JPH0147160B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to an immobilized glucose oxidase membrane (hereinafter referred to as an immobilized enzyme membrane or simply an enzyme membrane).
The present invention relates to an amylase analysis method and apparatus for analyzing the amount of amylase in a sample such as blood (human or animal whole blood, plasma or serum), saliva or urine. It is desirable that this type of analysis method and device be easy to operate and capable of accurate measurement in a short period of time for reasons such as an increase in the number of specimens or revision of health scores.

Conventionally, this type of analysis method uses the amyloclastic method, which measures the process by which the substrate for amylase, that is, starch, is gradually decomposed under the action of amylase using an iodostarch reaction. There are the Saccharodienik method, which measures the reducing power of maltose, and the chromodienik substrate method, which colorimetrically measures the soluble pigment liberated by the action of amylase (for example, the blue starch method). Coexisting substances (sugar, protein, urea, etc.) can interfere with measurement, which not only reduces measurement accuracy but also requires too much effort and time.

The present invention has been made under the above circumstances, and an object of the present invention is to provide an amylase analysis method and an apparatus therefor, which are not affected by substances that interfere with measurement and are capable of carrying out highly accurate measurements in a short period of time.
Still another objective is to reduce the cost of expensive amylase substrates by preventing wasteful consumption of them.

In the present invention, when a specimen containing amylase and a decomposition auxiliary enzyme are injected into an immobilized enzyme membrane electrode part which is supplied with an amylase substrate solution of a predetermined concentration, the current generated in the electrode part is initially mainly caused by the immobilized enzyme membrane. The value rises steeply due to the oxidation of glucose in the sample by enzymes, then becomes approximately constant after a predetermined period of time, after which glucose is gradually produced by the hydrolysis action of amylase and auxiliary enzymes in the sample. The output current from the electrode section is monitored by taking advantage of the characteristic that it gradually increases in response to being oxidized by the immobilized enzyme, and when the output current becomes approximately constant. The amylase analyzer according to the present invention is characterized by a method of determining the amount of change after a predetermined time from the amount of glucose and measuring the amount of amylase from the amount of change. an enzyme membrane electrode part, an injection part for injecting a sample and a decomposition-assisting enzyme into the electrode part, a buffer supply means for supplying a buffer solution containing a predetermined concentration of amylase substrate to the electrode part, The present invention is characterized in that it includes a calculation means for monitoring the output current and calculating the amount of change after a predetermined period of time after the output current value becomes substantially constant.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 is a block diagram showing an embodiment of this invention.
FIG. 2 is a block diagram showing another embodiment, FIG. 3 A is a waveform diagram to explain the operation of the liquid pump in FIG. 1, and FIGS. 3 B and C illustrate the operation of the liquid pump in FIG. FIG. 4 is a waveform diagram for explaining the output of the enzyme membrane electrode (hydrogen peroxide electrode) in FIG. 1 or 2.

In Figure 1, 1 is an enzyme membrane electrode [hydrogen peroxide electrode (platinum-silver electrode)] that measures the oxidation current flowing when glucose is oxidized by an immobilized enzyme membrane to oxidize hydrogen peroxide, and 2 is an enzyme membrane electrode [hydrogen peroxide electrode (platinum-silver electrode)]. A liquid pump sucks a predetermined amount of a buffer solution containing amylase substrate (starch) and supplies it to the reaction cell, and also cleans the inside of the cell; A reaction cell into which glucosidase is injected, 4 is the reaction cell 3
5 is a bottle, and 6 is an arithmetic control unit consisting of an amplifier A, an analog/digital converter A/D, and a digital processing device CPU such as a microcomputer.
Note that FIG. 2 is almost the same as FIG. 1, but includes a bottle 5' filled with only a buffer solution and a bottle 5' for sending the buffer solution to the reaction cell 3 to wash the inside of the cell. The difference is that a liquid pump 2' is provided.

The operation will now be explained with reference to FIGS. 1, 3, and 4.

The pump 2 shown in FIG. 1 is turned ON and OFF, for example as shown in FIG. Do this. Thereafter, the specimen and the decomposition assisting enzyme are injected into the reaction cell 3 simultaneously or sequentially.

The hydrogen peroxide electrode 1 immediately responds to the glucose initially present in the sample, and its output current
As shown in Figure A, it rises steeply and becomes approximately constant after a predetermined time _t1 . This reaction formula is well known and is expressed as: Glucose + O ₂ + H ₂ O Glucose oxidase――――――――――→ Gluconic acid + H ₂ O ₂ Hydrogen peroxide H ₂ O ₂ is oxidized on the platinum cathode of electrode 1 in FIG. 1 according to the reaction formula shown below, and at this time an oxidation current proportional to the amount of glucose flows.

H ₂ O ₂ →2H ⁺ +O ₂ +2e _-At the same time, in the reaction cell 3, a substrate, such as maltopentaose G5, mixed in a buffer solution at a constant concentration, is absorbed by amylase in the sample.
For example, it is hydrolyzed into maltose G2 and maltotriose G3 as shown in the following formula.

Maltopentaose (G5) + H ₂ O amylase――――→ Maltose (G2) + Maltotriose (G3) Next, α-glucosidase, which hydrolyzes maltose G2, is used as a decomposition auxiliary enzyme to convert the glucose that is present from the beginning. When the output of the hydrogen peroxide electrode becomes constant, for example at time t ₁ in Figure 4 A, G2 + H ₂ O α-α glucose oxidase――――――――――――――→ 2G1 Maltose G2 is sequentially converted to glucose G1 as follows. In this case, maltotriose G3
G2 is also converted to glucose, but its reaction rate is much slower than that of maltose G2, so we ignored it here. In proportion to the amount of glucose converted in this manner, the output of the electrode portion gradually increases as shown from time _t1 to time _t2 in FIG. 4A. Therefore, in this invention, the amount of glucose originally present in the sample is determined.
Measure GL ₁ , then measure the total amount of glucose GL ₂ , which includes the amount of glucose newly produced by amylase reaction and auxiliary enzyme reaction, for a certain period of time (late assay), and measure the difference ΔG to determine the amount of amylase. decide. Note that this determination is made by the calculation control section 6 shown in FIG. In other words, since a current proportional to the amount of glucose is obtained from the electrode 1, this is amplified by the amplifier A, converted to a digital signal by the converter A/D, and the above difference calculation is performed by the digital processing device CPU.
It can be found by doing it in In addition, this measurement is performed when the pump 2 is in the OFF state as shown in Fig. 3A, and then in the ON state following the OFF state in Fig. 3A.
At this time, the inside of the reaction cell 3 is washed with a buffer solution. Incidentally, FIG. 4 shows how the electrode output changes when amylase is not contained in the sample.

In the above explanation, α-
Glucosidase was injected at a time when the electrode output became stable, for example, at time _t1 in FIG. 4A. However, in this case, the newly added α-glucosidase inside the reaction cell changes the temperature or capacity inside the reaction cell, which adversely affects the hydrogen peroxide electrode for measurement, and as a result, the accuracy of measurement decreases. There is a risk that it will decrease. Furthermore, since the reaction by amylase and auxiliary enzymes is slow, it is difficult to obtain a sufficiently large output within a predetermined time. Therefore, in order to reduce the effect on the electrode output and to have an incubation (reaction acceleration) effect within the reaction cell, it was found from experiments that it is possible to inject a degrading auxiliary enzyme at the same time as the sample injection. It's getting old. Note that the reaction process and measurement method in this case are exactly the same as above.

In addition, washing after measuring the amount of amylase should be done first.
In the figure, a buffer solution containing a substrate is used. However, this amylase substrate is generally an expensive substance whose purity and other factors are carefully considered, and if it is consumed in excess of what is necessary, a problem arises in that running costs become high. In other words, this expensive amylase only needs to exist in the reaction cell during analysis and is not needed during cleaning after analysis. This is shown in Figure 2. That is, a bottle 5' filled with only a buffer solution and a pump 2' for supplying only the buffer solution into the reaction cell 3 are provided, and the pump 2' and the pump 2 for supplying the substrate are connected as shown in FIG.
By driving as shown in (c), the substrate is not wasted. In this case, although the control operation of the arithmetic control unit 6 that performs the control becomes complicated, the advantage is that the running cost can be reduced.

As described above, according to the present invention, based on the measurement of glucose using the immobilized enzyme membrane electrode, the amount of glucose newly produced by the amylase reaction and the auxiliary enzyme reaction is measured using one hydrogen peroxide electrode. By measuring the amount of amylase in a sample easily and quickly,
Moreover, it has the advantage of being able to be measured without being influenced by coexisting substances.

The present invention can be applied as an analysis method or apparatus for general reaction systems that produce glucose or hydrogen peroxide from a substance to be measured using the action of an auxiliary enzyme. Furthermore, there is a high correlation (number of samples = 96, correlation coefficient = 0.96) between the results of analysis using the analysis method according to the present invention and the results of analysis using, for example, the conventionally well-known blue starch method. has been confirmed.

[Brief explanation of drawings]

FIG. 1 is a block diagram showing an embodiment of this invention.
2 is a block diagram showing another embodiment of the present invention, FIG. 3A is a waveform diagram for explaining the operation of the liquid pump in FIG. 1, and FIGS. 3B and 3C are the liquid pumps in FIG. 2. FIG. 4 is a waveform diagram showing the output of the enzyme membrane electrode in FIG. 1 or 2. FIG. Description of symbols, 1... Enzyme membrane electrode (hydrogen peroxide electrode), 2, 2'... Liquid pump, 3... Reaction cell,
4...Air pump, 5,5'...Bottle, 6...
...Arithmetic control unit, A...Amplifier, A/D...Analog/digital converter, CPU...Data processing device such as a microprocessor.

Claims (1)

[Scope of Claims] 1. A sample containing amylase and α-glucosidase are injected into one immobilized glucose oxidase membrane electrode portion which is supplied with an amylase substrate solution of a predetermined concentration, and the immobilized glucose oxidase and α-glucosidase in the sample are injected. In an amylase analysis method in which the amount of amylase in a sample is measured from the amount of current related to the amount of glucose generated as a result of the hydrolytic action of amylase and α-glucosidase, the amount of amylase in the sample is initially measured mainly by the immobilized glucose oxidase. It rises sharply due to the oxidation of glucose, then becomes a substantially constant value after a predetermined period of time, and thereafter is based on the oxidation of glucose by the immobilized glucose oxidase, which is gradually produced by the hydrolytic action of the amylase and α-glucosidase. An amylase analysis characterized in that the amount of amylase in the sample is measured by monitoring the output from the electrode section and calculating the amount of change after a predetermined period of time after the output current amount becomes approximately constant. Method. 2. The amylase analysis method according to claim 1, wherein the α-glucosidase is injected substantially simultaneously with the specimen. 3. The amylase analysis method according to claim 1, characterized in that the α-glucosidase is injected at a time when the output of the electrode section becomes substantially constant after a rise. 4. One immobilized glucose oxidase membrane electrode part that generates a current related to the amount of glucose, an injection part that injects a sample and α-glucosidase into the electrode part, and a buffer solution containing an amylase substrate at a predetermined concentration in the electrode part. a buffer solution supply means for supplying a buffer solution, and a calculation means for monitoring an output current from the electrode section and calculating an amount of change after a predetermined period of time from when the output current value becomes approximately constant; An amylase analyzer characterized in that washing is performed with a buffer solution from the buffer solution supply means, and the amount of amylase in the sample is measured from the calculation result. 5. In the amylase analyzer according to claim 4, another buffer supply means for supplying only the buffer is provided, and the electrode section is washed with the buffer from the other buffer supply means. An amylase analyzer characterized by: