JPS59228160A - Amylase analyzing method - Google Patents

Abstract

PURPOSE:To make it possible to simultaneously measure the density of glucose and activity of amylase by such an arrangement wherein a hydrogen peroxide detection type polarograph is equipped with one kind of enzyme electrode which is equipped with an enzyme membrane in which two kinds of enzyme are fixed, and the speed of change of a current of the electrode is measured. CONSTITUTION:As a specimen including amylase and glucose is injected into a buffer solution including polysaccharide, an enzyme electrode composed of a membrane which selectively transmits hydrogen peroxide, wherein alpha-glucosidase and glucose oxidase are simultaneously fixed and a hydrogen peroxide electrode produces hydrogen peroxide, and due to the produced hydrogen peroxide, the primary increase A in electrode current which is proportional to the density of glucose takes place. After a period of 15-30sec, the secondary increase B in electrode current which is proportional to the activity of amylase takes place. Since the speed of the primary and secondary increase in electrode current is proportional to the density of glucose and the activity of amylase in each of (1)-(6) specimens respectively, it becomes possible to simultaneously measure the activity of glucose and amylase by one kind of enzyme electrode.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an enzyme membrane and an analysis method for measuring amylase activity using an enzyme electrode method.

For more details, see 1.
The present invention relates to an enzyme membrane and an analytical method that can simultaneously measure glucose concentration and/or amylase activity based on the rate of change in electrode current using different types of enzyme electrodes.

Conventional enzyme electrode method for measuring serum amylase as disclosed in JP-A-156-97863 or JP-A-57-1
There is a method described in Publication No. 77699 and the like.

The former method uses two electrodes, an immobilized glucose oxidase membrane electrode and an immobilized α-glucosidase/glucose oxidase membrane electrode, and uses one system to measure the glucose concentration and the maltose produced by α-amylase in a solution form. α-glucosidase is applied, the resulting glucose is detected with an immobilized glucose oxidase membrane electrode, and α-amylase activity is determined from the increase in one electrode current.

However, these methods require two electrodes, ■ The detectable electrode current value is small, ■ They require expensive and special substrates and unstable enzyme reagents, and ■ The measurement system is complicated and takes a long time to measure. It has disadvantages such as requiring

The present invention was made in order to improve these current measurement methods into a simpler and faster measurement method.The purpose of the present invention is to achieve simple operation in a short measurement time without being affected by measurement interfering substances. An object of the present invention is to provide an enzyme membrane and an analytical method for accurately measuring amylase activity and/or glucose concentration in body fluids.

That is, the present invention measures the concentration of a buffer solution containing an amylase substrate on an enzyme electrode consisting of a hydrogen peroxide permselective membrane and a hydrogen peroxide electrode on which α-glucosidase and glucose oxidase are simultaneously immobilized, and then This is an amylase analysis method characterized by measuring amylase activity from the rate of increase in secondary current based on hydrogen peroxide produced.

The present invention uses an enzyme membrane on which two types of enzymes are immobilized as an enzyme electrode, and the principle of measuring amylase activity and glucose concentration is the enzyme reaction described below.

Polysaccharide-7i2! Ni 12, H, O, maltose + maltotriose + maltotetraose + maltopentaose + oligosaccharide ヱヱ - 4 Satyo ≧
76-LH, Heguru Suk? ≦Shini Otoma Hikono 612 ± 4μi danh-gluconic acid + H2O2ゝ2H20 → 4H” +20.+ 4e- (anode)
4H+02+4e-12E, O (cathode), that is, in the enzyme electrode of the present invention, the secondary V of the electrode current due to hydrogen peroxide generated in a series of enzymatic reactions from polysaccharide to amylase, α-glucosidase, and glucose oxidase = polar current It is possible to measure amylase activity from the rate of increase. Furthermore, if necessary, the endogenous glucose concentration contained in the sample from the beginning can be determined from the rate of increase in the primary electrode current until the amylase action reaches a steady state.

The present invention will be described in more detail.

For example, the enzyme membrane of the present invention forms a dense skin layer on the side of the membrane facing the electrode, allowing only hydrogen monoperoxide to pass therethrough, and has a porous layer on the side of the membrane that comes into contact with the substance to be measured. Two types of enzymes are immobilized. Low-molecular-weight saccharides produced by the hydrolytic action of glucose or amylase already present in the sample react with enzymes immobilized on the membrane to generate hydrogen peroxide.

As raw materials for the membrane used in the present invention, hydrophilic polymeric substances such as polyamide and cellulose acetate-1-polyvinyl alcohol are preferred, and cellulose acetate is particularly suitable. The film is manufactured by casting a dope made by dissolving a polymer material as a raw material in a good solvent onto a smooth substrate, allowing the solvent to evaporate for a certain period of time, and then casting the dope onto the film surface on the opposite side of the substrate. After forming a hydrogen peroxide selectively permeable skin layer, the polymer material is immersed in a poor solvent to remove the solvent in the membrane and form a porous layer following the skin layer.Solvent evaporation The method is simple.

A method for simultaneously immobilizing α-glucosidase and glucose oxidase on the porous side of the membrane obtained by the above method is a crosslinking method using a bifunctional reagent such as 1 glutaraldehyde 1 hexamethylene diisocyanate - amino group, aldehyde group 1 isocyanate group There are covalent bonding methods in which enzymes are bonded to carriers into which acid azide groups have been introduced, and methods for enclosing enzymes with polymers such as polyacrylamide-collagen 1 gelatin and chitosan.The enlarged enzyme membrane was treated with glutaraldehyde. Most preferred is a covalent bonding method in which the second atom is bonded.

In this case, if it is necessary for the immobilization operation, there is no problem in coexisting a molecular electrolyte or the like.

As the hydrogen peroxide electrode used in the present invention, JP-A-54-1
Either the polarographic type or the galvanic type described in Publication No. 02193 may be used. The enzyme electrode is constructed by attaching these hydrogen peroxide electrodes so that the skin layer of the immobilized α-glucosidase/glucose oxidase membrane is in contact with the electrode surface, and the immobilized enzyme layer is in contact with the sample solution side.

The enzyme membrane of the present invention has two types of enzymes immobilized on the membrane surface at the same time, completely blocking low-molecular-weight interfering substances, and also allows for good contact between the deenzyme and their substrates, so it can accurately target target substances. Not only can it be detected as a change in electrode current, but also
High membrane strength and easy handling; ■ High membrane uniformity and excellent reproducibility of measured values. Glucose concentration and amylase activity can be measured simultaneously with 01 types of enzyme electrodes. ■ Low manufacturing and measurement costs. ■ Advantages include good permeability of the substrate and diffusibility of the detection substance, short response time, and rapid continuous measurement.

Although it is useful for measuring amylase in any body fluid, it can also be applied to measuring amylase activity in extremely thick samples such as pig innards, Bacillus subtilis, etc.

A method for simultaneously analyzing amylase activity and, if necessary, glucose concentration using an enzyme electrode using the enzyme membrane of the present invention will be described with reference to FIG. 1, which shows the response curve of the enzyme electrode.

When a sample containing amylase and glucose is injected in the presence of a buffer containing polysaccharide, the enzyme electrode experiences a primary electrode current increase proportional to the glucose concentration. After about 15-30 seconds there is an increase in secondary electrode current proportional to amylase activity. Since the primary and primary electrode current increase rates are proportional to the glucose concentration and amylase activity in the sample, respectively, it is possible to measure glucose and amylase activity simultaneously with one type of enzyme electrode.

Although there is no limitation on the type of buffer used in the present invention, a phosphate buffer with a concentration of 0.01 to 0.5 mol/l is suitable from the viewpoint of stability and cost. The buffer used in the present invention contains sodium chloride as an activator for amylase, and stabilizers for α-glucosidase and glucose oxidase. DT
A % As a preservative or as an inhibitor of catalase contained in the enzyme or in the hemolyzed sample, sodium azide 1 and as a substrate for amylase with an average molecular weight of 66
Contains 6 or more water-soluble polysaccharides. These additive substances are chloride) IJum 0.1-2.5% (wt/vol), ]IcDTA O,1-5 II mo
l/J! Sodium azide 0.1 to 1 mol/
/ and polysaccharides from 0.5 to 59/l are preferred, especially sodium chloride 0.876%, BCDTA 0.
5-2m 4o1/muadzide is sufficient to indicate a zero-order reaction.

There is nothing wrong with allowing an amylase inhibitor to coexist in the buffer solution for the purpose of differentially quantifying amylase derived from Borisatsukato saliva as a substrate for the amylase of the present invention.

The present invention, which has excellent substrate permeability, stability, and enzyme activity, does not require special substrates or expensive and unstable solution enzyme reagents, and can detect blood and urine using a small amount of sample using a single electrode detector. , amylase contained in tissues, etc., and glucose concentration, if necessary, can be measured easily, accurately, and at low cost in a short time.

Next, the present invention will be explained with reference to Examples.

Example L 2 g of cellulose acetate was dissolved in 50 of a mixed solvent consisting of 30 of acetone and 20 of cyclohexanone, and cast onto a glass plate using a knife coater to form a film with a thickness of 300 μm. After leaving it in the air for 10 minutes, Sn-
The solvent was extracted by immersion in hexane. After air drying, it was peeled off from the glass plate to obtain a white translucent film with a thickness of 13 μm. The resulting membrane consists of a dense skin layer and a porous layer.

This membrane is readily permeable to hydrogen peroxide, as confirmed by uric acid and peroxidity tests. An enzyme solution was prepared by adding 50 μl of an aqueous solution of α-glucosidase 950% glucose oxidase 250% U1 bovine aldehyde.

This enzyme solution was permeated through the porous side of the membrane and heated to 4℃ for 1 hour.
The immobilization reaction was completed by allowing the mixture to stand for some time.

After treatment with 1M glycine solution, the plate was thoroughly washed with -0.05M phosphate buffer. It was covered with a porous polycarbonate membrane and air dried at 4°C. α-glucosidase activity 0.1
0 engineering[/c++1. Glucose oxidase activity 0.0
An immobilized composite enzyme membrane of 4 U/C- was obtained. The obtained enzyme membrane was attached to a Clark-type hydrogen peroxide electrode so that the skin layer side of the enzyme membrane was in contact with the electrode surface to form an enzyme electrode.

This enzyme electrode was heated to 37°C, p! -1'r, o of 0.876
% Sodium Chloride - 1111M EDTA 5111M
0 containing sodium azide and 19/l soluble starch
．． The cell was immersed in 0.5-ml of 1M phosphate buffer. Next, glucose 90-450 m? /61 and human α-amylase o-2oootr/a! 25 μm of a standard solution containing caraway units was injected into the cell with a micropipette while stirring.

Figure (1) shows the electrode current and time characteristics generated by the oxidation of hydrogen peroxide produced by a series of enzymatic reactions. There was a sharp increase in primary electrode current proportional to glucose concentration, and an increase in secondary electrode current proportional to amylase activity was observed at about 30 seconds.

The rate of increase in secondary electrode current detected using a differential circuit shows a good linear relationship with glucose concentration.

The rate of increase in primary electrode current shows a good linear relationship with amylase activity. The range of linearity is for each glucose O~45
0 W/dt, amylase activity 0-2000 U/dl
(in caraway units).

Example 2 Using the enzyme electrode of Example 1, amylase activity and glucose concentration were simultaneously measured in exactly the same manner as in Example 1, using three types of human serum samples instead of the glucose/amylase standard solution. - At this time, the glucose standard solution is 90
Glucose concentration and amylase activity in serum were determined by calculation from α-amylase derived from human eyelid 3oou, "a" as a W/dl amylase standard solution. The measurement results are shown in Table 1. The simultaneous reproducibility of glucose concentration and amylase activity was good.

[Brief explanation of the drawing]

FIG. 1 shows the relationship between electrode current value (vertical axis) and time (horizontal axis) when measuring glucose and amylase standard solutions. This is a graph (~ is the increase in the primary electrode current, (B) is the increase in the second electrode current.
The following shows an increase in electrode current. (1) is glucose 90■/d/amylase OU/
d/(2) is glucose 90 I+97d1 amylase 400 U/61(8) is glucose 180η/e
Ll amylase 800U/dl (4) glucose 270 cape/dl amylase 1gooU/dt (5)
is glucose 360”f/dt amylase 1600
U/dl(6) is glucose 450η7dt amylase 2000U/d7! shows the response curve. Patent applicant: Toyobokyo Co., Ltd.

Claims (1)

[Claims] A sample is applied to an enzyme electrode consisting of a permselective hydrogen peroxide membrane on which α-glucosidase and glucose oxidase are immobilized and a hydrogen peroxide electrode in a buffer containing a substrate for amylase, and peroxide is generated as necessary. measuring the glucose concentration from the hydrogen-based primary current increase rate;
An amylase analysis method characterized by measuring amylase from the rate of increase in secondary current based on subsequently generated hydrogen peroxide.