JP4030186B2 - Carbon electrode for chip-type quantitative analysis using enzyme and its production method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a carbon electrode and a method for producing the same, which can electrochemically and rapidly measure the amount of a specific substance in a sample solution using enzyme activity.
[0002]
[Prior art]
Conventionally, as a working electrode for measuring the amount of a specific substance in a sample solution by an electrochemical method, a mercury dropping electrode, a stationary mercury electrode, platinum, gold, gold amalgam, copper, carbon (glassy carbon, carbon fiber, carbon Paste, pyrographite) and the like have been studied, but among them, the use of glassy carbon has become mainstream. However, glassy carbon has low current sensitivity, and when a potential is applied through the electrolyte at a high positive potential, the reproducibility of the electrode surface becomes poor. Therefore, the applicable potential range was up to around +1.0 V. In addition, the influence of pretreatment such as oxidation treatment was great, and there was a problem in measurement reproducibility.
[0003]
Graphite is a useful electrode material because it has a wide potential window, electrode reaction activity, and no toxicity to the living body, but it has poor mechanical strength, and it alone impregnates oil and resin because the electrolyte penetrates into the tissue. In a system containing even a small amount of organic solvent, the impregnated material is eluted, so that there are many variations in electrochemical characteristics, and data reproducibility cannot be obtained. In spite of this, the graphite crystal end face (edge face) is rich in electrode reaction activity, has a large potential window, is easy to pretreat, has high stability without deterioration over time, and does not elute and has no toxicity. Based on this property, graphite / glassy carbon composite obtained by using graphite-like carbon material in which graphite is oriented in one direction using glassy carbon showing impermeability as a matrix Carbon electrodes do not require oil or resin impregnation, are impervious by themselves, and have high mechanical strength.
[0004]
However, the electrode made of graphite / glassy carbon composite carbon has a disadvantageous property for enzyme activity measurement due to the strength of its end face, that is, a large amount of substances that hinder the measurement contained in the sample solution. It had the disadvantage of adsorbing. Therefore, a method for eliminating the influence of the inhibitor without losing the characteristics of the electrode, by coating the surface with a polymer permeable thin film without being affected by the concentration of the inhibitor, The following carbon electrode for quantitative analysis was proposed (Japanese Patent Application No. Hei 8-21395) for a method capable of measuring the amount of a specific substance using an enzyme quickly and with high accuracy.
[0005]
This electrode is a cylindrical electrode made of a carbon material which is a graphite / glassy carbon composite material in which a graphite crystal is oriented in one direction so as to have an electrode reaction unique to graphite crystal in a glassy carbon matrix, and this electrode It is a carbon electrode for specific substance quantification using the enzyme comprised from the permeable thin film which coat | covers the surface of this.
[0006]
[Problems to be solved by the invention]
With such a conventional shape, the amount of sample required for the measurement is as relatively large as 1 ml, and it is difficult to reduce the size when the electrode is disposable.
[0007]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present invention provides a flaky form from a rod-like carbon material prepared by extruding a carbon material which is a graphite / glass-like carbon composite material in which a graphite crystal is oriented in one direction in a glassy carbon matrix. The chip electrode was prepared, and at least a hydrophilic polymer layer and a reaction layer containing an enzyme were provided on the electrode. By further including an electron carrier in the reaction layer, it is not affected by the shortage of dissolved oxygen caused by the fact that the measurement solution can no longer be stirred by chipping.
[0008]
That is, the principle of specific substance quantification using the electron carrier as described above is as follows. For example, when the specific substance is glucose, when glucose, which is a substrate, is oxidized by glucose oxidase, which is an oxidase, the active center of the enzyme changes from an oxidized form to a reduced form, thereby reducing the electron carrier and the enzyme itself Return to the oxidized form again. The electron carrier is electrolyzed by an appropriate applied voltage on the working electrode, and the glucose concentration can be measured by measuring the oxidation current obtained at that time.
[0009]
The graphite / glassy carbon composite material used for the working electrode in the present invention is a glassy carbon matrix in which 40% or more and 95% or less of the material composition is impervious, and the graphite is preferably powder of quiche graphite or natural graphite. Glassy carbon is a glassy non-graphitizable carbon obtained by firing an organic substance in an inert atmosphere or a non-oxidizing atmosphere. Specifically, an organic polymer substance and its monomer / oligomer , Tar pitches, carbonized pitches, thermoplastic resins, and carbonized products obtained from a mixture of two or more of thermosetting resin initial polymers.
[0010]
The method of orienting the graphite crystals in the present invention in the present invention can be performed as follows. Mix organic polymer substance and crystalline carbon fine powder according to the purpose and thoroughly disperse the powder with Henschel mixer. A dry-distilled pitch is blended here, and a plasticizer, a solvent, etc. are added to this, and the mixture is sufficiently mixed and dispersed using a kneader capable of applying a high shearing force such as a pressure kneader or two rolls. After granulation, the formed shape is formed by extruding at a high speed to a desired diameter with an extruder such as a screw type or plunger type, and applying an orientation operation so that the composite crystals of graphite are well aligned in the direction of extrusion. can get.
[0011]
Next, the molded body is treated for 10 hours in an air oven heated to 180 ° C. while being stretched to obtain a carbon precursor (precursor) material. Further, the carbonization is completed by gradually heating up to 1000 ° C. while controlling the rate of temperature rise in an inert gas atmosphere such as nitrogen or argon. Depending on the purpose, heat treatment is further performed up to 2200 ° C. in a vacuum or an argon atmosphere to densify the whole and increase the purity. In this manner, a rod-like graphite / glassy carbon composite material in which graphite is oriented in one direction as used in the present invention can be prepared. This carbon material is processed into a thin piece by a cutting device such as a diamond wheel. The obtained flaky carbon electrode is bonded onto an insulating resin substrate such as polyethylene terephthalate with an insulating adhesive. Further, an insulating coating layer is formed so that the exposed area of the carbon electrode is constant. After constituting the electrode portion, a hydrophilic polymer layer is formed. Next, a composite layer of an enzyme and an electron carrier is formed.
[0012]
As the hydrophilic polymer used here, carboxymethyl cellulose, other cellulose, vinyl alcohol, starch, maleic anhydride, acrylamide, or the like may be used. A solution of these hydrophilic polymers in an appropriate concentration or the like is dropped and dried to form a hydrophilic polymer layer having a required film thickness on the electrode.
[0013]
In addition, as materials that can be used as an electron carrier, any conventionally used materials can be used, such as potassium ferricyanide, p-benzoquinone, ferrocene, phenazine methosulfate, thionine, pyrroloquinoline quinone, naphthoquinone, and methylene blue. be able to.
Enzymes that can be used in the carbon electrode of the present invention are not particularly limited, and enzymes used in conventional enzyme sensors can be used alone or in combination. Specifically, glucose oxidase, cholesterol oxidase, lactate oxidase, uricase, alcohol oxidase, NADH oxidase, amine oxidase and other oxidases, glucose dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, 3-hydroxybutyrate dehydrogenase, fructose dehydrogenase, alcohol dehydrogenase, diaphorase , Urease, etc. can be used. As substances whose concentration can be measured using these enzymes, the concentration of various substances can be measured by appropriately selecting from the above-mentioned enzymes to be used. Typical examples include glucose, cholesterol, lactic acid, uric acid, alcohol, NADH, galactose, pyruvic acid, amine, fructose, glutamic acid, and any other substance that can be a substrate for enzyme reaction. It can be measured quantitatively.
[0014]
Next, an Example is shown and the production method of the carbon electrode of this invention and the result of the quantitative analysis using this are clarified.
[0015]
【Example】
A glucose sensor will be described as the first embodiment of the present invention. First, a method for producing a graphite / glassy carbon composite material will be described. As a glassy carbon source, 35 parts by weight of chlorinated vinyl chloride resin, 50 parts by weight of furan resin, 15 parts by weight of natural scaly graphite powder having an average particle size of 2 μm, and 20 parts by weight of diallyl phthalate monomer as a plasticizer are mixed with a Henschel mixer. After dispersion, the mixture is kneaded with two mixing rolls, the kneaded product is extruded with a screw type extruder, and this is treated for 10 hours in an air oven heated to 180 ° C. while being subjected to stretching operation, to obtain a carbon precursor. Body (pre-cursor) material. Further, the carbonization is completed by gradually heating up to 1000 ° C. while controlling the heating rate in a nitrogen gas atmosphere. Furthermore, a rod-like graphite / glassy carbon composite material in which graphite is oriented in one direction can be prepared by applying heat treatment up to 1400 ° C. in an argon atmosphere to densify the whole and increasing the purity. This carbon material is processed into a flake shape with a diamond wheel. After smoothing the electrode surface of the obtained flaky electrode 10 (see the plan view of FIG. 1 and the sectional view of FIG. 2) with 2000th silicon carbide abrasive paper, the obtained flaky carbon electrode was Then, the insulating resin substrate 14 made of polyethylene terephthalate is joined with an insulating adhesive. The lead 16 is formed, and the insulating coating layer 12 is formed so that the area of the exposed portion of the carbon electrode is constant. After constituting the electrode portion, a hydrophilic polymer layer is formed.
[0016]
A 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was dropped on the electrode and dried. Next, a solution in which glucose oxidase as an enzyme and potassium ferricyanide as an electron acceptor were dissolved in a phosphate buffer was dropped and dried to form a reaction layer. Measured by a two-electrode method using the electrode prepared as described above as a working electrode, a silver / silver chloride electrode as a counter electrode and a reference electrode, and using a Yanagimoto voltammetric analyzer P1000 and a Toa radio t-Y recorder EPR-151A Went. Glucose oxidase (EC 1.1.3.4 microorganism-derived, 1000 U / l) was manufactured by Sigma, and glucose was a glucose standard solution manufactured by Wako Pure Chemical. Further, a 0.1 M phosphate buffer (pH 6.8) was used as the buffer.
[0017]
Next, the measurement will be described. One minute after supplying the glucose solution with the micropipette onto the carbon electrode for quantifying the specific substance prepared as described above, a pulse voltage of 600 mV was applied to the working electrode in the anode direction based on the counter electrode of the electrode system. When the current value after 5 minutes was measured, a response current value proportional to the glucose concentration in the sample solution was obtained as shown in FIG. From the fact that it showed extremely good linearity, it was found that the concentration could be measured quickly and accurately from the current value obtained even when glucose of unknown concentration was used as a sample.
[0018]
A cholesterol sensor as a second embodiment of the present invention will be described. First, a method for producing a graphite / glassy carbon composite material will be described. As a glassy carbon source, 60 parts by weight of a furan resin, 20 parts by weight of a carbonization pitch, and 20 parts by weight of natural scaly graphite powder having an average particle size of 2 μm are mixed and dispersed with a Henschel mixer, and then kneaded with two mixing rolls. The product is extruded with a screw-type extruder, and this is treated for 8 hours in an air oven heated to 200 ° C. while being stretched to obtain a carbon precursor (precursor) material. Further, the carbonization is completed by gradually heating up to 1000 ° C. while controlling the heating rate in a nitrogen gas atmosphere. Further, a rod-like graphite / glass-like carbon composite material in which graphite is oriented in one direction can be prepared by performing a heat treatment up to 1600 ° C. in a vacuum firing furnace to densify the whole and increase the purity. This carbon material is processed into a flake shape with a diamond wheel. In the same manner as in the first example, after the electrode surface of the obtained flaky electrode 10 was smoothed with 2000th silicon carbide polishing paper, the obtained flaky carbon electrode was made of polyethylene terephthalate. Bonding is performed on the insulating resin substrate 14 with an insulating adhesive. The lead 16 is formed, and the insulating coating layer 12 is formed so that the area of the exposed portion of the carbon electrode is constant. After constituting the electrode portion, a hydrophilic polymer layer is formed.
[0019]
A 0.5 wt% aqueous solution of CMC as a hydrophilic polymer was dropped on the electrode and dried. Next, a solution obtained by dissolving cholesterol oxidase as an enzyme and potassium ferricyanide as an electron acceptor in a phosphate buffer was dropped and dried to form a reaction layer. Measured by a two-electrode method using the electrode prepared as described above as a working electrode, a silver / silver chloride electrode as a counter electrode and a reference electrode, and using a Yanagimoto voltammetric analyzer P1000 and a Toa radio t-Y recorder EPR-151A Went. Cholesterol oxidase (EC 1.1.3.6 microorganism-derived, 100 U / l) was manufactured by Toyobo, and cholesterol was a free cholesterol standard solution A (200 mg / dl) manufactured by Wako Pure Chemical Industries. The buffer used was a 0.1 M phosphate buffer (pH 6.8) to which Triton X-100 was added to 5 ml / l.
[0020]
A cholesterol standard solution was dropped as a sample solution on the surface of the working electrode configured as described above, and a pulse potential of 600 mV was applied 30 minutes after the dropping, using the reference electrode as a reference, and the current value after 5 minutes was taken as a measured value. The added cholesterol in the sample solution reacts with potassium ferricyanide supported on the reaction layer on the electrode to produce potassium ferrocyanide. Therefore, by applying the pulse potential, an oxidation current proportional to the generated potassium ferrocyanide concentration is obtained, and this current value corresponds to the concentration of cholesterol as a substrate.
[0021]
FIG. 4 shows the relationship between the current value obtained by the above series of reactions and the cholesterol concentration, and showed very good linearity. From this, it was found that even when cholesterol of unknown concentration was used as a sample, the concentration could be measured quickly and accurately from the obtained current value.
[0022]
【The invention's effect】
As described above, according to the present invention, there is provided a disposable type quantitative analysis electrode that can accurately quantify the substrate concentration and can be easily miniaturized.
[Brief description of the drawings]
FIG. 1 is a plan view of a carbon electrode for quantifying a specific substance according to the present invention.
FIG. 2 is a cross-sectional view of the electrode of FIG.
FIG. 3 is a graph showing the relationship between the glucose concentration and the current value obtained by the glucose quantitative experiment of the first example.
FIG. 4 is a graph showing the relationship between cholesterol concentration and current value obtained by cholesterol quantification experiment of the second example.
[Explanation of symbols]
10 ... Working electrode (end face)
12 ... Insulating coating film 14 ... Insulating resin substrate 16 ... Working electrode lead

Claims (4)

An electrode composed of a graphite / glassy carbon composite material in which graphite crystals are oriented in one direction in a glassy carbon matrix, and the shape of the electrode is a flaky chip type;
A carbon electrode for quantitative analysis comprising at least a hydrophilic polymer layer and a reaction layer containing an enzyme formed on the electrode. The carbon electrode for quantitative analysis according to claim 1, wherein the reaction layer further contains an electron acceptor. Made of a graphite / glassy carbon composite material in which a graphite crystal is oriented in one direction in a glassy carbon matrix, the electrode is made of a chip-like chip shape,
A method for producing a carbon electrode for quantitative analysis, comprising each step of forming a reaction layer containing at least a hydrophilic polymer layer and an enzyme on the electrode. The method for producing a carbon electrode for quantitative analysis according to claim 3, wherein the reaction layer further contains an electron acceptor.

Images