JPH0792138A - Lactic acid sensor - Google Patents

Abstract

PURPOSE:To make it possible to determine lactic acid and including L-lactic acid and D-lactic acid through a simple operation by detecting variation of concentration electrochemically using an enzyme having a function for racemizing lactic acid and an enzyme having function for oxidizing lactic acid. CONSTITUTION:A carboxymethylcellulose(CMC) layer of hydrophilic polymer is formed on an electrode system comprising a working electrode 4 and a pair electrode 5 formed on a substrate 1. An enzyme having function for oxidizing lactic acid, i.e., lactic acid oxidase, and an enzyme having function for racemizing lactic acid, i.e., lactic acid racemase, are dissolved into a phosphoric acid buffer and developed onto a CMC layer and then it is dried to form a reaction layer 11. When the reaction layer 11 is dissolved into a sample liquid, only one of L-lactic acid or D-lactic acid is oxidized by the lactic acid oxidase and the other lactic acid not oxidized by the enzyme is converted, by the lactic acid racemase, into D-lactic acid or L-lactic acid being oxidized by enzyme thus allowing simultaneous determination of L-lactic acid and D-lactic acid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor capable of quickly and accurately quantifying a lactic acid component in a sample.

[0002]

2. Description of the Related Art As a method for quantifying lactic acid using an enzyme electrode, a method in which lactate oxidase is used as an enzyme and combined with a hydrogen peroxide electrode is disclosed (for example, in medical engineering,
54, 68, 1984). A measuring device according to this method will be described. The enzyme electrode used is composed of an immobilized enzyme membrane in which lactate oxidase is immobilized on an acetylcellulose membrane and the surface is covered with a polycarbonate thin film, and a hydrogen peroxide electrode composed of platinum and silver. The measuring instrument has a flow line, and the sample solution is mixed and diluted with a buffer solution, mixed with air containing oxygen, and introduced into the enzyme electrode portion through a defoamer. At the same time that lactic acid in the sample solution reacts with lactate oxidase in the immobilized enzyme membrane, hydrogen peroxide is produced. Lactic acid is quantified by measuring the increased amount of hydrogen peroxide with a hydrogen peroxide electrode.

[0003]

In such a conventional measuring method, it is necessary to replace the enzyme membrane, and further a device equipped with a pump, a mixer, a defoamer and the like.
Therefore, maintenance such as cleaning of the flow line is required, and the operation is complicated, such as supplying a fixed amount of sample liquid,
And it lacked in simplicity. Further, the enzyme used in the conventional method has a specific oxidizing ability for L-lactic acid, but does not have an ability for oxidizing D-lactic acid. For example, in the food-related fields such as yogurt and lactic acid bacteria beverages, it is necessary to quantify lactic acid including both L-form and D-form, but it is impossible to measure D-lactic acid concentration by a method using a conventional device. Regarding the quantification of D-lactic acid concentration, an automatic analyzer has not been developed, and a method that combines an enzymatic reaction and spectroscopic analysis is generally used. This method requires a great deal of time and labor, and has the drawbacks that human error is likely to occur due to manual work by a person. Therefore, an object of the present invention is to provide a lactic acid sensor capable of quantifying lactic acid including L-form and D-form by a simple operation.

[0004]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present invention uses an enzyme capable of racemizing lactic acid together with an enzyme capable of oxidizing lactic acid, and a substance accompanying the oxidation reaction of lactic acid. It is intended to provide a lactate sensor having an electrode for electrochemically detecting a change in concentration. More specifically, the lactic acid sensor of the present invention comprises an electrode system including a working electrode and a counter electrode formed on an insulating substrate, and a reaction layer located on the electrode system, wherein the reaction layer is at least hydrophilic. Characteristic polymer, an enzyme capable of oxidizing lactic acid, and an enzyme capable of racemizing lactic acid.

In addition, the lactic acid sensor of the present invention comprises an electrode system including a working electrode and a counter electrode formed on an insulating substrate, a reaction layer located on the electrode system, and an auxiliary reaction located near the electrode system. A layer is provided, and the reaction layer contains at least a hydrophilic polymer and an enzyme having an ability to oxidize lactic acid, and the auxiliary reaction layer contains an enzyme having at least an ability to racemize lactic acid. Here, the reaction layer further includes an electron acceptor, or a combination of an enzyme having an ability to oxidize lactic acid with an enzyme having an ability to oxidize L-lactic acid and an enzyme having an ability to oxidize D-lactic acid is used. In addition, the reaction layer may further contain a coenzyme.

[0006]

According to the present invention, one enzyme having an ability to oxidize lactic acid oxidizes only one of L-form and D-form, but is not oxidized by the enzyme having an ability to ceramize lactic acid. The D-form or L-form will be converted to the L-form or D-form that is oxidized by the enzyme.
Therefore, according to the present invention, it is possible to obtain a lactic acid sensor capable of simultaneously quantifying L-lactic acid and D-lactic acid in a sample with a simple operation.

[0007]

EXAMPLES The present invention will be described below with reference to examples. [Embodiment 1] FIG. 1 is a perspective view of a lactic acid sensor manufactured as an embodiment of the present invention, excluding a reaction layer, and FIG. 2 is a schematic sectional view of the lactic acid sensor. The method for producing the lactate sensor will be described below. On an insulating substrate 1 made of polyethylene terephthalate, silver paste is printed by screen printing to form leads 2 and 3. Next, the working electrode 4 of the electrode system is formed by using a conductive carbon paste containing a resin binder, and then the insulating layer 6 made of an insulating paste is sequentially formed by printing. Insulating layer 6 is working electrode 4
The area of the exposed portion is constant and the leads 2 and 3 are partially covered. Finally, the counter electrode 5 of the electrode system is printed by using a conductive carbon paste containing a resin binder. The electrodes 4 and 5 are connected to the leads 2 and 3, respectively.

Next, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer is spread on the electrode system and dried to form a CMC layer. Then, lactate oxidase (EC1.1.EC) as an enzyme having an ability to oxidize lactic acid on the CMC layer.
3.2; hereinafter abbreviated as LOD) and lactate racemase (EC5.1.) As an enzyme having the ability to racemize lactic acid.
A solution obtained by dissolving 2.1) in a phosphate buffer (pH 7.0) is developed and dried in a warm air dryer to form the reaction layer 11. In the reaction layer forming step, when the mixed aqueous solution of the enzyme is dropped, the CMC layer is once dissolved, and the reaction layer 11 is formed in the form of being mixed with the enzyme in the subsequent drying process. However, since it is not accompanied by stirring or the like, it is not in a completely mixed state, and the electrode system surface is in a state of being covered only with CMC. That is, since it is difficult for the enzyme and the electron acceptor added in the examples described below to contact the surface of the electrode system, it is possible to obtain a lactate sensor having a highly accurate sensor response.

10 μl of an aqueous lactic acid solution was dropped onto the reaction layer 11 as a sample solution to the lactic acid sensor produced as described above,
After 5 minutes, a pulse voltage of +1.0 V was applied to the working electrode 4 in the anode direction based on the counter electrode 5 of the electrode system, and the current value after 5 seconds was measured. The response was proportional to the concentration of lactic acid in the sample solution. The current value was obtained. When the reaction layer 11 is dissolved in the sample solution, D-lactic acid in the sample solution is racemized by the lactate racemase to produce L-lactic acid. L-lactic acid previously present in the sample solution and L-lactic acid produced by the racemization
Both lactic acid is oxidized by LOD, and at the same time, oxygen in the solution is reduced to generate hydrogen peroxide. Next, by applying the above voltage, an oxidation current of the generated hydrogen peroxide is obtained,
This current value corresponds to the concentration of lactic acid which is a substrate.

[Embodiment 2] FIG. 3 is an exploded perspective view of the lactic acid sensor manufactured as one embodiment of the present invention, excluding the reaction layer and the auxiliary reaction layer. FIG. 4 is an exploded perspective view of the same lactic acid sensor.
FIG. 5 is a schematic cross-sectional view of the lactate sensor excluding a cover and a spacer. The method for producing the lactate sensor will be described below. In the same manner as in Example 1, the electrode system including the leads 2 and 3, the working electrode 4 and the counter electrode 5 shown in FIG. 3, and the insulating layer 6 are formed on the insulating substrate 1 by screen printing. Next, a 0.5 wt% aqueous solution of CMC as a hydrophilic polymer is spread on the electrode system and dried to form a CMC layer. Subsequently, a mixed aqueous solution of LOD and lactose racemase as an enzyme is developed on the CMC layer and dried in a warm air drier to form the reaction layer 11. Next, a solution in which lactase racemase is dissolved in a phosphate buffer is spread on the insulating layer 6 and dried to form the auxiliary reaction layer 12. After forming the reaction layer 11 and the auxiliary reaction layer 12 as described above, the cover 22 and the spacer 21 may be formed as shown in FIG.
The inner and inner parts are bonded with the positional relationship shown by the one-dot chain line.

When the cover is attached, the sample liquid is easily introduced into the reaction layer portion by a simple operation of bringing the sample liquid into contact with the sample supply hole 10 at the tip of the sensor due to the capillary action of the space portion generated by the cover and the spacer. In order to make the supply of the sample solution smoother, it is advisable to further spread an organic solvent solution of lecithin from the sample supply section onto the reaction layer 11 and dry it if necessary. Furthermore, when the treatment with the lecithin is carried out, the sample liquid can be supplied even when the space formed by the cover and the spacer has a size such that the capillary phenomenon cannot be expressed. Since the amount of the sample solution supplied depends on the space volume generated by the cover and the spacer, it is not necessary to quantify in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed.

When 3 μl of the lactic acid aqueous solution as a sample solution is supplied from the sample supply hole 23 to the lactic acid sensor manufactured as described above, the sample solution quickly reaches the air holes 24, and the auxiliary reaction layer 12 and the reaction layer 11 are sequentially formed. Dissolve. Example 1
In the same manner as described above, 4 minutes after supplying the sample solution, the working electrode 4 is +1.0 toward the anode with the counter electrode 5 of the electrode system as a reference.
When a pulse voltage of V was applied and the current value was measured 5 seconds later, a response current value proportional to the concentration of lactic acid in the sample solution was obtained. When the concentration of D-lactic acid in the sample solution is high, sufficient racemization does not occur within a fixed time only by the action of the lactate racemase in the reaction layer 11. By providing the auxiliary reaction layer 12 containing the lactate racemase, the lactic acid concentration can be quantified in a short time. Further, when a lactic acid sensor was produced by adding lactate racemase only in the auxiliary reaction layer 12 without containing the lactate racemase in the reaction layer 11, the effect of improving the storage reliability of the sensor was obtained. This is because it was possible to avoid a decrease in enzyme activity caused by the inclusion of a plurality of enzymes in the reaction layer 11.

[Embodiment 3] In the same manner as in Embodiment 1, the reel shown in FIG. 1 is formed on the insulating substrate 1 by screen printing.
The electrode system including the electrodes 2, 3, the working electrode 4, and the counter electrode 5, and the insulating layer 6 are formed. Next, a 0.5 wt% aqueous solution of CMC as a hydrophilic polymer is spread on the electrode system and dried to form C.
Form the MC layer. Then, a mixed aqueous solution of LOD and lactose racemase as an enzyme and potassium ferricyanide as an electron acceptor is developed on the CMC layer and dried in a warm air dryer to form a reaction layer 11. The lactic acid sensor produced as described above was added with a lactic acid aqueous solution 10 as a sample solution.
μl was dropped on the reaction layer 11, and 5 minutes later, the counter electrode 5 of the electrode system was
A pulse voltage of +0.5 V was applied to the working electrode 4 in the direction of the anode with reference to, and the current value after 5 seconds was measured.
A response current value proportional to the concentration of lactic acid in the sample solution was obtained.

When the reaction layer 11 is dissolved in the sample solution, D-lactic acid in the sample solution is racemized by the lactase racemase to give L-lactic acid.
-Lactic acid is produced. Both L-lactic acid and L-lactic acid that was previously present in the sample solution are oxidized by LOD, and at the same time potassium ferricyanide is reduced to potassium ferrocyanide. Next, by applying the pulse voltage, an oxidation current of the generated potassium ferrocyanide is obtained, and this current value corresponds to the concentration of lactic acid as a substrate.
By including an electron acceptor in the reaction layer, it became possible to quantify a higher concentration of lactic acid. Further, as shown in Example 2, when the auxiliary reaction layer containing lactate racemase is formed on the insulating layer, a lactic acid sensor capable of measuring higher concentration of lactic acid in a short time can be realized.

[Embodiment 4] In the same manner as in Embodiment 1, the reel shown in FIG. 1 is printed on the insulating substrate 1 by screen printing.
The electrode system including the electrodes 2, 3, the working electrode 4, and the counter electrode 5, and the insulating layer 6 are formed. Next, a 0.5 wt% aqueous solution of CMC as a hydrophilic polymer is spread on the electrode system and dried to form C.
Form the MC layer. Subsequently, D-lactate dehydrogenase (EC1.1.1.2) was added as an enzyme on the CMC layer.
8; hereinafter, abbreviated as D-LDH), LOD, lactate racemase, potassium ferricyanide as an electron acceptor, and a mixed aqueous solution of nicotinamide adenine dinucleotide (hereinafter, abbreviated as NAD) as a coenzyme, and the mixture is dried in a warm air dryer. The reaction layer 11 is formed by drying in the inside. The lactic acid sensor produced as described above was added with a lactic acid aqueous solution 10 as a sample solution.
μl was dropped on the reaction layer 11, and 3 minutes later, the counter electrode 5 of the electrode system was
A pulse voltage of +0.5 V was applied to the working electrode 4 in the direction of the anode with reference to, and the current value after 5 seconds was measured.
A response current value proportional to the concentration of lactic acid in the sample solution was obtained.

When the reaction layer 11 is dissolved in the sample solution, the D-lactic acid in the sample solution is racemized by the lactate racemase to form L.
-Lactic acid is produced. Both L-lactic acid and L-lactic acid that was previously present in the sample solution are oxidized by LOD, and at the same time potassium ferricyanide is reduced to potassium ferrocyanide. Further, D-lactic acid is also oxidized by D-LDH and coenzyme NAD is reduced to produce NADH. This NADH reacts with potassium ferricyanide, resulting in the production of potassium ferrocyanide. Next, by applying the pulse voltage, an oxidation current of potassium ferrocyanide produced by the above two reaction paths is obtained, and this current value corresponds to the concentration of the substrate lactic acid. Reaction system of LOD and Lactase racemase and D-
By using the LDH and NAD reaction systems in combination, the lactic acid concentration can be measured in a shorter time. on the other hand,
NADH produced by reduction of NAD does not necessarily have to be conjugated with an electron acceptor, and can be quantified by directly oxidizing it with an electrode system.

Although CMC was used as the hydrophilic polymer in Examples 1 to 4 above, the present invention is not limited to this, and other cellulose derivatives, specifically, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose are used. , Ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose may be used, and further, polyvinylpyrrolidone, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, maleic anhydride and The same effect can be obtained by using the salt.

On the other hand, as the electron acceptor, in addition to potassium ferricyanide shown in the above examples, p-benzoquinone, meldra blue, phenazine methosulfate, methylene blue, ferrocene and derivatives thereof which are conjugated with the oxidoreductase to be used. You can use it freely. Further, in the above-mentioned examples, the method of dissolving the enzyme and the electron acceptor in the sample solution was shown, but the method is not limited to this, and it can be applied to the case of being insolubilized in the sample solution by immobilization. it can.
Further, although the two-electrode system including the working electrode and the counter electrode has been described in the above embodiment, the three-electrode system including the reference electrode enables more accurate measurement.

[0019]

As described above, according to the present invention, a lactic acid sensor having excellent responsiveness can be obtained by a simple operation.

[Brief description of drawings]

FIG. 1 is a perspective view of a lactic acid sensor manufactured as an example of the present invention, excluding a reaction layer.

FIG. 2 is a schematic cross-sectional view of the lactate sensor.

FIG. 3 is an exploded perspective view of a lactic acid sensor manufactured as another embodiment of the present invention, excluding a reaction layer and an auxiliary reaction layer.

FIG. 4 is an exploded perspective view of the lactate sensor.

FIG. 5 is a schematic cross-sectional view of the lactate sensor excluding a cover and a spacer.

[Explanation of symbols]

 1 Insulating substrate 2, 3 Lead 4 Working electrode 5 Counter electrode 6 Insulating layer 11 Reaction layer 12 Auxiliary reaction layer 21 Spacer 22 Cover 23 Sample supply hole 24 Air hole

Claims (6)

[Claims] 1. An enzyme having the ability to oxidize lactic acid, an enzyme having the ability to racemize lactic acid, and an electrode for electrochemically detecting a change in the concentration of a substance associated with the oxidation reaction of lactic acid. Lactic acid sensor. 2. An electrode system including a working electrode and a counter electrode formed on an insulating substrate, and a reaction layer located on the electrode system, wherein the reaction layer oxidizes at least a hydrophilic polymer and lactic acid. A lactate sensor comprising an enzyme having the ability and an enzyme having the ability to racemate lactic acid. 3. An electrode system including a working electrode and a counter electrode formed on an insulative substrate, a reaction layer located on the electrode system, and an auxiliary reaction layer located in the vicinity of the electrode system. A lactic acid sensor, wherein the layer contains at least a hydrophilic polymer and an enzyme capable of oxidizing lactic acid, and the auxiliary reaction layer contains at least an enzyme capable of racemizing lactic acid. 4. The lactic acid sensor according to claim 3, wherein the reaction layer further contains an enzyme having the ability to racemize lactic acid. 5. The lactic acid sensor according to claim 2, wherein the reaction layer further contains an electron acceptor. 6. An enzyme having the ability to oxidize lactic acid is L
The lactic acid sensor according to claim 2 or 3, which comprises a combination of an enzyme capable of oxidizing lactic acid and an enzyme capable of oxidizing D-lactic acid, and further comprises a coenzyme in the reaction layer.