JPH05203607A - Glucose biosensor - Google Patents

Abstract

PURPOSE:To provide a glucose biosensor with which glucose quantity in solution including protain, lipoid, glycolipid, blood corpuscle, etc., can be measured without attachment of these components to a film surface, and with which, therefore, problems of prohibiting responsiveness or deterioration of durability can be eliminated. CONSTITUTION:Ethylenediamine tetra sodium acetate solution-enclosed polyamide microcapsules are mixed with water-soluble photo-crosslinkable resin solution, obtained matter is applied on a glucose oxidase stabilized polyvinyl chloride film-coated platinum electrode, and UV ray is radiated. A sensor using obtained matter for a working pole is connected to an ammeter, a counter pole is shortcircuitted to a reference pole of the ammeter, a potential is applied to the working pole, and response to the glucose solution is measured.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to glucose biosensors. More specifically, it relates to a glucose biosensor having improved durability.

[0002]

2. Description of the Related Art In conventional biosensors, when an attempt is made to further form a film on an enzyme-immobilized film, the material thereof is mainly cellulose acetate. The purpose of forming such a cellulose acetate-based film is to limit the diffusion of the substrate,
To expand the range of calibration. However, protein,
When trying to measure the amount of glucose in a solution containing lipids, sugars, blood cells, etc., such as blood, these components adhere to the surface of the membrane and coagulate, resulting in inhibition of response and durability. There was a problem of being inferior.

[0003]

The object of the present invention is to measure the amount of glucose in a solution containing proteins, lipids, sugars, blood cells, etc., even if these components are present on the membrane surface. (EN) Provided is a glucose biosensor which does not adhere to or coagulate on the surface of the body and therefore does not have a problem that the response is inhibited or the durability is poor.

[0004]

The object of the present invention is as follows.
This is achieved by a glucose biosensor in which sodium ethylenediaminetetraacetate aqueous solution-encapsulated microcapsules are immobilized on a glucose oxidase-immobilized electrode and used as a working electrode.

Sodium ethylenediaminetetraacetate has a function of capturing calcium ions and inhibiting coagulation of blood and the like. Sodium ethylenediaminetetraacetate having such a function is encapsulated in a microcapsule, and the sustained release of sodium ethylenediaminetetraacetate through the capsule wall prevents the coagulation of blood or the like at the glucose oxidase-immobilized electrode that has fixed it. The durability of the glucose sensor response is improved.

The microencapsulation is carried out by using a commonly used interfacial polymerization method, phase separation method or the like. The formed microcapsules are made of gum arabic-gelatin, polyamide, polyimide or the like, and the particle size thereof is not particularly limited to about 0.1 to 1000 μm, and a sodium ethylenediaminetetraacetate aqueous solution encapsulated therein is used. The concentration of is also not particularly limited.

Microcapsules containing an aqueous solution of sodium ethylenediaminetetraacetate are fixed on a glucose oxidase-immobilized electrode. As the electrode, a platinum electrode, a gold electrode, etc. are generally used. When immobilizing glucose oxidase on these electrode surfaces, it is preferable that the electrode surfaces are once coated with a vinyl chloride resin film and then immobilized.

The vinyl chloride resin film is formed on the electrode as follows. That is, polyvinyl chloride or a copolymer obtained by copolymerizing a small amount of ethylene with vinyl chloride, vinyl acetate or the like is used as a soluble solvent thereof, for example, dimethylformamide, cyclohexanone, tetrahydrofuran,
A solution of methyl ethyl ketone dissolved in a concentration of about 0.1 to 20% by weight is generally dropped directly on the electrode surface at room temperature and then air-dried, or the electrode surface is immersed in the solution,
After pulling up, it is air dried, washed with water and dried.

After coating the electrode surface with a vinyl chloride resin film in this manner, it is immersed in an aqueous glucose oxidase solution having a concentration of about 1 to 200 mg / ml at 4 ° C. for about 1 to 120 minutes, and then pulled up and washed with water. After drying, glucose oxidase is immobilized. Such a method for immobilizing glucose oxidase is not only simple, but also has good adhesiveness to the electrode surface of the vinyl chloride resin film and immobilizability of glucose oxidase, and has excellent results in terms of responsiveness. Shows.

Immobilization of the ethylenediaminetetraacetic acid sodium salt aqueous solution-encapsulated microcapsules on the glucose oxidase-immobilized electrode is carried out by using a suitable adhesive substance,
For example, it is carried out by a method in which microcapsules are dispersed in a solution of a water-soluble photocrosslinkable resin, albumin, fibrinogen, chitosan, etc., and then applied and cured.

To measure the amount of glucose, a sensor having the thus obtained one as a working electrode is connected to an ammeter, and a counter electrode (silver electrode, silver / silver chloride electrode) and a reference electrode (silver electrode) of the ammeter are connected. )
It is performed by short-circuiting and, applying a potential to the working electrode, and measuring the response to the glucose solution.

[0012]

According to the present invention, proteins, lipids, sugars,
A glucose biosensor having excellent durability is provided even when used in a solution containing blood cells and the like.

[0013]

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

EXAMPLE A dope solution (0.5 μl) consisting of a 10 wt% cyclohexanone solution of polyvinyl chloride was applied on a platinum electrode (1.0 × 1.5 mm), left at room temperature for 1 minute and then immersed in water for gelation. ,
A polyvinyl chloride film was formed.

This polyvinyl chloride film-coated platinum electrode was immersed in a 1 mg / ml aqueous solution of glucose oxidase (product of Sigma) at 4 ° C. for 24 hours to immobilize and fix glucose oxidase on the membrane surface.

Separately, microcapsules containing an aqueous solution of sodium ethylenediaminetetraacetate were produced as follows. 0.4 mol of 1,6-hexanediamine, 0.4
After adding an equal amount of water to 7.5 ml of an aqueous solution in which 5 mol of sodium carbonate and 0.2 mol of sodium ethylenediaminetetraacetate are dissolved, 15 ml of this diluted aqueous solution is used as a surfactant.
(Span 85) is added to 75 ml of a chloroform-cyclohexane (volume ratio 1: 4) mixed solvent solution in which 5% by volume is dissolved, and well stirred to emulsify to form a W / O type emulsion.

Five minutes after the emulsification, 0.6 g of terephthalic acid dichloride dissolved in 75 ml of the above mixed solvent was added while continuing stirring (400 rpm) at 4 ° C. As a result, a polycondensation reaction occurs between equimolar amounts of terephthalic acid dichloride and 1,6-hexanediamine on the surface of water droplets in the emulsion,
A polycondensation product is obtained. Sodium carbonate is a neutralizing agent for the hydrogen chloride produced in this reaction. The produced microcapsules (5 μm diameter) were centrifuged and collected.

0.5 ml of the microcapsules containing the sodium ethylenediaminetetraacetate aqueous solution and 0.5 ml of the water-soluble photocrosslinkable resin aqueous solution (11.1% aqueous solution of styrylpyridinium group-containing polyvinyl alcohol containing Toyo Gosei Co., Ltd.) were mixed, and 0.5 μl of this mixed solution Is applied to the glucose oxidase-immobilized polyvinyl chloride film-coated platinum electrode at 25 ° C.
After being dried for 1 hour, the sample was irradiated with ultraviolet rays having a wavelength of 360 nm for 10 seconds, washed with water, and further irradiated with ultraviolet rays for 30 seconds.

The thus obtained one was used as a working electrode, the counter electrode composed of a silver / silver chloride electrode was short-circuited with a reference electrode of an ammeter at an applied voltage of 0.7 V, and the steady current value was measured by a BAS ammeter. The response to glucose was measured by measuring with LC-4B.

Specifically, the response steady-state current value to fresh whole pig blood immediately after blood collection was measured, and the measured value (150 nA) was 100%.
And After the measurement, the sensor is washed with physiological saline and 4 ℃
Saved in. This kind of operation was performed once a day.
The steady-state current value after day still kept 95% of the initial value.

Comparative Example In the examples, microcapsules enclosing an aqueous solution of sodium ethylenediaminetetraacetate were not used, but only an aqueous solution of a water-soluble photocrosslinkable resin was applied onto a glucose oxidase-immobilized polyvinyl chloride film-coated platinum electrode, followed by drying and UV irradiation. Irradiated.

When the response to glucose was similarly measured using the working electrode thus obtained, the steady-state current value decreased to 10% of the initial value on the second day, and no response was exhibited on the third day. It was

Claims (2)

[Claims] 1. A glucose biosensor in which a sodium ethylenediaminetetraacetate aqueous solution-encapsulated microcapsule is immobilized on a glucose oxidase-immobilized electrode to serve as a working electrode. 2. The glucose biosensor according to claim 1, wherein the glucose oxidase-immobilized electrode is a glucose oxidase-immobilized vinyl chloride resin film-coated electrode.