JPS61262652A - Oxygen sensor - Google Patents

Abstract

PURPOSE:To enable mass production by making it possible to simultaneously forming a large number of minute sensors on the same substrate, by arranging an enzyme- immobilized film, of which the enzyme is immobilized by a photocrosslinked polymer, to the former electrode of the anode and cathode each comprising a thin metal film formed to an insulating substrate. CONSTITUTION:The patterning of a positive type photoresist 3 is applied to an insulating substrate 1 so as to form electrode parts within the range of a substrate exposed parts and, subsequently, a chromium membrane and a gold or platinum membrane 5 are successively formed by a vacuum vapor deposition process. Thereafter, the resist is removed and an anode and a cathode each comprising a thin metal film are formed to predetermined parts on the insulating substrate 1. Next, an aqueous enzyme solution containing a photocrosslinkable polymer is applied to the surface of an electrode 5 to form a coating layer 6 which is, in turn, naturally dried and covered with a photomask 7. Then, the coating layer 6 is irradiated with ultraviolet rays to photocrosslink the photocrosslinkable polymer to form an enzyme-immobilized film 8 while electrode exposed surfaces are formed to the parts dissolved by a solvent and lead wires 10, 10' are mounted to the electrode exposed surfaces 5, 5'.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an enzyme sensor. More specifically, the present invention relates to an enzyme sensor that is compact and can be mass-produced.

[Conventional technology]

Recently, various biosensors that utilize biological reactions such as enzyme reactions and immune reactions have been developed, and especially in the clinical field, there is a growing demand for smaller, higher performance, and lower priced biosensors. Enzyme sensors are one type of such biosensors.

For example, glucose sensors that utilize the catalytic action of glucose oxidase are used to measure glucose concentrations in blood and urine, and are practically important for clinical tests on diabetic patients.

However, when these enzyme sensors are put into practical use,
Most commercially available products have a structure in which the enzyme immobilization membrane 11 is pressed against the electrode surface via the circle ring 12 or the soft plastic electrode membrane cover 13, as shown in FIG. has become complex and expensive. In addition, in FIG. 4, reference numeral 14 indicates a platinum cathode, and 15
16 indicates a glass cathode support, 16 indicates a silver anode, 17 indicates a plastic outer cylinder, and 18 indicates a potassium chloride electrolyte.

Further, as can be seen from this drawing, the electrode and the enzyme-immobilized membrane are not integrated, which hinders the achievement of miniaturization and cost reduction through mass production.

[Problem that the invention seeks to solve]

In view of these problems with commercially available enzyme sensors, research has recently progressed to immobilize enzymes directly on electrodes, and as a result, considerably smaller versions have been developed. However, if a large number of sensors are to be formed on the same substrate at the same time and mass production is possible, for example, the connecting part with the external electrode is exposed and the enzyme is immobilized only on the detection part. , it is necessary to immobilize the enzyme only on the necessary portions of the electrode substrate.

The present invention aims to solve these problems,
We have found that this problem can be solved by forming an enzyme-immobilized membrane using a photocrosslinked polymer.

[Means for Solving the Problem] and [Operation] Accordingly, the present invention relates to an enzyme sensor, which comprises:
An enzyme-immobilized film immobilized with a photocrosslinked polymer is installed on at least the former electrode of an anode electrode and a cathode electrode, which are made of a metal thin film formed on an insulating substrate.

Examples of combinations of a substrate detectable by this enzyme sensor and an enzyme as a catalyst that reacts with this substrate are as follows, in which both electrodes act as hydrogen peroxide electrodes.

]I■■0 glucose glucose oxidase galactose galactose oxidase - amino acid
L-amino acid oxidase ethanol alcohol oxidase phospholipid phospholipase choline When making a choline oxidase enzyme sensor, first a glass plate is used.

Hard resin plates such as vinyl chloride resin and polyimide resin, S
On a flat insulating substrate such as a silicon wafer with an insulating film of iO□, Si, N4, etc. formed on the surface.

Forming an anode electrode and a cathode electrode is performed. The electrodes are formed using gold scrap materials such as gold, platinum (for the anode) or silver, gold, platinum (for the cathode), and by the foot-off method as shown in FIG. 1 of the drawings.
The photo-etching method involves etching and removing a metal thin film deposited on a substrate for patterning, the mask evaporation method involves placing a mask with an electrode-shaped window on the substrate and depositing an electrode-forming material through the mask, and the mask-evaporation method, in which an electrode-forming material is deposited through the mask. This can be carried out by a screen printing method in which a conductive paint as a conductive material is printed in the shape of an electrode, or a plating method in which electroless plating of an electrode forming material is performed instead of vapor deposition in the above photoetching method or mask vapor deposition method.

In the embodiment shown in FIG. 1, a lift-off method is used. First, a positive photoresist 3 is patterned on a cleaned flat insulating substrate, for example, a glass plate 1, so that the electrode portion becomes the substrate exposed portion 2 (step a). Next, a chromium thin film 4 (about 500 mm thick) and a gold or platinum thin film 5 (about 0.2 mm thick) are deposited on this substrate by vacuum evaporation.
μm) are sequentially formed. Here, the chromium thin film is provided to improve the adhesion between the gold or platinum thin film forming the electrode and the glass plate substrate (step b).Then, the whole is immersed in a resist stripping solution such as acetone. The resist was removed, and the deposited thin film 5 remaining on the exposed substrate portion 2 was used as an electrode surface (Step C).

After forming anode electrodes and cathode electrodes made of metal thin films at predetermined locations on the insulating substrate in this way,
An enzyme-immobilized membrane is installed on at least the former electrode, and the installation is carried out using photolithography, for example, on the second electrode.
This is done as shown in the figure.

First, an enzyme aqueous solution containing a photocrosslinkable polymer is uniformly coated 6 on the electrode surface 5 on the insulating substrate 2 formed as shown in FIG. Step d).

As the photocrosslinkable polymer, a water-soluble polymer is generally used because it is dispersed in an enzyme aqueous solution. Polyvinyl alcohol having a group is used as an aqueous solution.

The above photocrosslinkable polymer aqueous solution (concentration 8° 5-12% by weight)
An aqueous enzyme solution prepared by dissolving 3 to 72 ml of enzyme in 0.8 mQ of distilled water is added to 1 g, and the mixture is stirred and mixed for several minutes before being used for coating.

The coating liquid is coated on the electrode surface on the insulating substrate, and when it dries naturally, it is covered with a film 7 having a negative or positive image, and the photocrosslinkable polymer is photocrosslinked by irradiation with ultraviolet rays, leaving the photocrosslinkable polymer uncrosslinked. The enzyme-immobilized membrane 8 is fixed with a photocrosslinked polymer on the photocrosslinked portion by eluting the portion with pure water.
and an electrode exposed surface 9 is formed in the solvent eluted portion (step e). This is irradiated with ultraviolet light again, dried, divided into each element, and lead wires 10 and 10' are attached to the electrode exposed surfaces 5 and 5' (step f). The enzyme sensor thus produced is One embodiment is shown in plan view in FIG.

When such an enzyme sensor is used as a glucose sensor, it functions as follows. First, the glucose sensor is immersed in a buffer solution that does not contain glucose, and a voltage of 0.8 V is applied to the electrode surface, for example, in the case of a gold electrode. When glucose is added to this, the glucose is immobilized by the glucose oxidase enzyme. It diffuses into the membrane and reacts as follows due to the catalytic action of the immobilized enzyme.

Hydrogen peroxide generated during this reaction is oxidized on the anode electrode as follows, and from the current value flowing at this time,
The hydrogen peroxide concentration is measured, and from this the glucose concentration can be determined (see Figure 4).

H, O, → 2H" + O□+ 2e- [Effect of the invention] Using a photolithography method, the connection part with the external electrode is exposed by means of immobilizing the enzyme with a photocrosslinked polymer,
By immobilizing the enzyme only on the detection part, it is possible to simultaneously form many microscopic sensors on the same substrate, making mass production possible.

〔Example〕

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

Example Photocrosslinkable polyvinyl alcohol (photocrosslinkable stilbazolium group content: 1.4 mol%, saponification degree: 88%).

0.4 mQ of distilled water in which 30 mg of glucose oxidase enzyme was dissolved was added to 0.5 g of an 11.7% by weight aqueous solution with a degree of polymerization of 1400), and the mixture was stirred and mixed for several minutes to prepare a coating solution. This coating liquid was applied onto a gold electrode formed on a glass plate at 4000 rpm.
Spin code for 20 seconds. After the coating solution had air-dried, it was covered with a film having a negative image, irradiated with ultraviolet rays (output 250 W) for 15 seconds, developed by washing with pure water, and irradiated with ultraviolet rays again.

Regarding the glucose sensor obtained in this way,
When a glucose calibration curve showing the relationship between glucose concentration and output current value was measured, the results shown in Figure 5 were obtained, and a good linear relationship was observed within the glucose concentration range of 0 to 2゜lIg/dll. It was done.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view sequentially showing the steps of forming an electrode on an insulating substrate. FIG. 2 is a cross-sectional view showing the steps of installing an enzyme-immobilized membrane immobilized with a photocrosslinked polymer on an electrode formed on an insulating substrate, and FIG. FIG. 2 is a plan view of one embodiment of the produced enzyme sensor. FIG. 4 is a cross-sectional view of a conventional enzyme sensor. Also. FIG. 5 is a graph of a calibration curve of the enzyme sensor according to the present invention. (Explanation of symbols) 1... Insulating substrate 2... Substrate exposed portion 5... Electrode 6... Enzyme aqueous solution containing photocrosslinkable polymer 7...
・Film with image 8... Enzyme immobilization membrane 9... Electrode exposed surface

Claims (1)

[Scope of Claims] 1. An enzyme-immobilized film immobilized with a photocrosslinked polymer is installed on at least the anode electrode and the cathode electrode, which are made of a metal thin film formed on an insulating substrate. enzyme sensor. 2. The enzyme-immobilized film immobilized with a photocrosslinkable polymer is formed by applying a photolithography method to an enzyme aqueous solution containing the photocrosslinkable polymer. enzyme sensor. 3. The enzyme sensor according to claim 1, wherein both electrodes constitute hydrogen peroxide electrodes.