JPS6260080B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field of the Invention] The present invention relates to a method for measuring enzyme activity using an electrochemical measurement technique. [Technical background of the invention and its problems] Conventionally, enzyme activity is measured by adding a sample solution containing a certain enzyme, a solution containing the substrate of the enzyme, and other enzymes, coenzymes, coloring reagents, etc. It is carried out by causing an enzyme substrate to undergo a chemical reaction in a reaction system, and measuring the absorbance of the reaction system at that time. For example, in the case of the enzymatic activity of glutamic pyruvate transaminase (GPT), when GPT is brought into contact with its substrate, pyruvate is produced by the enzymatic action of GPT, and the pyruvate is reduced by the enzymatic action of lactate dehydronase (LDH). do.
At this time, if a predetermined concentration of nicotinamide adenine nucleotide (NADH) is allowed to coexist in this reaction system, the NADH decreases over time and its concentration decreases. The concentration change is at a wavelength of 340nm
This can be determined by measuring the absorbance of NADH in . Therefore, the enzyme activity of GPT can be measured indirectly. However, in this absorbance measurement method,
Measurement costs are extremely high because expensive reagents such as NADH, LDH, or various coloring reagents are used, and these must be discarded after measurement. Furthermore, since absorbance measurement is not possible for reaction systems containing suspended matter, such samples require pretreatment such as removal of suspended matter prior to measurement. In order to solve these drawbacks of absorbance measurement, an apparatus using an electrochemical measurement method has been proposed (see JP-A-56-97864). This is a flow system in which two enzyme electrodes are placed apart from each other and each has a membrane attached with a certain enzyme that acts on a substance (sensing substance) produced by the enzyme in the sample. The electrical signal given by the detection substance originally contained in the sample is used as the base value, and the electrical signal given by the substance (detection substance) produced in response to the activity of the enzyme by the action of the enzyme in the sample is further measured. The activity of the enzyme in the sample is measured 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. That is, it is extremely difficult to manufacture enzyme electrodes that have exactly the same electrode characteristics such as sensitivity, response speed, and gain, and also have the same change in characteristics over time. Therefore, it is necessary to adjust the output of each electrode during measurement, but even in this case, the linearity and linear region of the obtained calibration curve differ between each electrode, which may cause errors in measurement. invite In addition, if two electrodes are placed apart from each other in the liquid flow path of a flow system, a dilution phenomenon due to sample diffusion will occur in the liquid flow path, and the concentration of the detected substance will decrease at the downstream electrode position. As a result, it becomes extremely difficult to adjust the output difference between the two electrodes, or to adjust it during differential amplification. In particular, it is extremely difficult to measure small amounts of samples containing enzymes with low activity. In addition, an enzyme-substrate-containing buffer solution is passed through the liquid channel at a constant flow rate, a sample solution is injected into the solution, and the enzyme and substrate are brought into contact while flowing through the channel, causing the enzyme to decrease or increase due to the action of the enzyme. A method has also been proposed in which enzyme activity is measured by detecting the electrical signal given by the detected substance using an electrode placed downstream (Japanese Patent Publication No. 1983).
-92445, 92446). However, in this method, the liquid flow path between the sample injection port and the electrode is the site of the reaction between the enzyme and the substrate, so the liquid flow path becomes longer in the case of a low-activity enzyme that requires a long reaction time. Additionally, the longer the flow path becomes, the more the injected sample is diluted within the flow path, and as a result, the amount of sample injected increases, but this has the disadvantage of requiring a longer flow path to lengthen the reaction time. occurs. [Object of the Invention] An object of the present invention is to provide a method for measuring enzyme activity with a novel structure that solves the above-mentioned problems in conventional devices using electrochemical techniques. [Summary of the Invention] That is, the method of the present invention supplies a sample liquid and an enzyme substrate-containing liquid from a sample liquid supply section and an enzyme substrate-containing liquid supply section to a reaction section, and combines the sample liquid and the enzyme substrate-containing liquid. After introducing the reaction solution obtained by the reaction into a flow cell equipped with one electrode for detecting electrochemical signal changes and connected to the reaction section via a liquid flow path to obtain an electric signal, Furthermore, the method is characterized in that a reaction solution obtained by continuing the reaction in the reaction section is introduced into the flow cell to obtain an electric signal. The apparatus according to the present invention will be explained in detail based on the schematic diagram shown in FIG. In the figure, 1 is a reaction section, 2 is a flow cell, and both are connected by a liquid flow path 3. A supply section 4 for a sample solution containing an enzyme and a supply section 5 for an enzyme substrate-containing solution containing an enzyme substrate are each independently connected to the reaction section 1 via piping 6, 6', and each is connected to A predetermined amount of the sample liquid and the enzyme substrate-containing liquid are supplied to the reaction section 1 by the metering pumps 7, 7', where they are mixed and brought into contact by, for example, a magnetic stirrer. At this time, the reaction system is adjusted to an appropriate pH for the reaction between the enzyme and the substrate. Specifically, this is carried out by adding an appropriate buffer to the enzyme substrate-containing solution. The flow cell 2 is sensitive to a detection substance whose concentration changes over time by being consumed or produced by the enzyme action in the enzyme-substrate reaction system, and converts the concentration change into a change in electrochemical signal (e.g., current value). One electrode 8 is built-in. The electrode 8 may be any electrode that can detect changes in the concentration of the detection substance. For example, glass electrode for PH,
ammonium ion electrode, ammonia gas electrode,
Examples include various enzyme electrodes for detecting organic substances such as glucose and pyruvic acid. The signal from the electrode 8 is guided to the measuring section 9, where it is processed and calculated, and the enzyme activity value is displayed. 7'' is a pump for introducing a predetermined amount of the reaction liquid from the reaction section 1 into the flow cell 2 at a constant flow rate.Pumps 7, 7', and 7'' may include plunger type, peristaltic type, etc. can. The method of the present invention will be explained below. First, by operating the pumps 7 and 7', a predetermined amount of the standard enzyme solution is supplied from the sample liquid supply section 4 and a substrate-containing solution containing the substrate in the buffer solution is supplied to the reaction section 1 from the substrate-containing solution supply section 5. The enzyme is injected, and the two are brought into contact and reacted for a predetermined period of time, and the resulting reaction solution is introduced into the flow cell 2 by a pump 7'' to obtain an electric signal (base signal).Then, in the reaction section 1, the enzyme and the substrate react. While continuing to do so, the reaction liquid is sequentially introduced into the flow cell 2 through the liquid channel 3 by the pump 7'' at predetermined time intervals. In the flow cell 2, a change in the concentration of a detection substance produced or consumed based on a chemical reaction between an enzyme and a substrate is detected as a change in an electrical signal by an electrode 8, which is measured by a measuring section 9. Therefore, the difference between these electrical signals and the base signal corresponds to a change in the concentration of the detected substance. Therefore, by creating a calibration curve based on the relationship between the concentration of the detection substance and the electrical signal, the concentration of the detection substance and, ultimately, the enzyme activity can be measured from this electrical signal.
For example, if the enzyme is GPT and therefore the enzyme substrate is L
-GPT for alanine and α-ketoglutarate
Pyruvate is produced by the enzymatic action of GPT, and the amount produced is proportional to the activity of GPT. In this case, pyruvic acid is the detection substance. Pyruvate consumes oxygen and generates hydrogen peroxide by pyruvate oxidase, changing its concentration. Therefore, the pyruvate concentration is measured using an oxygen electrode or a hydrogen peroxide electrode. Therefore, if a calibration curve is prepared in advance from the pyruvate concentration and the electric signal from the electrode, the concentration of pyruvate and the enzyme activity of GPT can be measured using the signal from the electrode. Also, for example
Regarding the enzymatic activity of LDH, the pyruvate concentration is

[Embodiments of the invention]

Example 1 The enzyme activity of glutamate pyruvate transaminase (GPT) was measured using the apparatus shown in FIG. The entire piping system in the apparatus was composed of tetrafluoroethylene pipes with an inner diameter of 0.5 mm. The electrode is a membrane with pyruvate oxidase immobilized in collagen (film thickness 60 μm, enzyme activity
40 IU/cm ² ) was attached to the sensitive part of an oxygen electrode with polarographic behavior, and was further coated with an asymmetrically structured ultrafiltration membrane made of cellulose diacetate with a film thickness of 35 μm. First, from the enzyme substrate-containing liquid supply section 5, 0.05M phosphate buffer (PH7.6), 0.01mM flavin adenine nucleotide (FAD), 0.05mM manganese chloride,
Inject 900μ of a substrate-containing solution containing 0.1mM thiamine pyrophosphate (cocarboxylase), 2mM α-ketoglutarate, and 30mML-alanine into reaction section 1, and simultaneously inject 100μ of a sample solution containing GPT, and stir and mix both at 30℃. did. After 10 seconds and 5 minutes had elapsed after the sample solution was injected, 450μ of the reaction solution was drawn out by the pump 7'' at a flow rate of 0.4 ml/mm, respectively, and introduced into the flow cell 2.
The current value indicated by the electrode 8 was measured by the measurement unit 9.
Figure 3 shows the relationship between the electrode indicated current value (μA) and the GPT activity (IU/) when the GPT activity was determined from the difference between the two. Example 2 Cholinesterase activity was measured using the apparatus shown in FIG. The electrode consists of an immobilized enzyme membrane in which cholinesterase is immobilized in collagen, which is attached to the sensitive part of a polarographic electrode for detecting hydrogen peroxide, and an ultrafiltration membrane (thickness: A material coated with the dense layer (40 μm) facing outward was used. 200 serum samples are added to the reaction section 1 from the sample liquid supply section 4.
700 μ of a 0.05 M phosphate buffer (PH7.4) and 100 μ of a 15 mM acetylcholine chloride solution were injected from the enzyme substrate-containing liquid supply section 5 and stirred and mixed at 37°C. The solution supply section 11 contains 0.05M phosphate buffer (PH
8.5) was stored. Every 3 minutes, 200μ of the mixed solution was introduced into the channel 3 at a flow rate of 400μ/mm using the pump 7″, and 0.05M phosphate buffer (PH8.5) was added to this at the injection part 10 by operating the pump 7. The mixture was mixed by flowing at a flow rate of 400 μ/mm, and the electrical signal was measured using flow cell 2.
When this operation was repeated and the cholinesterase enzyme activity was measured from the difference in the electrical signals obtained, it was found to be 0.
A good calibration curve could be created in the range of ~3000IU/. [Effects of the Invention] The method of the present invention is useful because only one electrode is used, eliminating the need for complicated and difficult electrode output adjustment as in the conventional method, and making handling extremely simple.

[Brief explanation of the drawing]

Figures 1 and 2 are both schematic diagrams for explaining the device according to the present invention, and Figure 3 shows the GPT activity and electrode indicated current value measured using the device of the present invention (Figure 1). It is a relationship diagram. 1... Reaction section, 2... Flow cell, 3... Liquid flow path, 4
... Sample liquid supply section, 5... Enzyme substrate-containing liquid supply section,
6, 6', 6''... Piping, 7, 7', 7'', 7... Pump, 8... Electrode, 9... Measuring section, 10... Injection section, 11
...Buffer supply section, 12...Mixing piping.

Claims (1)

[Scope of Claims] 1. A reaction obtained by supplying a sample solution and an enzyme substrate-containing solution from a sample solution supply section and an enzyme substrate-containing solution supply section to a reaction section, and causing the sample solution and the enzyme substrate-containing solution to react. After introducing the liquid into a flow cell equipped with one electrode for detecting electrochemical signal changes and connected to the reaction section via a liquid flow path to obtain an electric signal,
Furthermore, the method for measuring enzyme activity is characterized in that the reaction solution obtained by continuing the reaction in the reaction section is introduced into the flow cell to obtain an electrical signal. 2. The method for measuring enzyme activity according to claim 1, wherein the liquid flow path is provided with a buffer supply section for adjusting the pH of the reaction solution.