JPS6052375B2 - How to measure enzyme activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring enzyme activity using electrochemical redox of coenzymes.

One of the new methods for quantifying in vivo enzyme activity in clinical chemistry analysis is to bring the enzyme to be measured into contact with a conjugated coenzyme and a substrate, and to use the oxidation or reduction of the coenzyme to electrolyze it. A method has been developed to detect it. In this analysis method, a pair of electrodes is provided in the sample passage, and a substrate and enzyme sample are passed through the sample passage with a coenzyme present between the two electrodes, and a potential is applied. As a result of the coenzyme reacting with the substrate, the coenzyme is oxidized or reduced, and this oxidized or reduced coenzyme is reduced or oxidized again by the electrochemical circulation action of the current, and the sample is measured by reading the change in the current value. It is designed to quantify the enzyme activity in the sample.

However, the coenzyme used in the above analysis method is an extremely unstable substance outside of living tissue, and it is impossible to store it for a long time, especially in a solution state. When the coenzyme is brought into contact with the coenzyme, the expensive coenzyme is discarded, resulting in high testing costs per unit sample and low electrode sensitivity. On the other hand, a method has also been proposed in which the coenzyme is immobilized on agarose beads, the beads are suspended near the electrode, and the beads are covered with a porous diaphragm to prevent them from scattering from the electrode. However, the above method is difficult to attach to the electrode and troublesome to handle, and like the above method, the activity of the coenzyme itself is low, so accurate measurements cannot be made, and the pot life is short. shellfish.

Therefore, in either method, the instability of the coenzyme and problems with the immobilization method have been obstacles to the practical application of this type of analytical method. The present inventors conducted various studies and experiments to overcome the above-mentioned problems, and as a result, they constructed an immobilized coenzyme membrane by immobilizing a coenzyme on a porous polymer membrane, and this coenzyme membrane The above problem was solved by coating the tip of the measuring electrode with the material, and the present invention was thus completed. In other words, a porous polymer membrane with a coenzyme immobilized has strong activity and is stable. Coating the surface of the working electrode provided on the surface of the working electrode simplifies the structure of the analyzer and makes it more convenient to handle. Combined with the characteristics of the coenzymes mentioned above, this reduces measurement time and provides highly reproducible measurement results. Furthermore, it has excellent properties such as durability, long pot life, and continuous measurement.

In the measurement method of the present invention, a reference electrode and a working electrode are provided in a measurement chamber, and the surface of the working electrode is covered with an immobilized coenzyme membrane formed by immobilizing a coenzyme on a porous polymer membrane. A sample to be measured and a substrate are introduced into the sample and brought into contact with the coenzyme membrane to cause an enzymatic reaction.As a result, the enzyme in the sample reacts with the substrate and becomes an oxidized or reduced form. The enzyme activity in the sample to be measured is quantified by electrochemically circulating the coenzyme and measuring the oxidation or reduction current at the time.First, we will explain the analytical equipment used in the above measurement method. do.

FIG. 1 shows an analyzer for carrying out the present invention, which includes a reference electrode block 1 and a working electrode block 2.
are placed facing each other, a space is provided between these two blocks 1 and 2 to form a sample passage 3, and these two blocks 1 and 2 are connected with screws 4.
It is fixed airtight.

The reference electrode block 1 has an inlet port 5 communicating with the sample passage 3 and an outlet port 6.

Each of the ports 5 and 6 has a supply and discharge path 7,
8 is connected, and the supply side 7 includes a sample tank 9 and a tank 10 for accommodating a buffer solution containing a substrate.
The sample to be measured and the buffer solution in each of these tanks 9 and 10 can be mixed and switched by a three-way tank 11.

Further, a metering pump 12 is disposed on the discharge side 8, and the pump 12 causes the sample and buffer solution to flow through the sample passage 3 at a constant speed. A comparison electrode 15 is provided between each of the ports 5 and 6.

The reference electrode 15 includes a porous member 17 made of a dialysis membrane or a ceramic diaphragm disposed in an opening 16 facing the sample path, an electrolyte 18 sealed in the opening 16, and It consists of an Ag-AgCl electrode 19 inserted into the electrolytic solution 18, and a cap 20 is attached to this to prevent leakage of the electrolytic solution 18. A working electrode 21 is provided on the working electrode block 2, facing the reference electrode 15. The working electrode 21 includes a glassy carbon plate 23 stuck to the surface of a copper plate 22 embedded in the working electrode block 2.

On the side of the plate 23 opposite to the passage, there is an immobilized coenzyme membrane 24 formed by immobilizing coenzymes (described later) with a porous polymer membrane.
The coenzyme membrane 24 is tightly attached to the plate 23 by fixing its periphery with an O-ring 25. A measuring device 26 is connected to the ends of the electrodes 15 and 21 via a lead wire 27, applies a voltage between the electrodes 15 and 21, and measures changes in the current value.

Therefore, in order to perform quantitative analysis of a sample using this device, it is necessary to apply a voltage between both electrodes 9 and 13 while
1 is opened to allow the sample to be measured to pass into the sample passage 3 together with the buffer solution.

If an enzyme is present in this sample, this enzyme will promote the reaction between the substrate in the buffer and the coenzyme, converting the coenzyme into an oxidized or reduced form, and this coenzyme will undergo electrochemical circulation. It changes back to its original form by action. Since the substrate and the coenzyme are constant factors, the enzyme activity in the sample can be quantified by reading the change in current value at this time using the measuring device 26. In addition, the sensitivity, stability, and reproducibility for enzymes in the measurement method using the above analyzer depend on the activity and stability of the immobilized coenzyme membrane.Next, this coenzyme membrane will be explained in more detail. .

The above-mentioned coenzyme membrane is one in which a coenzyme is chemically bonded to a cellulose-based porous polymer membrane such as regenerated cellulose or cellulose ester.

In this case, rather than simply physically adsorbing, there is a method of chemically covalently bonding using the functional groups of the polymer membrane,
A method of crosslinking using a crosslinking agent and a combination of these methods are advantageous in terms of coenzyme activity.

The coenzyme to be immobilized is a coenzyme that performs an enzymatic reaction with the substrate by conjugating with the enzyme to be measured, that is, when the analyte is GDH (glucose dehydrogenase) or LDH (lactate dehydrogenase), NAD+ (nicotinamide adenine dinucleotide) and its reduced form NADH, ICDH (isocitrate dehydrogenase), G-6-PD
NA for (glucose-6-phosphate dehydrogenase)
DP+ (nicotinamide adenine dinucleotide phosphate) and its reduced form NADPH can be used, as well as FMN (flavin mononucleotide), FA
D (frapin adenine dinucleotide) and the like can also be used depending on each enzyme.

Therefore, these coenzymes can be arbitrarily selected depending on the target enzyme to be analyzed. Next, a specific example of immobilizing the above-mentioned coenzyme on a porous polymer membrane will be explained.

The time and temperature for the reaction of the coenzyme with the porous polymer are not particularly limited, but the reaction is usually carried out within a range that does not deactivate the coenzyme.

Production Example (1) In order to activate the regenerated cellulose membrane or cellulose ester membrane, it is immersed in an IM-sodium metaperiodate solution at about 50° C. for 3 hours to react.

After the reaction, wash with pure water. (2) Also, as an activation method other than the above (1), the membrane is immersed in an approximately 5% bromcyan solution and reacted for 1 hour to 1 hour.

At this time, highly concentrated sodium hydroxide was added to the solution to adjust the pH.
The reaction temperature should be maintained at 11 to 12 degrees Celsius and should not exceed 30°C. After the reaction, the membrane is quickly washed with pure water.
(3) Adding a crosslinking agent to the membrane obtained in step (1) or (2).

Examples of this crosslinking agent include diamines represented by the chemical formula NH2/(CH2)n-NH2, and NH2/NH4eOC.
● Select the optimal dihydrazide represented by (CH2)n●CO●NHINH2, prepare a large excess solution, and soak the membrane activated in step {1) or (2) in it. If the reaction is allowed to proceed for a long time at °C, the functional group at one end of the crosslinking agent will bond to the activated portion of the membrane surface.

(4) After the above reaction is completed, the membrane is heated to 0. ? Wash and store with sodium chloride solution.

{5) Adjustment of coenzyme solution.

NAD or its reduced form NADHl or NADP
or its reduced form NADPH in a cold neutral solution with 0.0
Dissolve at a concentration of 1 to 0.0'2M, add sodium metaperiodate to a concentration of 0.01 to 0.02M, and react in the dark at 0°C for 1 hour.

(6) If the membrane obtained in steps (1) to {4) is immersed in this coenzyme solution and reacted at 4°C for 3 hours or more, the coenzyme will be attached to the functional group on the other end of the crosslinking agent bound to the membrane. are combined.

The pH of the solution at this time is adjusted to 5 by adding a buffer solution. (7) After the reaction? An immobilized coenzyme membrane is obtained by washing with a sodium chloride solution.

Next, the immobilized coenzyme membrane obtained in the above production method example is set in the working electrode obtained in the above example, and a specific quantitative method using this will be described in detail.

Example 1 A GDH measurement buffer was adjusted to the optimal pH for the enzyme reaction, and glucose, which reacts with the coenzyme in the presence of the GDH as a substrate, was mixed into the buffer to give the optimal concentration. It is stored in the tank 10.

In addition, the coenzyme membrane 24 contains NAD or its reduced form, which performs an enzymatic reaction by conjugating with the GDH obtained in the above production method example.
A porous polymer membrane in which H is immobilized is used, and this is set on the working electrode 21 as shown in FIG. Then, they are connected to the measuring device 26 with the working electrode 21 side set as (+) and the comparison electrode 15 side set as (-), and +0
．． A voltage of 5V to +1.0V is applied in advance.

At this time, the coenzyme in the coenzyme membrane 24 has become oxidized NAD+ due to electrochemical circulation between the two electrodes. Also, before making measurements, remove the three-way tank 11 from the buffer side tank 10.
side, and operate the pump 12 to flow the buffer solution into the sample passageway 3 at a constant speed in advance to stabilize the potential between the two electrodes.

Next, the three-way switch 11 is switched to introduce the buffer solution and the sample to be measured into the passageway 3.

The enzyme in the measurement sample and the glucose in the buffer contact the coenzyme membrane 24 to perform the following reaction.

Formula 1 Glucose + N7V) + 9ρ NADH + H++ Gnoreconolactone The reduced form of NADH due to this reaction is electrode 1
5,21 is oxidized again by the electrochemical cycling action of N.
Changes to AD+.

Equation 2 Therefore, by reading the value of the oxidation current at this time, it is possible to know the amount of live bamboo ρDH in the measurement sample. : In Figure 2, various concentrations of ICDH were measured using the same device using the above method, and the results were used to create a calibration curve.The vertical axis shows the current value (μA), and the horizontal axis shows the concentration of ICDH (μA). 1-U/m
l) is shown.

Example 3 Gluconate-6 phosphate lactone as the substrate in the G-6-PD measurement buffer and NADP conjugated with the above G-6-PD as the coenzyme membrane
Alternatively, a coenzyme membrane in which reduced NADPH is immobilized on a porous polymer membrane is used.

Contrary to the first and second embodiments, a voltage of -1.0 to -2.5 V is applied between the electrodes 15 and 21. Therefore, the coenzyme is usually reduced NADPH. After that, the measurement sample and buffer solution are introduced into the sample passage 3 by the same operation as in Examples 1 and 2. Due to the presence of G-6PD in the measurement sample, NADPH is mixed with gluconic acid-6 phosphate lactone as follows. It reacts with water and performs electrochemical circulation.

Equation 4 Equation 5 Therefore, the amount of active G-6-PD in the sample to be measured can be determined by reading the value of the reduction current at this time.

Example 41J) Measurement of H The measurement conditions are the same as in Example 3, except that pyruvate is used as the substrate and NAD or 8 is used as the coenzyme.

Example 2 Measurement of ICDH The conditions and method for measuring GDH are the same as in Example 1, except that isocitrate is used as the substrate in the buffer and conjugated with ICDH obtained by the above production method example as the coenzyme used in the coenzyme membrane. DoN
ADP or its reduced form NADPH is used.

The reaction formula is shown below. Formula 3 NADH in its reduced form was immobilized on a porous polymer membrane.

Use coenzyme. The reaction formula is shown below. Formula 6
--- Figure 3 shows a calibration curve created by measuring LI)H at various concentrations using the above method, and the vertical axis shows the current value (N
A), the horizontal axis shows the concentration of LDH (1-U/ml).

In each of Examples 1, 2, 3, and 4, the current value and sample concentration had a linear relationship over a fairly wide range as shown in Figures 2 and 3, and very good measurement results were obtained. Obtained.

This suggests that the above-mentioned immobilized coenzyme has extremely high activity and stability, and also that it has extremely high sensitivity to enzymes because it performs electrochemical circulation while in close contact with the electrode. This shows that.

Furthermore, the analysis method according to the present invention is economical because the coenzyme can be used repeatedly, and it is also easy to operate and significantly shortens measurement time because it allows rapid and continuous analysis by simply injecting the sample while flowing the buffer solution. can. In addition, cleaning after a single measurement can be easily performed by simply switching the three-way switch to the buffer side, and the cleaning performance is also good. In addition, although each example uses NAD and NADP as coenzymes,
Coenzymes that transfer hydrogen by conjugating with other redox enzymes, such as FMN (flavin mononucleotide), FAD
It goes without saying that a coenzyme such as (flapin atenine dinucleotide) can also be used to obtain the same effect as in the above example.

[Brief explanation of the drawing]

FIG. 1 is a sectional view schematically showing an analyzer for carrying out the present invention, and FIGS. 2 and 3 are graphs showing the relationship between the concentration of ICDH and LDH and the current value, respectively.

Claims (1)

[Claims] 1 A reference electrode and a working electrode are provided in the measurement chamber, and the surface of the working electrode is covered with an immobilized coenzyme membrane formed by immobilizing a coenzyme on a porous polymer membrane, and a sample to be measured and a substrate are placed in the measurement chamber. are introduced and brought into contact with the above-mentioned coenzyme membrane to perform an enzymatic reaction, and as a result, the enzyme in the above-mentioned sample to be measured reacts with the substrate, and the coenzyme becomes oxidized or reduced form. A method for measuring enzyme activity, wherein the enzyme activity in the sample to be measured is determined by circulating the sample through the sample and measuring the oxidation or reduction current at that time.