JPH04215054A - Glucose biosensor - Google Patents

Abstract

PURPOSE:To provide a sensor having non-dependency on oxygen by a method wherein a photocrosslinked film of glucose hydrogenase water soluble photocrosslinked resin mixture and a ferrocene compound are adhered on an electrode to produce an working electrode, and a pair electrode forms a silver or silver/silver chloride electrode CONSTITUTION:A glucose biosensor is formed in such a way that a photocrosslinked film of a glucose dehydrogenase water soluble photocrosslinked resin mixture and a ferrocene compound are adhered on an electrode to produce a working electrode, and silver or silver/silver chloride electrode is used as a pair electrode. Since the glocose biosensor has non-dependency on dissolved oxygen while does need oxygen during oxidation of glucose, even when it is used in a area, such as a blood vessel, where the feed of oxygen is unstable, there is no need to correct a dissolved oxygen amount to a response value. The working electrode has excellent durability.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a glucose biosensor. More specifically, the present invention relates to a dissolved oxygen-independent glucose biosensor with improved durability.

0002

2. Description of the Related Art In conventional glucose biosensors, glucose oxidase is used as an enzyme immobilized on an electrode. When using this enzyme, 2
This is a substrate reaction and requires oxygen when measuring the amount of glucose.

[0003] Furthermore, when this glucose sensor is used in a location where oxygen supply is unstable, such as in a blood vessel, it is necessary to correct the amount of dissolved oxygen with respect to the response value.

0004

SUMMARY OF THE INVENTION An object of the present invention is to provide a dissolved oxygen-independent glucose biosensor that does not require oxygen when determining the amount of glucose.

Another object of the present invention is to provide such a glucose biosensor with improved durability.

[0006]

[Means for Solving the Problems] The purpose of the present invention is to
This is achieved by a glucose biosensor in which a photocrosslinked film of a glucose dehydrogenase-water-soluble photocrosslinkable resin mixture and a ferrocene compound are respectively attached on an electrode to serve as a working electrode, and a silver or silver/silver chloride electrode is used as a counter electrode.

[0007] As the electrode, a platinum electrode, a gold electrode, a carbon fiber electrode, etc. are used. Platinum electrodes and gold electrodes are excellent in sensitivity, while carbon fiber electrodes, which are made by bundling one or more carbon fibers with a diameter of about 0.1 to 1 μm, can be used to miniaturize the sensor body (about 1 μm in diameter). (below) and is effective in achieving cost reduction.

Formation of a working electrode using these electrodes is as follows:
This is done as follows.

[0009] Glucose dehydrogenase approximately 5-90m
g, preferably 30 mg (in the case of activity 6.3 Unit/mg) and a concentration of about 5 to 50 mg of a water-soluble photocrosslinkable resin such as polyvinyl alcohol or polyethylene glycol containing a stilbazolium group, styrylpyridinium group, etc. as a photocrosslinkable group. % aqueous solution, and in the case of a platinum electrode (surface area 0.02 cm2), apply 0.5 μl of the aqueous solution on it, or in the case of a carbon fiber electrode, immerse it in the aqueous solution and pull it up. After that, it is left to dry at room temperature for about 30 to 120 minutes. Next, it is irradiated with light of a wavelength that does not impair the activity of glucose dehydrogenase, preferably ultraviolet light with a wavelength of 360 nm, for a predetermined time, for example, about 10 seconds, and after washing with water, it is further irradiated for a predetermined time, for example, 3
A photocrosslinked film is formed by irradiating for about 0 seconds.

[0010] Thereafter, the photocrosslinked film has a concentration of about 1 to 50%,
Preferably about 1% in a 10% ferrocene compound solution.
Immerse for ~60 seconds, generally about 5 seconds, and then pull it out.
Dry at room temperature and store at 4°C. Examples of ferrocene compound solutions include organic solvent solutions such as ferrocene [bis(cyclopentadienyl)iron(II)], 1,1'-dimethylferrocene, vinylferrocene, ferrocenecarboxylic acid, and dimethylaminomethylferrocene, generally 1, 1
An acetone solution of '-dimethylferrocene or the like is used.

[0011] In this way, a photocrosslinked film of glucose dehydrogenase-water-soluble photocrosslinkable resin mixture and a ferrocene compound were attached on the electrode, and this was used as a working electrode, and a counter electrode was formed of a silver or silver/silver chloride electrode. In the glucose biosensor, a voltage of +100 to 600 mV is applied to the working electrode, and the counter electrode is used by shorting it to the reference electrode of the ammeter.

At this time, if a cellulose ester film is further formed on the working electrode surface, the calibration range can be significantly expanded. Formation of a cellulose ester film involves depositing a ferrocene compound and then adding cellulose esters such as cellulose acetate and cellulose acetate butyrate to their soluble solvents such as dimethylformamide, dimethylacetamide, acetone, etc. at a concentration of about 5 to 30% by weight. This is done by dissolving the solution, applying the solution, drying it at room temperature, and then immersing it in water to gel it.

[0013]

[Operation] The glucose biosensor according to the present invention reacts with glucose as follows. Glucose + glucose dehydrogenase (oxidized type) → gluconolactone + glucose dehydrogenase (reduced type) Glucose dehydrogenase (reduced type) + 1,1'-dimethylferrocene (oxidized type) → glucose dehydrogenase (oxidized type) + 1,1'-
Dimethylferrocene (reduced form)

[0014] Therefore, this reaction does not require oxygen, and the amount of glucose can be determined by detecting the movement of electrons to the working electrode accompanying the oxidation of glucose as a change in current value. At this time, the ferrocene compound acts as an electron acceptor for glucose dehydrogenase, causing electron transfer, that is, a reaction.

[0015]

[Effects of the Invention] The glucose biosensor according to the present invention is independent of dissolved oxygen and does not require oxygen when oxidizing glucose, so it can be used in locations where oxygen supply is unstable, such as in blood vessels. However, the response value does not require correction of the amount of dissolved oxygen.

[0016] Furthermore, by using a combination of an electrode for the working electrode and a silver or silver/silver chloride electrode for the counter electrode, the drift of the current value (baseline drift) can be almost zero.
A stabilized nA/h was obtained, which is an essential condition when using glucose dehydrogenase with low enzymatic activity.

[0017] Furthermore, as an enzyme immobilization technique, a method using albumin and glutaraldehyde is conventionally known, but the enzyme-immobilized electrode prepared by this method loses its properties after long-term use. Although peeling may occur, the working electrode according to the present invention is also excellent in durability.

Furthermore, a cellulose ester membrane further formed on the working electrode surface has the great practical advantage of expanding the calibration range by an order of magnitude because the diffusion of glucose is restricted.

Furthermore, when carbon fiber electrodes are used, not only does the carbon fiber itself generally have a low drift rate, but also miniaturization and cost reduction of the sensor can be achieved.

[0020]

[Example] Next, the present invention will be explained with reference to an example.

Example 1 Glucose dehydrogenase (manufactured by Sigma, 6.3Un
0.5 μl of a mixture of 30 mg of water-soluble photocrosslinkable resin (Toyo Gosei product, styrylpyridinium group-containing polyvinyl alcohol) dissolved in 0.5 ml of an 11.1% by weight aqueous solution of a platinum electrode (surface area 0.02cm
2) After coating on top and drying at 25℃ for 1 hour, wavelength 3
It was irradiated with 60 nm ultraviolet light for 10 seconds, washed with water, and then further irradiated with ultraviolet light for 30 seconds. Then 10% by weight 1
, 1'-dimethylferrocene acetone solution for 5 seconds, then pulled up and dried at 25°C.

[0022] The sensor having the working electrode thus manufactured was connected to an ammeter (LC-4B manufactured by BAS),
At this time, the counter electrode (silver electrode) and the reference electrode (silver electrode) of the ammeter were short-circuited, and the potential of the working electrode was set to +500 mV.

The measurement solution had a dissolved oxygen content of 6.4 ppm (0.
2 mmolar) phosphate buffer, pH 7.4.
The response to glucose was measured at an ionic strength of 0.15 and a temperature of 37° C. while stirring with a stirrer.

The 90% response time to a glucose aqueous solution with a concentration of 10 mg/dl is 30 seconds, the baseline drift is 0 nA/hr, and the calibration range is 1 to 50 mg/dl.
The steady-state current for an aqueous glucose solution of dl increases linearly in proportion to the concentration from 0.2 to 17 nA, and from 50 to 10 nA.
At 0 mg/dl, a gradual increase was shown until it reached 20 nA.

[0025] This sensor was used for 10 days at a glucose concentration of 1
00 mg/dl, pH 7.4, ionic strength 0.15, and a temperature of 37°C, after which the concentration was 10 mg.
/dl of the glucose aqueous solution was measured and showed a value equivalent to the value on the first day.

Example 2 In Example 1, measurements were carried out under stirring with nitrogen gas bubbling. The sensor characteristics were the same as in Example 1.

Example 3 In Example 1, after attaching 1,1'-dimethylferrocene, 0.5 μl of a 20% by weight cellulose acetate dimethyl formaldehyde solution was applied to the attached surface, and 2
It was dried at 5° C. for 1 minute, and then immersed in water for 1 minute to form a gel.

Using the sensor having the working electrode thus manufactured, the response to glucose was measured in the same manner as in Example 1.

The 90% response time to a glucose aqueous solution with a concentration of 10 mg/dl was 60 seconds, the baseline drift was 0 nA/hr, and the calibration range was 1 to 500 mg.
/dl of glucose aqueous solution increases linearly in proportion to the concentration from 0.2 to 17 nA, and from 550 to 17 nA.
At 600 mg/dl, it showed a constant value of 19 nA.

Example 4 A carbon fiber electrode (0.7 μm in diameter) was immersed for 1 minute in a mixed solution of the glucose dehydrogenase used in Example 1 dissolved in an aqueous solution of a water-soluble photocrosslinkable resin.
After pulling it up, it was left to dry at 25° C. for 1 hour.

Thereafter, the cells were treated in the same manner as in Example 1, and the response to glucose was measured.

The 90% response time to a glucose aqueous solution with a concentration of 10 mg/dl is 25 seconds, the baseline drift is 0 nA/hr, and the calibration range is 1 to 50 mg/dl.
The steady-state current for a glucose aqueous solution of dl increases linearly from 0.002 to 0.1 nA in proportion to the concentration, and
At ~100 mg/dl, it showed a gradual increase until it reached nA.

[0033] This sensor was used for 10 days at a glucose concentration of 1
00 mg/dl, pH 7.4, ionic strength 0.15, and a temperature of 37°C, after which the concentration was 10 mg.
/dl of the glucose aqueous solution was measured and showed a value equivalent to the value on the first day.

Example 5 In Example 4, measurements were carried out under stirring with nitrogen gas bubbling. The sensor characteristics were the same as in Example 4.

Claims (2)

[Claims] [Claim 1] A glucose biomolecule comprising a working electrode on which a photocrosslinked film of glucose dehydrogenase-water-soluble photocrosslinkable resin mixture and a ferrocene compound are respectively attached, and a silver or silver/silver chloride electrode as a counter electrode. sensor. 2. The glucose biosensor according to claim 1, further comprising a cellulose ester film formed on the working electrode.