JPH0694672A - Biosensor and production thereof - Google Patents

Abstract

PURPOSE:To realize a biosensor capable of rapidly and quantitatively measuring the specific component in a sample with high accuracy and high reliability by simple operation. CONSTITUTION:A biosensor consists of the electrode system based on the measuring electrode 4 and opposed electrode 5 formed on an insulating substrate 1 and a reaction layer 7 containing a hydrophilic polymer, lactic acid oxidase and an electron acceptor and is produced by providing the electrode system based on the measuring electrode 4 and the opposed electrode 5 on the insulating substrate 1 and providing a hydrophilic polymer layer on the electrode system and subsequently dripping the ag. soln. containing lactic acid oxidase and the electron acceptor on the electrode system to dry the ag. soln. layer to form the reaction layer 7.

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 easily and rapidly quantifying a specific component in a sample, and a method for producing the biosensor.

[0002]

2. Description of the Related Art As a method for quantifying lactic acid using an enzyme electrode, a method in which lactate oxidase (hereinafter abbreviated as LOD) is used as an enzyme and combined with a hydrogen peroxide electrode has been reported (Medical Engineering, 54 Vol. 68, 1984). A measuring method using the above equipment will be described. An enzyme electrode is composed of an immobilized enzyme membrane in which LOD is immobilized on an acetyl cellulose membrane and the surface is covered with a polycarbonate thin film, and a hydrogen peroxide electrode composed of platinum and silver.

The above 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.
Hydrogen peroxide is produced at the same time when lactic acid in the sample solution reacts with LOD in the immobilized enzyme membrane. Lactic acid is quantified by measuring the increased amount of hydrogen peroxide with a hydrogen peroxide electrode.

[0004]

In such a conventional measuring method, it is necessary to replace the enzyme membrane.
Equipment equipped with a pump, mixer, deaerator, etc. is required. Therefore, maintenance such as cleaning of the flow line is required, and it is complicated and lacks in simplicity such as supplying a fixed amount of sample liquid.

An object of the present invention is to provide a biosensor capable of performing highly accurate quantitative determination of lactic acid in a sample solution quickly and extremely easily.

[0006]

In order to achieve the above object, the present invention provides an electrode system comprising at least a measurement electrode and a counter electrode on an insulating substrate, and the hydrophilic polymer and lactic acid oxidation are provided on the electrode system. This is a structure in which a reaction layer containing an enzyme and an electron acceptor is installed.

[0007]

According to the present invention, the reaction layer is dissolved in the sample solution and the lactate oxidase contained therein causes an oxidation-reduction reaction in the electrode system to obtain a response current corresponding to the concentration of lactic acid. . Therefore, a highly reliable biosensor can be obtained by a simple operation.

[0008]

EXAMPLES The present invention will be described below with reference to examples.

(Example 1) FIG. 1 is a cross-sectional view of a lactate sensor produced as an example of the biosensor of the present invention, and FIG. 2 is an exploded perspective view of the lactic acid sensor seen from an obliquely upper direction of FIG. .

A method for producing a lactic acid sensor will be described below. An insulating substrate 1 made of polyethylene terephthalate is printed with a silver paste by screen printing and then reprinted.
Dos 2 and 3 were formed. Next, using a conductive carbon paste containing a resin binder, the measuring electrode 4 of the electrode system was measured.
Then, an insulating layer 6 made of an insulating paste was sequentially formed by printing. The insulating layer 6 keeps the exposed area (about 1 mm ² ) of the measuring electrode 4 constant and partially covers the leads 2 and 3. Finally, the counter electrode 5 of the electrode system was printed by using a conductive carbon paste containing a resin binder.

Next, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was spread on the electrode system and dried to form a CMC layer. Subsequently, a mixed aqueous solution of LOD as an enzyme and potassium ferricyanide as an electron acceptor was developed on the CMC layer and dried in a warm air drier to form a reaction layer 7.

In the above reaction layer forming step, when a mixed aqueous solution of LOD and potassium ferricyanide is dropped, the CMC layer is once dissolved and the reaction layer 7 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 the enzyme and the electron acceptor do not come into contact with the surface of the electrode system, the characteristics of the electrode system do not change due to the adsorption of proteins on the surface of the electrode system, and as a result, a biosensor having a highly accurate sensor response can be obtained. Obtainable.

After forming the reaction layer 7 as described above,
The cover 9 and the spacer 8 were adhered so as to have a positional relationship shown by a dashed line in FIG. If a transparent material is used for the cover, the state of the reaction layer and the introduction state of the sample solution can be confirmed very easily from the outside.

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 capillarity phenomenon in the space created 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 on the surface from the sample supply part to the reaction layer and dry it, if necessary.

Further, when the treatment with lecithin is carried out, the sample liquid can be supplied even when the space portion formed by the cover and the spacer has a size such that the capillary phenomenon cannot be expressed.

Since the supply amount of the sample liquid depends on the space volume generated by the cover and the spacer, it is not necessary to quantify it in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed.

As a sample solution, 3 μl of a solution of lactic acid adjusted to pH 7 with 0.2 M phosphate buffer was supplied to the lactic acid sensor manufactured as described above from the sample supply hole 10. The sample solution quickly reached the air holes 11 and the reaction layer on the electrode system was dissolved.

One minute after supplying the sample solution, the counter electrode 5 of the electrode system is used as a reference and the measuring electrode 4 is +0.5 V in the anode direction.
When the pulse voltage was applied and the current value was measured 5 seconds later, a response current value proportional to the lactic acid concentration in the sample solution was obtained.

When the reaction layer 7 is dissolved in the sample solution, lactic acid in the sample solution is 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.

In the above embodiment, LOD was added to the reaction layer.
4 U / cm ² , potassium ferricyanide 0.65 m
g / cm ^{2 is} included. When the contents of this enzyme and electron acceptor were examined, the following results were obtained.

When the amount of the enzyme was decreased, the reaction rate was lowered, and it took a long time for measurement. As a result of studying in consideration of these points, the LOD content was preferably 0.2 U / cm ² or more.

When the content of potassium ferricyanide is more than 4.1 mg / cm ^2, there is an effect such that a reaction layer having a smooth or sufficient strength cannot be formed. From this, the content of potassium ferricyanide was preferably 4.1 mg / cm ² or less.

Although the reaction layer 7 is formed in contact with the surface of the electrode system in the above embodiment, it is not always necessary. When integrated with the cover 9 and the spacer 8 as shown in FIG. 2, a space is formed in the upper part of the electrode system by the cover 9, the spacer 8 and the insulating substrate 1. The reaction layer can be formed at an appropriate place as long as it is a portion corresponding to the wall of the space of the cover 9, the spacer 8 and the insulating substrate 1. Since the sample solution supplied to the sensor fills the space, the reaction layer can be dissolved.

In the above embodiment, CMC was used as the hydrophilic polymer.
Although not limited thereto, other cellulose derivatives, specifically, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose may be used, and further The same effect was obtained by using polyvinyl pyrrolidone, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, maleic anhydride and its salts.

On the other hand, as the electron acceptor, in addition to potassium ferricyanide shown in the above examples, p-benzoquinone, phenazine methosulfate, methylene blue, ferrocene derivative and the like can be used.

Further, in the above-mentioned examples, the method of dissolving the enzyme and the electron acceptor in the sample solution has been shown, but the invention is not limited to this, and the invention can be applied to the case where the enzyme and the electron acceptor are insolubilized in the sample solution. be able to.

Further, in the above embodiment, the two-electrode system consisting of the measuring electrode and the counter electrode is described, but the three-electrode system including the reference electrode enables more accurate measurement.

[0028]

As is apparent from the above description, an electrode system including at least a measuring electrode and a counter electrode is provided on an insulating substrate, and a hydrophilic polymer, a lactate oxidase and an electron acceptor are provided on the electrode system. By providing the containing reaction layer, a response current corresponding to the concentration of lactic acid can be obtained, so that a biosensor with high reliability can be provided by a simple operation.

[Brief description of drawings]

FIG. 1 is a sectional view of a biosensor according to an embodiment of the present invention.

FIG. 2 is an exploded perspective view of the biosensor according to one embodiment of the present invention excluding the reaction layer.

[Explanation of symbols]

 1 Insulating Substrate 2, 3 Lead 4 Measurement Electrode 5 Counter Electrode 6 Insulation Layer 7 Reaction Layer 8 Spacer 9 Cover 10 Sample Supply Hole 11 Air Hole

Claims (2)

[Claims] 1. An electrode system mainly composed of a measuring electrode and a counter electrode formed on an insulating substrate, and a reaction layer formed on the electrode system, wherein the reaction layer is at least a hydrophilic polymer, an enzyme, and an electron acceptor. A biosensor comprising a body, wherein the enzyme has an ability to oxidize lactic acid. 2. An electrode system mainly comprising a measuring electrode and a counter electrode is provided on an insulating substrate, a hydrophilic polymer layer is provided on the electrode system, and then at least on the hydrophilic polymer layer. A method for producing a biosensor, which comprises developing an aqueous solution containing lactate oxidase and an electron acceptor and drying the solution to form a reaction layer.