JP3127599B2 - Biosensor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to, for a specific component in a sample, about the biosensor service which can be conveniently carried out quickly and accurate quantification.

[0002]

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

[0003] The above-mentioned measuring instrument has a flow line, and after mixing and diluting a sample solution with a buffer solution, air containing oxygen is mixed therein and introduced into the enzyme electrode portion through a defoamer.
Lactic acid in the sample solution reacts with LOD in the immobilized enzyme membrane, and hydrogen peroxide is generated at the same time. Lactic acid is quantified by measuring the amount of increase in hydrogen peroxide with a hydrogen peroxide electrode.

[0004]

In such a conventional measuring method, it is necessary to replace the enzyme membrane.
Equipment with pumps, mixers, defoamers, etc. is required. Therefore, maintenance such as cleaning of the flow line is required, and a complicated and simple operation such as supplying a certain amount of sample liquid has been lacking.

[0005] It is an object of the present invention to provide a biosensor capable of quickly and extremely simply performing high-precision quantification of lactic acid in a sample solution.

[0006]

For the present invention SUMMARY OF THE INVENTION The To achieve the above object, provided with at least the measurement electrode and the electrode system consisting of a counter electrode on an insulating substrate, and a hydrophilic polymer on the electrode system, lactic acid A reaction layer containing lactate oxidase as an oxidase and potassium ferricyanide as an electron acceptor , wherein the lactate oxidase in the reaction layer is provided.
And the content of zero is 0.2 U / cm ² or more, the content of the potassium ferricyanide in the reaction layer is 4.1mg
/ Cm ² or less.

[0007]

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

[0008]

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

(Example 1) FIG. 1 is a cross-sectional view of a lactic acid sensor manufactured as an example of the biosensor of the present invention, and FIG. 2 is an exploded perspective view of FIG. .

Hereinafter, a method for manufacturing a lactic acid sensor will be described. A silver paste is printed on the insulating substrate 1 made of polyethylene terephthalate by screen printing, and
, And were formed. Next, using a conductive carbon paste containing a resin binder, the measuring electrode 4 of the electrode system was used.
Subsequently, an insulating layer 6 made of an insulating paste was sequentially formed by printing. The insulating layer 6 keeps the area (about 1 mm ² ) of the exposed portion of the measuring electrode 4 constant and partially covers the leads 2 and 3. Finally, the counter electrode 5 of the electrode system was formed by printing 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 developed and dried on the electrode system to form a CMC layer. Subsequently, a mixed aqueous solution of LOD as an enzyme and potassium ferricyanide as an electron acceptor was spread on the CMC layer, and dried in a hot-air drier to form a reaction layer 7.

In the above-mentioned reaction layer forming step, when a mixed aqueous solution of LOD and potassium ferricyanide is dropped, the CMC layer is dissolved once and forms a reaction layer 7 in a form mixed with an enzyme or the like in a subsequent drying process. However, since no stirring or the like is involved, a completely mixed state is not obtained, and the surface of the electrode system is in a state covered with only 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 to the surface of the electrode system. Obtainable.

After forming the reaction layer 7 as described above,
The cover 9 and the spacer 8 were adhered in a positional relationship as indicated by a chain line in FIG. When a transparent material is used for the cover, the state of the reaction layer and the state of introduction of the sample solution can be checked 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 contacting the sample supply hole 10 at the tip of the sensor due to the capillary action of the space generated by the cover and the spacer. Note that, in order to further smoothly supply the sample liquid, it is preferable that an organic solvent solution of lecithin is spread on the surface from the sample supply section to the reaction layer and dried as needed.

Further, when the treatment with lecithin is performed, the sample liquid can be supplied even when the space created by the cover and the spacer is too small to exhibit the capillary phenomenon.

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 determine the amount beforehand. 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 prepared by adjusting lactic acid to a pH of about 7 with 0.2 M phosphate buffer was supplied from the sample supply hole 10 to the lactic acid sensor prepared as described above. The sample liquid quickly reached the air holes 11 and the reaction layer on the electrode system was dissolved.

One minute after the supply of the sample solution, the measurement electrode 4 is set to +0.5 V in the anode direction with respect to the counter electrode 5 of the electrode system.
When the pulse voltage was applied and the current value after 5 seconds was measured, 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, the lactic acid in the sample solution is oxidized by LOD, and at the same time, potassium ferricyanide is reduced to potassium ferrocyanide. Next, by the application of 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 is contained in the reaction layer.
Is 4 U / cm ² , and potassium ferricyanide is 0.65 m
g / cm ² . When the contents of the enzyme and the electron acceptor were examined, the following results were obtained.

[0021] When the amount of the enzyme was reduced, the reaction rate was reduced, and the effect of taking a long time for the measurement was observed. Investigations taking these points into consideration revealed that 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, an effect such as the inability to form a smooth or sufficiently strong reaction layer was observed. Thus, the content of potassium ferricyanide was preferably 4.1 mg / cm ² or less.

Although the method of forming the reaction layer 7 in contact with the surface of the electrode system has been described in the above embodiment, it is not always necessary. When the cover 9 and the spacer 8 as shown in FIG. 2 are integrated, a space is formed above the electrode system by the cover 9, the spacer 8 and the insulating substrate 1. A reaction layer can be formed at an appropriate place as long as it corresponds to the wall surface of the space portion of the cover 9, the spacer 8, and the insulating substrate 1. Since the sample liquid supplied to the sensor fills the space, the reaction layer can be dissolved.

In the above embodiment, CMC was used as the hydrophilic polymer.
It was used, but is not limited thereto, other cellulose derivatives, specifically, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethylethyl cellulose may be used. Similar effects were obtained by using polyvinylpyrrolidone, polyvinyl alcohol, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch and its derivatives, and maleic anhydride and its salts.

[0025]

In the above embodiment, the method of dissolving the enzyme and the electron acceptor in the sample solution has been described. However, the present invention is not limited to this, and the present invention is also applicable to the case where the enzyme and the electron acceptor are insolubilized in the sample solution by immobilization. be able to.

Further, in the above-described embodiment, a two-electrode system including a measurement electrode and a counter electrode has been described. However, a three-electrode system including a reference electrode enables more accurate measurement.

[0028]

As is apparent from the above description, an electrode system including at least a measurement electrode and a counter electrode is provided on an insulating substrate, and a hydrophilic polymer, 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 highly reliable biosensor can be provided by a simple operation.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a biosensor according to one embodiment of the present invention.

FIG. 2 is an exploded perspective view of a biosensor according to an embodiment of the present invention, from which a reaction layer is removed.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 insulating substrate 2, 3 lead 4 measuring electrode 5 counter electrode 6 insulating layer 7 reaction layer 8 spacer 9 cover 10 sample supply hole 11 air hole

Continuation of the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 27/327 G01N 27/28 331

Claims (1)

(57) [Claims] An electrode system mainly comprising a measurement electrode and a counter electrode formed on an insulating substrate, and a reaction layer formed on the electrode system, wherein the reaction layer comprises at least a hydrophilic polymer and lactic acid oxide.
Consists oxidase and potassium ferricyanide, or One content of the lactate oxidase of the reaction layer is 0.2 U /
cm ^{2 or} more, the ferricyanide in the reaction layer
A biosensor having a potassium content of 4.1 mg / cm ² or less.