JPH0614020B2 - Enzyme electrode activation method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for activating an immobilized enzyme electrode (hereinafter abbreviated as an enzyme electrode).

(Prior Art) The use of an enzymatic reaction for quantifying a measurement target substance contained in a biological substance, food, etc. is widely performed because of its advantages such as a high reaction rate and substrate specificity. In particular, the measuring method using an enzyme electrode opens the possibility of utilizing a small amount of enzyme in a repetitive manner, and its application range is expanding to medical treatment, food, drug analysis and the like. For the enzyme electrode, an enzyme is directly immobilized on a substrate such as platinum or graphite, or Japanese Patent Application No.
No. 14648, there is one in which an enzyme is immobilized through a polymer membrane such as a permselective membrane. Usually, in these enzyme electrodes, a protein such as albumin and a crosslinking agent are mixed with the enzyme for the purpose of giving the enzyme-containing layer sufficient strength.
A mixed immobilization layer is formed.

However, the mixed immobilization layer thus obtained is a crosslinked protein having a higher-order structure and does not allow the substance to be measured to permeate freely, and therefore tends to interfere with the contact between the substance to be measured and the enzyme. An enzyme electrode having the required measurement sensitivity cannot be obtained. On the other hand, if the amount of protein in the mixed immobilization layer is reduced in order to increase the sensitivity, there is a drawback that sufficient layer strength cannot be obtained.

(Problems to be Solved by the Invention) An object of the present invention is to improve measurement sensitivity in an enzyme electrode having a mixed immobilization layer of an enzyme and a protein without impairing the strength of the immobilization layer.

(Means for Solving the Problem) The present invention relates to a method in which an enzyme electrode having a mixed immobilization layer (3) of an enzyme and a protein formed on a substrate (1) directly or through a polymer membrane (2) is treated with water or It is a method for activating an enzyme electrode, which comprises heat-treating at 40 ° C. or higher in a buffer solution.

(Function) The enzyme electrode used in the present invention has a mixed immobilization layer containing an enzyme. For example, one in which a mixed immobilization layer containing one or more kinds of enzymes is formed on the surface of a substrate such as gold, platinum, silver, or graphite, and mixed immobilization of enzymes on a polymer membrane (2) such as acetylated cellulose. Forming the layer (3) and
There are those fixed to (1) (Fig. 1), those in which a selective permeation film of a polymer such as albumin is directly formed on a substrate, and a mixed immobilization layer is formed on the selective permeation film.

Such a mixed immobilization layer of an enzyme electrode is formed by applying an enzyme solution containing a protein other than an enzyme and a cross-linking agent such as glutaraldehyde to a substrate or a polymer film and drying it. As the enzyme, glucose oxidase, invertase, amylase, alcohol oxidase, etc. are used, and as the protein other than the enzyme, serum albumin, ovalbumin, globular protein such as globulin, etc. can be used.

The present invention is characterized in that the enzyme electrode having such a mixed immobilization layer is heat-treated in water or a buffer solution to improve the measurement sensitivity. If the heat treatment temperature is low, the effect of the present invention cannot be obtained, so it is necessary to perform the heat treatment at 40 ° C. or higher. However, if the temperature is too high, the enzyme may be inactivated, so the temperature is preferably 70 ° C. or lower.

For example, in the case of a mixed immobilization layer of glucose oxidase and albumin or globulin, 40 to 70 ° C, preferably 43 to 60 ° C, more preferably 45 to 55 ° C, for 1 minute to
Heat treatment is performed for 2 hours, preferably 5 minutes to 60 minutes. It also contains invertase and glucose oxidase,
When a mixed immobilization layer with albumin or globulin is formed, heat treatment is performed at 40 to 60 ° C, preferably 43 to 60 ° C, more preferably 45 to 55 ° C for 1 minute to 2 hours, preferably 5 minutes to 60 minutes. .

Such heat treatment, with the electrode attached to the measuring device,
Alternatively, it is removed and carried out in water or a buffer solution. As the buffer solution, a phosphate buffer solution, a citrate buffer solution, a Tris-hydrochloric acid buffer solution or the like is used.

The reason why such an effect is obtained is not necessarily clear, but proteins other than the enzyme in the mixed immobilization layer are denatured by heating, and the measurement target substance is easily permeated. It is considered that the reaction by is accelerated.

The present invention can also be applied to a reactor having a mixed immobilization layer containing an enzyme. For example, in the case of an immobilization carrier in which a mixed immobilization layer of invertase and albumin is provided on glass beads or the like, it can be activated by heat treatment at 40 to 60 ° C for 1 minute to 2 hours.

Examples are shown below, but of course the present invention is not limited to these.

Example 1 The end of a platinum wire having a diameter of 2 mm was polished with emery paper and the side surface was coated with heat-shrinkable Teflon to prepare an electrode substrate. A 2% bovine serum albumin (Sigma; fraction V) aqueous solution containing 0.2% glutaraldehyde was applied onto a substrate at 5 μm and dried to form a polymer having selective permeability.

1% glucose oxidase (manufactured by Sigma; Type
eII) and 1% bovine serum albumin (manufactured by Sigma;
0.2% in sodium phosphate buffer containing fraction V)
The above-mentioned polymer membrane was coated with 2.5 μl of a liquid to which glutaraldehyde had been added so that the enzyme electrode was obtained.

The enzyme electrode prepared in this way was used in 0.1M phosphate buffer (p
H70.0) was incorporated into a three-electrode type cell having a platinum counter electrode, silver, and a silver chloride reference electrode, and set as a flow type measuring device.

A voltage of +0.6 V against the silver / silver chloride reference electrode was applied to the enzyme electrode, 10 μm of a glucose aqueous solution of 5 mM to 50 mM was successively injected from the sample inlet, and the output current value was recorded (FIG. 2).

Similarly, an ascorbic acid aqueous solution of 10 mM to 50 mM was injected, and the output current value was recorded (FIG. 2).

Next, the enzyme electrode was removed and placed in a beaker containing 0.1 M phosphate buffer (pH 7.0), which was kept at 50 ° C. for 60 minutes in a water bath for heat treatment. Set the enzyme electrode to the measuring device, apply a voltage of +0.6 V to the silver / silver chloride reference electrode to the enzyme electrode, and sequentially inject 5 μm to 50 mM glucose aqueous solution 10 μm from the sample injection port to determine the output current value. Recorded (Fig. 3).

Similarly, 10 mM-50 mM ascorbic oxygen solution was injected and the output current was recorded (FIG. 3).

By the heat treatment, the output current value of the enzyme electrode for glucose 20 mM was changed from 0.28 μA to 0.44 μA, and the sensitivity was improved about 1.5 times. The output current value of ascorbic acid, which becomes a measurement interfering substance during glucose determination, increased by about 1.5 times due to the heat treatment, but since it is almost the same as the improvement rate of sensitivity to glucose, the amount of ascorbic acid contained in the sample was increased. The rate of measurement error due to acid was the same as before treatment.

Comparative Example 1 The measurement was performed in the same manner as in Example 1 except that the heat treatment temperature was 35 ° C.

The output current value before the heat treatment is shown in FIG. 4, and the output current value after the heat treatment is shown in FIG.

No effect of heat treatment was obtained at 35 ° C.

(Effect) As described above, according to the activation method of the present invention, the enzyme electrode having the mixed immobilization layer can be improved in extremely excellent measurement sensitivity without impairing the strength of the mixed immobilization layer.

[Brief description of drawings]

FIG. 1 is a cross-sectional view illustrating an enzyme electrode used in the present invention, and shows an enzyme electrode in which a mixed immobilization layer of enzymes is formed on a polymer membrane and the polymer membrane is immobilized on a substrate. FIG. 2 shows the output current value for glucose and ascorbic acid obtained by the oxygen electrode before the heat treatment in Example 1, and FIG. 3 shows the output current value after the heat treatment according to the present invention. Represent FIG. 4 shows the output current value for glucose and ascorbic acid obtained by the enzyme electrode before the heat treatment in Comparative Example 1, and FIG. 5 shows the output current value after the heat treatment at 35 ° C. . (1) …… Substrate (2) …… Polymer membrane (3) …… Mixed immobilization layer

Claims (1)

[Claims] 1. An enzyme electrode having a mixed immobilization layer (3) of an enzyme and a protein formed on a substrate (1) directly or through a polymer membrane (2) in water or a buffer solution at 40 ° C. or higher. A method for activating an enzyme electrode, which comprises heat treatment.