JPS59162898A - Method and apparatus for determination of solute using immobilized enzyme - Google Patents

Abstract

PURPOSE:To prevent the deactivation of enzyme with heavy metal (e.g. Ag) in an analyzer utilizing an SH enzyme, by contacting the immobilized enzyme constantly with a low molecular SH compound during the pause of the measurement. CONSTITUTION:The cell 19 is furnished with an enzyme electrode (having an enzyme membrane immobilized with an enzyme such as urease, as a sensitive membrane), a reference electrode 2 and an earth electrode 3 in the flow path in the order. The specimen supplied from the injector 18 is carried by the carrier buffer liquid 15 and transferred to the cell 19 through the thin tube 14 and the feed pump 17. A signal corresponding to the concentration of the material (e.g. urea) to be determined is generated by the electrode, and is transferred via the preamplifier 20 and recorded with the recorder 21. The carrier buffer solution in the above process is the one containing about 0.1-100mM of a compound containing SH group (e.g. mercaptoethanol, glutathion, etc.).

Description

Detailed Description of the Invention [Field of Application of the Invention] The present invention relates to a method for measuring substances dissolved in a solution using an enzyme electrode or a reactor in which an enzyme is completely immobilized.
The present invention relates to a measuring method and a measuring device suitable for measuring substances dissolved in body fluids such as blood using enzyme electrodes or reactors using enzymes.

[Prior art]

Enzyme electrodes, which combine enzyme membranes with immobilized enzymes with high reaction specificity to substrates (substances to be measured) and ion electrodes, were developed over 10 years ago (G, G).

Quilbault, Handbook of
EnZymaticMethod of Ana
lysis, Marcel Dekker.

NeW 'York (1976), some of which have recently reached the practical stage. However, problems such as short life still remain. In particular, SH enzymes that require SH groups for catalytic action are easily deactivated by trace amounts of heavy metals. In general, chelate compounds can be easily removed using gold, ethylenediaminetetraacetic acid, etc., but some ions, such as silver, are difficult to remove and are responsible for shortening the life of enzyme electrodes.

[Purpose of the invention]

Is the purpose of the present invention an SH enzyme? The purpose is to prevent the enzyme electrode used or the reactor in which the enzyme is immobilized from being inactivated by heavy metals, and to improve the lifespan of the enzyme electrode and the like.

[Summary of the invention]

SH enzyme cord (denoted as E-8H), i.e. 8H for enzyme activity
For example, when a negative heavy metal ion (denoted by M+) acts on an enzyme in which the group is involved, the heavy metal ion easily binds to the enzyme as shown in the following formula.

M++E-8H4E-8M+H'' If the SH group in the above formula is involved in catalytic action, the activity of the enzyme is lost due to the above reaction.

On the other hand, when a compound having another SH group (denoted by R-8H) with high reaction specificity for M+ comes into contact with M+, a similar reaction occurs as shown in the following formula, and therefore the activity of the SH enzyme is Never get caught.

Furthermore, R-8H' is added to the enzyme that has been inactivated by binding to M+.
By contacting with , it is possible to restore the enzyme activity as shown in the following formula.

E-8M+R-8H-+E-8H+R-8M The present invention will be explained below by taking as an example the measurement of blood urea nitrogen using an immobilized urease enzyme electrode.

FIG. 1 is a schematic diagram of the electrode portion of the measurement system used in this example. In the figure, 1 is an enzyme electrode, 2 is a reference electrode, 3 is a ground electrode, 4 is an immobilized urease membrane, 5 is an ammonium selectively permeable membrane, 6 and 7 are ion permeable membranes, 8.9 and 1
0 is an Ag-AgCt electrode, 11, 12 and 13 are electrolyte solutions, 14 is a capillary, 15 is a buffer solution, and 16 is an arrow indicating the flow direction of the buffer solution.

Now, when a small amount (for example, 10 μt) of a sample such as blood is flowed through the thin tube 14 using a buffer solution as a carrier, the urea contained in the sample is hydrolyzed when it comes into contact with the immobilized urease membrane of the enzyme electrode, producing ammonium ions. . Ammonium ions change the membrane potential of the ammonium permselective membrane,
As a result, a potential change occurs between the enzyme electrode and the reference electrode depending on the urea concentration in the sample.

Generally, in this type of measurement, it is known that an Ag-AgCt electrode has the best stability as an electrode for emitting a potential. Although the solubility of hgct in water is small (solubility product 1.7 (
For example, it is dissolved as [Ag (NH3)t ]"cz).

Since the ammonium permselective membrane 5 lacks hydrophilicity, it is thought that Ag + dissolved in the electrolyte solution 11 is difficult to exit through the membrane, but a trace amount of Ag + does permeate. Ag4' dissolved in the electrolyte solutions 12 and 13 passes through the ion permeable membranes 6 and 7 and easily diffuses to the outside.

In this measurement system, as long as the buffer solution 15 is flowing, Ag+ originating from the electrodes 2 and 3 flows away and is difficult to be captured by the enzyme membrane 4, but when the flow of buffer solution 15 stops, Ag0 naturally diffuses into the enzyme membrane. and inhibits urease activity.

According to experiments conducted by the present inventors, electrodes 1 and 2 and electrodes 3 were all 1 cr.
I! When arranged at intervals, the activity of the immobilized urease membrane 4 was completely lost within 15 h after the apparatus was stopped. Therefore, when the tubule 14 was filled with a 50 mM mercaptoethanol solution and remeasured after being left for 4 hours, the activity of the urease membrane was recovered by about 80%. Furthermore, when the membrane was kept in contact with a 50 mM mercaptoethanol solution of urease gf, which had been completely inactivated, for 8 hours, the activity of the membrane was completely recovered. On the other hand, immediately after stopping the device, the thin tube 14
was filled with mercaptoethanol solution, and the activity of the urease membrane was measured 15 hours later, and no attenuation was observed.

The same effect as above was also obtained when an immobilized enzyme reactor and an ammonium electrode were used as a pair in place of the immobilized enzyme membrane electrode.

Apart from experiments using the apparatus shown in Figure 1, several S
A fundamental experiment was conducted on the H enzyme.

That is, alcohol dehydrogenase, asparakinase, β-amylase, urease, fructo-striped phosphatase, and α-glucosidase were each immobilized on a cellulose acetate membrane and used as experimental samples. A container partitioned into two chambers with a creofilter was prepared, and each chamber was filled with 10 mt of 60mM IJ phosphate buffer, 100 μt of 10 μM silver nitrate solution was added to one, and a small piece of the immobilized enzyme membrane was added to the other.

After 4tll' and 15h of standing, the enzyme activity was measured.
In both cases, activity had disappeared. On the other hand, 100 μt' of 1M mercaptoethanol solution was added to the chamber to which the immobilized enzyme membrane was added.
2 Add in advance and add 4C for 15h in the same way as above.
After standing, enzyme activity was measured. Separately, as a control, an immobilized enzyme membrane was placed in a buffer solution and left at 4tr for 15 hours. No decrease in the enzyme activity of the enzyme membrane was observed compared to the control.

In an analyzer using an 8H enzyme (an analyzer equipped with an enzyme electrode, an enzyme reactor, etc.), as mentioned above, if the immobilized enzyme is brought into contact with a low-molecular-weight SH compound while the measurement is paused, heavy metals, In particular, it has been found that enzyme deactivation caused by monovalent heavy metals can be prevented. Here, being on hiatus includes a hiatus of about one night or a hiatus of several days.

Generally, the activity of immobilized enzyme membranes gradually decreases over time, and one of the reasons for this is thought to be deactivation due to heavy metals. As mentioned above, the origin of heavy metals is clear, such as Ag0, which originates from various electrodes, but there are also heavy metals that are contained as impurities in the reagents used. These trace amounts of heavy metals are also a factor that reduces the lifespan of enzymes in the long term. Trace amounts of heavy metals that enter the measurement system as impurities can also be captured by the compound having an SH group and removed from the system.

A compound having an SH group is a reducing substance, but if it does not affect the intended measurement, it can be added to the buffer solution. In this case, the concentration of the compound having an SH group is preferably in the range of 0.1 to 100 mM, more preferably in the range of 5 to 50 mM. 100mM
Although it is acceptable to have a concentration exceeding 1, it is not desirable because it is uneconomical and gives off a bad odor. Also 0,1mM
If it is less than that, it is necessary to flow a large amount of solution, which is similarly uneconomical.

The compound having an SH group may be any compound as long as it dissolves in the buffer solution, but compounds with a low molecular weight are particularly preferred since they are easily soluble.

As such compounds, many SH compounds such as mercaptoethanol, glutathione, cysteine, thiocricholic acid, 2-mercagutoethylamine, benzenethiol, parathiocresol, and dithiothreitol can be used.

Another method of the present invention is to place a trap filled with a monovalent heavy metal scavenger in the liquid flow path between the enzyme electrode and the reference electrode. - As the antistatic metal scavenger, there are insoluble substances having SH groups, such as SH-modified polystyrene fine particles. The fine particles are placed in the flow path as described above,
In order to completely prevent the outflow of fine particles, a porous membrane such as a hydrophilically treated polyester nonwoven fabric is placed before and after the membrane. This method also allows the enzyme electrode to be protected from monovalent heavy metals flowing out from the reference electrode and the ground electrode.

The same applies when an enzyme reactor and an ammonium electrode are used instead of an enzyme electrode. In this case, since the outflow of A g 4- from the ammonium electrode is extremely low compared to that of the reference electrode, a trap filled with an antistatic metal scavenger is placed in the flow path between the reactor and the reference electrode. if,
It can be used upstream or downstream of the ammonium electrode. However, it is usually preferable to provide it between the reactor and the ammonium electrode.

When the measurement pause time is long, that is, the trapping agent is used for a long time, and the trapping agent has adsorbed a large amount of heavy metals, by flowing a buffer solution containing a compound having an SH group, such as mercaptoethanol, into the trap, the above-mentioned formula As can be seen from the above, the function as a heavy gold handle scavenger can be easily regenerated.

Still another method of the present invention is a method in which the buffer solution is intermittently flowed through the flow channel even during the suspension of measurement. As already mentioned,
Since heavy metals mainly flow out from the reference electrode and the ground electrode, the enzyme electrode, reference electrode, and ground electrode are arranged from the upstream side of the buffer flow path as shown in Figure 1, and the buffer solution is kept flowing even during measurement suspension. The enzyme electrode will not be deactivated if it is kept flowing at all times. However, this is not practical from an economic standpoint. Therefore, as described above, it is sufficient to intermittently flow the buffer solution into the channel.

The length of the flow path between the enzyme electrode and the reference electrode is usually 1
It is preferable that it is 5 to 150 rran, and 20 to 50 rran
It is especially desirable to be the heir. When both electrodes are arranged at such an interval, a new volume of 1 to 20 times, more preferably 2 to 10 times, the volume of the buffer solution present in the flow path between the two electrodes is generated at intervals of 5 to 10 hours. All you have to do is run a suitable buffer solution. For example, when the thin tube has a diameter of about 1φ, a liquid of 10 to 50 mA may be applied.

Of course, it is possible to flow at shorter intervals than the above-mentioned interval or to flow a larger amount of liquid, but it is not economical.

[Embodiments of the invention] The present invention will be explained below using examples.

FIG. 2 shows an embodiment of the apparatus of the present invention.・Figure 2 (a
) is a diagram of the principle of an analysis device using an enzyme electrode, and FIG. 2(b) is a diagram of the principle of an analysis device using an Fi enzyme reactor. In the figure, 1, 2.3 are an enzyme electrode, a reference electrode, and a ground electrode, respectively. Further, 22 is an enzyme reactor in which urease is immobilized, and 23 is an ammonium electrode.

14 is a thin tube, 15 is a carrier buffer, 17 is a liquid pump, 18 is a sample injector, 19 is a cell holding an electrode, 20
is a preamplifier, and 21 is a recorder. While flowing the carrier buffer into the cell, add a small amount (for example, 10 to 2
When a sample (0 μt) is injected into the entire flow path system, the sample is delayed to the electrode, or is decomposed in a reactor before reaching the electrode, and a signal corresponding to the concentration is obtained. Here, if mercaptoethanol (5mMk) is added to the buffer as a compound having an SH group f:, the heavy metals flowing out or mixed into the channel system will always be captured by the compound having an SH group, and the immobilized enzyme will not be degraded by the heavy metals. It can be prevented. In this way, not only when the measurement is stopped, but also during the measurement, the 18H group? Even if the compound containing urease is present in the buffer solution, it does not adversely affect the enzyme electrode or reactor that has a sensitive membrane on which urease is immobilized.

Figure 2 shows an example of a device that measures urea sound using immobilized urease, but it is also possible to install electrodes using many immobilized enzymes in the same channel system to perform multi-item simultaneous measurements. The apparatus is shown in FIG. In this case, for example, the electrode for measuring glucose is affected by the reducing substance, and if the substance is added to the carrier buffer, the measurement accuracy will be reduced. In the figure, 24 is the more S of urease.
H cell for enzyme electrode; 25 is a cell equipped with a reference electrode, etc.;
26 indicates a cell equipped with other electrodes, such as for glucose measurement. In addition, 27 is a buffer containing SH compound, 28 and 2
9 is a flow path switching valve.

During the measurement, a carrier buffer containing no SH compound is allowed to flow through the entire channel system. When the measurement is suspended or stopped, the valves 28 and 29 are switched to drain the buffer solution 27 containing the SH compound.
After flowing the buffer solution for a short period of time, the pump is completely stopped and the liquid is filled into this channel system.

In this way, S without affecting other electrodes
Deterioration of the H enzyme electrode can be prevented.

The buffer solution 27 may be replaced as appropriate.

In addition, instead of mercaptoethanol, glutathione,
Similar results were obtained when cysteine, thioglycolic acid, 2-mercaptoethylamine, be/zenthiol, parathiocresol, dithiothreitol, etc. were used.

Still another embodiment is shown in FIG. 4, in which a trap filled with gold, a scavenger for monovalent heavy metals, is provided. Average particle size approximately 0.3
Polystyrene fine particles of ranφ were provided with SH groups on the surface by a known method to obtain SH-modified polystyrene fine particles. This SH-modified polystyrene fine particle 41 has a diameter of 2W+I.
It was filled into a φ thin tube to a length of 10 mm, and porous membranes 42 and 43 of hydrophilic nonwoven polyester fabric were placed before and after the tube to prevent the outflow of fine particles. Although FIG. 4 shows a part of the analyzer, the provision of a buffer solution container, liquid feed pump, sample injector, etc. on the upstream side is the same as in FIG. 2(a). After measuring the sample using this device, the liquid feeding was stopped for 15 hours, and the sample was measured after the liquid feeding was started again, but no deactivation of the enzyme electrode was observed.

On the other hand, for comparison, when a similar experiment was conducted except for the use of SH-modified polystyrene fine particles, the enzyme electrode was completely inactivated after 15 hours of stopping measurement.

Next, an example will be shown in which the buffer solution is intermittently supplied. Figure 2 (a
) using an analyzer with the structure shown in 6.
omM (pH 7,4) (7) phosphoric acid water bath flow, and a tube with an inner diameter of 0.5 inches was used as the capillary 14. However, the flow path in the cell 19 had an inner diameter of 1flD11.

1 enzyme electrode, 2 reference electrodes, 3 earth electrodes, 20 each
They were arranged at intervals of mm, and the pump was configured to flow the buffer solution at a speed of 'l m t/m, and a sample (serum) lOμtf was injected from the sample injector 18 and analyzed. After the measurement continued for 8 hours, the bong was completely stopped, and for the remaining 16 hours, the pump was operated for 5 minutes every 6 hours using an electronic device (not shown). In this way, measurements were repeated for 60 days and approximately 5,000 samples were analyzed, but no deterioration of the enzyme electrode was observed.

On the other hand, for comparison, the pump was not operated at all during the 16-hour measurement pause, and even after the pump was operated 16 hours later, the enzyme electrode was still inactivated.

〔Effect of the invention〕

According to the present invention, the lifetime of an enzyme electrode using an enzyme membrane or a reactor in which the enzyme is immobilized can be significantly increased.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of an electrode portion of an analyzer using an enzyme electrode according to an embodiment of the present invention, and FIG. 2(a). (b) is a principle diagram of an analyzer using an enzyme electrode and an analyzer using an enzyme reactor, Figure 3 is a principle diagram of a multi-item simultaneous analyzer using an enzyme electrode, and Figure 4 is a diagram showing the principle of an analyzer using an enzyme electrode and an enzyme reactor. Partial schematic diagram of an analyzer equipped with a trap 1... Enzyme electrode, 2
...Reference electrode, 3... Earth electrode, 4... Immobilized enzyme membrane, 5... Ammonium selectively permeable membrane, 6.7...
・Ion permeable membrane, 8,9.10...Ag-AgCA electrode, 11,12.13...Electrolyte solution, 14...tubule, 15...buffer solution, 16...flow direction, 17 ...
Pump, 18... Sample injector, 19... Cell, 20
... preamplifier, 21 ... recorder, 22 ... enzyme reactor, 23 ... ammonium electrode, 24 ... s
H Enzyme electrode cell, 25...Reference electrode cell, 26.
...electrode cell, 27...buffer containing a compound having an 8H group, 28.29...switching valve, 41...SH
Polystyrene fine particles, 42.43 Porous membrane. Atsushi 1 Figure 8132 Figure 2 (1) Sample ↓ 1 (Continued from page 1 of page 0 Inventor Yoshiharu Karasawa Inside the Central Research Laboratory, Hitachi, Ltd., 1-280 Higashikoigakubo, Kokubunji City

Claims (1)

[Scope of Claims] 1. In a method for measuring the substrate of the enzyme in a solution using an enzyme electrode having an enzyme membrane on which an enzyme is immobilized as a sensitive membrane or a reactor on which an enzyme is immobilized, A method for measuring a solute, which comprises keeping the sensitive membrane of an enzyme electrode or the above-mentioned reactor 'k in contact with a solution of a compound having an SH group. 2. The method for measuring a solute according to claim 1, wherein the enzyme is urease, alcohol dehydrogenase, asparakinase, β-amylase, fructos dihomephatase, or α-glucosidase. 3. The method for measuring a solute according to claim 1 or 2, wherein the method for measuring the enzyme substrate uses an enzyme electrode whose sensitive membrane is an enzyme membrane on which an enzyme is immobilized. 4. The method for measuring a solute according to claim 1 or 2, wherein the method for measuring the substrate of the enzyme involves immobilizing the enzyme or using an acta. 5. The method for measuring a solute according to any one of claims 1 to 4, wherein the solution of the compound having an SH group has a concentration of the compound in the range of 0.1 to 100 mM. 6. A container for holding a buffer solution, a buffer solution kept in the container, a sample injection part, and an enzyme electrode or a reactor with an enzyme immobilized thereon, whose sensitive membrane is an enzyme membrane on which an enzyme is immobilized, and a sample decomposed by the enzyme. A biochemical device consisting of an electrode for measuring the decomposition product of a substrate, a reference electrode, a ground electrode, the container mentioned above, a sample injection part, an enzyme electrode or a reactor, and a capillary for connecting the electrode, the reference electrode and the ground electrode, and transporting a buffer solution. 1. A measuring device for measuring equipment, characterized in that the buffer solution is a solution in which a compound containing an SH group is dissolved. 7. The measuring device according to claim 6, wherein the solution of the compound containing the SH group has a concentration of the compound in the range of 0.1 to 100 mM. 8. The enzymes mentioned above are urease, alcohol dehydrogenase, asparakinase, and β-amylase. The measuring device according to claim 6 or 7, which is fructo-streak phosphatase or α-glucosidase. 9. A container for holding a buffer solution, a buffer solution kept in the container, a sample injection part, and an enzyme electrode or a reactor with an enzyme immobilized thereon, whose sensitive membrane is an enzyme membrane with an immobilized enzyme, and a sample decomposed by the enzyme. An electrode for measuring the decomposition product of the substrate, a reference electrode, a ground electrode, the container, a sample injection part, an enzyme electrode or a reactor, a capillary tube for connecting the enzyme electrode or the reactor, a buffer Wi for connecting the reference electrode and the ground electrode, and transporting the buffer Wi. a second container for storing a second buffer solution in which a compound containing a compound is dissolved; a second buffer solution is supplied from the second container to bring the second buffer solution into contact with the enzyme electrode or the reactor when the measurement is stopped; A biochemical measurement device characterized by having a switching valve for inflowing and outflowing into the thin tube. 10. The measuring device according to claim 9, wherein the concentration of the compound containing an SH group in the second buffer is in the range of 0.1 to 100 mM. 11. The above enzymes are urease, alcohol dehydrogenase, asparakinase, and β-amylase. The measuring device according to claim 9 or 10, which is fructo-streak phosphatase or α-glucosidase. 12. A biochemical measurement device having an enzyme electrode and a reference electrode, the sensitive membrane being an enzyme membrane on which an enzyme is immobilized, characterized in that a heavy metal trap is provided in the flow path connecting the enzyme electrode and the reference electrode. Measuring device.