JPS62277549A - Device for quantitatively determining alpha-amylase - Google Patents

Abstract

PURPOSE:To permit exact measurement with a simple operation by providing a porous film impregnated with a substrate of alpha-amylase in such a manner that said membrane can be freely attached to and detached from an enzyme electrode. CONSTITUTION:A hydrogen peroxide electrode 1, an immobilized enzyme film 2, the porous film 3, an O-ring 4 and a lead wire 4' are provided. The enzyme electrode is stored by being immersed in a phosphate buffer soln. Said electrode can be preserved stably for a long period of time when a constant voltage is kept impressed thereto at all times. The enzyme electrode is taken out of the phosphate buffer soln. in the stage of measurement. The porous film 3 impregnated with the substrate of the alpha-amylase is mounted on the enzyme film 2 and the specified amt. of the liquid to be examined is dropped onto the porous film 3. The formed amt. of the hydrogen peroxide based on the enzyme reaction is detected by detecting the reaction current by a polarographic method using the electrode 1 or by combining a current-voltage converter therewith. The alpha-amylase is thus exactly quantitatively determined by the simple operation.

Description

[Detailed Description of the Invention] 3. Detailed Description of the Invention [Technical Field to which the Invention Pertains] This invention provides an enzyme membrane comprising an enzyme membrane on which glucose oxidase and glucoamylase are immobilized on the electrode part of a hydrogen peroxide electrode. The present invention relates to a device for quantifying α-amylase in a test liquid using an electrode.

[Conventional technology and its problems] In recent years, various clinical testing devices using enzyme methods have been researched and developed. Enzyme methods have higher selectivity and sensitivity than non-enzyme methods, and enzyme electrode methods also have short measurement times. α-Amylase is essential for diagnosis of diseases such as the pancreas, and automatic analyzers using an enzyme method are commercially available and are used in hospital laboratories and the like. However, in recent years, interest in self-diagnosis has increased mainly in Europe and America, and there has been an increasing demand for measuring devices that can be easily operated by individuals or at home. On the other hand, conventional vertical devices require preparation of reagents during measurement, as well as stirring or mixing mechanisms, cleaning mechanisms, etc., making them large and expensive, and they also have drawbacks such as complicated maintenance. have

[Purpose of the invention]

SUMMARY OF THE INVENTION An object of the present invention is to eliminate the above-mentioned drawbacks, to provide a measuring device that can quantify α-amylase in a test liquid with simple operation and high accuracy, and is small and easy to maintain.

[Key points of the invention]

This invention consists of an enzyme electrode in which an enzyme membrane on which glucose oxidase and glucoamylase are immobilized is attached to the electrode part of a hydrogen peroxide electrode. A membrane is provided to cover the enzyme membrane of the electrode section, and by contacting the sample liquid with this porous membrane, α-amylase in the sample liquid is quantified. That is, using an enzyme electrode equipped with a porous membrane impregnated with sodium starch glycolate, the following enzymatic reaction proceeds due to the action of α-amylase in the test solution to generate hydrogen peroxide.

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(1) Glucose oxidase glucose gluconolactone + H20 It is intended to be quantified. This results in
There is no need to prepare reagents for each measurement, and the operation is as simple as replacing the porous membrane and dropping the test solution.Also, since the test solution only comes into contact with the porous membrane, the surface of the enzyme electrode is There is little contamination, and cleaning can be done by simply removing the porous membrane and immersing the enzyme electrode in a phosphate buffer solution. Furthermore, since no stirring or mixing mechanism is required, the apparatus can be made compact and inexpensive.

[Embodiments of the invention]

FIG. 1 shows an embodiment of the quantitative determination apparatus of the present invention.

1 is a hydrogen peroxide electrode, 2 is a glucose oxidase/glucoamylase immobilized enzyme membrane attached to the electrode part of this hydrogen peroxide electrode, 3 is a porous membrane attached to the immobilized enzyme membrane 2, and 4 is a porous membrane attached to the immobilized enzyme membrane 2. An O-ring 4 for attaching the immobilized enzyme membrane and porous membrane to the electrode is a wire drawn out from the hydrogen peroxide electrode.

The enzyme electrode is stored immersed in phosphate buffer at pH 7.
．． By constantly applying a constant voltage of 6v to 0.75V, it is possible to maintain stability for a long period of time.

At the time of measurement, remove the enzyme electrode from the phosphate buffer.

A porous membrane 3 impregnated with sodium starch glycolate, which is a substrate for α-amylase, is mounted on the immobilized enzyme membrane 2, and a certain amount of the test liquid is dropped onto the porous membrane. The amount of hydrogen peroxide produced due to the enzymatic reaction can be detected by detecting the reaction current by a polarographic method using the hydrogen peroxide electrode, or by using a combination of a current-voltage converter.

(Example 1) When urine was used as the test liquid and the flow of the reaction was measured by the polarographic method, as the enzyme reaction proceeded after dropping the test liquid, an increase in the current value due to the production of hydrogen peroxide was observed. Furthermore, after the measurement is completed, the substrate-impregnated porous membrane 3 is removed,
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Within seconds, the current value reached the value before dropping the test liquid, indicating that cleaning was completed.

FIG. 2 shows, using an α-amylase standard solution, the relationship between the current value and the α-amylase activity value a certain time after dropping the standard solution. As is clear from FIG. 2, good linearity was exhibited, indicating that the increase in current value was due to the action of α-amylase. Therefore, by preparing a calibration curve in advance, a sample with unknown α-amylase activity can be accurately quantified.

(Example 2) In the above-mentioned Example 1, the case where the test liquid did not contain glucose was shown, but in the case where the test liquid such as blood contains glucose from the beginning.

A porous membrane that is not impregnated with the substrate of α-amylase is attached to the enzyme electrode, and after measuring the current value based on the glucose initially contained in the test solution, the enzyme electrode is impregnated with the substrate in the same manner as in Example 1. By attaching a porous membrane and subtracting it from the measured current value, α-amylase in the sample solution can be accurately measured (
This can be quantified. Also in this case, cleaning is completed within 30 seconds by removing the porous membrane 3 and immersing it in a phosphate buffer solution.

As the porous membrane 3 used in the present invention, in addition to polycarbonate, cellulose acetate, polyvinyl chloride, etc., porous ceramics, paper, etc. can be used, and the substrate can be impregnated with it by a normal impregnation method.

〔Effect of the invention〕

As is clear from the above description, according to the present invention, since a porous membrane pre-impregnated with an α-amylase substrate is detachably attached to the enzyme electrode, there is no need for reagent preparation during measurement, and the process is simple. Accurate quantification is possible through simple operations, and since the test liquid only comes into contact with the porous membrane, there is little contamination of the surface of the enzyme electrode, and the enzyme electrode can be used stably for long periods of time without the need for special cleaning operations. can do. Also.

Expensive enzymes can be used repeatedly, the cost for one measurement is low, and since no washing mechanism, stirring or mixing mechanism is required, it can be made small and inexpensive.
Not only can it be used for emergency testing in hospitals or diagnosis at the pedside, but it can also be used for general home use.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view of an enzyme electrode showing an embodiment of the present invention, and FIG.
The figure is a graph showing the relationship between α-amylase activity and measured current value in an example of the present invention. 1...Hydrogen peroxide electrode, 2.Glucose oxidase/glucoamylase immobilized enzyme membrane, 3...Porous membrane,
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Claims (1)

[Scope of Claims] 1) An enzyme electrode in which an enzyme membrane on which glucose oxidase and glucoamylase are immobilized is attached to the electrode part of a hydrogen peroxide electrode, wherein the electrode part has a porous impregnated with a substrate for α-amylase. 1. An α-amylase quantification device characterized by having a removable membrane. 2) The apparatus for quantifying α-amylase according to claim 1, wherein the α-amylase substrate is sodium starch glycolate.