JPH02120655A - Glucose sensor - Google Patents

Abstract

PURPOSE:To facilitate the immobilization of enzyme and manufacturing by a method wherein a glucose oxidaze immobilizing flat plate-shaped acting electrode is bonded to the cut surface of a hollow needle like opposed electrode through an insulating layer in such a state that the enzyme immobilizing surface of the acting electrode is turned toward the inside. CONSTITUTION:A hollow needle like opposed electrode 1 is formed by obliquely cutting the leading end part of the half cut body of a platinum hollow needle. Glucose oxidase (GOD) 7 is immobilized on the single surface of a GOD immobi lizing flat plate-shaped acting electrode 5. The acting electrode 5 is bonded to the electrode 1 through the epoxy resin adhesive layer applied to the cut surface of the electrode 1 so that the enzyme immobilizing surface of the acting electrode 5 is turned toward the inside. By this method, a sensor easy in the immobilization of enzyme and manufacturing, easily inserted in a living body and suitable for measuring a sugar level in blood is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a glucose sensor. For more details,
The present invention relates to an amperometric glucose sensor.

[Conventional technology]

In conventional amperometric glucose sensors,
Two electrodes are required, one of which is a counter electrode and the other is a working electrode, and glucose oxidase enzyme (COD) is immobilized on the four working electrodes. The principle of this sensor is that the glucose concentration can be determined by quantifying the electrode detection substance generated on the working electrode as a result of the reaction between glucose and COD.

Incidentally, one type of glucose sensor is one with a needle-like structure, but in the conventional amperometric glucose sensor, these two electrodes are constructed by arranging flat plate-shaped objects such as glass plates in parallel. A needle-shaped electrode that is easy to insert into a living body and can be suitably used for measuring blood sugar levels, etc., has not yet been put into practical use. This is thought to be due to the difficulty in manufacturing due to the shape itself and the difficulty in immobilizing the enzyme.

[Problem to be solved by the invention]

An object of the present invention is to overcome such problems and to provide an amperometric glucose sensor that allows enzyme immobilization and ease of production.

[Means to solve the problem]

The glucose sensor of the present invention that achieves this object has a flat working electrode with glucose oxidase enzyme immobilized on the cut surface of a hollow needle-shaped counter electrode that has been cut in half in the length direction with an insulating layer interposed therebetween, with the enzyme-immobilized surface side facing inside. Glue it to the right direction.

FIG. 1 of the drawings shows one embodiment of the glucose sensor according to the present invention as a perspective view in a state before adhesion. That is, the acicular counter electrode 1 is generally made of platinum, gold, or gold having an outer diameter of about 1 m or less.
It is a longitudinal half-cut piece 2 of a hollow needle such as titanium, and its tip 3 is cut obliquely into the shape of a hypodermic needle so that it can be easily inserted. The half-cut surface is generally coated with an insulating layer 4, 4' which also serves as an adhesive layer, such as an epoxy resin adhesive, a silicone adhesive, or glass.

On the other hand, the glucose oxidase enzyme-immobilized flat working electrode 5, which is adhered to the hollow needle-like counter electrode through this insulating layer, is a flat plate 6 made of platinum, titanium, etc. with a thickness of approximately 0.05 to 1 m, and is generally GOD is immobilized on one side of which the length is less than the length of the opposite electrode cut surface and the width is equal to the cut surface interval 7
It's forcing me.

When immobilizing COD on the working electrode, a film-forming material,
For example, celluloses such as cellulose acetate.

Synthetic polymeric substances such as polyvinyl butyral and polysulfone, or natural polymeric substances such as cellulose, chitin, albumin, sodium alginate, agarose, and carrageenan are used, and after preparing them as a dope solution by dissolving them in a soluble solvent, immersion is performed. method, drop method,
Apply it on the working electrode surface using the spinner method or the like. Further, as a film-forming material, a water-soluble photocrosslinkable polymer such as polyvinyl alcohol having a photosensitive group such as a stilbazolium group or a diazo group as a photocrosslinkable group in the molecule can also be used in the form of an aqueous solution.

GOD on the eye formed by these film-forming materials
The immobilization is carried out by commonly used methods such as a covalent bonding method using glutaraldehyde, carbodiimide, etc., an ionic bonding method, an adsorption method, and a crosslinking method. Alternatively, GOD can be mixed in a solution of a film-forming material or the like, and then immobilized by one of these immobilization methods during film formation.

In this case, when a photocrosslinkable polymer is used, its photocrosslinking occurs at a wavelength of about 350 to 350 nm, which is a wavelength that does not deactivate COD.
This is carried out by light irradiation with near ultraviolet light of 450 nm.

As a result of such a reaction of quantifying glucose using immobilized COD, the generated 1(20□) is detected as a change in the oxidation current value at the working electrode.

Also, a ferrocene compound which is an electron carrier (electron carrier), specifically ferrocene [bis(cyclopentagenyl)iron (■)] or a derivative thereof.

For example, using l,1'-dimethylferrocene, etc., it is vacuum-deposited on the working electrode surface, and then COD is applied to this vapor-deposited surface.
is immobilized by a commonly used immobilization method,
Alternatively, after immobilizing COD, a ferrocene compound is layered thereon by a dropping method or the like.

j＠ In this case, for one molecule of glucose to be oxidized, 2
Since electrons move to the working electrode, this can be detected as a change in current value.

The GOD-immobilized plate-like working electrode is adhered to the hollow needle-like counter electrode with the enzyme-immobilized surface facing inward. Note that lead wires 8 and 8' are drawn out from the counter electrode and the working electrode, respectively, and a reference electrode is also used in addition to these electrodes during measurement.

〔Effect of the invention〕

In the needle-shaped amperometric glucose sensor according to the present invention, since the glucose oxidase enzyme is immobilized on the plate-shaped working electrode surface, there is no difficulty therein, and the needle-shaped i'l! There are no particular difficulties in manufacturing due to the polar shape, and the glucose sensor is provided as a glucose sensor that can be easily inserted into a living body and can be suitably used for measuring blood sugar levels and the like.

〔Example〕

Next, the present invention will be explained with reference to examples.

Example A glucose sensor of the embodiment shown in FIG. 1 was manufactured.

The hollow needle-shaped counter electrode has an inner diameter of 0.8 mm and an outer diameter of 1. Onwn,
A platinum hollow needle with a length of 31 mm is cut in half and the tip is cut obliquely.As a flat working electrode, 1. OX2.5X0
．． A size of 2+nm is used, and one side of it is coated with CO.
D was immobilized.

For immobilization of GOD, GO030■ and bovine serum albumin 30■ were dissolved in 0.4 mΩ of 5 InM tris-maleate buffer at pH 7.0, 4 μQ of the solution was dropped onto the plate-like working electrode surface, and the solution was incubated at 4°C for 4 hours. After leaving it for a while, there 1,1'
-Drop 4μQ of dimethylferrocene acetone solution,
This was carried out by leaving the mixture at 4° C. for 4 hours again.

This COD-immobilized flat working electrode was adhered to a hollow needle-like counter electrode with the enzyme-immobilized side facing inward through an epoxy resin adhesive layer applied to its cut surface, and left at 4°C for 48 hours. did.

Connect each of these electrodes to a potentiostat.

Potential between counter electrode and working electrode 50 mV, working electrode and reference electrode (Ag/
The response to glucose was measured at a potential of 200 mV between the AgCQ electrodes. Measurements were carried out at 37°C and pH 7.0.
5mM Tris-maleate buffer.

As shown in the graph of FIG. 2, the results showed a linear correlation with the steady-state value of the response current within the glucose concentration range of 50 to 300 mg/dQ.

[Brief explanation of drawings]

FIG. 1 is a perspective view showing one embodiment of the glucose sensor of the present invention in a state before adhesion. Moreover, FIG. 2 is a graph showing the relationship between glucose concentration and output current when this glucose sensor is used. (Explanation of symbols) 1... Needle-like counter electrode 2... Half-cut hollow needle 4... Insulating layer 5... GOD-immobilized plate-like working electrode 6...
・Flat plate 7...immobilized COD

Claims (1)

[Claims] 1. A glucose sensor in which a flat plate-like working electrode with glucose oxidase enzyme immobilized is adhered to the cut surface of a hollow needle-like counter electrode that has been cut in half in the length direction via an insulating layer, with the enzyme-immobilized surface facing inward.