JPH05215710A - Glucose biosensor - Google Patents

Abstract

PURPOSE:To obtain a glucose biosensor which can coagulate a glucose oxidase immobilization film readily when a liquid solution for immobilizing glucose oxidase is dropped on the surface of an electrode and is dried. CONSTITUTION:0.5mul equivalent mixture liquid solution of aprotinin liquid solution of fibrinogen and glucose oxidase and calcium chloride aqueous solution of thrombin is applied to a platinum electrode surface, is dried by wind for one minute, and then is coagulated. The glucose oxidase immobilization film formation platinum electrode is used as an operation pole of a glucose biosensor.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to glucose biosensors. More specifically, the present invention relates to a glucose biosensor in which a glucose oxidase-immobilized film is formed on an electrode surface and used as a working electrode.

[0002]

2. Description of the Related Art In the production of a glucose biosensor having a glucose oxidase-immobilized membrane formed on the electrode surface as a working electrode, serum albumin or the like is used to immobilize glucose oxidase (for example, a special Kaihei 2
-120,655 publication). In this method, a step of dropping an aqueous solution of a mixture of glucose oxidase and serum albumin on the electrode surface and drying at low temperature is taken,
There was a problem that this drying process takes time.

[0003]

An object of the present invention is to provide a glucose biosensor capable of rapidly coagulating a glucose oxidase-immobilized film when a solution for immobilizing glucose oxidase is dropped on an electrode surface and dried. To provide.

[0004]

The object of the present invention is as follows.
This is achieved by a glucose biosensor in which a glucose oxidase-immobilized film immobilized with fibrinogen and thrombin is formed on the electrode surface and used as a working electrode.

In immobilizing glucose oxidase, about 10 to 200 mg of fibrinogen is mixed in 1 ml of an aprotinin solution (a preparation using bovine lung as a raw material, and fibrinogen is stably dissolved), and the mixture is heated to 37 ° C. to dissolve it. Solution of glucose oxidase in about 5 to 50 mg and calcium chloride (activator of blood coagulation factor XIII, used to crosslink fibrin polymer to obtain a stabilized fibrin clot. ) Thrombin (about 0.5 to 10 U) is mixed with 1 ml of an aqueous solution in which about 1 to 10 mg is dissolved, and the solution and the solution heated to 37 ° C. are mixed in equal amounts for about 10 seconds. 0.5 μl of this mixed solution is applied on the electrode surface of a platinum electrode, a gold electrode, etc., and air-dried to solidify in about 1 minute.

The glucose-immobilized membrane thus formed can be used as it is, but is preferably used after being crosslinked with glutaraldehyde. The cross-linking treatment is performed by a method of immersion in an aqueous solution of about 0.5 to 10% by weight of glutaraldehyde at room temperature for about 1 to 60 minutes, or a method of exposing to glutaraldehyde vapor for about 1 to 60 minutes.

A glucose biosensor is constructed by using an electrode having a glucose oxidase-immobilized film formed on the electrode surface in this way as a working electrode and using a silver or silver / silver chloride electrode in combination as a counter electrode. .. At the time of measurement, short the counter electrode and the reference electrode (unmodified electrode),
An applied voltage of about 0.65 to 0.75V is applied to it, and the steady-state current value is measured.

[0008]

INDUSTRIAL APPLICABILITY When immobilizing a glucose biosensor, fibrinogen and thrombin are used as immobilizing agents.
(Protein-degrading enzyme that has a catalytic action when fibrinogen is converted to fibrin) is used, the mixture solution is dripped on the electrode surface, and when air-dried, the coagulation of the mixture rapidly takes about 1 minute. It can be carried out.

[0009]

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

Example: To 1 ml of an aprotinin solution (3000 U), 90 mg of fibrinogen (manufactured by Immuno AG, human coagulation protein) was mixed, and the mixture was incubated at 37 ° C.
It was heated to dissolve. To this, 30 mg of glucose oxidase was added.

Separately, thrombin (4 U) was mixed with 1 ml of an aqueous solution in which 4.44 mg of calcium chloride was dissolved, and the mixture was heated to 37 ° C.

0.5 μl of each of these two types of solutions was taken and mixed for 10 seconds. After 30 seconds, 0.5 μl of this mixed solution was dropped on a platinum electrode (1.0 × 1.5 mm) and air-dried for 1 minute to coagulate.

Two such sensors were prepared, and one of them was immersed in a 2 wt% glutaraldehyde aqueous solution at room temperature.
Crosslink by immersing for 5 to 10 minutes, preferably about 10 minutes, leave the other one as it is, put it in a phosphate buffer at 4 ° C before use, and soak under stirring at 4 ° C for 48 hours did.

To measure the response to glucose, glucose was prepared as a phosphate buffer solution having a pH of 7.4, an ionic strength of 0.15 and a temperature of 37 ° C., and a counter electrode composed of a silver / silver chloride electrode was used as a reference electrode at an applied voltage of 0.7 V. Short-circuit and set the steady-state current value to BA
It was performed by measuring with an ammeter LC-4B manufactured by S company. The response of the two sensors to a glucose solution with a concentration of 100 mg / dl is 60 nA (crosslinking treatment) and 45 nA at steady current values, respectively.
(Untreated).

Claims (2)

[Claims] 1. A glucose biosensor in which a glucose oxidase-immobilized film immobilized with fibrinogen and thrombin is formed on an electrode surface to serve as a working electrode. 2. The glucose biosensor according to claim 1, wherein a glucose oxidase-immobilized membrane cross-linked with glutaraldehyde is used.