JPH0332741B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an electrode for measuring amylase activity.

Among the currently used diagnostic methods for pancreatic diseases, there is a method of testing for pancreatic enzyme deviation, which involves measuring pancreatic enzymes in serum or urine. In particular, amylase is extremely important for the diagnosis and follow-up of pancreatic diseases due to its advantages over other enzymes, such as its stable activity and the fact that there are no activity inhibitors or similar enzymes in the body, making it easy to measure. .

Conventional methods for measuring amylase activity include turbidimetry, iodine titration, and enzymatic methods. The nephelometric method correlates the turbidity of an aqueous starch solution after hydrolysis of amylose with the concentration of α-amylase produced. The iodine titration method utilizes the fact that a blue starch-iodine solution is hydrolyzed by α-amylase and the blue color becomes lighter. However, this method has low reliability because there is no direct relationship between color change and α-amylase.

In the enzymatic method, an attempt has been made to measure α-amylase activity by hydrolyzing starch with α-amylase, coexisting with an enzyme that decomposes the product into glucose, and measuring the produced glucose. Makes good use of specificity. However, this approach ultimately
Since glucose generated through the involvement of amylase is measured, the influence of glucose already present in the sample (endogenous glucose) becomes a problem. or,
Since enzymes are expensive, measurement costs are high.

Therefore, an object of the present invention is to provide an electrode that can easily remove endogenous glucose and quickly and repeatedly measure α-amylase activity.

The present invention is characterized by an enzyme that decomposes a reaction product produced by amylase and its substrate into glucose;
A first electrode for detecting H ₂ O ₂ formed by immobilizing glucose oxidase (hereinafter referred to as GOD);
It consists of a second electrode for electrolytically removing substances that interfere with the detection of H ₂ O ₂ by the first electrode. Since the second electrode is a porous membrane that immobilizes GOD and has a platinum layer, endogenous glucose is absorbed into GOD.
H ₂ O ₂ generated as gluconic acid is also oxidized by the platinum layer and can be removed. GOD is also an enzyme (glucoamylase, or α-
glucosidase, etc.) is also immobilized, α-amylase activity can be measured repeatedly and quickly.

An example of a reaction when serum is added to the amylase activity measuring electrode according to the present invention is shown below. α−
As an enzyme that uses sodium starch glycolate as a substrate for amylase and decomposes the reaction product produced by amylase and its substrate into glucose,
Glucoamylase was used.

(intrinsic)
Glucose + O ₂ Glucose――――――→ Oxidase Gluconolactone + H ₂ O ₂ (1) H ₂ O ₂ →2H ⁺ +2e＋O ₂ (2) Sodium starch glycolate α-amylase―――――― ―――→ Glucoamylase glucose (3) Glucose + O ₂ glucose ――――――→ Oxidase gluconolactone + H ₂ O ₂ (4) H ₂ O ₂ →2H ⁺ +2e+O ₂ (5) First, Endogenous glucose is converted to gluconolactone by the immobilized GOD of the second electrode as shown in equation (1) above, and the generated H ₂ O ₂ is converted to gluconolactone as shown in equation (2). It is removed by electrolysis.
Similarly, ascorbic acid and uric acid, which interfere with measurement, are also electrolytically removed by the platinum layer of the second electrode. Sodium starch glycolate is decomposed by α-amylase in serum as shown in equation (3), and further decomposed into glucose by glucoamylase immobilized on the first electrode. The generated glucose is oxidized by the immobilized GOD of the first electrode as shown in equation (4) to generate H ₂ O ₂ , and this H ₂ O ₂ is then oxidized by the platinum of the first electrode as shown in equation (5). It is oxidized in the layer, and the concentration of glucose can be detected from the obtained oxidation current value, thereby making it possible to measure the α-amylase activity.

The amylase measurement electrode according to the present invention will be explained below using Examples.

Example 1 FIG. 1 shows the structure of an electrode in which glucoamylase is used as an enzyme that decomposes a reaction product produced by α-amylase and its substrate into glucose. Polycarbonate porous membranes (diameter 10 mm, pore diameter 2000 Å, membrane thickness 10 μm, pore density 3×10 ⁸ /cm ² ) 1 and 2 were used as electrode carriers, and a platinum layer 3 was deposited on one side of these membranes by sputtering. 4 (thickness of several hundred to several thousand angstroms) was formed. GOD was developed on membrane 2, and GOD and glucoamylase were developed on membrane 1, and after installing them on an electrode holder, they were immobilized in glutaraldehyde vapor. GOD 7 is immobilized on the surface and pores of the second electrode 9 on the test liquid side, and GOD 5 and glucoamylase 6 are immobilized on the surface and pores of the first electrode 8.

FIG. 2 schematically shows a cross section of a cylindrical electrode holder equipped with the above-mentioned thin film electrode and an electrode system. In the figure, reference numeral 10 denotes an enzyme electrode on which the above enzyme is immobilized, and it is attached to the main body 12 through an outer tube 11 so that the membrane 1 is on the inside of the holder. The platinum layer 4 is in contact with the inner platinum lead 13, and the platinum layer 3 is in contact with the outer platinum lead 14. The Ag/AgCl reference electrode 15 and counter electrode 16 for the first electrode 8 are placed inside the electrode holder, and the Ag/AgCl reference electrode 15 and counter electrode 16 for the second electrode 9
An Ag/AgCl reference electrode 17 and a counter electrode 18 are arranged outside the electrode holder to constitute an electrode system. Further, the inside of the electrode holder is filled with an electrolytic solution 19. 1st
The electrode is set to +0.6V with respect to the reference electrode 15,
The following measurements were performed. The voltage was similarly set and measured for the second electrode.

The above electrode was immersed in a buffer solution sufficiently containing sodium starch glycolate as a substrate for α-amylase, and α-amylase was added thereto. In Figure 3, the horizontal axis shows the amount of α-amylase added (unit/100
ml), and the vertical axis shows the current increase rate of the first electrode (μA/
minutes). A good correlation was observed between the two values, and it was confirmed that amylase activity measurement was sufficiently possible. Furthermore, when glucose was added to examine the influence of endogenous glucose, almost no increase in the oxidation current value was observed. This is the second
This is thought to be because glucose was removed by the electrode. Furthermore, no increase in oxidation current was observed when ascorbic acid or uric acid was added.
This is considered to be because the effect was removed by electrolytic oxidation of the second electrode.

Next, the above amylase measurement electrode according to the present invention was designated as A, and the following electrodes B and C were prepared for comparison. B is the same as A except that GOD is not immobilized on the second electrode; C is that GOD is not immobilized on the second electrode and lucoamylase is not immobilized on the first electrode. In other words, GOD is immobilized only on the first electrode of electrode A.

Serum was added to each of A, B, and C, and the oxidation current value (response current μA) was measured. In FIG. 4, the horizontal axis shows time (sec) after addition of serum, and the vertical axis shows response current (μA). In contrast to A, the current increased quickly and linearly after the addition of serum, B and C increased in a curved manner for about 30 seconds, and then in B the current increased parallel to A, and in C the current stopped increasing. . C
Because it only uses GOD-immobilized electrodes, it measures the amount of glucose in serum. B is GOD to the second electrode
Endogenous glucose cannot be removed because it is not immobilized. Therefore, in order to measure α-amylase activity in B, there is the inconvenience that the measurement must be performed after approximately 30 seconds after the addition of α-amylase, when the current starts to increase linearly.
Also, the error becomes large due to endogenous glucose.
Therefore, by installing a second electrode with immobilized GOD like electrode A of the present invention, the activity of α-amylase can be measured more accurately and quickly.

Example 2 An electrode similar to that in Example 1 was prepared using maltopentose as the substrate for α-amylase and α-glucosidase as the enzyme that decomposes the reaction product generated by α-amylase and maltopentose into glucose. An α-amylase addition experiment and a serum addition experiment were conducted. As in Example 1, the activity of α-amylase could be measured accurately and quickly.

Although the above two examples have been given, the present invention can also be applied to other amylase activity measurement methods (enzymatic methods) by simply changing the combination of substrate and immobilized enzyme.

All enzymes involved in measuring amylase activity are immobilized, allowing repeated measurements.
Since a porous thin film was used for the electrode, the response was quick.

As described above, the amylase activity measuring electrode of the present invention can easily remove endogenous glucose and can rapidly and repeatedly measure amylase activity.

[Brief explanation of drawings]

Figure 1 is an enlarged schematic cross-sectional view of an electrode for measuring amylase activity, which is an embodiment of the present invention. Figure 2 is a schematic diagram showing the cross-section of the electrode for measuring amylase activity and the electrode system. Figure 3 is a diagram showing the amount of amylase added and the current. FIG. 4 is a diagram showing the relationship between the rate of increase and the relationship between the time after serum addition and the current value. 1, 2... Porous membrane, 3, 4... Platinum layer, 5,
7... Immobilized glucose oxidase, 6... Immobilized glucoamylase, 8... First electrode, 9...
... Second electrode, 10 ... Enzyme electrode, 11 ... Outer tube, 12 ... Main body, 13, 14 ... Platinum lead, 15, 17 ... Ag/AgCl reference electrode, 16, 1
8... Counter electrode, 19... Buffer solution.

Claims (1)

[Scope of Claims] 1. A first electrode in which an enzyme and glucose oxidase are immobilized on a porous membrane on which a conductive layer is formed, and a porous membrane adjacent to the first electrode on which a conductive layer is formed. a second electrode in which glucose oxidase is immobilized on a plasma membrane;
An electrode for measuring amylase activity, wherein hydrogen peroxide is detected, and the second electrode electrolytically removes a substance that interferes with the detection of hydrogen peroxide by the first electrode. 2. The amylase activity measuring electrode according to claim 1, wherein the conductive layer is a platinum layer. 3. The amylase activity measuring electrode according to claim 1 or 2, wherein the enzyme is glucoamylase or a-glucosidase.