JPS6258717B2 - - 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 measured. The enzyme activity in the sample is then 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 that case, the linearity and linear region of the obtained calibration curve differ between each electrode, resulting in 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 flowed at a constant flow rate in a liquid flow channel, a sample solution is injected into the flow channel, and the enzyme and substrate are brought into contact while flowing in the flow channel. A method has also been proposed in which enzyme activity is measured by detecting the electrical signal given by the detection substance with an electrode placed downstream (Special Publication No. 56
-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, resulting in the inconvenience of having to increase the sample injection volume or lengthen the flow path to lengthen the reaction time. occurs. [Object of the Invention] An object of the present invention is to provide a novel enzyme activity measurement method 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 buffer solution from a buffer supply section attached to a liquid channel connecting a reaction section and a flow cell, and is equipped with one electrode for detecting electrochemical signal changes. After the sample liquid and the enzyme substrate-containing liquid are introduced into the flow cell and an electric signal is obtained, the sample liquid and the enzyme substrate-containing liquid are supplied to the reaction part from the sample liquid supply part and the enzyme substrate-containing liquid supply part to combine the sample liquid and the enzyme substrate-containing liquid. is reacted, and the resulting reaction solution is further introduced into the flow cell to obtain an electrical 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 incorporating an appropriate buffer substance into the enzyme substrate-containing solution. The flow cell 2 is sensitive to a detection substance that is consumed or produced by the enzyme action in the enzyme-substrate reaction system and whose concentration changes over time, and converts the concentration change into a change in electrochemical signal (for example, current value). 1
A book electrode 8 is built-in. As the electrode 8,
Any electrode may be used as long as it can detect a change in the concentration of the detection substance. Examples include glass electrodes for pH, ammonium ion electrodes, ammonia gas electrodes, and 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. Reference numeral 10 denotes a buffer solution supply section, and a flow path switching valve 11, preferably a solenoid valve, is connected via piping 6''.
It is connected to the liquid flow path 3 by. A buffer solution, such as a buffer solution obtained by removing the substrate from an enzyme substrate-containing solution, is stored in the supply section. 7″ is the reaction solution and buffer supply section 10 of the reaction section 1
This is a pump for introducing a predetermined amount of the buffer solution and a mixture thereof into the flow cell 2 at a constant flow rate. The pumps 7, 7', 7'' are plunger type,
Examples include peristaltic type. After the measurement is completed, the liquid flows into the drain section 12. The method of the present invention will be explained below. First, operate the flow path switching valve 11 to form a flow path between the buffer solution supply solution 10 and the flow cell 2, operate the pump 7'' to introduce a predetermined buffer solution into the flow cell 2, and use the electrode 8 to generate a base signal. Then,
Switch the valve 11 to form a flow path between the reaction section 1 and the flow cell 2, and operate the pumps 7 and 7' to supply the standard enzyme solution from the sample solution supply section 4 and the buffer solution from the substrate-containing solution supply section 5. A predetermined amount of each substrate-containing solution containing a substrate is injected into the reaction section 1, the two are brought into contact and reacted for a predetermined time, and the obtained reaction solution is introduced into the flow cell 2 by a pump 7'' to obtain an electrical signal. The difference between the electrical signal and the base signal at this time becomes a concentration signal of the detection substance corresponding to the standard enzyme.
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, when the enzyme is GPT, and therefore the enzyme substrates are L-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 created in advance from the pyruvate concentration and the electrical signal from the electrode, the concentration of pyruvate can be determined by the signal from the electrode.
Furthermore, the enzyme activity of GPT can be measured.
In addition, for example, regarding LDH enzyme activity, pyruvate concentration is

[Embodiments of the invention]

Example 1 Measurement of GPT enzyme activity A pyruvate oxidase entrapping immobilization membrane (film thickness 60 μm, enzyme activity
40IU/cm ² ), and applied this to the electrode sensitive part of the hydrogen peroxide electrode using a cellulose membrane (film thickness) for contamination prevention.
40 μm) with an O-ring to form an electrode according to the present invention. The buffer was a 0.05M phosphate buffer (PH7.4) containing 0.01mM flavin adenedinucleotide (FAD), 0.2mM manganese chloride, and 0.01mM thiamine pyrophosphate (cocarboxylase). This was stored in the buffer supply section 10. The enzyme substrate-containing solution contains 2mM α-ketoglutarate and the above buffer.
50mM of L-alanine was further added and the pH was adjusted to 7.4 with 1N sodium hydroxide. This was stored in the supply section 5. Pumps 7, 7', and 7'' are all peristaltic pumps, and the piping system is all made of Tygon tubes with an inner diameter of 0.5 mm, and the entire flow path is kept at 37°C. First, operate the valve 11 to connect the supply section 10 to the flow cell 2. A flow path is formed and the flow rate is increased by pump 7″.
A buffer solution was pumped at a rate of 0.8 ml/min, and pyruvic acid at various concentrations was injected into the buffer solution to create a calibration curve of pyruvic acid for the electrode. The results were as shown in Figure 2. Next, the valve 11 was switched to form a flow path between the reaction section and the flow cell. 100μ of the GPT-containing sample solution and 900μ of the substrate-containing solution were injected into reaction section 1 and mixed using a magnetic stirrer. The reaction solution was introduced into the flow cell at the above flow rate, and the signal from the electrode was measured. The results are shown in Figure 3 as a relationship between signal value (current) and GPT activity. FIG. 3 shows good agreement with the results of absorbance measurement using the conventional method. Example 2 Measurement of LDH enzyme activity Add 50ml of the buffer of Example 1 as an enzyme substrate-containing solution.
Except that a solution containing M lactic acid and 1 mM nicotinamide adenine dinucleotide (NAD) and adjusted to pH 8.4 with 1N sodium hydroxide was used, and that the sample solution contained LDH. The enzymatic activity of LDH was measured in the same manner as in Example 1. The results are shown in Figure 4. The results shown in the figure were in good agreement with the results of absorbance measurements. [Effects of the Invention] As is clear from the above explanation, according to the method of the present invention, only one electrode is used, eliminating the need for the complicated and labor-intensive work of adjusting the output of the electrode, which is extremely easy to handle. This is extremely useful.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram for explaining the device according to the present invention, Figure 2 is a calibration curve for pyruvic acid of the electrode used in Example 1, Figures 3 and 4 are respectively
This is a calibration curve for the enzyme activities of GPT and LDH. 1... Reaction section, 2... Flow cell, 3... Liquid channel, 4... Sample liquid supply section, 5... Enzyme substrate containing liquid supply section, 6, 6', 6''... Piping, 7, 7 ′,7″…
...pump, 8...electrode, 9...measuring section, 10...
Buffer supply unit, 11...flow path switching valve, 12
...Drainage tank.

Claims (1)

[Scope of Claims] 1. A buffer solution is introduced from a buffer solution supply section attached to a liquid flow path connecting the reaction section and the flow cell into the flow cell equipped with one electrode for detecting electrochemical signal changes. to obtain an electrical signal, supply the sample liquid and the enzyme substrate-containing liquid from the sample liquid supply part and the enzyme substrate-containing liquid supply part to the reaction part to cause the sample liquid and the enzyme substrate-containing liquid to react, and further . A method for measuring enzyme activity, which comprises introducing the obtained reaction solution into the flow cell to obtain an electrical signal. 2. The enzyme activity measuring method according to claim 1, wherein the electrode is a pyruvate sensor. 3. The method for measuring enzyme activity according to claim 1, wherein the enzyme is glutamate pyruvate transaminase or lactate dehydronase.