JPH1164271A - Current amplification type oxyzen sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve detection sensitivity by placing on a comb-shaped electrode a membrane in which a mediator slightly soluble in a peroxidase and water is immobilized, reducing the oxidant of the mediator which occurs due to an oxygen reaction in the membrane at one electrode, and oxidizing the oxidant of the mediator at the other electrode. SOLUTION: An immobilization film 5 in which a mediator slightly soluble in a peroxidase and water is immobilized in a film is adhered on a comb-shaped electrode. At the time of measurement, a generator electrode 3 is set at a potential that reduces an oxidized-type mediator, and a collector electrode 4 is set at a potential that oxidizes a reduced-type mediator. In the case of adding hydrogen peroxide into a measurement liquid, an oxygen reaction occurs in a membrane in which a mediator and a peroxidase coexist to generate an oxidized-type mediator. The oxidized-type mediator is reduced at the generator 3 and again oxidized at the collector electrode 4. A redox cycling reaction occurs between the generator electrode 3 and the collector electrode 4 to enable the amplification of the output current of a sensor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an enzyme sensor, and more particularly, to an enzyme sensor using peroxidase as an enzyme and immobilized on an electrode together with a mediator.

[0002]

2. Description of the Related Art Conventionally, an enzyme sensor technology in which an enzyme-immobilized membrane is mounted on an electrode and an electrode active material generated by the enzyme reaction in the membrane is detected as an electric signal by the electrode reaction has been known. Various sensors, such as a glucose sensor and an alcohol sensor, have been put to practical use as a device that skillfully utilizes high substrate specificity.

[0003] In the field of enzyme sensors, it has heretofore been known to use peroxidase, which is an enzyme using hydrogen peroxide as a substrate, to add a substance serving as a mediator to a solution, or to knead a mediator into a carbon paste electrode. A featured hydrogen peroxide sensor has been developed and published (eg, Biosens. Bi).
oeletron. , 421-428 (1992),
Electroanalysis, 281-285 (1
991), Electroanalysis, 575-
579 (1993)).

In these techniques, a mediator causes a redox reaction between an enzyme reaction and an electrode reaction to detect a current caused by the reaction, and oxidizes hydrogen peroxide directly on the electrode. Since the set potential can be lowered as compared with the above, there is an advantage that it is hardly affected by interfering substances.

[0005] However, when the concentration of hydrogen peroxide in this sensor is low, there is a disadvantage that the measurement current becomes small and the measurement becomes difficult.

[0006]

SUMMARY OF THE INVENTION In view of such circumstances, an object of the present invention is to provide a hydrogen peroxide sensor using a peroxidase and a mediator, which is dramatically more sensitive than conventional ones. It is.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies on increasing the sensitivity of a hydrogen peroxide sensor using an immobilized enzyme membrane coexisting with peroxidase and a mediator. It has been clarified that the sensitivity of the sensor can be dramatically improved by performing electrochemical amplification of the electrode reaction using a shaped electrode. That is, a membrane in which a peroxidase and a water-insoluble electron-donating mediator are immobilized is placed on an electrode (hereinafter referred to as a “comb electrode”) in which two minute comb electrodes are alternately engaged as shown in FIG. 2), the oxidized form of the mediator generated by the enzymatic reaction in the membrane causes a redox reaction in which the mediator is reduced at one electrode and oxidized at the other electrode, thereby increasing electrochemical current amplification. It has been found that the detection sensitivity (see FIG. 3) can dramatically improve the detection sensitivity of hydrogen peroxide. Hereinafter, the present invention will be described in detail.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The enzyme sensor according to the present invention comprises a comb-shaped electrode having two electrodes in close proximity, and a membrane (5) in which a peroxidase and a water-insoluble mediator are immobilized in the membrane. The immobilization film (5) has a structure in which it is closely attached to the comb-shaped electrode (FIG. 2). In this case, the method of adhering the immobilizing film (5) and the comb-shaped electrode is not particularly limited. For example, a method in which the immobilizing film is covered with a nylon mesh or the like and fixed using a magic tape is used. Can be.

In the measurement, a comb-shaped generator electrode (3) is operated by a dual potentiostat.
Is set to the potential where the oxidized mediator is reduced,
The collector electrode (4) is set at a potential at which the reduced mediator is oxidized.

FIG. 3 shows the operation principle of the enzyme sensor according to the present invention. When hydrogen peroxide is added to the measurement solution, an enzymatic reaction occurs in the membrane where the mediator and peroxidase coexist, and an oxidized mediator is generated. This is reduced by the comb-shaped generator electrode (6) and returns to the reduced type. This regenerated reduced mediator is reoxidized at the collector electrode (7) immediately next to it. For this reason, a redox cycling reaction occurs between the generator electrode (6) and the collector electrode (7) of the comb electrode, and the output current of the sensor can be amplified.

In this case, since the oxidized form and the reduced form of the mediator move between the electrodes by diffusion, the amplification factor increases as the distance between the collector electrode and the generator electrode decreases.

FIG. 4 shows an actual measuring method using the enzyme sensor according to the present invention. The sensor (12), the counter electrode (13), and the reference electrode (14) according to the present invention are mounted on the measurement cell. At the time of measurement, the generator electrode of the sensor is set to the potential at which the mediator is reduced by the dual potentiostat (15),
The collector electrode is set to the potential at which the mediator is reduced. At this time, the concentration of the substrate can be known by measuring the current flowing through the generator electrode.

[0013] In the enzyme sensor according to the present invention, the type of the membrane on which the enzyme and the mediator are immobilized is not particularly limited. For example, a method of ultraviolet curing using photosensitive polyvinyl alcohol, a crosslinking reaction of albumin with glutaraldehyde, and the like. For example, it can be produced using a conventionally known method for producing an enzyme-immobilized membrane. In addition, the type of mediator is an electron donor that can reduce hydrogen peroxide by peroxidase, and is a substance that is reversibly oxidized and reduced by an electrode reaction, and further prevents elution into the measurement solution. Therefore, the material is not particularly limited as long as the material is hardly soluble in water. As a mediator satisfying such conditions, for example, ferrocene derivatives such as ferrocene, cyclohexylferrocene, and dimethylferrocene can be used.

In the present invention, if a composite sensor in which an immobilized membrane of an oxidase-based enzyme (20) is provided on a membrane (19) on which peroxidase and a mediator are immobilized, a substance serving as a substrate of the oxidase-based enzyme is used. It can be a highly sensitive sensor (FIG. 5). That is, in the enzymatic reaction of the oxidase enzyme, hydrogen peroxide is generated in the immobilized membrane (20) of the oxidase enzyme because hydrogen peroxide is generated by the reaction between the substrate and oxygen.
This hydrogen peroxide is further reacted with the mediator in the underlying peroxidase and mediator-immobilized membrane (19), whereby the substrate of the oxidase-based enzyme reaction is detected with high sensitivity.

The type of oxidase-based enzyme in such a composite sensor is not particularly limited, but it can be converted into highly sensitive glucose when combined with glucose oxidase, and can be converted into highly sensitive fructose when combined with fructose oxidase. It can be a sensor.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Highly sensitive hydrogen peroxide sensor using ferrocene as a mediator 10 mg of peroxidase was dissolved in 100 μL of 0.1 M phosphate buffer at pH 7, 10 mg of ferrocene powder was added, and bis-sulfosuccinate ( 50 μL of aqueous solution of sodium salt of 2-ethylhexyl) (1 g / L)
Add and suspend. 200 mg of a 11.5% aqueous solution of photosensitive polyvinyl alcohol (manufactured by Toyo Gosei Co., Ltd.) is added thereto, and the mixture is poured on a Teflon plate and dried in a cool and dark place.
The dried product was irradiated with ultraviolet light by a high-pressure mercury lamp for 10 minutes to perform a photocrosslinking reaction, thereby obtaining a polyvinyl alcohol film on which peroxidase and ferrocene were immobilized.

This membrane is placed on a comb-shaped carbon electrode (manufactured by BAS Co., Ltd., electrode width 5 μm, electrode interval 5 μm), and mounted on the electrode using a tetron mesh and magic tape, and a highly sensitive hydrogen peroxide sensor is provided. Was prepared.

In this sensor, if the generator electrode of the comb electrode is set to 0 mV with respect to the Ag / AgCl electrode by the dual potentiostat and the collector electrode is set to +400 mV, it operates as a current amplification type hydrogen peroxide sensor. I do. If the generator electrode of the comb electrode is set at 0 mV with respect to the Ag / AgCl electrode and the collector electrode is kept at the natural potential, the device operates as a hydrogen peroxide sensor using a usual mediator. FIG. 6 shows a comparison between the two measurement results. As can be seen, the output of the current amplification type sensor was 10 times or more that of the normal type sensor, and the sensitivity was dramatically improved.

Example 2 A high-sensitivity hydrogen peroxide sensor using 1,1'-dimethylferrocene as a mediator Except that a powder of 1,1'-dimethylferrocene was used instead of ferrocene, the same as in Example 1 High sensitivity hydrogen peroxide sensor was fabricated by the method. When a comparative test similar to that of Example 1 was performed on this sensor, the output of the current amplification type sensor was three times or more that of the normal type sensor, and the sensitivity was dramatically improved (FIG. 7).

EXAMPLE 3 Highly Sensitive Hydrogen Peroxide Sensor Using Cyclohexenyl Ferrocene as Mediator A highly sensitive hydrogen peroxide sensor was prepared in exactly the same manner as in Example 1 except that powder of cyclohexenyl ferrocene was used instead of ferrocene. Was prepared. When a comparative test similar to that of Example 1 was performed on this sensor, the output of the current amplification type sensor was twice or more that of the normal type sensor, and the sensitivity was dramatically improved (FIG. 8).

Example 4 Highly Sensitive Glucose Sensor Combining Immobilized Membrane with Peroxidase and Ferrocene and Immobilized Membrane with Glucose Oxidase 10 mg of glucose oxidase and 200 mg of a 11.5% aqueous solution of photosensitive polyvinyl alcohol (Toyo Gosei Co., Ltd.) The solution is added to 100 μL of 0.1 phosphate buffer at pH 7, dissolved, poured on a Teflon plate, and dried in a cool and dark place. The dried product was irradiated with ultraviolet rays by a high-pressure mercury lamp for 10 minutes to perform a photocrosslinking reaction, thereby obtaining a glucose oxidase-immobilized film. This membrane was superimposed on the immobilized membrane coexisting with peroxidase and ferrocene prepared by the method of Example 1, and a comb-shaped carbon electrode (manufactured by BAS Co., Ltd .; (Electrode width: 5 μm, electrode interval: 5 μm), and mounted on the electrodes with tetron mesh and magic tape to obtain a highly sensitive glucose sensor. When a comparative test was performed on this sensor, the output of the current amplification type sensor was 10 times or more as compared with the case where it was not, and the sensitivity was dramatically improved (FIG. 9).

[0022]

According to the present invention, it is possible to provide a sensor with much higher sensitivity than a conventional hydrogen peroxide sensor using peroxidase and a mediator. In addition, by combining a peroxidase-immobilized membrane and an oxidase-based enzyme-immobilized membrane, a highly sensitive sensor for a substance (eg, glucose or fructose) serving as a substrate for an oxidase-based enzyme can be provided.

[Brief description of the drawings]

FIG. 1 is a schematic view of a comb electrode used in the present invention.

FIG. 2 is a schematic view of a current amplification type hydrogen peroxide sensor according to the present invention.

FIG. 3 is a conceptual diagram illustrating the operation of a current amplification type hydrogen peroxide sensor according to the present invention.

FIG. 4 is a diagram of an example of a measurement method using the sensor of the present invention.

FIG. 5 is a schematic diagram of a cross section of a current amplification type sensor in combination with an oxidase enzyme.

FIG. 6 is a comparison of responsiveness between a current amplification type hydrogen peroxide sensor and a normal type hydrogen peroxide sensor when ferrocene is used as a mediator.

FIG. 7 is a comparison of the responsiveness of a current amplification type hydrogen peroxide sensor and a normal type hydrogen peroxide sensor when 1,1′-dimethylferrocene is used as a mediator.

FIG. 8 is a comparison of the responsiveness of a current amplification type hydrogen peroxide sensor and a normal type hydrogen peroxide sensor when cyclohexenyl ferrocene is used as a mediator.

FIG. 9 is a comparison of the responsiveness of a current amplification type hydrogen peroxide sensor and a normal type hydrogen peroxide sensor when a glucose oxidase-immobilized membrane is combined with a ferrocene and peroxidase-immobilized membrane.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Generator electrode 2 ... Collector electrode 3 ... Generator electrode 4 ... Collector electrode 5 ... Peroxidase + mediator coexistence immobilization film 6 ... Generator electrode 7 ... Collector electrode 8 ...・ Peroxidase + mediator coexisting immobilized membrane 9 ・ ・ ・ Reduced mediator 10 ・ ・ ・ Oxidized mediator 11 ・ ・ ・ Measurement liquid 12 ・ ・ ・ Sensor 13 ・ ・ ・ Reference electrode 14 ・ ・ ・ Counter electrode 15 ・ ・ ・ Dual potency Ostat 16 ・ ・ ・ Recorder 17 ・ ・ ・ Generator electrode 18 ・ ・ ・ Collector electrode 19 ・ ・ ・ Peroxidase + mediator coexisting immobilization membrane 20 ・ ・ ・ Oxidase immobilization membrane

Claims (4)

[Claims] 1. A hydrogen peroxide sensor comprising a comb-shaped electrode in which two adjacent electrodes are alternately engaged with each other, and an immobilized membrane in which a peroxidase and a water-insoluble mediator coexist. 2. The hydrogen peroxide sensor according to claim 1, wherein the mediator is ferrocene or a ferrocene derivative. 3. A structure in which an immobilized membrane in which peroxidase and a mediator coexist is mounted on a comb-shaped electrode in which two adjacent electrodes are alternately engaged with each other, and an oxidase-immobilized membrane is stacked thereon. Enzyme sensor. 4. The method according to claim 1, wherein the oxidase is glucose oxidase,
Fructose oxidase, alcohol oxidase,
The enzyme sensor according to claim 3, wherein the enzyme sensor is selected from the group consisting of lactate oxidase and cholesterol oxidase.