JP3319008B2 - Measuring device and measuring method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides a multifunctional measuring apparatus and a measuring method with a simple structure using an immobilized enzyme.

[0002]

2. Description of the Related Art Immobilized enzyme technology is used in a wide range of fields such as production of foods by bioreactors, production of fine chemicals, and solving environmental problems. In addition, applications to the field of analytical chemistry are being advanced by utilizing the advanced reactions and substrate specificity, high-speed reaction, and high sensitivity characteristics of enzymes. An analyzer using an immobilized enzyme is generally referred to as a biosensor, and has been actively researched.

[0003] Biosensors can be classified into an electrode system, a field effect transistor system, a light detection system, a heat detection system, and the like according to the detection system. Among these, various types of devices have been particularly developed because the electrode-type device can simplify the mechanism and can add selectivity by electrodes in addition to the specificity of the enzyme. Methods using electrodes are further broadly classified into amperometric sensors using an oxygen electrode or a hydrogen peroxide electrode, and potentiometric sensors using an ion-selective electrode such as a pH electrode or an ammonia electrode. Amperometric sensors are frequently used because the substance concentration and the output current value are in a linear relationship, so that data processing is easy, and a high-precision and high-sensitivity device can be easily formed with a simple device.

[0004] As described above, the measurement method using a biosensor is excellent in terms of accuracy and sensitivity, and its use is expanding due to simplicity of operation and data processing.
However, since most biosensors measure oxygen decreasing or hydrogen peroxide increasing due to the action of oxidase, a simple measurement system limits the measurement target only to substances that serve as substrates for oxidase. I will. For this reason, a method of immobilizing not only an oxidase but also a hydrolase, an isomerase, a dehydrogenase, and the like and using the same together with an immobilized oxidase for measurement has been performed. Use of these enzyme-immobilized products has advantages such as an increase in the number of measurement targets, an increase in measurement sensitivity, and an increase in the reaction rate.

[0005] Among them, a system for measuring lactic acid and pyruvic acid has attracted particular attention because of its high importance in fields such as fermentation control and clinical examination. For example, a reaction in which the oxidase-immobilized product and the dehydrogenase-immobilized product are simultaneously applied to a sample, the substrate oxidized by the oxidase is reduced by the dehydrogenase, and the substrate is oxidized again by the oxidase, is repeated. There is known a method of increasing the sensitivity by utilizing the fact that oxygen of more molecules than hydrogen is converted into hydrogen peroxide, that is, a method utilizing enzyme cycling (Japanese Patent Publication No. 3-65492). This method has advantages in terms of increasing sensitivity.

However, as a drawback of the cycling system combining enzymes, there is a drawback that once the enzymatic reaction starts, both the oxidized form and the reduced form of the substrate for dehydrogenase react. More specifically, when L-lactate dehydrogenase and L-lactate oxidase are immobilized and enzymatic cycling is performed, when NADH (reduced nicotinamide adenine dinucleotide) coexists, pyruvate in the sample is first removed. It is converted to L-lactic acid, and at the same time, L-lactic acid is converted to pyruvate by an oxidase. If enough NADH
This cycle is repeated as long as the presence of and the amount of dissolved oxygen is sufficient, and the hydrogen peroxide generated during the oxidation of lactic acid is accumulated. Therefore, when the decrease in oxygen or the increase in hydrogen peroxide is electrochemically detected, the sensitivity is improved as compared with the case without cycling. However, L
-If lactic acid is present, there is no way to distinguish it from pyruvate. That is, the amount of pyruvic acid cannot be measured accurately.

[0007]

However, the conventional measurement example using an immobilized enzyme has the above-mentioned problem, and no effective apparatus has been proposed for a reaction system using a dehydrogenase or the like.
The present invention relates to a measuring apparatus and a measuring method using an immobilized form of dehydrogenase and an oxidized enzyme, wherein two components of a substrate oxidized form or coenzyme reduced form of dehydrogenase and a substrate reduced form are combined with one component. It is an object of the present invention to provide a multifunctional measuring device and a measuring method capable of measuring simultaneously by one injection.

[0008]

DISCLOSURE OF THE INVENTION The present invention provides a dehydrogenase which catalyzes a reaction for producing a coenzyme oxidant Aox and a substrate reductant Bred from a coenzyme reductant Ared and a substrate oxidant Box, and a substrate reductant Bred. A measuring device using an oxidase that oxidizes oxidase, comprising a mechanism for continuously supplying a buffer, and a supply mechanism for supplying a sample in the buffer, and an oxidase immobilized downstream of the supply mechanism. A mechanism including a first electrode for detecting an electrode active substance that increases or decreases by the body and the oxidase reaction, and a dehydrogenase-immobilized body, an oxidase-immobilized body, and an electrode that increases or decreases by the oxidase reaction downstream of the mechanism A second electrode for detecting the active substance is arranged in this order from the upstream side.
And a measuring device in which is disposed .

[0009] distribution in parallel above the mechanism and the mechanism described above
In this case, when the shunt ratio changes, a measurement error may occur. Therefore, a device connected in series is advantageous .
Also, as a preferred combination, a measurement device is disclosed in which the dehydrogenase is L-lactate dehydrogenase and the oxidase is L-lactate oxidase.

The immobilized dehydrogenase is obtained by introducing an amino group onto the surface of a carrier with an aminosilanating reagent, performing formylation with a polyfunctional aldehyde, and then immobilizing the dehydrogenase by contact. The above-mentioned measuring device which is a dehydrogenase-immobilized product is disclosed.

Further, the present invention relates to a method for preparing coenzyme reductant A in a buffer.
red, substrate reduced form Red and substrate oxidized form Bo
A measurement method using an immobilized enzyme, wherein x is simultaneously measured is disclosed. Similarly, the oxidized substrate B is added to the buffer.
ox, the coenzyme reduced form Ared and the substrate reduced form Br
A measurement method using an immobilized enzyme, wherein ed is simultaneously measured, is disclosed. Also, as a specific example,
The coenzyme reduced form Ared is NADH, and the substrate oxidized form B
ox is pyruvic acid and the substrate reduced form Bred is L-lactic acid.

The pH of the buffer is preferably about 6 to 9,
Examples thereof include a phosphate buffer and a Tris-HCl buffer. In the present invention, as the electrode active substance, hydrogen peroxide, oxygen, or a mediator such as p-benzoquinone,
Examples include ferricyanide, tetrathiafulvalene, and ferrocene. NAD is used as coenzyme reductant Ared
H, NADPH, FADH ₂ , FMNH _{2 and the} like. Ethanol, L-
Natural L-amino acids such as glutamic acid, glucose, L
-Lactic acid, galactose and the like can be exemplified. Examples of the dehydrogenase include alcohol dehydrogenase, L-amino acid dehydrogenase, L-glutamate dehydrogenase, glucose dehydrogenase, L-lactate dehydrogenase, and galactose dehydrogenase.

[0013]

FIG. 7 shows the reaction between dehydrogenase and oxidase. Ared + Box → Aox by dehydrogenase
+ Bred reaction proceeds. (For example, NADH + H ⁺
+ Pyruvic acid → NAD ⁺ + L-lactic acid) The reaction of Bred + O ₂ → Box + H ₂ O ₂ proceeds by the oxidase. (For example, L-lactic acid + O ₂ → pyruvic acid + H ₂ O ₂ )

In the present invention, simultaneous analysis of the oxidized substrate Box and the reduced substrate Bred, or the simultaneous analysis of the reduced substrate Bred and the reduced coenzyme Ared is possible. First,
In order to measure the concentration of oxidized substrate Box (or coenzyme reduced form Ared) in the dehydrogenase reaction, a necessary and sufficient amount of coenzyme reduced form Ared (or oxidized substrate Box) is added to a buffer solution. , To generate a substrate reductant, Bred. The substrate reductant Bred reacts with oxygen in the buffer solution by the oxidase-immobilized product to generate a substrate oxidant Box and hydrogen peroxide. Next, these electrode active substances which increase or decrease by the oxidase reaction are detected. Specifically, the concentration of the oxidized substrate Box (or the reduced coenzyme Ared) can be determined by electrochemically quantifying the amount of the decreasing oxygen or the increasing hydrogen peroxide.

Here, the substrate reduced form Br is initially contained in the sample.
If ed is contained, this amount is also added, and thus appears as a sum of the substrate oxidant Box and the substrate reductant Bred generated by the reaction of the dehydrogenase from the substrate oxidant Box according to the above reaction formula. Or coenzyme reductant Are
The quantity of d) cannot be determined accurately. Therefore, in the present invention, the amount of the substrate oxidant Box (or coenzyme reductant Ared) is determined accurately by correcting the contribution of the substrate reductant Bred contained in the sample from the beginning. Further, if the substrate reductant Bred is also measured at the same time, the above-mentioned two components can be simultaneously measured.

FIG. 1 illustrates a flow-type measuring apparatus according to the present invention. A mechanism having an oxidase-immobilized body (4) and a measuring section (5) having a first electrode set downstream thereof, and a dehydrogenase-immobilized body (6) and an oxidase-immobilized body (7) further downstream. , And a mechanism having a measuring unit (8) on which a second electrode is set is arranged in series. The relationship between the output values of the substrate reductant Bred and the substrate oxidant Box in the first measurement unit and the second measurement unit is determined by injecting a standard solution of the substrate reductant Bred and the substrate oxidant Box whose concentration is known before the sample measurement. Determined by creating a line. Since an output value can be obtained from both the mechanism (first measuring unit) and the mechanism (second measuring unit) from the standard solution of the substrate reduced form Bred, two calibration curves No1 (first measuring unit) and No2 ( The second measurement section) is obtained, but with the standard solution of the oxidized substrate Box, an output value is obtained only in the second measurement section, so that one calibration curve No. 3 is obtained and a total of three calibration curves are obtained.

For example, an oxidized substrate Box and a reduced substrate B
When measuring red, the value obtained by the first mechanism (first measuring unit) is substituted into the calibration curve No. 1 to detect the substrate reduced form Bred contained in the sample. That is, a buffer containing a coenzyme reductant Ared in the buffer is used to detect an oxidase-immobilized body (4) disposed downstream of the sample injection mechanism and an electrode active substance that increases or decreases due to the oxidase reaction. With the first electrode, the substrate reduced form Bred contained in the sample from the beginning can be detected, and the substrate reduced form Bred can be directly quantified.

Next, the dehydrogenase-immobilized body (6) and the oxidase-immobilized body (7) of the mechanism (second measuring section) are used to detect the electrode active substance which increases or decreases due to the oxidase reaction in the final stage. 2
The substrate reduced form Bred generated by the reaction of the deoxidase from the substrate oxidized form Box contained in the sample and the coenzyme reduced form Ared contained in the buffer, and the substrate reduced form initially contained in the sample. The total amount of Bred was detected, and the amount of the substrate reduced form Bred included from the beginning in the sample analyzed earlier than the value was subtracted by the calibration curve No. 2,
By substituting the remaining output value into the calibration curve No. 3, the oxidized substrate Box in the sample can be analyzed. In this manner, the substrate reduced form Bred and the substrate oxidized form Box can be simultaneously analyzed.

Next, the simultaneous analysis of the substrate reduced form Bred and the coenzyme reduced form Ared will be described. Using a buffer solution containing a substrate oxidant Box in the buffer solution, an oxidase-immobilized body (4) arranged downstream of the sample injection mechanism and an electrode active substance that increases or decreases due to the oxidase reaction are detected. First
Substrate reductant Br initially contained in the sample by the electrode
Analyze ed.

Next, the immobilized dehydrogenase (6) and the immobilized oxidase (7), which are sequentially arranged downstream, and a second electrode for detecting an electrode active substance which increases or decreases due to the oxidase reaction in the final stage. By the mechanism of including, the coenzyme reductant Ared contained in the sample and the substrate reductant Bred generated by the reaction of the deoxidase from the substrate oxidant Box in the buffer and the substrate reductant Bred initially contained in the sample Analyze the sum. By correcting the amount of the substrate reduced form Bred contained in the sample analyzed earlier than the measured value, simultaneous analysis of the substrate reduced form Bred and the coenzyme reduced form Ared in the sample is possible.

In the above mechanism, for example, dehydrogenase and oxidase can be mixed and immobilized, or each can be immobilized on a carrier, and both carriers can be mixed and included in the same reactor. In this case, a cycle is formed in which the oxidized substrate Box is converted to the reduced substrate Bred, and the reduced substrate Bred is converted to the oxidized substrate Box.
Therefore, although the sensitivity is improved, the output value largely fluctuates due to the decrease or fluctuation of the activity of the immobilized dehydrogenase or the oxidized enzyme, which results in large daily fluctuation. Therefore, in the above mechanism, the immobilized dehydrogenase and the immobilized oxidase are included in another reactor without mixing, and the immobilized dehydrogenase, the immobilized oxidase, and the second electrode are mixed with each other. It is preferable to arrange in this order in series from the upstream side.

The system to which the measuring device and the measuring method according to the present invention can be applied include (1) a combination of alcohol dehydrogenase and alcohol oxidase, acetaldehyde and ethanol, or ethanol and NADH or NADP.
A system for measuring H (the pH of the buffer is preferably about 7 to about 9), (2) a combination of L-amino acid dehydrogenase and L-amino acid oxidase, and 2-keto acid corresponding to L-amino acid; Or a system for measuring L-amino acid and NADH (the pH of the buffer is preferably about 7 to about 8); (3) a combination of L-glutamic acid dehydrogenase and L-glutamic acid oxidase; A system for measuring glutamic acid, or L-glutamic acid and NADH or NADPH (reduced nicotinamide adenine dinucleotide phosphate) (the pH of the buffer is preferably about 6 to about 9), and (4) glucose dehydrogenase. A system for measuring glucose and glucono-δ-lactone or glucose and NADPH using glucose oxidase (buffer p
H is preferably from about 7 to about 9. ), (5) A system for measuring L-lactic acid and pyruvate or L-lactic acid and NADH by combining L-lactic acid dehydrogenase and L-lactic acid oxidase (the pH of the buffer is preferably about 7 to about 8) ), (6) A system for measuring galactose and galactono-δ-lactone or galactose and NADPH by combining galactose dehydrogenase and galactose oxidase (the pH of the buffer solution is about 8 to about 1).
0 is preferred. ), And the like.

The method for immobilizing the dehydrogenase or oxidase is not particularly limited, and known methods can be used. That is, an adsorption method, a chemical bonding method, an inclusive method, or the like can be used. Above all, a chemical bonding method capable of producing a strong immobilized body is desirable. As an application form of the immobilization, for example, in order to improve the sensitivity at the time of measurement, it is desirable to use an immobilized enzyme in which the enzyme is immobilized on a fine-powder-like carrier and the immobilized body is incorporated in a column-shaped reactor.

The carrier used for immobilization is diatomaceous earth,
Silica gel, glass beads, alumina, ceramic, carbon, activated carbon, molecular sieve, silicon rubber,
Examples include cellulose, agarose, and amino acid-based polymers. As the chemical bonding method, a method in which an amino group is introduced to the surface of a carrier with an aminosilanating reagent, formylation is performed using a polyfunctional aldehyde such as glutaraldehyde, and then immobilized by contacting with an enzyme is preferable. An example is given. The carrier is preferably a silicon-containing material having a hydroxyl group, and examples thereof include diatomaceous earth, calcined diatomaceous earth, porous glass, silica gel, and acid clay. Aminosilation is 3-aminopropyltriethoxysilane,
This is performed by bringing a reagent such as aminoethyltrimethoxysilane into contact with a carrier in a solvent such as anhydrous benzene or toluene. Glutaraldehyde, glyoxal, succinyl dialdehyde and the like can be used as the polyfunctional aldehyde.

Hereinafter, the case where L-lactate dehydrogenase is used as the dehydrogenase and L-lactate oxidase is used as the oxidase will be described in more detail as examples (see FIG. 1). First, L-lactic acid dehydrogenase or L-lactic acid oxidase is respectively immobilized on a fine powder by a chemical bonding method to prepare immobilized bodies. The immobilized body is packed in a plastic or stainless steel column having an inner diameter of about 1 to 10 mm and a length of about 2 to 50 mm, for example, to form respective reactors. Each reactor can be of different size to change the concentration range to be measured.

Next, as a buffer near the optimum pH of these enzymes, for example, a 1 to 500 mM phosphate buffer having a pH of 7.0 can be used. As a pump used for liquid sending, a pump capable of sending liquid at a flow rate of, for example, about 1.0 ml / min is prepared. Various pumps such as a plunger type and a peristalic type can be used as this pump.
The buffer may contain other salts, stabilizers, surfactants and the like. The temperature at the time of measurement is preferably about 25 to 35 ° C.

As a sample supply mechanism, an injector for a high-speed liquid chromatograph utilizing a six-way valve can be used. An oxidase-immobilized reactor is arranged downstream of the injector, and first, L-lactic acid in the sample is oxidized.
At this time, hydrogen peroxide is an increasing electrode active substance, and oxygen is a decreasing electrode active substance. Therefore, if a first hydrogen peroxide electrode or an oxygen electrode is placed downstream of the oxidase-immobilized reactor, an output value corresponding to the amount of L-lactic acid can be obtained.

The dehydrogenase-immobilized reactor and the oxidase-immobilized reactor are arranged in this order further downstream of the oxidase-immobilized reactor and the electrode. A second hydrogen peroxide electrode or an oxygen electrode is placed downstream thereof. When measuring L-lactic acid and pyruvic acid, add NADH to the buffer. The amount of NADH added depends on how much the sample injected from the injector is diluted by mixing with the buffer before reaching the dehydrogenase immobilized reactor. That is, in the reduction reaction from pyruvic acid to L-lactic acid, NADH and pyruvic acid proceed one to one. Generally, a concentration of about 1/2 to 1/100 of the pyruvic acid concentration in the sample is sufficient. NADH was added to the sample, and NAD
It is possible to measure by injecting into a measuring device in which a buffer solution containing no H is flowed, but NADH is also diluted at the same time as pyruvate as the measurement target, so that the required amount of NADH is a practically usable solution volume. It is not a cost-preferred method because it increases.

For the injected sample, L-lactic acid measurement is first performed in the first reactor and the first electrode. Then L
-Both pyruvate generated from lactic acid and pyruvate present in the sample from the beginning are reduced inside the dehydrogenase-immobilized reactor. This reaction proceeds at a high speed because the reaction equilibrium of the dehydrogenase is remarkably inclined in the direction of producing L-lactic acid.

Naturally, it is possible to carry out the reaction by a general solution reaction, but it is difficult to control the reaction time, and it is not suitable for analyzing a large amount of sample because expensive dehydrogenase is disposable. is there. In a measuring device in which a column-shaped reactor is arranged in a continuous flow, the reaction time depends only on the flow rate of the buffer, in other words, the contact time between the sample and the enzyme immobilized body. Therefore, there is an advantage that the reaction time can be easily defined by precisely controlling the flow rate.

L-lactic acid produced from pyruvate by the action of dehydrogenase is subsequently oxidized by an oxidase and can be quantified from the output value of a hydrogen peroxide electrode or an oxygen electrode in proportion to its concentration. By injecting the L-lactic acid standard solution before the sample measurement, the relationship between the two electrode outputs before and after and the L-lactic acid concentration can be grasped. Next, by injecting a pyruvic acid standard solution, a relationship between the second electrode output value and the pyruvic acid concentration is obtained. As a result, two calibration curves are obtained for L-lactic acid and one calibration curve for pyruvic acid.

In the mixture analysis, the concentration of L-lactic acid is first determined, and the effect of pyruvic acid generated from L-lactic acid on the second electrode is predicted. Then, by subtracting the contribution of pyruvic acid generated from L-lactic acid, the amount of pyruvic acid contained in the sample from the beginning can be calculated. These processes can be easily performed by A / D converting the two electrode outputs and transferring them to a microcomputer. In addition, L-lactic acid and NA
When DH is analyzed simultaneously, pyruvate is included in the buffer instead of NADH. These series of operations are also applied to other combinations of enzymes.

A hydrogen peroxide electrode or an oxygen electrode can be used as an electrode for detecting an increase or decrease in the concentration of the electrode active substance.
As a substrate of the hydrogen peroxide electrode, carbon on the anode side,
Platinum, nickel, palladium or the like can be used.
It is desirable to use platinum because the overvoltage is low, high sensitivity is obtained, and the potential window is wide. Further, a permselective membrane such as a polysiloxane, an acrylic resin, a protein membrane, or an acetylcellulose membrane may be provided on the electrode surface. On the cathode side, carbon, gold, platinum, lead, or the like can be used. The anode and the cathode may be contained in a single tube and may have an electrolyte such as potassium chloride as an internal solution. It may be inserted into the flow of the buffer, and an electrolyte such as potassium chloride may coexist in the buffer. Further, a three-electrode type having a working electrode, a reference electrode, and a counter electrode can be used. A known type of oxygen electrode can also be used. Among these electrodes, it is desirable to use a three-electrode type hydrogen peroxide electrode in view of stability, response speed, and accuracy. The electrodes are usually set in a flow cell and constitute a measuring unit.

Further, in the apparatus shown in FIG. 8, the second measuring unit and the first measurement unit and that are arranged in series in FIG. 1, a modification to the first measurement unit and the second measurement unit in parallel is there. That is, a first measuring section including the oxidase fixed body (4) and the first electrode, and a second measuring section including the dehydrogenase fixed body (6), the oxidase fixed body (7), and the second electrode. Are arranged in parallel. However, the embodiment of FIG. 1 has the advantage that the shunt ratio does not vary and the measurement can be performed with higher accuracy than the embodiment of FIG.

Further, the present invention can be implemented with a flow-type measuring apparatus shown in FIG. FIG. 9 omits the first measurement unit including the oxidase-immobilized body (4) and the first electrode in FIG. Then, for example, an oxidized substrate Bo
When measuring x and the substrate reductant Bred, without adding the coenzyme reductant Ared in the buffer, the dehydrogenase reaction was prevented from occurring, and the same as the measurement in the first measurement unit was performed. The substrate reductant Bred in the sample is measured. Then, if the coenzyme reduced form Ared is added to the buffer, the same measurement as in the second measuring section can be performed. This method has the advantage of not using a complicated apparatus, but the sample is injected twice, and the coenzyme reduced form Ar
It is necessary to measure when ed is added and when ed is not added. Of course, the addition of the coenzyme reductant is the same even if it is performed in the sample. The above is the measurement of the oxidized substrate Box and the reduced substrate Bred.
The same applies to the measurement of red and the substrate reduced form Bred.

[0036]

EXAMPLES The contents of the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 An apparatus for measuring L-lactic acid and pyruvic acid was constructed, and the characteristics of each enzyme-immobilized reactor were examined. (1) Method for producing L-lactic acid oxidase-immobilized reactor 150 mg of refractory brick (30 to 60 mesh), which is a diatomaceous earth-based silicate carrier, is thoroughly dried, and then dried in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane. After immersing for 1 hour, it is thoroughly washed with toluene and dried at 120 ° C. for 2 hours. After the aminosilane-treated carrier is immersed in a 5% glutaraldehyde aqueous solution for 1 hour, the carrier is thoroughly washed with distilled water and finally pH
Replace with 7.0, 100 mM sodium phosphate buffer and remove this buffer as much as possible. 50 L-lactate oxidase (manufactured by Sigma) was added to this formylated refractory brick at pH 7.0 and 100 mM sodium phosphate buffer.
200 μl of a solution dissolved at a concentration of unit / ml is brought into contact with the solution, and the solution is allowed to stand at 10 ° C. or lower for one day to be fixed. This enzyme-immobilized support is packed in an acrylic resin column having an inner diameter of 3.5 mm and a length of 30 mm to form an L-lactic acid oxidase-immobilized reactor.

(2) Method for Producing L-Lactate Dehydrogenase Immobilized Reactor In the same manner as in the method for immobilizing L-lactic acid oxidase, pigs were immersed in refractory bricks formalized at pH 7.0 and 100 mM sodium phosphate buffer. 200 μl of a solution obtained by dissolving L-lactic acid dehydrogenase derived from Boehringer Mannheim at a concentration of 4500 units / ml,
Leave at 10 ° C or lower for 1 day to fix. This enzyme-immobilized carrier is packed into a column having an inner diameter of 3.5 mm and a length of 30 mm, and
-Lactate dehydrogenase immobilized reactor.

(3) Production method of hydrogen peroxide electrode 2 m in diameter
The side of the m. platinum wire is covered with heat shrink Teflon, and one end of the wire is smoothed with a file and # 1500 emery paper. Using the platinum wire as a working electrode, a 1 cm square platinum plate as a counter electrode, and a saturated calomel electrode as a reference electrode, an electrolytic treatment is performed for 10 minutes at +1.4 V in 0.1 M sulfuric acid. Thereafter, the platinum wire is thoroughly washed with water, dried at 40 ° C. for 10 minutes,
The substrate is immersed in an anhydrous toluene solution of% γ-aminopropyltriethoxysilane for 1 hour and then washed. 20 mg of bovine serum albumin (Fraction V, manufactured by Sigma) is dissolved in 1 ml of distilled water, and glutaraldehyde is added to the solution in 0.1 ml.
Add to 2%. 5 μl of this mixed solution was quickly placed on the previously prepared platinum wire, and dried and cured at 40 ° C. for 15 minutes to form a hydrogen peroxide selectively permeable membrane. This is used as the working electrode of the hydrogen peroxide electrode.

A silver-silver chloride reference electrode is used as a reference electrode, and is mounted on a flow cell. On the outlet side of the flow cell, a conductive pipe was used as a counter electrode. To detect hydrogen peroxide, a voltage of +0.6 V was applied to the working electrode with respect to a silver-silver chloride reference electrode.

(4) Simultaneous measurement device for L-lactic acid and pyruvic acid This will be described with reference to FIG. First, 0.5 from buffer solution tank (1)
A sample containing pyruvate and L-lactic acid injected from a sampler (3) and a buffer containing mM NADH is continuously fed by a feed pump (2) is sent to a thermostat (9). Pyruvic acid and hydrogen peroxide are generated from L-lactic acid in the sample and oxygen in the buffer solution by the reactor containing the first L-lactic acid oxidase immobilized body (4) in the thermostat. This increase or decrease is detected by the measuring section (5) having the first hydrogen peroxide electrode.

Further, pyruvate which was previously present in the sample and pyruvate generated by L-lactate oxidase reacts with NADH in the buffer solution and reacts with the reactor containing the immobilized L-lactate dehydrogenase (6). -Converted to lactic acid and NAD. Then, hydrogen peroxide is similarly generated by the reactor containing the second L-lactic acid oxidase-immobilized body (7). This increase or decrease is sensed by the measuring section (8) having the second hydrogen peroxide electrode. The change in the current value at these two measuring units is converted into a voltage by a detector (10), for example, a potentiostat, and the voltage is A / D converted and sent to a single board computer (11). Finally, the data was sent to a personal computer (13) via an RS-232C cable (12) to perform data processing. Sample injection and pump control were performed by a personal computer. (14) in FIG. 1 is a pump control signal, (1)
5) is a sampler control signal, and (16) is a waste liquid.

(5) Measurement method Measurement was carried out using the above-mentioned L-lactic acid / pyruvic acid measuring apparatus. Sample injection volume is 5 μl, water as blank, L-lactic acid standard solution (dissolve lithium L-lactate in distilled water) 1.0 mM, 2.5 mM, pyruvate standard solution (dissolve lithium pyruvate in distilled water) 0m
M, 2.5 mM and 5.0 mM standard solutions were measured. A calibration curve was created from these measured values.

The composition of the buffer was 100 mM phosphoric acid, 50 mM potassium chloride, 1 mM sodium azide and NA
It contained 500 μM of DH and was measured at pH 7.0. The flow rate of the buffer was 1.0 ml / min, and the temperature of the thermostat was 30 ° C. FIG. 2 shows the output values obtained from the two measuring units at pH 7.0 as a calibration curve. Calibration curve 1 is the output from the first measurement unit for lactic acid, calibration curve 2 is the output from the second measurement unit for lactic acid, and calibration curve 3 is the output for pyruvic acid, and good linearity is obtained. You can see that there is.

Next, a calibration curve was similarly prepared by changing the pH of the buffer solution, and the change in the output value at pH 7.0 was examined from the gradient. FIG. 3 shows the result. 1 in the drawing shows the pH dependence of the response value to lactic acid. When the relative ratio was changed, the behavior of the output value with respect to lactic acid in the first and second measurement units was the same, and therefore, it was represented by one line. The output value for pyruvic acid is 2. pH7.
A maximum value was observed around 25.

FIG. 4 shows the change in the gradient as a relative value by further changing the temperature of the thermostatic bath. The change in the response value to lactic acid was indicated by a single line as in the case of FIG. The change in the response value to pyruvic acid in the measurement unit having the second electrode is 2. An optimum temperature is observed around 39 ° C. The time required for each measurement is 90 seconds, and high-speed measurement is possible.

From the above experiment, the pH was set to 7.0 and the temperature was set to 30.
° C. The measurement was carried out at 30 ° C. for 8 hours a day at this measuring apparatus, and at other times, the long-term stability when the enzyme-immobilized product was left at room temperature (25 ° C.) was confirmed. FIG. 5 shows the daily fluctuation of the output value when the output value immediately after fixing is 100. 5 relates to lactic acid and 2 relates to pyruvic acid. It was found to be stable for about two months.

Example 2 A mixture of a standard solution of L-lactic acid and pyruvic acid and a filtrate of sake moromi were analyzed. (1) Production Method of Enzyme-Immobilized Reactor The same method as in Example 1 was used for both dehydrogenase and oxidase. (2) Production method of hydrogen peroxide electrode A hydrogen peroxide electrode was produced in the same manner as in Example 1. (3) Measuring device The same measuring device as in Example 1 was used. (4) Measurement method The analysis was carried out in the same manner as in Example 1 except that the pH of the buffer solution was 7.0 and the temperature was 30 ° C.

The concentrations of the standard mixture are shown in Table 1. (5) Measurement Results Table 1 shows the measurement results of the standard solution mixture.

[0050]

[Table 1]

The calibration curve of L-lactic acid of the first electrode in the measuring section (5) is as follows, where X is the concentration of L-lactic acid (mM) and Y is the change in current value (nA): Y = 32.6X + 0.13
(Correlation coefficient 1.000). The calibration curve of L-lactic acid of the second electrode in the measuring section (8) is as follows, where X is the concentration of L-lactic acid (mM) and Y is the amount of change in current value (nA).
46.2X-0.31 (correlation coefficient 1.000).

The calibration curve of pyruvate of the second electrode is as follows, where X is the pyruvate concentration (mM) and Y is the change in current value (nA): Y = 23.4X + 0.03 (correlation coefficient 0.9
99), and the L-lactic acid and pyruvic acid concentrations determined from these calibration curves were correctly obtained as described above.

The sake mash filtrate, the concentration of lactic acid and pyruvic acid determined in advance by the solution enzyme method, was analyzed without dilution. Table 2 shows the results.

[0054]

[Table 2]

As shown in Table 2, good agreement with the solution enzyme method is observed. Further, it is shown that the measurement apparatus and the measurement method of the present invention are useful when operations such as dilution are unnecessary and automation of analysis is considered.

Example 3 Standard solutions of L-lactic acid and NADH were analyzed. (1) Production Method of Enzyme-Immobilized Reactor The same method as in Example 1 was used for both dehydrogenase and oxidase. (2) Production method of hydrogen peroxide electrode A hydrogen peroxide electrode was produced in the same manner as in Example 1.
(3) Measuring device The same measuring device as in Example 1 was used.
(4) Measurement method 0.5 mM instead of NADH in buffer
Was analyzed according to Example 2 except that pyruvic acid was contained.
(5) Measurement results The same results as in Example 1 were obtained for L-lactic acid. FIG. 6 shows a calibration curve of NADH. Thus, it was shown that NADH can be measured. Table 3 at the same time
Shows the results of analyzing the mixture.

[0057]

[Table 3]

[0058]

Industrial Applicability According to the present invention, it has become possible to measure a deoxidase substrate oxidant and a substrate reductant simultaneously or a coenzyme reductant and a substrate reductant simultaneously and simply and at high speed.

[Brief description of the drawings]

FIG. 1 shows an example of a measuring device using an immobilized enzyme according to the present invention used in Example 1, which is a simultaneous measuring device for L-lactic acid and pyruvic acid.

FIG. 2 shows a calibration curve obtained in Example 1.

FIG. 3 shows a calibration curve when pH was changed in Example 1.

FIG. 4 shows a calibration curve when the temperature is changed in Example 1.

FIG. 5 shows the daily fluctuation of the measurement result of Example 1.

FIG. 6 shows a calibration curve of NADH in Example 3.

FIG. 7 shows a reaction between an oxidase and a dehydrogenase.

FIG. 8 shows one embodiment of the measuring device of the present invention.

FIG. 9 shows another embodiment of the measuring device of the present invention.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 buffer tank 2 liquid feed pump 3 sampler 4 oxidase immobilized body 5 first measurement unit 6 dehydrogenase immobilized body 7 oxidase enzyme immobilized body 8 second measurement unit 9 constant temperature bath 10 detector 11 single board Computer 12 RS232C cable 13 Personal computer 14 Pump control signal 15 Sampler control signal 16 Waste liquid

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ identification code FI G01N 27/46 D 336H (58) Investigation field (Int.Cl. ⁷ , DB name) G01N 27/416 G01N 27/27 JICST file (JOIS)

Claims (6)

(57) [Claims] 1. A coenzyme reduced form Ared and an oxidized substrate Box
Produces coenzyme oxidized form Aox and substrate reduced form Bred
Dehydrogenase that catalyzes the reaction of
A measuring device that uses an oxidizing enzyme that oxidizes
And a mechanism for supplying a sample into the buffer.
An electrode which has a supply mechanism, and which is increased or decreased by an oxidase reaction downstream of the supply mechanism.
A mechanism including a first electrode for detecting an active substance; and
Downstream of the mechanismImmobilized dehydrogenase, immobilized oxidase
Body and electrode active substances that increase or decrease due to oxidase reaction.
Know the second electrodeMechanisms arranged in this order from the upstream side
And placedmeasuring device. 2. The dehydrogenase is L-lactate dehydrogenase,
The measuring device according to claim 1, wherein the oxidase is L-lactate oxidase. 3. The immobilized dehydrogenase is prepared by introducing an amino group into the surface of a carrier with an aminosilanating reagent, performing formylation with a polyfunctional aldehyde, and then contacting the dehydrogenase to immobilize the immobilized product. The measuring device according to claim 1, wherein the measuring device is a fixed dehydrogenase. 4. An immobilized enzyme, characterized in that a coenzyme reductant, Ared, is contained in a buffer solution and a substrate reductant, Bred, and a substrate oxidant, Box, are simultaneously measured using the measuring device according to claim 1. Measuring method. 5. An immobilized enzyme, characterized in that an oxidized substrate Box is contained in a buffer and a coenzyme reduced form Ared and a substrate reduced form Bred are simultaneously measured using the measuring device according to claim 1. Measuring method. 6. The coenzyme reductant Ared is NADH,
The oxidized substrate Box is pyruvate, and the reduced substrate Br is
The measurement method using the immobilized enzyme according to claim 4 or 5, wherein ed is L-lactic acid.