JPH0618478A - Measurement device and measurement method - Google Patents

Abstract

PURPOSE:To provide a method for accurately and concurrently measuring a substrate oxidant and a substrate reductant, using a measurement device where immobilized oxidase and dehydrogenase are used. CONSTITUTION:This measurement device uses dehydrogenase for catalyzing reaction to generate a coenzyme oxidant and a substrate reductant from a coenzyme reductant and a substrate oxidant, and oxidase for oxidizing the substrate reductant. In addition, the device has a mechanism for feeding a buffer solution continuously and a feed mechanism for supplying a sample to the solution. Furthermore, the device has a mechanism including immobilized oxidase 4 and the first electrode 5 for detecting an electrode active material to increase or decrease, depending upon oxidase reaction, and another mechanism including immobilized dehydrogenase 6, immobilized oxidase 7, and the second electrode 8 for detecting an electrode active material to increase or decrease, depending upon oxidase reaction, respectively at the downstream side of the feed mechanism.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides a measuring device and a measuring method which use an immobilized enzyme and have a simple structure and which are multifunctional.

[0002]

2. Description of the Related Art Immobilized enzyme technology is used in a wide range of fields such as food production by bioreactors, production of fine chemicals, and solution of environmental problems. Further, application to the area of analytical chemistry is also progressing by making use of the high degree of reaction, substrate specificity, high-speed reaction, and high sensitivity of enzymes. Analyzers using immobilized enzymes are generally called biosensors, and active research is being conducted.

The biosensor 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 them, various types of devices have been developed because the electrode type device can simplify the mechanism and can add the selectivity by the electrode in addition to the specificity of the enzyme. The method of using electrodes is roughly classified into an amperometric sensor using an oxygen electrode or a hydrogen peroxide electrode and a potentiometric sensor using an ion selective electrode such as a pH electrode or an ammonia electrode. Amperometric sensors are widely used because the substance concentration and the output current value are in a linear relationship to facilitate data processing and it is easy to produce a highly accurate and highly sensitive device with a simple device.

As described above, the measuring method using the biosensor is excellent in terms of accuracy and sensitivity, and its range of use is expanding due to the ease of operation and data processing.
However, most biosensors are methods of measuring oxygen that decreases or hydrogen peroxide that increases due to the action of oxidase, so in simple measurement systems, the measurement target is limited to only substances that are substrates for oxidase. I will end up. Therefore, not only oxidase but also hydrolase, isomerase, dehydrogenase, etc. are immobilized and used together with the immobilized oxidase for measurement. By using these enzyme-immobilized products, there are advantages that the number of measurement targets increases, the measurement sensitivity increases, the reaction rate increases, and the like.

Among them, the lactic acid and pyruvic acid measuring system has been particularly attracting attention because of its high importance in the fields of fermentation control, clinical examination and the like. For example, the oxidase-immobilized body and the dehydrogenase-immobilized body are simultaneously acted on the sample, the substrate oxidized by the oxidase is reduced by the dehydrogenase, and the reaction is again oxidized by the oxidase. There is known a method of increasing sensitivity by utilizing the fact that oxygen of more molecules than molecules 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 increased sensitivity.

However, a drawback of the cycling system in which enzymes are combined is that once the enzymatic reaction starts, both the oxidant and the reductant react with the dehydrogenase substrate. More specifically, when L-lactate dehydrogenase and L-lactate oxidase are immobilized and enzyme cycling is performed, when NADH (reduced nicotinamide adenine dinucleotide) coexists, pyruvic acid in the sample first becomes It is converted into L-lactic acid, and at the same time, L-lactic acid is converted into pyruvic acid by an oxidase. If enough NADH
If hydrogen is present and the amount of dissolved oxygen is sufficient, this cycle is repeated, and hydrogen peroxide generated during the oxidation of lactic acid is accumulated. Thus, electrochemically detecting a decrease in oxygen or an increase in hydrogen peroxide results in improved sensitivity compared to no cycling. However, L from the beginning
-If lactic acid is included, there is no way to distinguish it from pyruvic acid. That is, the amount of pyruvic acid cannot be accurately measured.

[0007]

The conventional measurement example using an immobilized enzyme has the above-mentioned problems, and an effective apparatus has not been presented for a reaction system utilizing a dehydrogenase or the like.
TECHNICAL FIELD The present invention relates to a measuring device and a measuring method using an immobilized form of dehydrogenase and an immobilized form of oxidase, which comprises a substrate oxidant or coenzyme reductant of dehydrogenase and two components of a substrate reductant. It is an object of the present invention to provide a multi-functional measuring device and a measuring method that can perform simultaneous measurement with one injection.

[0008]

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. It is a measuring device using an oxidase that oxidizes, and has a mechanism for continuously supplying a buffer solution and a supply mechanism for supplying a sample into the buffer solution, and the oxidase-immobilized downstream of the supply mechanism. A mechanism that includes a first electrode that detects an electrode active substance that increases or decreases due to an oxidase reaction with the body, and a mechanism that detects a dehydrogenase-immobilized product, an oxidase-immobilized product, and an electrode active substance that increases or decreases due to an oxidase reaction A measuring device having a mechanism including two electrodes.

According to the present invention, the above-mentioned mechanism is arranged in series downstream of the above-mentioned mechanism, and the dehydrogenase-immobilized body and the oxidase-immobilized body in the above-mentioned mechanism are not mixed with each other and separate reactors are used. It is immobilized inside, the reactor containing the dehydrogenase-immobilized body, and the reactor containing the oxidase-immobilized body were arranged in series from the upstream side in this order. Disclose.

It is also possible to arrange the above mechanism and the above mechanism in parallel. However, if the two are connected in parallel, a measurement error may occur when the diversion ratio changes, so a device connected in series is more preferable. Further, as a preferable combination, a measuring device is disclosed in which the dehydrogenase is L-lactate dehydrogenase and the oxidase is L-lactate oxidase.

A dehydrogenase-immobilized product is obtained by introducing an amino group on the surface of a carrier with an aminosilanizing reagent, further performing formylation using a polyfunctional aldehyde, and then contacting with a dehydrogenase for immobilization. Disclosed is the above-mentioned measuring device which is a dehydrogenase-immobilized body.

Furthermore, the present invention provides that the buffer solution contains coenzyme reductant A.
Red is included, and the substrate reduced form Bred and the substrate oxidant Bo are included.
Disclosed is a measuring method using an immobilized enzyme, which is characterized by simultaneously measuring x. Similarly, the substrate oxidant B was added to the buffer solution.
ox, coenzyme reductant Ared and substrate reductant Br
Disclosed is a measuring method using an immobilized enzyme, which is characterized by simultaneously measuring ed. Also, as a concrete example,
The coenzyme reductant Ared is NADH, and the substrate oxidant B is
ox is pyruvic acid, and the substrate reductant Bred is L-lactic acid.

In the present invention, the coenzyme reductant Ared and the substrate oxidant Box are compared with the coenzyme oxidant Aox and the substrate reductant Bre.
This is a measurement method using a measurement device that uses a dehydrogenase that catalyzes the reaction that produces d and an oxidase that oxidizes the substrate reductant Bred. Of the substrate reduced reductant Bred in the sample by converting the substrate reduced reductant Bred into the substrate oxidant box and detecting the electrode active substance that increases or decreases by this reaction, (b) the sample is a dehydrogenase-immobilized substance And converting the substrate oxidant Box in the sample into the substrate reductant Bred, and detecting the total amount of the produced substrate reductant Bred and the substrate reductant Bred originally present in the sample by the same operation as (a). And (b) correcting the value obtained by (b) with the value of the originally existing reduced substrate Bred obtained by (a) to measure the oxidized substrate Box. And base It discloses a method of measuring oxidant Box.

In the present invention, the coenzyme reductant Ared and the substrate oxidant Box are compared with the coenzyme oxidant Aox and the substrate reductant Bre.
Substrate oxidant Box that uses a dehydrogenase that catalyzes a reaction that produces d and an oxidase that oxidizes a substrate reductant Bred
(Or coenzyme reductant Ared) and substrate reductant Bre
A method for measuring d, comprising: (i) introducing a sample together with a buffer solution into an oxidase-immobilized body, and reducing the substrate reductant Bre in the sample.
a step of converting d into a substrate oxidant Box, and detecting the substrate reductant Bred in the sample by detecting an electrode active substance that increases or decreases by this reaction, (ii) coenzyme reductant Ar
The sample is introduced into a dehydrogenase-immobilized body together with a buffer solution containing ed (or substrate oxidant Box), and the substrate oxidant Box in the sample is reduced (or the coenzyme reductant Ared is oxidized) and a substrate reductant An electrode active substance that causes Bred to be generated and is further led to an oxidase-immobilized body, and that the generated substrate reductant Bred and the substrate reductant Bred originally present in the sample are converted into a substrate oxidant Box, and the reaction amount is increased or decreased by this reaction. And (iii) the value obtained in step (ii) is used as the substrate reductant Bre that was originally present in step (i).
a step of quantifying the substrate oxidant Box (or the coenzyme reductant Ared) by correcting the value of d, and a method for measuring the substrate oxidant Box (or the coenzyme reductant Ared) and the substrate reductant Bred in a sample. Is disclosed. In the present invention, either step (i) or step (ii) may be performed first.

The present invention includes (1) a first immobilized oxidase and a first electrode for detecting an electrode active substance that increases or decreases due to an oxidase reaction.
Measurement having a first measurement unit including an electrode, and (2) a dehydrogenase-immobilized product, an oxidase-immobilized product, and a second measurement unit including a second electrode that detects an electrode active substance that increases or decreases due to an oxidase reaction Disclosed is the above method, wherein the step (i) is performed by the first measuring unit and the step (ii) is performed by the second measuring unit using an apparatus.

Further, the present invention employs a measuring device having an immobilized dehydrogenase, an immobilized oxidase and an electrode for detecting an electrode active substance which increases or decreases due to an oxidase reaction, wherein the step (i) is The sample is introduced into the above apparatus together with a buffer solution containing no coenzyme reductant Ared (or substrate oxidant Box), and the substrate reductant Bre in the sample is detected by detecting the electrode active substance that increases or decreases due to the action of the oxidase-immobilized body.
In the step (ii), the sample is introduced into the above apparatus together with a buffer solution containing the coenzyme reductant Ared (or the substrate oxidant Box) to immobilize the dehydrogenase-immobilized product and the oxidase-immobilized product. Disclosed is the above method, which detects the substrate reductant Bred by detecting an increasing or decreasing amount of the electrode active substance due to the action of the compound.

The pH of the buffer solution is preferably about 6-9,
Examples thereof include a phosphate buffer solution and a Tris-hydrochloric acid buffer solution. Book
In the invention, as the electrode active substance, hydrogen peroxide, acid
Element or a mediator such as p-benzoquinone,
Ferricyanide, tetrathiafulvalene, ferrocene, etc.
Can be illustrated. NAD as the coenzyme reductant Ared
H, NADPH, FADH₂, FMNH ₂Etc. can be exemplified
It Further, as the substrate reductant Bred, ethanol, L-
Natural L-amino acids such as glutamic acid, glucose, L
-Lactic acid, galactose, etc. can be illustrated. Further dehydrogenase
As, alcohol dehydrogenase, L-amino acid dehydrogenation
Enzyme, L-glutamate dehydrogenase, glucose dehydrogenation
Enzyme, L-lactate dehydrogenase, galactose dehydrogenase, etc.
Can be illustrated.

[0018]

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

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

Here, the substrate reductant Br was added to the sample from the beginning.
If ed is included, this amount is also added, and therefore it appears as the sum of the substrate oxidant Box and the substrate reductant Bred produced by the reaction of the dehydrogenase according to the above reaction formula, and the substrate oxidant Box ( Or coenzyme reductant Are
The amount of d) cannot be obtained accurately. Therefore, in the present invention, the contribution of the substrate reductant Bred contained in the sample from the beginning is corrected to obtain an accurate amount of the substrate oxidant Box (or coenzyme reductant Ared). Further, if the substrate reductant Bred is also measured at the same time, the above-mentioned objective simultaneous measurement of the two components becomes possible.

FIG. 1 exemplifies a flow type measuring apparatus of the present invention. Mechanism having an oxidase-immobilized body (4) and a measurement part (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 the 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 with known concentrations before measuring the sample. Calculated by creating a line. Since the 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 reductant Bred, 2
The calibration curves No1 (first measurement part) and No2 (second measurement part) of the book are obtained, but the standard solution of the substrate oxidant Box is second
Since the output value can be obtained only from the measurement part, one calibration curve No3
Is obtained, and a total of 3 calibration curves are obtained.

For example, the oxidized substrate Box and the reduced substrate B
When measuring red, the value obtained by the first mechanism (first measurement unit) is substituted into the calibration curve No1, and the substrate reductant Bred contained in the sample is detected. That is, a buffer solution containing the coenzyme reductant Ared in the buffer solution is used to detect the oxidase-immobilized body (4) arranged downstream of the sample injection mechanism and the electrode active substance increased or decreased by the oxidase reaction. Substrate reductant Bred originally contained in the sample by the first electrode
Can be detected to directly quantify the substrate reductant Bred.

Next, the dehydrogenase-immobilized body (6) and the oxidase-immobilized body (7) of the mechanism (second measuring section) and the electrode active substance which increases or decreases depending on the final stage oxidase reaction are detected. By the electrode of 2, the substrate reductant Bred generated by the reaction of dehydrogenase from the substrate oxidant Box contained in the sample and the coenzyme reductant Ared contained in the buffer, and the substrate reduction originally contained in the sample The total amount of the body Bred is detected, and the amount of the substrate reduced body Bred contained in the sample analyzed earlier than that value is subtracted from the calibration curve No2.
By substituting the remaining output value into the calibration curve No3, the substrate oxidant Box in the sample can be analyzed. In this way, the substrate reductant Bred and the substrate oxidant Box can be analyzed simultaneously.

Next, the simultaneous analysis of the substrate reductant Bred and the coenzyme reductant Ared will be described. Using a buffer solution containing the substrate oxidant Box in the buffer solution, the oxidase-immobilized body (4) of the mechanism arranged downstream of the sample injection mechanism and the electrode active substance increased or decreased by the oxidase reaction are detected. Substrate reductant B originally contained in the sample by the first electrode
Analyze red.

Next, a dehydrogenase-immobilized body (6), an oxidase-immobilized body (7) and a second electrode for detecting an electrode active substance which increases or decreases due to the oxidase reaction at the final stage are arranged in the downstream order. By the mechanism of inclusion, the coenzyme reductant Ared contained in the sample, the substrate reductant Bred generated by the reaction of dehydrogenase from the substrate oxidant Box in the buffer, and the substrate reductant Bred originally contained in the sample Analyze the sum. If the amount of the substrate reductant Bred contained in the sample analyzed earlier than this measurement value is corrected, the substrate reductant Bred and the coenzyme reductant Ared in the sample can be simultaneously analyzed.

In the above mechanism, for example, a dehydrogenase and an oxidase may be mixed and immobilized, or each may be immobilized on a carrier and both carriers may be mixed and included in the same reactor. In this case, the substrate oxidant Box is converted into the substrate reductant Bred, and the substrate reductant Bred is also converted.
Is formed into a substrate oxidant Box. Therefore, although the sensitivity is improved, the output value greatly fluctuates due to the decrease or fluctuation of the activity of the dehydrogenase-immobilized product or the oxidase-immobilized product, resulting in large daily fluctuation, which is not preferable. Therefore, in the above-mentioned mechanism, the dehydrogenase-immobilized body and the oxidase-immobilized body are included in another reactor without being mixed, and the dehydrogenase-immobilized body, the oxidase-immobilized body and the second electrode are It is preferable to arrange them in series in this order from the upstream side.

As the system to which the measuring apparatus and the measuring method according to the present invention can be applied, (1) a combination of alcohol dehydrogenase and alcohol oxidase, acetaldehyde and ethanol, or ethanol and NADH or NADP is used.
A system for measuring H (pH of the buffer solution is preferably about 7 to about 9), (2) L-amino acid dehydrogenase and L-amino acid oxidase are combined, and 2-keto acid corresponding to L-amino acid, Alternatively, a system for measuring L-amino acid and NADH (pH of the buffer is preferably about 7 to about 8), (3) L-glutamate dehydrogenase and L-glutamate oxidase are combined, and 2-ketoglutarate and L are combined. -A system for measuring glutamic acid, or L-glutamic acid and NADH or NADPH (reduced nicotinamide adenine dinucleotide phosphate) (pH of the buffer solution 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 (p of buffer solution).
H is preferably about 7 to about 9. ), (5) A system for measuring L-lactic acid and pyruvic acid, or L-lactic acid and NADH by combining L-lactate dehydrogenase and L-lactate oxidase (the pH of the buffer solution 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. ), Etc. can be illustrated.

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

As a carrier used for immobilization, diatomaceous earth,
Silica gel, glass beads, alumina, ceramics, carbon, activated carbon, molecular sieves, silicone rubber,
Examples thereof include cellulose, agarose and amino acid polymers. Further, as the chemical bonding method, a method in which an amino group is introduced into the carrier surface with an aminosilanization reagent, formylation is further performed using a polyfunctional aldehyde such as glutaraldehyde, and then an enzyme is brought into contact to immobilize it is preferable. As an example. The carrier is preferably a silicon-containing substance having a hydroxyl group, and examples thereof include diatomaceous earth, calcined diatomaceous earth, porous glass, silica gel, and acid clay. Aminosilanization is performed by 3-aminopropyltriethoxysilane, 3-
It is carried out by bringing a reagent such as aminoethyltrimethoxysilane into contact with the carrier in a solvent such as anhydrous benzene or toluene. As the polyfunctional aldehyde, glutaraldehyde, glyoxal, succinyldialdehyde and the like can be used.

The case where L-lactate dehydrogenase is used as the dehydrogenase and L-lactate oxidase is used as the oxidase is described below as an example (see FIG. 1). First, an immobilization body is prepared by immobilizing L-lactate dehydrogenase or L-lactate oxidase on fine powder by a chemical bonding method. This immobilization body is packed in a column made of plastic or stainless steel having an inner diameter of about 1 to 10 mm and a length of about 2 to 50 mm, and used as each reactor. Each reactor can be of different sizes to change the concentration range measured.

Next, as a buffer solution of these enzymes in the vicinity of the optimum pH, for example, a 1 to 500 mM phosphate buffer solution having a pH of 7.0 can be used. As a pump used for liquid feeding, for example, a pump that can feed liquid at a flow rate of about 1.0 ml / min is prepared. As the pump, various types such as a plunger type and a peristaltic type can be used.
The buffer solution 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 performance liquid chromatograph using a 6-way valve can be used. An oxidase-immobilized reactor is arranged on the downstream side of the injector, and first, L-lactic acid in the sample is oxidized.
At this time, hydrogen peroxide is the electrode active substance that increases and oxygen is the electrode active substance that decreases. Therefore, if the first hydrogen peroxide electrode or the oxygen electrode is placed downstream of this 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 oxygen electrode is placed downstream thereof. When measuring L-lactic acid and pyruvic acid, NADH is added to the buffer solution. The amount of NADH added depends on how much the sample injected from the injector is diluted by being mixed with the buffer solution before reaching the dehydrogenase-immobilized reactor. That is, in the reduction reaction from pyruvic acid to L-lactic acid, NADH and pyruvic acid proceed in a one-to-one relationship. Generally, a concentration of about 1/2 to 1/100 of the concentration of pyruvic acid in the sample is sufficient. Add NADH to the sample and add NAD
It is possible to measure by injecting into a measuring device in which a buffer solution containing no H is allowed to flow, but since NADH is diluted at the same time as pyruvic acid, which is the measurement target, the necessary amount of NADH is practically available. This is not a preferable method in terms of cost because it increases.

L-lactic acid measurement is first carried out on the injected sample at the first reactor and the first electrode. Then L
-Pyruvic acid produced from lactic acid and pyruvic acid present in the sample from the beginning are both reduced inside the dehydrogenase-immobilized reactor. This reaction proceeds at high speed because the reaction equilibrium of dehydrogenase is remarkably inclined toward the production of L-lactic acid.

Naturally, it is possible to carry out the reaction by a general solution, but it is difficult to control the reaction time, and it is not suitable for analyzing a large amount of sample because the 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 solution, 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 pyruvic acid by the action of dehydrogenase is subsequently oxidized by an oxidase and can be quantified from the output value of the hydrogen peroxide electrode or the oxygen electrode in proportion to its concentration. By injecting the L-lactic acid standard solution before measuring the sample, the relationship between the two front and rear electrode outputs and the L-lactic acid concentration can be understood. Then, by injecting the pyruvic acid standard solution, the relationship between the second electrode output value and the pyruvic acid concentration can be obtained. As a result, two calibration curves for L-lactic acid and one calibration curve for pyruvic acid are obtained.

The mixture analysis first determines the L-lactic acid concentration and predicts the effect of pyruvic acid produced from L-lactic acid on the second electrode. Then, the amount of pyruvic acid produced from L-lactic acid can be subtracted to calculate the amount of pyruvic acid contained in the sample from the beginning. 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 at the same time, pyruvic acid is contained in the buffer instead of NADH. These series of operations are also applicable to other enzyme combinations.

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

The present invention can also be implemented by the device shown in FIG. FIG. 8 shows a configuration in which the first measuring unit and the second measuring unit are arranged in series in FIG. 1, but the first measuring unit and the second measuring unit are changed in parallel. That is, a first measurement part including an oxidase-immobilized body (4) and a first electrode, and a second measurement part including a dehydrogenase-immobilized body (6), an oxidase-immobilized body (7) and a second electrode. Are arranged in parallel. However, the aspect of FIG. 1 has an advantage that the diversion ratio does not change and the measurement can be performed with higher accuracy than the aspect of FIG.

Furthermore, the present invention can be implemented with the flow type measuring apparatus shown in FIG. FIG. 9 omits the first measurement part including the oxidase-immobilized body (4) and the first electrode in FIG. 1. Then, for example, the substrate oxidant Bo
When x and the substrate reductant Bred are measured, the coenzyme reductant Ared is not added to the buffer solution so that the dehydrogenase reaction does not occur, and the same measurement as in the first measurement unit is performed. The substrate reductant Bred in the sample is measured. If the coenzyme reductant Ared is added to the buffer solution, the same measurement as in the second measurement section can be performed. This method has the advantage of not requiring a complicated device, but the sample is injected twice and the coenzyme reductant Ar is added to the buffer solution.
It is necessary to measure whether ed is added or not. Of course, the addition of the coenzyme reductant is the same even if it is carried out in the sample. The above is the measurement of the substrate oxidant Box and the substrate reductant Bred.
The same applies to the measurement of red and the reduced substrate Bred.

[0041]

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

Example 1 The characteristics of each enzyme-immobilized reactor were examined by constructing a measuring apparatus for L-lactic acid and pyruvic acid. (1) Method for producing L-lactate oxidase-immobilized reactor Refractory brick (30-60) that is a diatomaceous earth silicic acid carrier
150 mg of (mesh) is thoroughly dried, immersed in a 10% γ-aminopropyltriethoxysilane anhydrous toluene solution for 1 hour, thoroughly washed with toluene, and dried at 120 ° C. for 2 hours. In this way, the aminosilane-treated carrier is
% Glutaraldehyde aqueous solution for 1 hour, washed thoroughly with distilled water, and finally replaced with 100 mM sodium phosphate buffer of pH 7.0 to remove this buffer as much as possible. The pH of this formylated refractory brick
200 μl of a solution of L-lactate oxidase (manufactured by Sigma) dissolved in 7.0 and 100 mM sodium phosphate buffer at a concentration of 50 units / ml is brought into contact with the solution and left at 10 ° C. or lower for 1 day for immobilization. This enzyme-immobilized carrier has an inner diameter of 3.5 m
An acrylic resin column having a length of m and a length of 30 mm is packed into a L-lactate oxidase-immobilized reactor.

(2) Method for producing L-lactate dehydrogenase-immobilized reactor As in the method for immobilizing L-lactate oxidase, formylated refractory bricks were adjusted to pH 7.0, and 100 mM sodium phosphate buffer was added to pig muscle. A solution of L-lactate dehydrogenase derived from Boehringer Mannheim at a concentration of 4500 units / ml is brought into contact with 200 μl of the solution and left at 10 ° C. or lower for 1 day for immobilization. This enzyme-immobilized carrier has an inner diameter of 3.
A column having a length of 5 mm and a length of 30 mm is packed into a L-lactate dehydrogenase-immobilized reactor.

(3) Method for producing hydrogen peroxide electrode A side surface of a platinum wire having a diameter of 2 mm is coated with heat-shrinkable Teflon,
One end of the wire is smoothed with a file and a No. 1500 emery paper. Using this 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, 0.1M
Electrolyte in sulfuric acid at +1.4 V for 10 minutes. After that, the platinum wire was washed well with water and then dried at 40 ° C for 10 minutes.
It is immersed in an anhydrous toluene solution of 10% 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 to make the concentration 0.2%. 5 μl of this mixed solution was quickly put on a platinum wire prepared previously, and dried and cured at 40 ° C. for 15 minutes to form a hydrogen peroxide selective permeable membrane. This is the working electrode of the hydrogen peroxide electrode.

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

(4) Simultaneous measurement apparatus for L-lactic acid and pyruvic acid will be described with reference to FIG. First, 0.5 from the buffer tank (1)
A sample containing pyruvic acid and L-lactic acid, into which a buffer solution containing mM NADH was continuously sent by a solution sending pump (2) and injected from a sampler (3), is sent to a constant temperature bath (9). Pyruvic acid and hydrogen peroxide are produced from L-lactic acid in the sample and oxygen in the buffer solution by the reactor containing the first immobilized L-lactate oxidase (4) in the thermostat. This increase / decrease is detected by the measuring unit (5) having the first hydrogen peroxide electrode.

Furthermore, the pyruvic acid pre-existing in the sample and the pyruvic acid produced by L-lactate oxidase react with NADH in the buffer solution to give L by a reactor containing the L-lactate dehydrogenase-immobilized body (6). -Converted to lactic acid and NAD. Then, hydrogen peroxide is similarly produced by the reactor containing the second immobilized L-lactate oxidase (7). This increase / decrease is detected by the measuring unit (8) having the second hydrogen peroxide electrode. The change in the current value at these two measuring portions 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 the personal computer (13) through the RS-232C cable (12). Injection of the sample and control of the pump were performed by a personal computer. Note that (14) in FIG. 1 is a pump control signal, and (1)
5) shows the sampler control signal, and (16) shows the waste liquid.

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

The composition of the buffer solution is as follows: phosphoric acid 100 mM, potassium chloride 50 mM, sodium azide 1 mM and NA.
It was measured at pH 7.0 containing DH 500 μM. The flow rate of the buffer solution was 1.0 ml / min, and the temperature of the constant temperature bath was 30 ° C. FIG. 2 shows the calibration curve based on the output values obtained from the two measuring parts at pH 7.0. The calibration curve 1 is the output from the first measuring section for lactic acid, the calibration curve 2 is the output from the second measuring section for lactic acid, and the calibration curve 3 is the output for pyruvic acid, and good linearity was obtained. You can see that

Next, a calibration curve was prepared in the same manner by changing the pH of the buffer solution, and the change with respect to the output value at pH 7.0 was examined from the gradient. The result is shown in FIG. 1 in the drawing shows the pH dependence of the response value to lactic acid. When the relative ratio was changed, the behaviors of the output values with respect to lactic acid in the first and second measurement parts were the same, and therefore, they were represented by one line. The output value for pyruvic acid is 2. pH 7.
A maximum value was observed around 25.

Further, FIG. 4 shows changes in gradient as relative values by changing the temperature of the constant temperature bath. The change in the response value to lactic acid is shown by one, as in the case of FIG. The change in the response value in the measuring section having the second electrode for pyruvic acid is 2. The optimum temperature is recognized 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 7.0 and the temperature was 30.
℃ was made. With this measuring device, measurement was performed for 8 hours a day at 30 ° C., and at other times, long-term stability was confirmed when the enzyme-immobilized body was left at room temperature (25 ° C.). FIG. 5 shows the daily variation of the output value when the output value immediately after being fixed is 100. In FIG. 5, 1 is lactic acid and 2 is pyruvic acid. It was found to be stable for about 2 months.

Example 2 A mixture of standard solutions of L-lactic acid and pyruvic acid and sake moromi filtrate were analyzed. (1) Method for producing enzyme-immobilized reactor The same method as in Example 1 was used for both dehydrogenase and oxidase. (2) Method for manufacturing hydrogen peroxide electrode The same method as in Example 1 was used for manufacturing. (3) Measuring device The same measuring device as in Example 1 was used. (4) Measuring method 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 liquid mixture.

[0055]

[Table 1]

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

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

Sake moromi filtrate, whose lactic acid and pyruvic acid concentrations were previously determined by the solution enzyme method, was analyzed without dilution. The results are shown in Table 2.

[0059]

[Table 2]

As shown in Table 2, good agreement with the solution enzyme method is observed. Further, it has been shown that the measuring apparatus and the measuring method of the present invention are useful in consideration of automation of analysis and the like, since operations such as dilution are unnecessary.

Example 3 Standard solutions of L-lactic acid and NADH were analyzed. (1) Method for producing enzyme-immobilized reactor The same method as in Example 1 was used for both dehydrogenase and oxidase. (2) Method for manufacturing hydrogen peroxide electrode The same method as in Example 1 was used for manufacturing. (3) Measuring device The same measuring device as in Example 1 was used. (4) Measurement method Analysis was performed according to Example 2 except that the buffer solution contained 0.5 mM pyruvic acid instead of NADH. (5) Measurement results Regarding L-lactic acid, the same results as in Example 1 were obtained.
FIG. 6 shows a calibration curve of NADH. Thus, it was shown that NADH can be measured. At the same time, Table 3 shows the results of analyzing the mixture.

[0062]

[Table 3]

[0063]

EFFECTS OF THE INVENTION According to the present invention, it becomes possible to measure the substrate oxidant and the substrate reductant of the dehydrogenase at the same time, or the coenzyme reductant and the substrate reductant at the same time easily and at high speed.

[Brief description of drawings]

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

FIG. 2 shows the 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 was changed in Example 1.

FIG. 5 shows the daily variation 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 apparatus of the present invention.

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

[Explanation of symbols]

 1 Buffer solution tank 2 Liquid feed pump 3 Sampler 4 Oxidase immobilization body 5 1st measurement part 6 Dehydrogenase immobilization body 7 Oxidation enzyme immobilization body 8 2nd measurement part 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

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12Q 1/32 6807-4B G01N 27/27 7235-2J G01N 27/46 336 H

Claims (10)

[Claims] 1. A coenzyme reductant Ared and a substrate oxidant Bo
dehydrogenase which catalyzes the reaction of producing coenzyme oxidant Aox and substrate reductant Bred from x, and substrate reductant Bred
It is a measuring device using an oxidase that oxidizes, and has a mechanism for continuously supplying a buffer solution and a supply mechanism for supplying a sample into the buffer solution, and the oxidase-immobilized downstream of the supply mechanism. A mechanism that includes a first electrode that detects an electrode active substance that increases or decreases due to an oxidase reaction with the body, and a mechanism that detects a dehydrogenase-immobilized product, an oxidase-immobilized product, and an electrode active substance that increases or decreases due to an oxidase reaction A measuring device having a mechanism including two electrodes. 2. The mechanism of is arranged in series downstream of the mechanism of, and the dehydrogenase-immobilized body and the oxidase-immobilized body in the mechanism are arranged in series in this order from the upstream side. The measuring device according to claim 1. 3. The measuring device according to claim 1, wherein the dehydrogenase is L-lactate dehydrogenase and the oxidase is L-lactate oxidase. 4. A dehydrogenase-immobilized product is prepared by introducing an amino group on the surface of a carrier with an aminosilanization reagent, and further performing formylation using a polyfunctional aldehyde, and then contacting with a dehydrogenase to immobilize it. The measuring device according to claim 1, which is a fixed body of dehydrogenase. 5. The measuring apparatus according to claim 1, wherein the buffer solution contains a coenzyme reductant Ared, and a substrate reductant Bred.
And a substrate oxidant Box are simultaneously measured, which is a measuring method using an immobilized enzyme. 6. An immobilized enzyme comprising the measuring apparatus according to claim 1, wherein the buffer solution contains a substrate oxidant Box, and the coenzyme reductant Ared and the substrate reductant Bred are simultaneously measured. Measuring method. 7. The immobilization according to claim 5 or 6, wherein the coenzyme reductant Ared is NADH, the substrate oxidant Box is pyruvic acid, and the substrate reductant Bred is L-lactic acid. Measuring method using activated enzyme. 8. A coenzyme reductant Ared and a substrate oxidant Bo
dehydrogenase which catalyzes the reaction of producing coenzyme oxidant Aox and substrate reductant Bred from x, and substrate reductant Bred
A method of measuring a substrate oxidant Box (or a coenzyme reductant Ared) and a substrate reductant Bred using an oxidase that oxidizes cesium, comprising: (i) introducing a sample together with a buffer solution into an oxidase-immobilized body, The substrate reductant Bred of the sample is converted into the substrate oxidant Box and the substrate reductant Bre in the sample is detected by detecting the electrode active substance that increases or decreases by this reaction.
a step of detecting d, (ii) introducing the sample to a dehydrogenase-immobilized body together with a buffer solution containing a coenzyme reductant Ared (or a substrate oxidant Box), and reducing the substrate oxidant Box in the sample (or A substrate reductant Bred is produced by oxidizing the coenzyme reductant Ared), and is further led to an oxidase-immobilized body, and the generated substrate reductant Bred and the substrate reductant Bred originally present in the sample are converted to the substrate oxidant. A step of detecting the substrate reductant Bred by converting into Box and detecting the electrode active substance that increases or decreases by this reaction, (i
ii) The value obtained by the step (ii) is corrected by the value of the original substrate reductant Bred obtained by the step (i) to correct the substrate oxidant Box (or the coenzyme reductant Are).
d) the step of quantifying,
(Or coenzyme reductant Ared) and substrate reductant Bre
A method of measuring d. 9. (1) A first measuring unit including an oxidase-immobilized body and a first electrode for detecting an electrode active substance that increases or decreases due to an oxidase reaction, and (2) a dehydrogenase-immobilized body, an oxidase. Using a measuring device having a second measuring part including an immobilized body and a second electrode that detects an electrode active substance that increases or decreases due to an oxidase reaction, the step (i) is performed in the first measuring part, and The method according to claim 8, wherein the step ii) is performed in the second measuring unit. 10. A measuring device having a dehydrogenase-immobilized body, an oxidase-immobilized body, and an electrode for detecting an electrode active substance that increases or decreases due to an oxidase reaction, wherein the step (i) is a coenzyme reductant. Ared (or substrate oxidant Box)
The sample is introduced together with a buffer solution not containing the above into the above-mentioned device, and the substrate reductant Bred in the sample is detected by detecting the electrode active substance that increases or decreases due to the action of the oxidase-immobilized body. The process is coenzyme reductant Ar
The sample is introduced into the above apparatus together with a buffer solution containing ed (or substrate oxidant Box), and the substrate reductant Bred is detected by detecting an increasing or decreasing electrode active substance by the action of the dehydrogenase-immobilized body and the oxidase-immobilized body. 9. The method according to claim 8, which is for detecting