JPH0557538B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an enzyme sensor, and particularly to an enzyme sensor that measures a change in the concentration of a substance involved in a biochemical reaction or an enzymatic reaction as a change in the amount of current to measure a substrate concentration. [Prior Art] Conventionally, sensors for glucose, urea, uric acid, etc. are known as enzyme sensors. The substrate concentration was measured by measuring changes in the oxygen concentration consumed in the enzyme membrane by the enzyme reaction using an oxygen sensor.
However, in such a sensor, since the enzyme membrane is bonded onto the gas permeable membrane of the oxygen sensor, the size of the sensor depends on the oxygen sensor, and miniaturization has been difficult. [Problems to be Solved by the Invention] The present invention provides a minute and highly sensitive enzyme sensor that measures changes in substrate concentration as changes in current amount. [Means and effects for solving the problem] As a means for solving this problem, the enzyme sensor of the present invention includes an electrically conductive substrate and an oxygen reducing agent having an oxygen reduction function that coats the electrically conductive substrate. It includes a catalyst layer and an oxygen-containing enzyme layer covering the oxygen reduction catalyst layer, and measures the oxygen concentration consumed by the oxygen reaction using an oxygen reduction current. [Examples] Example 1 A schematic structural diagram of a glucose sensor 10 produced in this example is shown in FIG. The method for creating it will be explained below. (1) Preparation of carbon fiber electrode Cut a bundle of carbon fiber 1 (Carboron: manufactured by Nippon Carbon Co., Ltd.) to a length of 2.0 cm, and attach conductive adhesive 2 (Sicilon B:
(manufactured by Amicon) to connect the lead wire 3. The electrode was insulated by being covered with an epoxy adhesive 4 and a Teflon (registered trademark) tube 5 so that only the cross section 1a of the carbon fiber became the electrode surface. Electrode area is 1.2×10 ^-4
cm2 . (2) Cobalt-porphyrin film coating Using a three-electrode cell with the carbon fiber electrode prepared in (1) as the working electrode, the Ag/AgCl electrode as the reference electrode, and the platinum winding as the counter electrode, meso-tetra An electropolymerized film 6 of (o-aminophenyl)cobalt porphyrin was deposited. <Electrolyte composition> Meso-tetra(o-aminophenyl)cobaltoporphyrin...1m mol/ Sodium perchlorate...0.1 mol/ Acetonitrile...Solvent Potential sweep from 0V to +1.8V (vs.Ag/AgCl) three times ( 30 at +1.8V after sweeping speed 50mV/sec)
Electrostatic electrolysis was carried out for 1 minute to complete the deposition. (3) Enzyme membrane coating Using a three-electrode cell in which the carbon fiber electrode coated with the cobalt porphyrin membrane prepared in (2) is used as the working electrode, the Ag/AgCl electrode as the reference electrode, and the platinum winding as the counter electrode, the following method is used. A glucose oxidase membrane was deposited. <Electrolyte composition> 1.2-diaminobenben...20m mol/Glucose oxidase 50mg/ml 50mM PH7.38 phosphate buffer...Solvent 0V to +1.5V (vs. After sweeping the potential three times (sweep speed 51 mV/sec) to +1.5V (vs.Ag/AgCl),
Constant potential electrolysis was performed for 30 minutes using AgCl), the enzyme membrane 7 was attached, and the glucose sensor 10 was completed. Experimental Example 1 Using the glucose sensor 10 produced in Example 1, the following experiment was conducted. PH with known concentration of glucose in electrolytic cell
6. Add 20 5mM phosphate buffer and use it as the test solution. The glucose sensor 10 prepared in Example 1 and the Ag/AgCl electrode are inserted into the test liquid. The potential of the glucose sensor 10 is set to -0.6V with respect to the Ag/AgCl electrode, and at that time, the potential of the glucose sensor 10 and
Measure the current value flowing between Ag/AgCl electrodes. Next, similar measurements are performed by changing the glucose concentration in the test liquid. In addition, as a method of changing the glucose concentration in the test solution, for the first measurement, the test solution was diluted (30%
mg/dl), and for the second and subsequent measurements, drop a certain amount of a high concentration solution (1000 mg/dl) into the test liquid.
A method of measuring by increasing the concentration of the test liquid was used. Measurement was performed at 32±0.1℃, and the current value was approximately 2 after the start of measurement.
The stable value after a minute elapsed was recorded. The glucose concentrations and current values obtained as a result of the measurements are shown in Table 1, and a plot of these values is shown in FIG. The current value decreases as the glucose concentration increases. Therefore, it has been found that by drawing a calibration curve in advance, the glucose concentration of the test liquid can be estimated from the observed current value. Example 2 A schematic structural diagram of the glucose sensor 20 produced in this example is shown in FIG. The method for creating it will be described below. A carbon fiber electrode is produced in the same manner as in Example 1, and coated with the cobalt porphyrin membrane 6 and further with the enzyme membrane 7 in the same manner. Next, a cellulose acetate membrane 8 was coated on the coated electrode to produce a glucose sensor 20 in which outflow of the enzyme and arrival of the substrate (glucose) to the electrode during measurement were controlled. The cellulose acetate membrane 8 was coated by a dipping method using a dichloromethane solution of 10% cellulose acetate as the dipping liquid. Experimental Example 2 Using the glucose sensor 20 produced in Example 2, the same measurements as in Experimental Example 1 were performed, and as a result, the values shown in Table 2 were obtained, and the plot thereof is as shown in FIG. Compared to the glucose sensor 10 of Example 1,
The linearity between glucose concentration and current value increases,
It was found that the sensitivity was also increased, providing a better glucose sensor. Although a glucose sensor has been described in this embodiment, the technical idea of the present invention can also be applied to other biosensors such as enzyme sensors or microbial sensors. Further, the conductive substrate or oxygen reduction catalyst layer described in this embodiment is not limited to this, and the conductive substrate may be graphite or a metal such as platinum or gold in addition to carbon fiber. The oxygen reduction catalyst layer may be a polymer film such as a cobalt phthalocyanine complex or a cobalt porphyrin complex. As described above, in the enzyme sensor of the present invention, (1) since the membrane is formed using an electrolytic polymerization method, an electrode of any shape can be produced, and in particular, a minute enzyme sensor can be produced. And it is easy to multitask. In addition, since polymer film formation can be performed under relatively mild conditions, deactivation of enzyme activity is suppressed, and high sensitivity can be expected. (2) Since it is an amperometric method, the response time is significantly faster than the several minutes required by the potential method.

【table】

[Table] [Effects of the Invention] The present invention can provide a minute and highly sensitive enzyme sensor that measures changes in substrate concentration as changes in current amount. In detail, (1) Since the membrane is formed using an electrolytic polymerization method, electrodes of arbitrary shapes can be produced, and in particular, minute enzyme sensors can be produced. And it is easy to multitask. In addition, since polymer film formation can be performed under relatively mild conditions, deactivation of enzyme activity is suppressed, and high sensitivity can be expected. (2) Since it is an amperometric method, the response time is significantly faster, taking several minutes, compared to several tens of minutes for the potential method.

[Brief explanation of drawings]

Figure 1 is a schematic diagram of the glucose sensor produced in Example 1, Figure 2 is a diagram showing the relationship between the glucose concentration and current density obtained in Experimental Example 1, and Figure 3 is Example 2.
FIG. 4 is a diagram showing the relationship between the glucose concentration and current density obtained in Experimental Example 2. In the figure, 1... carbon fiber, 2... conductive adhesive, 3... lead wire, 4... epoxy adhesive, 5... Teflon tube, 6... cobalt porphyrin membrane, 7... enzyme membrane , 8... is a cellulose acetate membrane.

Claims (1)

[Claims] 1. A conductive substrate comprising: an oxygen reduction catalyst layer having an oxygen reduction function covering the conductive substrate; and an enzyme layer containing an enzyme covering the oxygen reduction catalyst layer; An enzyme sensor characterized by measuring consumed oxygen concentration using oxygen reduction current. 2. Claim 1, wherein the oxygen reduction catalyst layer is selected from polymer films such as cobalt phthalocyanine complexes and cobalt porphyrin complexes.
Enzyme sensor described in section. 3. The enzyme sensor according to claim 1, wherein the enzyme layer has an enzyme immobilized on the oxygen reduction catalyst layer by an electrolytic polymerization method. 4. The enzyme sensor according to claim 1, wherein the conductive substrate is selected from carbon fiber, graphite, or metals such as platinum and gold.