JPS59180353A - Measuring device for pyruvic acid - Google Patents

Abstract

PURPOSE:To maintain stably enzyme activity for a long time in a measuring device for a pyruvic acid using immobilized pyruvic acid oxidase by bringing a specific concn. range of flavin adenine dinucleotide (FAD) soln. into the immobilized oxidase. CONSTITUTION:An FAD soln. is supplied through a piping 10 having a small inside diameter at a measured supplying rate of 10<-9>-10<-6>mol/min from a storage tank 12 by using a micro metering pump to an electrode part 15 of a measuring device for a pyruvic acid using a film 6 formed by fixing pyruvic acid oxidase (POP) to collagen, polyacrylamide, etc. A sample is supplied from a supplying part 14 and a buffer or substrate soln. is supplied from a storage tank 11, respectively to the piping 9 of the part 15. The FAD soln. is brought into the POP immobilized film 16 through the ultrafilter film 2 and buffer layer 3 of the part 15 in the above-mentioned way, by which the activity of POP is maintained stably for a long time. The same result is obtainable by bringing the FAD soln. into contact with said film in the stage of non-measuring or washing the part 15 in place of doing the same in the stage of measurement.

Description

[Detailed Description of the Invention] [Technical Field of the Invention] The present invention relates to a pyruvate measuring device, and more particularly to a pyruvate measuring device in which the enzymatic activity of immobilized pyruvate oxidase is stabilized.

[Technical background of the invention and its problems] In recent years, clinical analysis has rapidly developed, and it has become possible to accurately determine pathological conditions by measuring the activities of various enzymes present in the blood. For this reason, clinical analysis has come to make a significant contribution to the advancement of medicine.

Conventionally, enzyme activity is measured by carrying out an enzymatic reaction in an enzymatic reaction system consisting of an enzyme, a substrate, a coenzyme, and if necessary a coloring reagent, and measuring the change in absorbance before and after the reaction for colorimetric determination. was. For example, glutamate pyruvate transaminase (
When measuring the enzymatic activity of GPT, GPT acts on a substrate to generate pyruvate, and then the pyruvate is reduced by lactate dehydrogenase (LDH), which removes the nicotine coexisting in the reaction system. Since amide adenine dimerotide (NAD) I) is oxidized to form NAII, the NADH at a wavelength of 340 r+m
The enzymatic activity of GPT can be indirectly measured by measuring the absorbance of GPT. However, in this absorbance measurement method, NAD)1. LDH and various coloring reagents are expensive, and since these reagents are discarded after measurement, the cost of measurement is extremely high. Furthermore, if suspended substances such as platelets, blood cells, and dust are mixed in the reaction system, it becomes difficult to measure the absorbance, so pretreatment to remove the non-measurable substances is required prior to measurement.

Therefore, in order to overcome these drawbacks of the absorbance measurement method, a method was proposed for quantifying pyruvic acid, which is an enzymatic reaction product of GPT, using an electrode method (Japanese Patent Application Laid-Open No. 122-1989).
947). An immobilized enzyme membrane consisting of pyruvate oxidase immobilized on a porous polymer membrane is attached to the surface of an oxygen electrode, and the amount of pyruvate is determined from the amount of dissolved oxygen decreased due to the decomposition of pyruvate by pyruvate oxidase. It is used to measure. However, since the activity of the prepared immobilized enzyme membrane is unstable, it has the disadvantage that it cannot be used for a long time (50 times at most).

In addition, since pyruvate oxidase is generally an unstable enzyme, its lifespan (half-life of activity) was at most 1 to 2 weeks even if various immobilization methods and carriers were used.

In addition, as a method to measure the enzyme activity of GPT, GPT
When glutamic acid, an enzymatic reaction product, is decomposed by dehydrogenase, the activity of GPT can be determined from the measured value of the current flowing when the coenzyme reduced by the enzymatic reaction is oxidized with an electrode to which a constant voltage is applied. There is a method for measuring it (Japanese Patent Laid-Open No. 58-92448), but this method is easily interfered with by other reducing substances (vitamins, etc.) present in the blood.
However, it still has the drawback of not being able to withstand long-term use.

[Object of the Invention] The object of the present invention is to provide a long-life and stable pyruvic acid measuring device that does not have the above-mentioned drawbacks.

[Summary of the Invention l Conventionally, pyrubin protein measurement was performed using a buffer containing 0.01mM FAD, but the present inventors added a high concentration flavin adenine dinucleotide solution of 0.11 or more to the electrode. It was discovered that the lifetime of the enzymatic activity of pyruvate oxidase can be stabilized by bringing it into contact with the immobilized pyruvate oxidase, and the present invention has been completed.

That is, the present invention is a pyruvate measuring device using immobilized pyruvate oxidase, which is characterized by having a mechanism for bringing a 0.1 to 50 mM flavin adenine dinucleotide solution into contact with the immobilized pyruvate oxidase.

The present invention will be explained in detail below.

The pyruvic acid measuring device according to the present invention is a conventionally used pyruvic acid measuring device newly equipped with the above-described mechanism, and the structure other than this mechanism may be the same as the conventional one.

The immobilized pyruvate oxidase used in the present invention refers to pyruvate oxidase (POP) immobilized on a carrier capable of holding the enzyme. The immobilization carrier may be any carrier as long as it can immobilize pyruvate oxidase. Specific examples include collagen, polyacrylamide,
Examples include carrageenan, agarose gel, porous glass beads, albumin, and the like. Usually, pyruvate oxidase is immobilized on a carrier in a range of 0.01 to 30% by weight. Note that the method for immobilizing pyruvate oxidase on a carrier is not particularly limited.

However, when incorporating immobilized pyruvate oxidase into the flow system of a pyruvate measurement device, it is preferable to immobilize it on a membrane, granular, or gel-like carrier by an entrapment method or a covalent bonding method, and further attach it to the electrode surface. In this case, it is preferable to apply a comprehensive method in which an enzyme membrane is created using a fibrous protein such as collagen as a carrier.

Next, a mechanism for bringing the flavin adenine dinucleotide solution into contact with immobilized pyruvate oxidase will be explained, but in the present invention, any mechanism that can supply the solution may be used. An example of this mechanism is shown in the first example.
Shown in the figure. The figure is a schematic diagram of an improved electrode section,
1 is a flow cell, 2 is an ultrafiltration membrane, 3 is a buffer layer, 4 is an O-ring, 5 is an oxygen or hydrogen peroxide permeable membrane, 8 is P
OP immobilization membrane, 7 platinum cathode, 8 lead anode, 9 piping,
10 is a small inner diameter pipe. The mechanism shown in the figure is a mechanism for bringing the flavin adenine dinucleotide (FAD) solution' and pyruvate oxidase into contact during measurement. An electrode section having the same mechanism as above except that it does not include the small inner diameter section 1210 may be used. As the electrode, an oxygen electrode (using an oxygen permeable membrane 5) is usually used to measure the amount of reduction in dissolved oxygen during the enzymatic decomposition process of pyruvic acid, or an oxygen electrode is used to measure the amount of hydrogen peroxide generated in this process. A hydrogen peroxide quantity electrode (using a hydrogen peroxide permeable membrane 5) is used.

In the present invention, a method for stabilizing the enzymatic activity of pyruvate oxidase using the above mechanism is a method of bringing the FAD solution into contact with immobilized pyruvate oxidase after measuring pyruvate (when not measuring). Can be mentioned. In this case, the usual method is to fill the pipe with the FAII solution after the pyruvic acid measurement is completed and to clean the pipe, and to store the pipe in this state overnight at room temperature. In addition, as another method, the FAD
An example of a method is to clean the inside of the pipe using a cleaning liquid containing the liquid, and then fill the inside of the pipe with the cleaning liquid each time a day's measurement is completed, and store the liquid in this state overnight at room temperature.

Still another method is to add FA at a rate of 10-9 to 10-6 mol/min while measuring pyruvate.
An example of this method is to supply solution D into the pipe. In this case, there is an advantage that the measurement system can be cleaned and the POP activity can be prevented from decreasing at the same time. In the above measurement method, it is necessary to use a 0.1 to 50 mM FAD solution or a washing solution containing FAD. The longer the contact time between these solutions and the immobilized pyruvate oxidase, the better the results will be obtained, and it is desirable to carry out the contact treatment for consecutive days rather than completing the contact treatment only once. Note that the above methods may be performed alone or in combination. Incidentally, if only the conventional buffer solution containing FAD at a low concentration of about 0.01 mM is used, FAD will not be able to sufficiently diffuse through the permeable membrane 5 and will be discharged outside the measurement system, so the effect of the present invention will not be achieved. .

Next, a specific example of the pyruvic acid measuring device will be shown and a method of using the device will be explained. An example of the pyruvate measuring device of the present invention, in which the enzymatic activity of pyruvate oxidase is stabilized by bringing the FAD solution into contact with immobilized pyruvate oxidase during non-measurement periods, is shown in a block diagram in FIG. In the figure, 11 is a storage tank for buffer solution or substrate solution, 12 is a storage tank for concentrated FAD solution, 13 is an automatic solvent changer, 14 is a sample injection port, 15 is an electrode part, 16 is a constant flow pump, 17
is an automatic three-way valve, +8a and 18b are piping, 18 is a drain tank, 20 is a recorder, 21 is a controller, 22 is a stirrer, and 23 is an agitator.

In this measuring device, measurements are normally performed as follows. First, a measurement command is issued from the controller 21 (in actual operation, simply press the measurement start switch on the display panel of the controller 21), and the automatic solvent changer 13 connects the B or F valve and A. is connected and the automatic three-way knob 17 is connected to the pipe 18a,
The constant flow pump 16 is operated to fill the measurement system with the buffer solution. When it is completely filled, the RUN display lights up on the display panel of the controller 21. The buffer solution is constantly flowing at a constant flow rate during measurement. In this state, sample injection port 1
When an appropriate amount of sample liquid is injected from No. 4 (in the figure, the arrow indicates the injection direction), pyruvic acid in the sample is detected by the electrode section 15, and the signal is sent to the recorder 20 and recorded. Usually, it takes about 5 minutes from sample injection to recording on the recorder (when the flow rate is 0.B+++l/min).

Note that the waste liquid that has been detected or quantified is stored in the waste liquid tank 18 via the pipe 18a.

Next, after the measurement is completed, press the measurement end switch on the display panel of the controller 21, and the controller 21 will issue a command to change the C, E, and G of the automatic solvent changer 13.
Either valve I or I is connected to A, and the buffer solution and sample solution filled in the system are all discharged. After a certain period of time (the time required for the buffer solution to be completely drained), the valve of the automatic solvent changer 13 is switched to D or H, and at the same time, the automatic three-way valve 17 is also switched, and the valve 17 is connected to the pipe. Connected to +8b. By the above operations,
The measurement system is filled with a concentrated FAD solution, and the enzyme membrane of the electrode section 15 is prevented from being deactivated.

FIG. 3 shows a block diagram of an example of the pyruvate measuring device of the present invention, in which the enzymatic activity of immobilized pyruvate oxidase is stabilized by containing FAD in the washing solution after measurement. In the figure, 12a is a liquid storage tank for a concentrated FAD-containing cleaning liquid, but the other configurations are almost the same as the apparatus shown in FIG.

In this measuring device, measurements are normally performed as follows. After the measurement is completed and the buffer solution and sample solution filled in the system are discharged, the valve of the automatic solvent changer 13 is switched to D or H, and then the FAD in the liquid storage tank 12a is
Deactivation of the enzyme membrane can be prevented by flowing the containing cleaning liquid into the measuring device.

An example of the pyruvate measuring device of the present invention, in which the enzymatic activity of pyruvate oxidase is stabilized by bringing the FAD-containing washing solution into contact with the immobilized pyruvate oxidase during measurement, is shown in a pro-nk diagram in FIG. In the figure, 1
2 is a storage layer for concentrated FA oral solution, 24 is a micro-liquid pump,
10 is a small inner diameter pipe, but the other configuration is almost the same as the device shown in FIG.

In this measuring device, measurements are normally performed as follows. After the constant flow pump 16 is activated and the buffer solution and concentrated FAD solution begin to flow into the measurement system at a constant flow rate, when an appropriate amount of sample solution is injected from the sample injection port 14, the electrode section 15 Pyruvate is detected.

[Effects of the Invention] According to the present invention, the enzymatic activity of immobilized pyruvate oxidase can be stabilized, so it is possible to provide a long-life and stable pyruvate measuring device.

Furthermore, in measuring pyruvic acid, the reproducibility of measured values is also significantly improved.

[Examples of the Invention] Example 1 Using the apparatus and reagents shown below, immobilized pyruvate oxidase was brought into contact with an FAD solution during non-measurement.

A) Equipment (see Figure 2) 1) Automatic solvent changer: Model 5 manufactured by Gascro Kogyo Co., Ltd.
V-5008A 2) Automatic three-way valve: Manufactured by Gascro Industries Co., Ltd., model M
PV-3A 3) Controller 2 manufactured by Tokyo Shibaura Electric Co., Ltd. (equipped with a microcomputer) B) Reagent Pyruvate oxidase manufactured by Toyo Jozo Co., Ltd. was used. Thiamine pyrophosphoric acid and FAD manufactured by Tokyo Kasei Co., Ltd. were used. Other reagents are commercially available (special grade)
was used as is without purification. In addition, collagen was prepared from cowhide, stored frozen, and thawed before use. Note that ion exchange water was used throughout the entire operation.

C) The preparation buffer for W sap, POP-immobilized collagen membrane, electrode part, and sample solution was 0.05M phosphate buffer (pH 7).
, 5), 0.01mN FAD, 0.04!11
A solution containing 1 mM thiamine pyrophosphate and 1 mM thiamine pyrophosphate was used.

A POP-immobilized collagen membrane was prepared as follows.

After stirring 0.6% collagen suspension (pH 4,0) Log well, POP (21 units/mg) 1
Mix 00mg x 5cm) and spread on 28°
It was air-dried at C for 3 hours.

Next, the film peeled off from the Teflon plate was placed in a 1cm x 1c
A POP-immobilized collagen membrane was prepared by cutting the membrane into pieces of 0.1% glutaraldehyde aqueous solution (p 18.0) and crosslinking it in the gas phase at 28°C for 10 minutes.

The electrode part is a polarographic hydrogen peroxide electrode consisting of a platinum electrode covered with a hydrogen peroxide permeable membrane.
00 TU/C11') and then a cellulose acetate ultrafiltration membrane (thickness: approx. 48 g)
The material coated with the coarse and dense layer of m) was used. This electrode part was attached to a plastic 70-cell, and this was incorporated into a measurement system.

The sample solution used was the above buffer solution to which 0.5 mM sodium pyruvate was added.

D) Operation After turning on the measurement start switch on the display panel of the controller and filling the system with buffer, the syringe pump was turned on and 200 Jj-1 sample solution was injected. The reaction temperature during measurement was 37°0, and the flow rate was 0.6ml/l1l.
It was in. Next, hydrogen peroxide produced by the reaction was detected with the electrode section, and the value was recorded with a recorder. Measurements were performed approximately 30 times a day. After the measurement was completed, the measurement end switch on the display panel of the controller was turned on, the buffer solution in the system was discharged, and 1 mM FAD solution was filled, and the system was stored overnight in this state at room temperature. The next day, after turning on the measurement start switch on the controller and collecting the FAD solution in the system into the storage tank, the buffer solution was allowed to flow into the system again and the same operation as above was performed.

The above operation was repeated for 5 days. The obtained electrode response values are shown in A of FIG. As is clear from the figure, almost no decrease in response value was observed. Furthermore, the deviation of the response values on day - was also within 5%. Although not shown, when the above measurements were carried out for one month, the electrode response value after one month was about 80% of the initial response value.

Example 2 Example 1 except that a 0.1 mM FAD solution was used.
Measurements were carried out in the same manner. The electrode response value after one week was approximately 90% of the initial response value.

Example 3 Glutamate oxaloacetate transaminase (GOT
) converts α-ketoglutarate to oxaloacetate, and oxaloacetate decarboxylase (OAC) decomposes this oxaloacetate to pyruvate. Therefore, OAG was immobilized on aminoalkyl porous glass beads, and this was attached to a glass column (diameter 3+Ila+, length 5Qn+m).
) to prepare an OAC-immobilized column. This column was inserted between the sample injection port and the electrode section of the apparatus shown in FIG. 2, and the enzyme activity of GOT was repeatedly measured.

In this example, 0-01mM FA-
A substrate solution containing D, Q, (145a+M chloride mafgan, 0, 1mM thiamine pyrophosphate, 2.1mM a-ketoglutarine, and 30mM L-alanine) was used and was stored in a storage tank.

In addition, as a sample solution, add 10.50% to the above substrate solution.
, 100, and 500 units/L GOT
, containing 0.8% NaCl and 1% albumin was used.

When GOT activity was measured for one month in the same manner as in Example 1, the electrode response value after one month was about 80% of the initial response value. Furthermore, the deviation of the response values on day - was also within 5%.

Comparative Example I Instead of FAD solution, 0,011 used in Example 1
Measurements were carried out in the same manner as in Example 1, except that a buffer solution containing FAD of Moreover, the electrode response value gradually decreased from the 3rd day after the start of the measurement, and after 5 days, the electrode response value showed only about 30% of the initial response value.

Comparative Example 2 The same results as in Comparative Example 1 were obtained when measurements were carried out in the same manner as in Example 1, except that the 0.05 mM FAD solution was filled into the system during non-measurement periods.

Example 4 Using the apparatus and reagents shown below, an FAD-containing washing solution was brought into contact with immobilized pyruvate oxidase after measurement.

A) Apparatus (see Figure 3) 1) Automatic solvent changer: Model 5 manufactured by Gascro Kogyo Co., Ltd.
V-5008A 2) Constant flow pump: Roller pump manufactured by Full Engineering Co., Ltd. 3) Controller, sample injector 2 homemade B) Reagent The same one as in Example 1 was used.

C) Preparation of buffer solution, POP-immobilized collagen membrane, electrode section, and sample solution The same buffer solution and POP-immobilized collagen membrane as in Example 1 were used. Except that an oxygen permeable membrane was used instead of a hydrogen peroxide permeable membrane for the electrode part.
The same material as in Example 1 was used. In addition, as sample solutions, whole car supplemented with 0.5 mM sodium pyruvinbutyrate and whole blood supplemented with an appropriate amount of GPT were used.

D) Operation The same procedure as in Example 1 was performed except that the enzyme activity was measured by measuring the amount of dissolved oxygen that decreased as a result of the enzymatic reaction. In addition, each time a measurement is completed, the inside of the system is cleaned with a cleaning solution containing 0.5+oM FAD (0.01% Triton solution).
Next, a buffer solution was allowed to flow down into the pipe, and the following measurements were performed. Further, after one day's measurement was completed, the system was filled with a cleaning solution and stored in this state overnight at room temperature.

The above operation was repeated for 5 days. The obtained electrode response values are shown as A in FIG. As is clear from the figure, almost no decrease in response value was observed. Furthermore, the deviation of the response values on day - was also within 5%. In addition, the above measurements were performed at 1
The electrode response value after one month was approximately 90% of the initial response value.

Example 5 Measurements were carried out in the same manner as in Example 4, except that 0.1 mM FAD washing solution was used. The electrode response value after 5 days of measurement was approximately 80% of the initial response value.

Example 6 Alanine Ketoglutarate Aminotransferase (G
PT) is an enzyme that converts L-alanine to pyruvate. Therefore, in this example, instead of the buffer solution, 0.05
0.01mM F in M phosphate buffer (pH 7,5)
A substrate solution containing AD, 0.045 mM manganese chloride, 0.1 mM thiamine pyrophosphate, 0-02)l α-ketoglutaric acid, and 0.3 M L-alanine was used.

This was stored in a storage tank, and GPT activity in whole blood was measured.

In addition, as a sample solution, add 10.50% to the above substrate solution.
, 100.200 and 500un it/L GP
Whole blood containing T was used.

When GPT activity was measured in the same manner as in Example 4, GPT activity was measured in the same manner as in Example 4.
PT activity and electrode response values had a linear relationship within the above range. Therefore, measurements were carried out for I months using the entire surface containing 100 units/L of GPT, and the electrode response value after one month was about 75% of the initial response value. Furthermore, the deviation of the response values on day - was also within 5%.

Comparative Example 3 Measurement was performed in the same manner as in Example 4, except that the FAD concentration in the cleaning solution was 0.0111 IM. The measurement results are shown in the 6th
In the figure, it is indicated by B. As is clear from the figure, the electrode response value gradually decreased from the 3rd day after the start of the measurement, and after 5 days, the electrode response value showed only about 25% of the initial response value.

Comparative Example 4 Measurements were carried out in the same manner as in Example 4, except that the FAD concentration in the washing solution was 0.05 mM. The same results as in Comparative Example 3 were obtained, and the electrode response value after 5 days was the same as the initial value. Approximately 30
%.

Example 7 Immobilized pyruvate oxidase was brought into contact with a flavin adenine dimecretide solution during measurement using the apparatus and reagents shown below.

A) Device (see Figure 4) Micro-liquid pump: Shimadzu Corporation, LC-5AB)
The same reagent as in Example 1 was used.

C) Preparation of buffer solution, POP-immobilized collagen membrane, electrode section, and sample solution The same buffer solution and POP-immobilized collagen membrane as in Example 1 were used. The electrode part was the same as in Example 1 except that an oxygen permeable Teflon membrane was used instead of the hydrogen peroxide permeable membrane. Further, the same sample solution as in Example 1 was used, and 100 g of each solution was injected into the sample injection port 4 using a microsyringe.

D) Operation First, the constant flow pump (flow rate: 0.8 ml/ml) and the micro-liquid pump (flow rate: 107z I/win) were started, and the buffer solution and ImM FAD aqueous solution were injected into the system. After confirming on the recorder that the response of the electrode part was stable, 1007Ll of the above sample solution was injected from the sample injection port. After one day's measurements were completed, each device was stopped and stored overnight in this state at room temperature.

The measurement was carried out at least 30 times in October 2010, and the following two operations were repeated for 7 days. The obtained electrode response values are shown as A in FIG. As is clear from the figure, almost no decrease in response value was observed. Furthermore, the deviation of the response values on day - was also within 5%. The above measurements were carried out for one month, and the electrode response value after one month was about 90% of the initial response value.

0.0 in place of ImH FAD solution. ]mM FAII solution (flow rate + 10 mol/ml) was used, but the measurement was carried out in the same manner as in Example 7. When measurements were repeated for 5 times, the electrode response value was about 80% of the initial value. Furthermore, the deviation of the response values on day - was also within 10%.

Example 9 In the same manner as in Example 3, OAC was immobilized on aminoalkyl porous glass beads, and this was applied to a glass column (diameter 3 m).
m, length 50 mm) to prepare an OAC-immobilized column. This ram was inserted between the sample injection port and the electrode section of the apparatus shown in FIG. 4, and the enzyme activity of GOT was repeatedly measured.

In this example, 0.01mM FAD in 0.05M phosphate buffer (pH 7.5) was used instead of the buffer.
, 0.045mM manganese chloride, 0.1mM thiamine pyrophosphate, 2.1. mM α-ketoglutaric acid and 30m
A substrate solution containing M L-alanine was used and stored in a storage tank.

In addition, as a sample solution, 10.50,
100. and 5QOunits/Lc7) coT,
Furthermore, a solution containing 0.8% NaCl and 1% albumin was used. When GOT activity was measured for one month in the same manner as in Example 7, the electrode response value after one month was about 75% of the initial response value. Also, the deviation of the response value on -day is 8
It was within %.

Comparative Example 5 Measurement was carried out in the same manner as in Example 7, except that the micro-liquid pump was not operated. As is clear from the measurement results in Figure 9, indicated by B in Figure 7, the electrode response value gradually decreased from the third day after the start of the measurement, and after one week it reached a value of about 25% of the initial response value. only showed.

Comparative Example 6 IIIM FAD Solution 0.05mM F
AD solution (flow rate: 5 x 1O-10ffiat/+i
When measurements were carried out in the same manner as in Example 7 except that n) was used, the same results as in Comparative Example 5 were obtained.

[Brief explanation of drawings]

Fig. 1 is a partial sectional view of the electrode section in the device of the present invention, Fig.
The figure is a block diagram of the apparatus of the present invention, which is equipped with a mechanism for bringing an FAD solution into contact with immobilized POP during non-measurement periods, and FIG. A block diagram, FIG. 4 is a block diagram of an apparatus of the present invention equipped with a mechanism for bringing the FAD solution into contact with immobilized POP during measurement, and FIGS.
FIG. 3 is a diagram showing the relationship between the number of days of measurement and the electrode response value when measurements were taken in Examples and Comparative Examples using the apparatus shown in the figure. 1...Flow cell 2...Ultrafiltration membrane 3...
Buffer layer 4...0-ring 5...Oxygen or hydrogen peroxide permeable membrane 6...POP immobilization membrane 7...Platinum cathode 8...
Lead anode 9...Piping 10...Small inner diameter piping 11...Buffer solution or substrate solution storage tank 12...Concentrated FAD solution storage tank 13...Automatic solvent changer 14... Sample injection port 15
...Electrode part 16...Constant flow pump 17.
...Automatic three-way pulp 18a and 18b...Piping 18...Drainage tank 20.
・Recorder 21 ・Controller 22 ・
... Starter 23 ... Stirrer 24 ... Trace amount liquid delivery bomb

Claims (4)

[Claims] (1) A pyruvate measuring device using immobilized pyruvate oxidase, characterized in that it is equipped with a mechanism for bringing a flavin adenine dinucleotide solution of 0.1 to 50a+H into contact with the immobilized pyruvate oxidase. Device. (2) The pyruvate measuring device according to claim 1, comprising a mechanism for bringing the flavin adenine dinucleotide solution into contact with the immobilized pyruvate oxidase during non-measurement. (3) The pyruvate measuring device according to claim 1, comprising a mechanism for bringing the washing solution containing flavin adenine dinucleotide into contact with the immobilized pyruvate oxidase after the measurement is completed. (4) At the time of measurement, 10 w 10 mol/sin
2. The pyruvate measuring device according to claim 1, comprising a mechanism for bringing the flavin adenine dinucleotide solution into contact with the immobilized pyruvate oxidase at a supply rate of .