paraffin, and then 5F graphite powder was added. The nine NAD-graphite paste thus obtained was filled into one end of a glass tube with an internal diameter of 3.4 m. This exposed surface was smoothed using wax vapor to set up a paste-like electrode having a surface area of 9.0 x 10''-.

Next, an ADH aqueous solution of a predetermined concentration was dropped onto the surface of the paste-like electrode using a syringe, and the solvent was evaporated. Thereafter, a thin film of nitrocellulose was formed on the electrode surface by casting and drying a 20 μt collodion-ethanol (volume ratio 1:4) mixture.

By attaching 9 lead-out electrodes made of platinum wire to the thus obtained electrode, and further attaching a nylon net and a Teflon tube for supporting the nitrocellulose membrane, the present invention as shown in FIG. Alcohol dehydrogenase electrode (ADH-immobilized NAD-graphite paste electrode) (1) was obtained. In addition, in the figure, (2) is NAD
- graphite paste electrode, (3) immobilized ADH, (
41 is a nitrocellulose thin film, (5) Ti platinum wire, (6
) indicates a glass tube, (7) indicates a nylon net, and (8) indicates a heat-shrinkable Teflon tube.0 Before using this electrode as a sensor, wash it several times with distilled water, and adjust the pH to 8. The sample was immersed and stored overnight in Tris-HCl buffer (3).

(2) Cyclic Portammetry The O measurement conditions for performing portammetry using the triode method on the alcohol dehydrogenase electrode described above are as follows.

Equipment O Botechostat (manufactured by Fuyo Co., Ltd.) O signal generator (HB-104: manufactured by Hokuto Denko Co., Ltd.) O counter electrode (platinum plate) O reference electrode (saturated calomel electrode) O recorder (x-y recorder; horizontal tube Electrolyte O (manufactured by Denki Co., Ltd.) Deoxygenated Tris-HCl (pH 8.3)
Temperature: 23±1°C Stirring speed: 500 rpmResult: 20.1oADH was immobilized on a graphite paste electrode containing 3% by weight of NAD. The electrode of the invention was immersed in the above Tris-HCl buffer, and a cyclic voltammogram was recorded at a potential scanning rate of 50 mV/s. The results are shown in FIG. 1 as a solid line.

This result was the same as a portangram similarly recorded for a similar electrode without NAD.

On the other hand, it was found that an electrode containing NADH, which is a reduced form of NAD, produced an anodic wave having a peak at 100.6V, as shown by the broken line in FIG.

In this way, it has been found that only NADH, which is the reduced form of NAD, exhibits an electrochemical response at the carbon paste electrode. That is, it was found that NADH irreversibly undergoes electrochemical oxidation at the paste electrode according to the following formula: % formula, and that NAD can be regenerated using this electrode.

(c) Electrochemical catalytic oxidation of ethanol When ethanol was added to the above buffer, it was observed that the anodic current increased at a potential more positive than +0.6V. It was found that as the ethanol concentration in the liquid to be measured increases, the steady-state electric current 8 increases. It has been found that this current I8 increases depending on the amount of NAD in the graphite paste and the amount of ADH on the electrode surface.

These results indicate that immobilized ADH maintains its activity, and therefore NAD dragged to the interface between immobilized ADH and the paste electrode is reduced to NADH as an electron acceptor in the enzymatic reaction of ADH. and that this reduced NADH is electrochemically regenerated, resulting in a steady current.

2Qttf ADH, NAD (313ii'%)
−M For electrodes immobilized on lead paste 10, S
The dependence of Is on ethanol concentration when applying V (vs. saturated calomel electrode) is shown in FIG. This figure is 1
It can be used as a calibration curve when measuring the ethanol concentration in a liquid to be measured using one electrode.

In addition, at an ethanol concentration of 10 mM or less, the current response of this electrode, which shows a linear response, is rapid and reaches a steady current value within 20 seconds.Example 2 (Glucose dehydrogenase electrode) Example 1
Glucose dehydrogenase (GDH; a crude extract isolated from Pseudomonas fluorescens) was used instead of ADH, and dichlorophenolindophenol (DCIP: manufactured by Gyudon Chemical Co., Ltd.) was used as a substitute for NAD. Example 1. A glucose dehydrogenase electrode similar to that shown in Figure 1 was prepared in the same manner as in (A).
The electrode is washed several times with distilled water until ready for use, p)
This electrode was immersed and stored overnight in an I5.0 acetate buffer, and portammetry was performed using the same three-electrode method as in Example 1(B). i.e. 100μV GDH
The electrode of the present invention, which was immobilized on a graphite paste electrode containing 3 wt. of DCIP, was immersed in an acetate buffer at pH 5.0, and the cyclic voltammogram was recorded at a potential scanning rate of 50 mV/s. The results are as shown in Figure 4, and the cathode wave was 10.2V.
, for the anodic wave, peaks were observed at +0 and 3v.0 When glucose was added to the above buffer, +0
．． An increase in anode current was observed at a positive potential of 4 V. It was found that the steady-state current Is increases as the glucose concentration in the liquid to be measured increases.

It was also found that this current Is increases depending on the amount of DCIP in the graphite paste and the amount of GDH on the electrode surface.

The response characteristics of this electrode to the glucose concentration in the liquid to be measured are shown in FIG. From the time when 10mM glucose was added to the test solution (arrow in the figure), +
It was found that an increase in the anodic current at O,SV occurred and reached a steady state current in about 1 minute.

Example 3 An electrode having a configuration in which the counter electrode was incorporated into the glass tube (1) was prepared in Example 1. It was produced according to (A). This configuration is shown in FIG. In the figure, OO1 indicates a counter electrode (cathode), which is a silver/chloride chloride electrode, which is wound and fixed on an internal support (6'), and (9) is a counter electrode (cathode) made from a Tris-HCl buffer containing potassium chloride. The dehydrogenase electrode of the present invention can be used without adding any other electron transfer medium to the solution to be measured.
It shows a large current response to substrates such as glucose. Therefore, it can be suitably used as a sensor for various substrates. Moreover, compared to conventional dehydrogenase electrodes in which NAD is immobilized, a graphite paste electrode is used that contains many electron-accepting compounds of NAII and can constantly supply these to the enzyme immobilization layer. The high enzymatic reaction activity of the dehydrogenase enzyme can be maintained almost constant over a long period of time (usually - weeks or more).

Furthermore, the electrode of the present invention has the advantage that the electron-accepting compound reduced by the enzymatic reaction is regenerated (reoxidized) by the electrode reaction, so that the compound is hardly consumed by the reaction. .

[Brief explanation of drawings]

FIG. 1 is a structural diagram showing an alcohol dehydrogenase electrode according to an embodiment of the present invention. FIG. 2 is a graph showing the cyclic voltammogram of the electrode in FIG.
The solid line indicates an electrode containing NAD in the paste, and the broken line indicates an electrode containing NADH. FIG. 3 is a graph showing the relationship between the steady current (18) of the alcohol dehydrogenase electrode in FIG. 1 when 0.8 V is applied and the ethanol concentration in the liquid to be measured. FIG. 4 is a graph showing a cyclic voltammogram of a glucose dehydrogenase electrode according to another embodiment of the present invention, and FIG. 5 is a graph illustrating the response characteristics between twisting glucose concentration and anode current. . FIG. 6 is a configuration explanatory diagram showing still another embodiment of the present invention. +1)・・・・・・・・・・Alcohol dehydrogenase electrode, (2)・・・・・・・・・NAD-graphite paste electrode, (3)・・・・・・・・・ADH, (4) ...
...Nitrocellulose thin ll, +51...
...Platinum wire, (6) ...Glass tube. , -P Figure 4 Figure 6

Claims (1)

[Claims] (1) A paste-like electrode containing an electron-accepting compound in graphite paste, a dehydrogenase enzyme immobilized on the surface of the paste-like electrode, and a permeable thin film of a substrate for the dehydrogenase enzyme covering its outer surface. A dehydrogenase electrode comprising a substrate-sensitive portion configured as follows.