JPH0850112A - Glucose biosensor - Google Patents

Abstract

PURPOSE:To prevent a glucose biosensor from being changed with the lapse of hours after the responsiveness reaches its peak by successively laminating on a working pole a cellulose acetate film, a mixture film of glucose oxidase and photocrosslinked polyvinyl alcohol, and a perfluore-sulfonic acid type cation exchange film. CONSTITUTION:A working electrode 1, a counter electrode 2, and a reference electrode 3 are formed on a base plate 4, and on the working pole 1, a cellulose acetate film 5, a mixture film 6 of glucose oxidase(GOD) and photocrosslinked polyvinyl alcohol(PVA), and a Nafion film 7 are successively laminated, and a large part of the respective electrodes is covered with an insulating film 8. Thus, the permeation of a reducing substance can be prevented, and only H2O2 is allowed to permeate. By providing a perfluore-sulfonic acid type ion exchange film on the reverse side thereof, the change with the lapse of hours after the responsiveness reaches its peak can be eliminated for enhancing the responsiveness.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to glucose biosensors. More specifically, the present invention relates to a glucose biosensor with improved response characteristics.

[0002]

2. Description of the Related Art When a biosensor is evaluated by a batch method, there is a method of taking a steady-state response as a calibration method (end point method). As an effective condition when applying this method, after the response of the biosensor reaches the peak value,
It is required that it does not change over time.

However, in the conventional biosensor, the response often changes after reaching the peak value, which is a practical problem, and the glucose biosensor is no exception.

The operating principle of the glucose biosensor is as follows. Glucose is glucose oxidase
Oxidized by (GOD). Since the generated H ₂ O ₂ is proportional to the glucose concentration, the glucose amount can be quantified by measuring the H ₂ O ₂ amount with a base sensor (H ₂ O ₂ sensor). In this case, GOD is entrapped and immobilized in a photocrosslinked polyvinyl alcohol (PVA) film.

In such a structure of the GOD-photocrosslinked PVA mixture film / H ₂ O ₂ sensor, it is difficult to secure selectivity because the H ₂ O ₂ sensor also responds to a reducing substance such as ascorbic acid. Becomes Therefore, it is necessary to provide a film that allows H ₂ O ₂ to permeate but does not allow a reducing substance to permeate.

[0006]

In order to solve these problems, a structure was studied in which a cellulose acetate film was first formed on the working electrode and then a GOD-photocrosslinked PVA mixture film was formed. The glucose biosensor having such a structure does not respond to a reducing substance, but the phenomenon that the output waveform for glucose gradually decreases after passing the peak value was observed.

[0007] Such a phenomenon is caused by the fact that H ₂ O ₂ generated in the GOD-photocrosslinked PVA mixture film initially permeates the cellulose acetate film and reaches the working electrode. It is considered that the output will decrease due to the despreading in ().

The object of the present invention is to provide a cellulose acetate membrane that allows H ₂ O ₂ generated when glucose is oxidized and converted into gluconolactone by the action of glucose oxidase to pass therethrough, but does not allow a reducing substance to pass therethrough. In the glucose biosensor formed on the working electrode and then formed with the glucose oxidase-photocrosslinked polyvinyl alcohol mixture film, after the response value reached a peak,
It is to provide a product that does not change over time.

[0009]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
This is achieved by a glucose biosensor in which a cellulose acetate membrane, a glucose oxidase-photocrosslinked polyvinyl alcohol mixture membrane and a perfluorosulfonic acid type cation exchange membrane are sequentially laminated on the working electrode.

Although the base sensor used in the present invention is an H ₂ O ₂ sensor, it should have a two-pole structure of a working electrode and a counter electrode or a three-pole structure of a working electrode, a counter electrode and a reference electrode structurally. 1 and 2, the working electrode 1, the counter electrode 2, and the reference electrode 3 have a three-pole structure.

As the working electrode (anode) material, platinum,
Gold, carbon, etc., platinum as the counter electrode (cathode) material,
Silver, carbon, etc., and silver / silver chloride etc. are used as the reference electrode material. The working electrode and the counter electrode are formed on a substrate made of ceramics, glass, plastic, paper or the like by a vapor deposition method, a sputtering method, a screen printing method or the like, and the reference electrode is generally formed by a screen printing method.

First, a cellulose acetate (which may be any of triacetyl cellulose, diacetyl cellulose and butyryl acetyl cellulose, preferably triacetyl cellulose is used) film is formed on the working electrode. The formation of a cellulose acetate film is carried out by adding cellulose acetate to at least one of its soluble solvents such as acetone, cyclohexanone, etc., at about 20-100 cps, preferably about 60-90 cps.
so that the viscosity (room temperature) is about 1 to 7 W / V%, preferably about 4 to 6 W / V% after preparing a dope by dissolving it in a concentration of
Apply this dope solution with a spinner, etc., dry at a temperature of about 10 to 30 ° C, and then remove it in n-hexane to remove the solvent.
Immersion is carried out for 0.5 to 2 hours, preferably about 1 to 1.5 hours, and a film having a dry film thickness of about 0.1 to 2 μm, preferably about 0.8 to 1.5 μm is formed thereon.

On the cellulose acetate membrane, GOD-photocrosslinked PVA
The mixture film is laminated. Such a mixture film, polyvinyl alcohol containing about 1 to 3.5 mol% of photocrosslinkable groups such as stilbazolium group and styrylpyridinium group (saponification value of about 70 to 100, degree of polymerization of about 500 to 2000) has a solid concentration of about. Five~
In an aqueous solution dissolved at a concentration of 50% by weight, preferably about 10 to 20% by weight, GOD (for example, about 100,000 unit / g solid GOD about 10 to 50 mg per 1 ml of 15% photocrosslinkable PVA aqueous solution). And distilled water were added to prepare a dope solution having a total volume of 1 ml, which was coated with a spinner or the like, dried at a temperature of about 10 to 30 ° C., and then
Wavelength that does not impair the activity of GOD, preferably by irradiating ultraviolet rays having a wavelength of 405 nm for a predetermined time to perform photocrosslinking, to form a photocrosslinking film, where the film thickness when dried is about 0.1 to 2 μm.
A mixed membrane film of m, preferably about 0.5-1 μm is formed.

On such a cellulose acetate membrane, a perfluorosulfonic acid type cation exchange membrane is further laminated. The perfluorosulfonic acid type cation exchange membrane is a dope solution (concentration about 0.5 to 10% by weight, preferably about 1% by weight) of a perfluorosulfonic acid type cation exchange resin, for example, an organic solvent solution of Nafion, a product of DuPont. (~ 5% by weight)
Is applied with a spinner or the like and dried at about 10 to 30 ° C., so that the dry film thickness is about 0.05 to 3 μm, preferably about 0.1 to
It is formed as a 0.5 μm cation exchange membrane.

Nafion has a structure in which a perfluoropolyalkylene ether side chain having a terminal sulfonic acid group is bonded to a perfluoromethylene main chain, and it is a chemistry conventionally used in the chlorine and caustic soda industry. Is a stable cation exchange resin, and its solution in an organic solvent, such as Nafion 117, with isopropanol and n
-A solution prepared by dissolving about 5 W / V% in a mixed solvent containing propanol as a main component is commercially available as Aldrich manufactured by DuPont, and it can be used as it is in the present invention.

1 and 2 are a plan view and an end view taken along line II of one embodiment of the glucose biosensor according to the present invention. As described above, the working electrode 1, the counter electrode 2 and the reference electrode 3 are It is formed on a substrate 4, and a cellulose acetate film 5, a GOD-photocrosslinked PVA mixture film 6 and a Nafion film 7 are sequentially laminated on a working electrode 1, and most of each electrode is formed by an insulating film 8. Is covered.

[0017]

INDUSTRIAL APPLICABILITY In the glucose biosensor according to the present invention, a cellulose acetate film is provided on the working electrode side of the glucose oxidase-photocrosslinked polyvinyl alcohol mixture film to prevent the permeation of reducing substances and to reduce H ₂ O _{2. By} allowing only ₂ to pass through and providing a perfluorosulfonic acid type cation exchange membrane on the opposite side, it is possible to eliminate the change over time after the response value reaches its peak and improve the response characteristics. The effect is obtained.

[0018]

Next, the present invention will be described by way of examples.

Example (Preparation of Electrode) After cleaning an alumina substrate (Kyocera product A-493) with ultrasonic waves, a platinum vapor deposition apparatus (ULBX EBX-12C) was used to obtain a film thickness of 0.4 μm (DEKTAK 3030ST). Measurement), sheet resistance 300
A platinum film of mΩ / □ (measured by Nakamura Seimitsu Shokai Resistest-6) was formed on the entire surface of the substrate. Then, a positive type resist (OFPR800 manufactured by Tokyo Ohka Kogyo Co., Ltd.) was used to form an electrode pattern using a spin coater 1H-DX manufactured by Mikasa and a mask aligner M-2L.

Plasma etching equipment (ULVAC RBH-303
(0) is used to perform plasma etching on the formed electrode portion, and then the conduction is confirmed, and the dicing saw (D
It was cut into a predetermined size by ISCO DAD-2H / 6T).

Of the three electrodes on the substrate, two were used as the working electrode and the counter electrode, and one was used as the lead for the reference electrode. A silver / silver chloride electrode was formed on this platinum electrode for reference electrode.
For silver / silver chloride electrode, first use a silver paste (LSA-504J, product of Asahi Chemical Laboratory) to form a silver electrode by coating and baking by screen printing, and then in a 0.1 M hydrochloric acid electrolytic solution.
Constant-current electrolysis was performed for 20 minutes at a current density of 0.6 mA / cm ² (HA-501 / HB-104 manufactured by Hokuto Denko) was used to form a reference electrode on the substrate.

(Formation of laminated film on working electrode) Triacetyl cellulose (1.5 g) was added to acetone (25.5 ml) -cyclohexanone (3 ml).
A dope solution (concentration 5 W / V%, viscosity 85 cps) prepared by dissolving in a mixed solvent was applied to the working electrode by a spinner (4000 rpm, 15
After allowing to stand for 30 seconds at 25 ° C., it was immersed in n-hexane for 1 hour to form a cellulose acetate film having a dry film thickness of 1.0 μm.

Photocrosslinkable polyvinyl alcohol (product of Toyo Gosei Co., Ltd .; stilbazolium group content: 3.1 mol%, polymerization degree: 6)
Glucose oxidase (product of Sigma; Type VII-S, G7016, 115,000) in 0.37 ml of 15% aqueous solution of 00, saponification value 73)
6.7 mg (unit / g solid) and 0.56 ml of distilled water were sequentially added to prepare a dope solution. 2.5 μl of this dope solution was applied onto a cellulose acetate film by a spinner (3000 rpm, 15 seconds), and left at room temperature for 1 hour, and then irradiated with ultraviolet rays having a main wavelength of 405 nm for 60 seconds to photocrosslink the glucose oxidase with polyvinyl alcohol. It was immobilized comprehensively in the membrane. The dry film thickness of this mixture film was 0.5 μm, and the film activity was 2.1 units.

A commercial Nafion solution was placed on this mixture film.
(Aldrich) 2.5 μl spinner applied (3000 rpm,
After drying for 15 seconds) at room temperature, a Nafion film having a dry film thickness of 0.33 μm was formed. Then, as shown in the figure, the silicone adhesive (Shin-Etsu Chemical KE44
It was insulated with T), and a film having an effective film area of 0.154 cm ² was formed on the working electrode.

(Measurement) The measurement of the glucose biosensor produced was carried out using a current detector (LC-4B manufactured by BAS) under the conditions of 30 ° C., pH 6.9, 0.6 V vs. Ag / AgCl, It was done in batch mode. The buffer solution is a neutral phosphate standard solution (0.025M KH ₂ PO ₄ , 0.025M Na ₂ HPO ₄ ) 1 specified in JIS K-0020.
In 500 ml, 0.5 g (8.2 mM) benzoic acid as preservative and KC
l The addition of 1.86 g (50 mM) was used.

Glucose has a final concentration of 25,50,75,1
The amounts used were 00,150,200,400,600,800 or 1000 mg / dl, and ascorbic acid was used in amounts of 100 mg / dl. The response and characteristics to glucose at 100 mg / dl concentration are shown in the graph of Figure 3 and in the table below.

Comparative Example In the example, no Nafion film was laminated. The response and characteristics to glucose at 100 mg / dl concentration are shown in the graph of Figure 3 and in the table below. Examples Comparative examples No output change Yes Ascorbic acid selectivity (% FS) 1.8 2.5 Coefficient of variation (n = 10) (%) 3.9 5.0 Calibration range (mg / dl) 01000 0-100

[Brief description of drawings]

FIG. 1 is a plan view of one embodiment of a glucose biosensor according to the present invention.

FIG. 2 is an end view taken along line I-I in FIG.

FIG. 3 is a graph showing response characteristics in Examples and Comparative Examples.

[Explanation of symbols] 1 working electrode 2 counter electrode 3 reference electrode 4 substrate 5 cellulose acetate film 6 GOD-photocrosslinked PVA mixture film 7 Nafion film

Claims (1)

[Claims] 1. A glucose biosensor in which a cellulose acetate film, a glucose oxidase-photocrosslinked polyvinyl alcohol mixture film, and a perfluorosulfonic acid type cation exchange film are sequentially laminated on a working electrode.