JPH10311817A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the measurement precision and operability by arranging a net body on a mixture layer through a space part. SOLUTION: A working electrode 2, a counter electrode 3 and a reference electrode lead, if necessary, are formed by screen printing carbon on an insulating base 1 of a ceramic or biodegrading material, and the center part of each electrode 2, 3 is covered with a resin-made insulating film 4. A mixture layer 5 of oxidoreductase and an electron transmitter is formed on the working electrode 2 and the counter electrode 3, and a spacer 7 having a hole part 6 having substantially the same size is adhered to the mixture layer 5 by an adhesive 8. A net body 9 consisting of a woven fabric or knit fabric subjected to hydrophilic treatment is put on the hole part 6 of the spacer 7, and a cover having a hole part 10 is adhered and integrated thereto by an adhesive 12, whereby a biosensor is constituted. Since the net body 9 is provided, the danger of the mixture layer 5 being accidentally touched with a hand in operation and damaged can be eliminated, and the operability is improved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a biosensor. More specifically, the present invention relates to a biosensor in which an enzyme-electron carrier mixture layer is applied and formed on a screen-printed carbon electrode formed on an insulating substrate.

[0002]

2. Description of the Related Art There is known a biosensor in which a hydrophilic polymer layer is formed on a screen-printed carbon electrode formed on an insulating substrate, and then an enzyme-electron carrier mixture layer is formed. In biosensors, a cover is placed directly above the mixture layer through a space to protect the enzyme, and the measurement liquid is introduced from the side of the mixture layer. A method has been adopted (JP-A-1-291153).

However, in such a method, since the introduction of the measuring solution is performed from the lateral direction, the dissolution of the mixture layer containing the enzyme varies, which causes a decrease in the accuracy of the measured value. There was a drawback to make it work. In some cases, a network is directly attached to a layer containing an enzyme (Japanese Patent Application Laid-Open No. 6-48256), but in this case, there is a drawback in that the measurement solution varies in dissolution.

Further, there is a biosensor in which a space is provided on a mixture layer containing an enzyme by using a spacer, and a reticulated body is arranged thereon. In this case, an electrode formed on an insulating substrate has a thiol surface treatment. Since the electrode is a palladium foil-attached electrode, there is a problem in that the measured values vary due to non-uniformity such as thiol treatment and attachment to a substrate.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a biosensor in which an enzyme-electron carrier mixture layer is formed by coating on a screen-printed carbon electrode formed on an insulating substrate. Another object of the present invention is to provide a biosensor that is excellent in the above point.

[0006]

SUMMARY OF THE INVENTION The object of the present invention is as follows.
In the above-described biosensor, this is achieved by a biosensor in which a reticulated body is arranged on a mixture layer via a space.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION On an insulating substrate 1 such as ceramics, glass, plastic, paper, or a biodegradable material (eg, a microbial polyester), a working electrode and a counter electrode constituting a biosensor electrode or a working electrode. A pole, a counter electrode and a reference pole are provided. The working electrode 2, the counter electrode 3 and the reference electrode lead are formed from screen-printed carbon, preferably screen-printed carbon having a polished surface;
The reference electrode is printed on the reference electrode lead by screen printing, vapor deposition,
After a silver electrode is formed once by a sputtering method, a film sticking method, or the like, a method of galvanostatic electrolysis or a method of dipping in a ferric chloride aqueous solution, and a method of coating and laminating silver chloride by a screen printing method are used. It is formed. Thereafter, the central portion of each electrode is covered with a resin insulating film 4 or the like. Note that a two-electrode structure without a reference electrode may be employed.

Generally, a mixture layer 5 of an oxidoreductase and an electron carrier is formed on the working electrode 2 and the counter electrode 3, and when a reference electrode is provided, a mixture layer is formed thereon. Will be Glucose oxidase (GOD), lactate oxidase, alcohol oxidase, fructose dehydrogenase, pyruvate oxidase and the like are generally used as oxidoreductases. Potassium ferricyanide, parabenzoquinone and ferrocene are generally used as electron carriers.

Glucose is oxidized in the presence of an enzyme by the action of GOD to produce gluconolactone, and H ₂ O ₂ generated at that time is oxidized on the working electrode, and the oxidation current value at that time is measured. The method of indirectly determining the glucose concentration is well known. However, when the measurement solution is a stock solution that is not diluted with water, the oxidation reaction is limited by the dissolved oxygen concentration, so that the linear calibration range is shown only up to a glucose concentration of about 100 mg / dl. Then, for example, in a disposable glucose biosensor or the like, in many cases, measurement is performed on a stock solution sample.

Therefore, instead of oxygen having a finite concentration in a solution, an electron carrier (mediator) is used together with GOD or the like. Mediator is potassium ferricyanide K ₃ Fe (C
N) In case ₆ , the reaction proceeds as follows. The ferrocyan ion generated at this time is oxidized at the working electrode to generate an oxidation current.

When parabenzoquinone is used as a mediator instead of potassium ferricyanide, hydroquinone is produced by the reaction of glucose and parabenzoquinone in the presence of GOD, and the resulting hydroquinone is oxidized at the working electrode. , An oxidation current is generated and its value is measured.

The formation of a mixture layer on each of these electrodes
About 1 to 50 mg, preferably about 1 to 20 mg of GOD per ml of water (165800
About 1-200 mg, and preferably about 50-180 mg or about 1-200 mg of potassium ferricyanide.
About 0.5 mg of an aqueous solution in which 100 mg, preferably about 10 to 60 mg is dissolved
1010 μl, preferably about 0.5 to 3 μl, is dropped on the working electrode by a dropping method, a spin coating method or the like to form a mixture layer having a thickness of about 0.05 to 10 μm, preferably about 0.1 to 2 μm. . In addition, as this solvent, an alcohol, an aqueous alcohol solution and the like are also used.

A spacer 7 having a hole 6 having a size substantially corresponding to the mixture layer 5 is adhered to the biosensor thus formed [FIG. 1 (a)] with an adhesive 8. 1 (b)], and then superimpose a mesh body 9 having an area sufficiently covering the hole 6 of the spacer [(c) of FIG. 1], and further attach the cover 11 having the hole 10 with an adhesive 12. "(D) of FIG. 1]
It is integrated as shown in FIG. The adhesive layer is
It can also be provided on both sides of the spacer 7. For both the spacer and the cover, a polyethylene terephthalate film or the like is preferably used.

As the net, a woven or knitted fabric is preferable. As the woven fabric, a plain weave woven with a warp yarn and a weft yarn is preferable, and among the plain weaves, those using only chemical fibers such as polyester and polyamide are preferable. Blended or woven fabrics of only natural fibers can be used. As the knitted fabric, any of a vertical knit and a horizontal knit can be used,
The material of the yarn may be a chemical fiber, a natural fiber or a mixed fiber thereof, and the yarn may be a filament yarn or a twisted yarn.

These reticulated bodies are preferably used after being subjected to a hydrophilic treatment. Hydrophilic treatment of the reticulated body is performed by a chemical treatment method using an organic substance such as a surfactant, a protein, or a coupling agent, or a physical treatment such as a plasma discharge treatment, a glow discharge treatment, a corona discharge treatment, a flame treatment, or an ultraviolet irradiation treatment. A processing method is appropriately used depending on the material of the mesh used.

As the surfactant, amphoteric, cationic,
Either anionic or nonionic ones can be used, but octylphenoxypolyethoxyethanol, isononylphenoxypolyglycidol, etc., which have little effect on the enzymatic activity, are preferred. When the measurement sample is blood, a sorbitan ester, a polyoxyethylene or polyglycerin alkyl allyl ether or a fatty acid ester among nonionic surfactants is preferably used to prevent hemolysis. Furthermore, lecithin or the like is used as a protein, and a silane-based or titanium-based coupling agent is used as a coupling agent.

The chemical hydrophilization treatment using these is carried out by a method of dipping the net in a solution thereof or a method of spray-coating the solution. The physical hydrophilization treatment is performed under appropriate conditions by appropriately selecting the temperature, pressure, electric power, time and the like.

For example, in the measurement of glucose concentration, an aqueous glucose solution having a predetermined concentration is dropped on the glucose biosensor thus prepared and reacted for about 5 to 180 seconds. Preferably about 0.5-1.0V
Was applied, for example, and the current value 10 seconds after the application was defined as a glucose concentration-dependent output current value. A potentiogalvanostat and a function generator are used for the measurement.

[0019]

Industrial Applicability The biosensor according to the present invention not only sufficiently achieves the original purpose of improving the measurement accuracy, but also erroneously touches and damages the mixture layer during operation due to the presence of the mesh. There is no need to worry about this, and operability is improved.

[0020]

Next, the present invention will be described by way of examples. Example A biosensor was made according to the illustrated embodiment.
First, a carbon working electrode and a carbon counter electrode were formed on a polyethylene terephthalate substrate by a screen printing method, and the central portion of each electrode was covered with a polyethylene terephthalate insulating film. On each electrode, a dope solution in which 10 mg of glucose oxidase (165800 units / g) and 48 mg of potassium ferricyanide were dissolved in 1 ml of water 1.
5 μl was dropped and dried at room temperature to form a mixture layer.

The biosensor configured as described above includes:
A polyethylene terephthalate perforated spacer, a polyethylene terephthalate twisted yarn plain weave that has been hydrophilized with a nonionic surfactant (Triton X-100, manufactured by UCC) and a polyethylene terephthalate cover film are sequentially adhered to each other to form a bio-based biopolymer of the present invention. Sensor.

For the measurement using this biosensor, a potentiogalvanostat (HA-501 manufactured by Hokuto Denko) and a function generator (HB-104 manufactured by the company) were used. The measurement was performed with 20 μl of glucose aqueous solution with a concentration of 250 mg / dl.
Was dropped on the mesh part of the sensor, and allowed to stand for 80 seconds.
A voltage of 0.9 V was applied between the electrodes, and a current value was measured 10 seconds after the application. The measurement was performed nine times, and the average value and CV value (the ratio of the standard deviation to the average value) were calculated, and the following results were obtained. 25.5 23.0 24.0 24.5 25.5 26.0 (average) 25.0 27.0 25.0 24.5 (CV) 4.7%

COMPARATIVE EXAMPLE 1 Referring to the biosensor described in JP-A-1-291153, in which a measurement sample was introduced from the side, a mixture layer was formed without a hydrophilic polymer layer, and the sample was placed from the side. The following results were obtained by using the structure for introducing 26.5 22.0 23.0 24.0 26.0 27.0 (average) 25.0 28.0 23.5 25.0 (CV) 6.4%

Comparative Example 2 The above-mentioned Japanese Patent Publication No. 6-4825 in which the mesh was brought into direct contact with the mixture layer.
With reference to the biosensor described in JP-A No. 6-2006, the following results were obtained by using a biosensor in which the mesh was directly in contact with the mixture layer. 25.0 27.0 20.0 22.5 24.0 26.0 (average) 24.0 27.5 21.0 23.0 (CV) 10.8%

Comparative Example 3 In the biosensor of Example, the following results were obtained by using a thiol surface-treated palladium electrode instead of the screen-printed carbon electrode. 24.0 23.0 22.5 20.0 25.0 30.0 (average) 24.4 26.0 27.5 22.0 (CV) 12.7%

[Brief description of the drawings]

FIG. 1 is a perspective view showing an assembled state of a biosensor according to the present invention.

FIG. 2 is a perspective view of a biosensor according to the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Insulating substrate 2 Carbon working electrode 3 Carbon counter electrode 5 Mixture layer 6 Hole 7 Spacer 9 Reticulated body 11 Cover

Claims (2)

[Claims] 1. A biosensor in which an enzyme-electron carrier mixture layer is applied and formed on a screen-printed carbon electrode formed on an insulative substrate, and a reticulated body is arranged above the mixture layer via a space. Biosensor 2. The biosensor according to claim 1, wherein a reticulated body is used.