JP3525588B2 - Glucose biosensor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a glucose biosensor. More specifically, it relates to a glucose biosensor having excellent linear calibration and reproducibility.

[0002]

2. Description of the Related Art In a conventional method for measuring glucose concentration using a glucose biosensor, a potential step for measuring a current value after a fixed time is given to the electrode after dropping or injecting a measurement sample on the electrode surface. Since the method is adopted and the sample solution is not diluted and continuously supplied, the amount of dissolved oxygen required for the enzyme reaction is insufficient, and there is a drawback that the calibration range is narrow.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a glucose biotechnology in which at least two electrodes, a working electrode and a counter electrode, are provided on an insulating substrate, and a glucose oxidase-containing photocurable resin film is formed on the working electrode surface or the like. It is intended to provide a sensor which has excellent linear calibration and reproducibility even when it is used for measurement of an undiluted solution.

[0004]

The object of the present invention is as follows.
In a glucose biosensor in which a working electrode and a counter electrode or a working electrode, a counter electrode and a reference electrode are provided on an insulating substrate,
(1) Glucose oxidase-containing photocurable resin film, parabe
Nzoquinone or potassium ferricyanide layer and chloride
Sodium or potassium chloride layer (2) Glucose oxidase and parabenzoquinone
Or a photocurable resin film containing potassium ferricyanide
And sodium chloride or potassium chloride layer (3) glucose oxidase and sodium chloride or
Is a photocurable resin film containing potassium chloride and paraben
Zoquinone or potassium ferricyanide layer (4) glucose oxidase, parabenzoquinone or
Potassium ferricyanide and sodium chloride or salt
This is achieved by a glucose biosensor in which a photocurable resin film containing potassium iodide is formed (sequentially).

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION As the insulating substrate, glass, ceramics, plastic, paper, biodegradable material (for example, microbial-produced polyester, etc.) and the like are used. The formation of the working electrode and the counter electrode on such an insulating substrate is
In any case, using an electrode forming material such as platinum, silver, gold, or carbon, vapor deposition, sputtering, screen printing,
It is performed by a general thin film forming method such as a plating method.

When the reference electrode is further formed,
Formed as a silver / silver chloride electrode. The silver / silver chloride electrode is formed by a commonly used method, that is, a silver electrode is once formed on the reference electrode lead made of platinum, gold, carbon, etc. by screen printing, vapor deposition, sputtering, etc. Then, constant current electrolysis, a method of immersing in a ferric chloride aqueous solution, a method of coating and laminating silver chloride by a screen printing method, and the like are performed.

At least one electrode of each of these electrodes,
Preferably, all the electrodes provided thereon have the above-mentioned embodiment.
Including the various glucose oxidases described in (1) to (4)
Yes photocurable resin layer is formed.

In the case of the above aspect (1), a glucose oxidase (GOD) -containing photocurable resin film is first formed.

The formation of such a GOD-containing photocurable resin film is carried out by using a polyvinyl alcohol containing a photocrosslinkable group such as a stilbazolium group or a styrylpyridinium group in an amount of about 0.5 to 3.5 mol% (saponification value: about 70 to 100, polymerization degree: about 500). ~ 4000) represented by about 5 to 50% by weight, preferably about
In an aqueous solution dissolved at a concentration of 10 to 20% by weight, GOD (e.g., 165,800 units / 3.6 g of 11.1% photocrosslinkable PVA aqueous solution).
(GOD of solids of about 10 to 200 mg) and distilled water are added to prepare a dope solution having a total amount of about 5 to 10 g, and an appropriate amount of this is dropped and applied so as to cover the electrode surface with a spinner or the like, About 10-30
Dry for about 0.5 to 2 hours at a temperature of ℃, then photocrosslink by irradiating with ultraviolet light for a predetermined time (about 0.5 to 10 minutes), wash with water and develop as necessary to obtain a film thickness of about 0.05
A photo-cured film having a thickness of about 10 μm, preferably about 0.1 to 2 μm is formed.

A para-benzoquinone layer is formed on the GOD-containing photo-curable resin film thus formed. The formation of the parabenzoquinone layer is about 1 as parabenzoquinone.
~ 200mM, preferably about 50-150mM parabenzoquinone dissolved in an aqueous solution is dropped onto the resin film, generally by drying at room temperature conditions, to form a parabenzoquinone-impregnated resin film Let

Further, in the case of the above aspect (2) , in order to form a parabenzoquinone layer as an impregnation layer on the GOD-containing photocurable resin film, about 1% of the parabenzoquinone layer is previously added to the dope solution used in (1).
About 0.5 to 10 μl of a dope solution in which an amount of 0 to 200 mM, preferably about 50 to 150 mM parabenzoquinone is dissolved is used to form a photocurable resin film containing glucose and parabenzoquinone on a predetermined electrode surface. Is done.

In the above aspects (1) and (2) ,
A sodium chloride or potassium chloride layer is further formed on the parabenzoquinone layer of (1) or the photocurable resin film of (2) . These chlorides are added dropwise as an aqueous solution and dried to form a sodium chloride or potassium chloride layer. The amount is such that the final concentration of the measurement solution at the time of addition is about 1 to 2000 mM, preferably about 10 to 100 mM. Note that these chlorides can also be used in a state of being impregnated and supported by paper, polyacrylamide gel, saponified product of vinyl acetate-acrylic acid copolymer, or the like.

Further, in the above aspect (3) , the GOD of the above (1)
The photocurable resin film containing glucose oxidase and sodium chloride or potassium chloride formed therein is prepared by adding sodium chloride or potassium chloride to the dope solution used for forming the photocurable resin film containing The formation of the parabenzoquinone layer is carried out in the same manner as in the case of (1).

Further, in the aspect of (4) , the dope liquid used in the formation of the photocurable resin film of (2) to which sodium chloride or potassium chloride is further added is used,
A photocurable resin film containing glucose oxidase, parabenzoquinone and sodium chloride or potassium chloride is formed.

The amount of sodium chloride or potassium chloride used in these embodiments (3) and (4) is such that the final concentration at the time of adding the measurement solution is about 1 to 200 mM, preferably about 10 to 10 mM.
The amount is 0 mM. Further, the amount of parabenzoquinone in the aspect of (4) is used in the same manner as in the case of (2) .

After any of (1) to (4) is carried out, the central portion of each electrode is covered with an insulating film such as a silicone resin, a polyimide resin or an epoxy resin. In addition, each of these embodiments is a mediator having the same effect as that of parabenzoquinone.
The same operation can be performed by using potassium ferricyanide (an electron carrier).

When measuring the glucose concentration using the glucose biosensor thus constructed,
About 1 to 100 μl of glucose solution was dropped on the working electrode membrane, and about 1
After allowing the reaction to proceed for about 120 seconds, a voltage of about 0.05 to 0.8 V, preferably about 0.4 to 0.7 V is applied, and, for example, the current value 20 seconds after the application is measured.

[0018]

[Operation] and

EFFECTS OF THE INVENTION Glucose is GO in the presence of oxygen.
It is generally known that the glucose concentration can be indirectly determined by oxidizing H ₂ O ₂ generated by D and generated at that time on the working electrode and measuring the oxidation current value generated at that time.

In this case, a batch method or FIA (flow injection analysis) in which the measurement solution is diluted and used
In this method, the dissolved oxygen concentration in the aqueous solution is 8 ppm (0.3
Even if the concentration is about 25 mM, it is possible to ensure sufficient calibratability by using a means such as setting a limiting permeation membrane.

However, in the case of the undiluted solution which is not diluted, the enzymatic reaction is rate-determined by the dissolved oxygen concentration, so that the linear calibration range can be ensured only up to a glucose concentration of about 100 mg / dl. Is. Therefore, in the present invention, instead of oxygen, which has a finite concentration in solution,
Parabenzoquinone is used as an electron carrier.

Parabenzoquinone reacts with glucose as follows, The hydroquinone generated at this time is oxidized at the working electrode to generate an oxidation current.

[0022] Here, since the addition amount of parabenzoquinone is arbitrary,
The calibration range of the sensor is no longer limited by dissolved oxygen. Potassium ferricyanide also acts as an electron carrier.

In this way, the glucose concentration can be indirectly measured by an enzymatic reaction without limiting the dissolved oxygen concentration to a rate-determining value. If sodium chloride or potassium chloride is allowed to coexist there, the measurement can be performed. The value of the coefficient of variation showing the reproducibility of the value can be reduced.

Therefore, the glucose biosensor according to the present invention is used as a disposable sensor for quick and simple measurement of a stock solution, for domestic medical examination (self-care), especially for self-management of diabetes by measuring blood glucose level and urine glucose level, and diabetes. It can be effectively used not only for the prevention of buttocks, etc., and also for the control of glucose amount during the food manufacturing process.

[0025]

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

Example 1 (drop of parabenzoquinone-drop of NaCl)
Bottom ) A platinum counter electrode, a platinum working electrode, and a platinum reference electrode lead were formed on an alumina substrate (Kyocera product A-493) with a film thickness of 4000 Å by vapor deposition. Next, a silver paste (LS-504J manufactured by Asahi Chemical Laboratory) was printed on the reference electrode lead by a screen printing method, and baked to form a silver electrode. This silver electrode part was immersed in 0.1M hydrochloric acid, and at a current density of 0.6mA / cm ^2.
Constant-current electrolysis was performed for 20 minutes to silver chloride the surface of the reference electrode lead to form a silver / silver chloride reference electrode. For this constant current electrolysis, potentiogalvanostat (Hokuto Denko HA-501)
Was used.

(1) 3.6 g of 11.1% aqueous solution of photocrosslinkable polyvinyl alcohol (product of Toyo Gosei Co., Ltd .; stilbazolium group content 1.3 mol%, degree of polymerization 1700, saponification value 88), glucose oxidase (Sigma product; type VII) -S, G7016, 1
65800 units / g solid) 16.6 mg and distilled water 3.46 g were sequentially added to prepare a dope solution. 2.5 μl of this dope solution was applied to the working electrode by spinner (200 rpm, 15 seconds), left at room temperature for 1 hour, and then irradiated with ultraviolet rays for 4 minutes to immobilize glucose oxidase in the photocrosslinked polyvinyl alcohol film. Let (2) Then, 150 mM parabenzoquinone aqueous solution 5
μl (0.08 mg as para-benzoquinone) was added dropwise and dried under room temperature conditions. (3) Furthermore, the working electrode, counter electrode (and
50 mM sodium chloride in at least one position
Aqueous solution was added dropwise and dried at room temperature. (4) After that, an insulating film made of polyester was formed so as to cover the central portion of each electrode.

To the glucose biosensor thus produced, 40 μl of 100 mg / dl glucose aqueous solution was dropped on the working electrode membrane, the reaction was allowed to proceed for 5 seconds, and then a voltage of 0.6 V was applied for 20 seconds. After measuring the current value ,
It was evaluated as a coefficient of variation (n = 10) showing reproducibility . A potentiogalvanostat (HA-501) and a function generator (Hokuto Denko HB-104) were used for the measurement.

Example 2 (dope solution containing parabenzoquinone)
-NaCl dripping) As the dope solution prepared in Example 1 (1),
Benzoquinone 226 mg was further added, which was used.
2.5 μl (0.08 mg as parabenzoquinone) of spinner applied
Glucose oxidase and parabenzox
Non-encapsulated in a photocrosslinked polyvinyl alcohol film.
Let Then, a sodium chloride layer was prepared in the same manner as in Example 1 (3).
Was formed and the reproducibility of the obtained sensor was evaluated in the same way.
Was done.

Example 3 (NaCl-doped dope-parabenzo)
Quinone addition) As the dope solution prepared in Example 1 (1), chloride
Further addition of sodium at a final concentration of 50 mM
Glucose is used by applying 2.5 μl of it using a spinner.
Sucrose oxidase and sodium chloride
It was entrapped and immobilized in a nyl alcohol film. Then carry out
A parabenzoquinone layer was formed in the same manner as in Example 1 (2),
The reproducibility of the resulting sensor was also evaluated.

Example 4 (parabenzoquinone-NaCl added
As a dope solution prepared in Example 1 (1),
Benzoquinone 226mg and sodium chloride final concentration 50m
2.5 μl of that added to the amount of M was used.
In the form of applying spinner, glucose oxidase, para
Photo-cross-linked polyvinyl benzoquinone and sodium chloride
It was immobilized comprehensively in an alcohol membrane. Of the obtained sensor
Reproducibility was similarly evaluated.

The reference electrodes obtained in Examples 1 to 4 were used.
The coefficient of variation with and without use is shown in the table below.
It The linear calibration range of glucose concentration is
The value was 0 to 1000 mg / dl in each case.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-2-62952 (JP, A) JP-A-6-213858 (JP, A) JP-A 61-262652 (JP, A) JP-A 63- 75552 (JP, A) (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 27/327 G01N 27/416

Claims (4)

(57) [Claims] 1. A glucose biosensor in which a working electrode and a counter electrode or a working electrode, a counter electrode and a reference electrode are provided on an insulating substrate, and a glucose oxidase-containing photocurable resin film and parabenzoquinone or potassium ferricyanide on at least one electrode surface. Layers are sequentially formed and on the layers
Form a layer of sodium chloride or potassium chloride on the
Glucose biosensor. 2. A working electrode and a counter electrode on an insulating substrate or
Glucose biocell with working, counter and reference electrodes
Glucose on at least one electrode surface
Oxidase and parabenzoquinone or ferricia
Forming a photocurable resin film containing potassium chloride on the film
Form a layer of sodium chloride or potassium chloride on the
Glucose biosensor. 3. A working electrode and a counter electrode on an insulating substrate or
Glucose biocell with working, counter and reference electrodes
Glucose on at least one electrode surface
Oxidase and sodium chloride or potassium chloride
A photocurable resin film containing and a parabenzoquinone or
Glue formed by sequentially forming potassium ferricyanide layers
Course biosensor. 4. A working electrode and a counter electrode on an insulating substrate or
Glucose biocell with working, counter and reference electrodes
Glucose on at least one electrode surface
Oxidase, parabenzoquinone or ferricyanation
Contains potassium and sodium chloride or potassium chloride
Glucose by forming a photocurable resin film having
O sensor.