JPS58201977A - Device for measuring enzyme activity - Google Patents

Abstract

PURPOSE:In a device of flow cell system for measuring enzyme activity wherein the enzyme activity is measured by one electrode, to improve measurement accuracy, by setting a liquid flow path part to reduce the concentration of substances responsive to the electrode except the enzyme contained in a specimen, connected to an inlet part for a specimen solution. CONSTITUTION:The constant delivery pumps 4' and 4'' are operated, a buffer solution is sent from the buffer solution tank 9 to the flow cell 5, and a substrate corresponding to an enzyme of a measuring object from the substrate solution tank 9 to it. The constant delivery pump 4 is operated, and an enzyme solution of measuring object is sent from the specimen tank 2 to the flow cell 5. The concentration of substances responsive to an electrode except the object enzyme is reduced with an immobilized enzyme, etc. immobilized to the inner wall of the liquid flow path part 8 while the enzyme solution being passed through the liquid flow path part 8. The enzyme is brought into contact with a substrate and reacted with it in a path from the inlet part 10 to the flow cell 5, and the change in concentration of a substance to be detected, decreasing extremely by the reaction, is measured by the electrode 6 of the flow cell 5.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field of the Invention] The present invention relates to an enzyme activity measuring device using electrochemical measurement fθ.

[Technical background of the invention and its problems]

Conventionally, enzyme activity has been measured using a reaction system consisting of a solution containing a certain enzyme, a substrate for the enzyme, and further addition of other enzymes, prosthetics, coloring reagents, etc. Substrate (chemical reaction is carried out in its entirety, and the absorbance of the reaction system at that time is measured).

For example, glutamate pyruvate transamipase (
In the case of the enzymatic activity of GPT), contacting GPT with its substrate produces pyruvate by the enzymatic action of the GPT,
The pyruvate is lactate dehydrogenated.

It is reduced by the enzymatic action of lonase (LDI). At this time, if a sufficient concentration of nicotinamide adenine nucleodes (NADH) is allowed to coexist in this reaction system, the NADH decreases over time and its concentration decreases.

The change in concentration can be determined by measuring the absorbance of NADH at a wavelength of 340 nm. Therefore, the enzyme activity of GPT can be measured indirectly.

However, in this absorbance measurement method, NADH
, LDH, or various coloring reagents are used, and these must be discarded after the measurement, making the measurement extremely costly. Furthermore, since absorbance measurement is not possible for reaction systems containing suspended matter, these samples require pretreatment such as removal of suspended matter prior to measurement.

In order to solve these drawbacks of absorbance measurement, a device using an electrochemical measurement method has been proposed (Japanese Patent Application Laid-Open No.
6-97864).

This is made by attaching a membrane with an enzyme immobilized on it that acts on a substance (sensing substance) that is produced or annihilated by the enzyme in the sample. It is a flow system that uses the electric signal given by the detection substance originally contained in the sample as a base value, and further generates a signal corresponding to the activity of the enzyme by the action of the enzyme in the sample. The electrical signal given by the annihilated substance (sensing substance) is measured, and the activity of the enzyme in the sample is determined from the difference between the two.

However, since this device uses two enzyme electrodes, it is not easy to adjust the output of both, and the operation thereof is extremely complicated and requires a great deal of effort.

In other words, it is extremely difficult to produce enzyme electrodes that have exactly the same electrode characteristics such as sensitivity, response speed, and gain, and also have the same changes over time in these characteristics.

Therefore, it is necessary to adjust the output of each electrode during measurement, but even in that case, the linearity and linear region of the obtained calibration curve differ between each electrode, and the -1 error may occur in the measurement. result in consequences.

Also, there are two in the liquid flow path of the flow system? When the 11 electrodes are placed apart from each other, a dilution phenomenon occurs due to sample diffusion in the liquid flow path, and the concentration of the detection substance decreases at the downstream electrode position, resulting in a decrease in the output between the two t'FMs. Adjustment becomes extremely difficult during differential adjustment and differential amplification. However, it is extremely difficult to measure small amounts of samples containing enzymes with low activity.

In addition, an enzyme substrate-containing buffer solution is flowed at a constant flow rate in the liquid flow channel, a sample solution is injected into the flow channel, and the enzyme and substrate are brought into contact while both flow in the flow channel to cause the enzyme action to occur. A method has also been proposed in which the enzyme activity kTJ41J is determined by directing the electric signal given by the detection substance, which decreases or increases, using an electrode placed completely downstream (Japanese Patent Publication No. 56-92445.924
46) 2゜However, in this method,
The liquid flow path between the sample injection port and the electrode is the center of the reaction between enzyme and substrate.
Therefore, in the case of a low-activity enzyme that requires a long reaction time, the liquid flow path becomes long. In addition, the longer the flow path becomes, the more diluted the injected sample is in the flow path, and as a result, the reaction time must be lengthened due to the sample injection M hole (1) and the flow path metal (1). This causes the inconvenience of not being able to do so.

On the other hand, enzyme activity can also be measured using a single electrode (see JP-A-56-92445).

That is, the enzyme sample solution of Shun Toreashi is injected into the substrate solution flowing toward the t70-cell in the solution flow channel at a constant flow rate, and the reaction between the two proceeds in the flow channel, and at that time, the enzyme is produced by the action of the enzyme. Or the concentration change of the detected substance that disappears'ft1
This is a method in which electric signals are transmitted using book electrodes.

However, in this case, if the sample solution contains in advance an electrode-sensitive substance other than the enzyme to be measured, the quadrupole will also be sensitive to this substance and its concentration will be the electrical tfIi. An error corresponding to the concentration of the substance occurs, making the measurement of the activity of the target enzyme inaccurate.

[Object of the Invention] The present invention solves the above-mentioned problems of conventional devices using electrochemical techniques, and solves the above-mentioned problems.
The purpose of the present invention is to provide a device for determining activation 1j111.

1. Summary of the Invention The inventors of the present invention have solved the above-mentioned problem with an enzyme activity measuring device that has a single electrode for measuring the concentration of a detection substance, namely, that the concentration of a detection substance is contained in the sample solution from the beginning. As a result of extensive research into removing the interfering effects of electrode sensitive substances, we found that before mixing the sample solution and substrate solution with gold and allowing the enzyme in the sample solution to react with the substrate in the substrate solution, M@k states that the influence of the electrode sensitive substance can be completely suppressed by reducing the concentration of the contained electrode sensitive substance T-ray.
1@, 9. Developing the device of the present invention.

That is, the device of the present invention is an enzyme activity 1'111I determination device of a flow cell system that measures enzyme activity sound using one acid solution, and a sample solution and a buffer solution are injected from a sample solution tank and a buffer solution tank, respectively. A sample liquid injection part that mixes both;
a liquid flow path connected to a sample liquid injection part for reducing the total concentration of an electrode sensitive substance previously contained in the sample liquid;
A substrate solution 7F is connected to the liquid flow path section and the substrate solution tank and mixed with the mixed liquid from the liquid flow path section as a substrate solution.
Tomobe; equipped with one electrode tube that is connected to the substrate solution inlet and converts into an electrical signal a change in the concentration of a detection substance produced or extinguished by the reaction between the enzyme in the sample solution and the substrate in the substrate solution; It is characterized by a structure in which the flow cells are arranged in this order.

In Figure 0, which will be explained below with reference to a block diagram of the apparatus of the present invention, reference numeral 1 is a sample liquid injection section, where the sample liquid is supplied from the sample liquid tank 2, and the buffer solution is supplied from the buffer liquid tank 3. A predetermined amount of each is injected by a metering pump 4.4', and both are mixed. The mixed solution is fed in the direction of arrow A while the reaction between the enzyme and the substrate progresses.

Reference numeral 5 denotes a flow cell, which contains a flow cell 1 that converts the concentration of a detection substance produced or extinguished by the action of an enzyme 1 or the concentration of an electrode sensitive substance previously contained in a sample into an electrical signal.
A book electrode 6 is attached, and the electrical signal of the electrode 6 is transmitted to a measuring system 7.
Measured and displayed by. The electrode 6 may be any electrode as long as it is sensitive to changes in the concentration of the detection substance, and is appropriately selected depending on the type of enzyme to be measured.For example, an electrode, an ammonium ion electrode, an ammonia gas electrode, Carbon dioxide electrodes, oxygen electrodes, hydrogen peroxide electrodes, and so-called fermentation electrodes that can detect organic substances such as glucose and pyruvic acid can be used.

Now, the device R of the present invention includes sample liquid injection section 1 to 70-cell 5.
The main feature is that a substrate solution injection section 1° connected to a liquid flow path section 8 and a substrate solution tank 9 via a fixing pump 4'' are arranged in this order.

The liquid flow path section 8 has a function of reducing the total concentration of the electrode sensitive substance contained in the sample liquid injected into the sample liquid injection section 1. In this case, without impairing the activity of the enzyme to be measured in the sample solution, it is possible to measure at a temperature close to the temperature at which p! 1
, it is necessary to reduce the S11 degree of the electrode sensitive material without changing the ionic strength.

For this purpose, in the device of the present invention, it is necessary to
A predetermined enzyme that transforms or decomposes into a substance that is not sensitive to the t-pole is fixed on the inner wall, and 1 g of the predetermined enzyme is immobilized on the surface of a skin-immobilized IW element tube or a fine carrier (e.g., glass beads). It is preferable that a solidified #element column filled with powder particles or the like is used as the liquid flow path section. Further, since the electrode-sensitive substance generally has a low molecular weight, the effect can be obtained even if the liquid flow path section is constructed of an ultrafiltration membrane capable of filtration or dialysis, and running water is passed around the outer circumference of the ultrafiltration membrane.

The device of the invention operates as follows. First, the metering pumps 4' and 4' are activated and continue to pump a predetermined buffer solution from the buffer solution tank 3 and a substrate solution containing a substrate for the enzyme to be measured by Fi from the substrate solution tank 9, respectively. Both are mixed and flow through the flow cell 5.

Next, the metering pump 4 is operated, and the enzyme to be measured and! A predetermined amount of a sample solution containing a highly sensitive substance is injected into the sample solution injection section 1. Here, the sample liquid and the liquid solution are mixed at a predetermined ratio, and a mixed liquid is formed inside the liquid flow path section 8.
The liquid is fed in the direction of arrow A. At this time, the electrode sensitive substance previously contained in the sample liquid is removed or decomposed by the action of the liquid flow path section 8, and its concentration is reduced.

The mixture of sample liquid and buffer that has passed through the liquid flow path section 8 joins and mixes with the substrate solution in the substrate solution injection section 10 , and is sent to the flow cell 5 . Thus, the injection sections 10 to 7
In the flow path leading to the 0-cell 5, contact between the enzyme and the substrate progresses, and a reaction occurs to generate or eliminate the detection substance. The length of the channel at this time is selected to be an appropriate length to cause the above reaction.

Therefore, the change in the concentration of the detection substance at this time is converted into an electrical signal by the electrode 6 and measured and displayed by the measurement system 7. As is clear from the above explanation, this measured value corresponds to the enzyme activity from which the electrode sensitive substance previously contained in the sample solution has been removed.

[Embodiments of the invention]

Example 1 The enzymatic activity of α-amylase was measured using the apparatus shown in the figure.

0.02 M phosphate buffer with pi-+ 6.0 as buffer, 0.11 M phosphate buffer with pH 7.0 containing soluble starch 5y/ls amylose 5 fi/l as substrate solution. , respectively, are stored in the buffer solution tank 3 and the substrate solution tank 9.

The electrode is a hydrogen peroxide electrode with a dense layer side of an asymmetric membrane of cellulose diacetate (25 μm thick, manufactured by Istoman Kodak Co., Ltd.) and about 40 tJ/l of glucose oxidase and about 1 sro/J of α. - A three-layer membrane is formed by pressing in this order a collagen membrane (thickness: 15 μm) on which glucosidase is completely immobilized and the coarse layer side of the treated membrane, which is obtained by heat-treating the above asymmetric membrane in hot water at 85°C for 2 minutes. An electrode with a structure in which the membrane was attached in close proximity to the platinum electrode was used. During the measurement, a voltage of 10.5 SV was externally applied to the platinum electrode with respect to the counter silver electrode.

Next, as a liquid flow path section, glucose oxidase (Type II, manufactured by Jig Corporation, USA) was placed on the inner wall of a nylon tube with an inner diameter of 1.0 and a length of 50α, approximately 551 U/IJ, and catalase (manufactured by Magma Corporation, USA), approximately 110 R IJ. /1m'
T-immobilize and then use the immobilized enzyme tube.

All other arrangements used Teflon tubes with an inner diameter of 1.5 m, metering pump 4 was a piston type pump, and 4' and '4'' were peristaltic pumps.

The inner diameter of the piping from the substrate solution injection part 1o to the electrode 6 is 1.5
Am 2m long Tear 0/Tube and friends.

Measurements were carried out at 11t and 35°C.

The sample solution was serum containing α-amylase as an enzyme, and the electrode sensitive substance at this time was glucose.

Under the above conditions, the flow rate of the buffer solution and the flow rate of the substrate solution were both set to 0.5 ml/min, and 5 μt of the sample solution was intermittently injected into the sample solution injection port 1 to measure the enzymatic activity of α-amylase. Next, it was possible to measure without error in the range of 20 to 2000 IU/l around the active level.

Example 2 The electrode was equipped with a polytetrafluoroethylene membrane having a thickness of 10βm, and the total glucose oxidase concentration was approximately 5%.
0 ■ U/112 immobilized collagen membrane (thickness 7 μm
) coated with history and on top of that? The procedure was carried out except that the asymmetric membrane of acetyl cellulose (thickness: 20 lAm) was covered with a dense layer side, and the liquid flow path was a nylon tube with an inner diameter of 2 mm and a length of 1 m fixed on the inner wall of α-glucosidase 251Uf. The enzymatic activity of α-amylase in serum was measured under the same conditions as in Example 1. Measurement was possible with a repeatability of 31 inches (α-amylase enzyme activity: 500 IU/1lI2).

〔Effect of the invention〕

As is clear from the above explanation, the device of the present invention measures enzyme activity with a single electrode, eliminating the need for complicated adjustment of gold electrodes, and at the same time eliminating the need for the electrode-sensitive substance pre-contained in the sample solution. Since enzyme activity can be measured with high precision by removing all influences, its industrial value is great.

[Brief explanation of drawings]

The figure is a block diagram of an example of the device of the present invention. 1... Sample liquid injection part, 2... Sample liquid tank, 3... Slow Ili liquid tank, 4.4', 4"... Metering pump. 5...
・Flow cell, 6... Electrode, 7... Measurement system, 8...
-Liquid flow path section, 9...substrate solution tank, 10...substrate solution injection section.

Claims (1)

[Claims] 1. An enzyme activity measuring device using a flow cell system that measures enzyme activity with one electrode, in which a sample solution and a buffer solution are injected from a sample solution tank and a buffer solution tank, respectively, and the two are mixed. a sample liquid injection part; a liquid flow path part connected to the sample liquid injection part and for reducing the concentration of an electrode sensitive substance previously contained in the sample liquid; the liquid flow path part and a substrate solution. a substrate solution injection section connected to the tank and configured to inject and mix a substrate solution into the mixed solution from the liquid flow path section; 1. Produced by reaction with a substrate. An enzyme activity measuring device characterized by having a structure in which a flow cell equipped with one electrode that converts a degree change in a detected substance to be annihilated into an electrical signal is arranged in this order. 2. The enzyme activity measuring device according to claim 1, wherein the liquid flow path section is either an immobilized enzyme tube or an immobilized enzyme column.