SU1180771A1 - Method of glucose electrochemical determination and electrode for accomplishment of same - Google Patents

Abstract

1. A method for the electrochemical determination of glucose, which involves imposing a potential on an enzyme electrode, is different. In order to increase the sensitivity, before applying the potential, the enzyme electrode is kept in the analyzed solution at 7.0 pH 6 7.5 for 1.5-12 minutes, after which the amount of electricity proportional to the glucose content is measured. 2. An electrode for the electrochemical determination of glucose containing a conductive layer connected to a current lead and an enzymatic layer, characterized in that, in order to increase the detection sensitivity, the electrode is additionally provided with a layer of soot paste placed between the conductor and the enzyme layer (O 3. Electrode according to claim 2, characterized in that the conductive layer is made of a complex of N-methylphenazini and tetradian-p-chinodimet;

Description

The invention relates to analytical chemistry and can be used in medicine to determine the concentration of glucose. The purpose of the invention is to increase the sensitivity of the method. Example 1. Determination of glucose on a modified carbon black electrode. A glassy carbon electrode with a working surface of 0.1 cm is obtained by applying 1 mg of PM-105 carbon black paste and water (1: 2 by weight) to its surface. Then, 10 µl of a glucose oxidase solution (60 µM) was applied to the electrode, the dialysis membrane was put on top and secured with a rubber ring. Next, the electrode is immersed in a 0.1 M phosphate buffer pH 7.0 in the electrolysis cell and set to a potential of 0.0 V relative to the Ag / AgCl reference electrode. After 10 minutes, 10 or 20 ppm of glucose is injected and accumulated for 1.5-8 minutes. After this time, the potential of 0.4 V is turned on with a bipolar key and the amount of accumulated electricity is determined for 10 s. In tab. Figure 1 shows the data on the dependence of accumulated electricity on the accumulation time in determining glucose (pP 7.0; 0.1 M phosphate buffer). Table 712 is ruled by adsorbing from a 052% aqueous solution of polyvinylpyrimidine redox polymer quaternized with 2-bromobenzoquinone for 5 minutes. Then, the electrode is washed with water, 10 µl of a solution of gl ocosoxidase (60 µM) is applied to its surface, and a dial phosphate membrane is put on top, which is fixed with a rubber ring. The electrode is immersed in a 0.1 M phosphate buffer (pH 7.0) in the Electrolysis cell and a potential of 0.03 V relative to the Ag / AgCl reference electrode is set. After 10 min, 20–200 mM are introduced (concentration range for plotting the calibration graph a) glucose and accumulate for 1.5 or 4 minutes. After this time, the bipolar switch 2 includes a potential of 0.4 V and the amount of accumulated electricity is determined for 30 s. Data for determination of glucose (pH 7.05 0.1 M phosphate buffer solution) is given in Table. 2. Table 2

p and M e p 2. Determination of glucose on the electrode, modified 55 and

Mr. Redox polymer.

Glass electrode with a surface of 0, 1 cm modification. Table. 2 that when the accumulation time is 1.5 or 4 min, the glucose determination graph is linear to 110

the metabolite determination count is 20 µM. The electrode remains operable for 5 days ≈ 70 μM, respectively. Lower npe 3 Example 3. Determination of glucose electrolyte accumulation depending on time and degree of electrode modification of the redox polymer. A glassy carbon electrode with a working surface of 0.1 cm is modified by adsorption from a 0.2% stadol solution of polyhydrochloride for 2 or 5 minutes. The electrode is then washed with ethanol and water, immersed in a 0.1 M phosphate buffer (pH 7.0) and the degree of modification of the electrode with redox groups of 0.3 and 0.5 nmol-cm, respectively, is determined by recording the cyclic voltammogram. Then, 10 µl of the glucose oxidase solution (60 µM) is applied to the electrode surface, covered with a dialysis membrane, which is fixed with a rubber ring. The electrode sets a potential of 0.0 V relative to the Ag / AgCl reference electrode. After 10 minutes, 80 µM of glucose is injected and accumulated for 1.5; 3; five; eight; 12 min After this time, the potential of 0.4 V is switched on by the bipolar ring 2 and the amount of accumulated electricity is determined for 30 s. The data on the amount of accumulated glucose electricity on time and degree of electrode modification (glucose concentration 80 µM in 0.1 M phosphate buffer, pH 7.0, t 25 s) is given in Table. 3. Table 3

From the above data it can be seen that the limit value of accumulated 71 electricity is reached at accumulation time of more than 12 minutes The amount of accumulated electricity increases with an increase in the degree of modification of the electrode by the redox polymer. Example 4. Determination of glucose on an electrode from a conductive organic complex. An electrode obtained by pressing a conductive complex of tetracyano-p-quinodimethane and N-methylphenazine with a working surface of 0.3 cm, containing a 10 µm glucose oxidase solution (60 µM) under the dialysis membrane, is immersed in 0.1 M phosphate buffer (pH 7 , 0) in the electrolysis cell. The electrode sets a potential of 0.3 V relative to the Ag / AgCl reference electrode and is held for 10 minutes. After that, 10200 µM (concentration range for plotting the calibration graph) of glucose are introduced and accumulated for 5-10 minutes. After this time, the potential of 0.0 V is turned on with a bipolar key and the amount of accumulated electricity is determined within 60 s. These dependences of the accumulated electricity in the determination of glucose (pH 7.0; 0.1 M phosphate buffer) are given in Table. 4. Table 4

Continued table. 4 From the above data it can be seen that the amount of accumulated electricity using conductive complexes is proportional to the amount of glucose and the time of accumulation. The lower limit of metabolite determination is 10 µM. Example 5. Determination of glucose in the blood. The studies were carried out analogously to example 4, except for the introduction of 0.1 ml of ten-fold diluted human blood buffer solution of glucose instead of glucose solution into a 5 ml electrochemical cell. With a storage time of 10 minutes, the amount of accumulated electricity is 2.35 µC. The glucose concentration in the blood, calculated on the basis of the calibration graph, is (including dilutions) 5.1 mM (91.9 mg.%, Which is close to the physiological level of the metabolite (80-100 mg.%). Example 6. Determination of lactate concentration. A graphite electrode with a working electrode area of 0.15 cm is immersed in 9.2 μM of cytochrome B solution in 0.1 M phosphate buffer (pH 7.0) for 2 hours. Then the electrode is washed with the same buffer and immersed in 2 ml of buffer solution. in the electrolysis cell. A potential of 0.2 V relative to the Ag / AgCl electrode is set on the electrode After 10 minutes, 10,004-; 0.2 mM L-lactate (the interval of concentration to build the calibration graph) are introduced and accumulated for 5 or 20 minutes. After this time, a potential of 0.2 B and determine the amount of accumulated electrical power for 2 s. The data for determining lactate (pH 7, 0.1 M phosphate buffer solution 25 ° C) are given in Table 5. From the given data, a calibration graph is plotted based on the amount of accumulated electricity on L-lactate in the cell.

Table 5 The calibration graph for determining lactate is linear to 0.16 and 0.44 µM at 5 and 20 min accumulation. The lower limit of metabolite determination is 0.020 (5 min of accumulation) or 0.032 µM (20 min of accumulation). The electrode remains operable for 7 days. Example 7. Determination of lactate in saliva. Analogously to Example 6 except for injecting 10 µl of ten times diluted with a buffer solution of saliva instead of a solution of lactate. With a storage time of 20 minutes, the amount of accumulated electricity is 5.9; 6.4 and 7.5 µC for different samples. The concentration of lactic acid in the saliva, calculated on the basis of the calibration graph, is 0.59; 0.64 and 0.76 mM, which is close to the physiological level of the metabolite (7.4 mg.% According to known data). Example 8. The determination of the concentration of H, 0 ,. On the surface of graphite; Electrode, impregnated paraffin7

(0.018 cm) is applied with 10 μl of a solution of vegetable deoxidase (10 µM) in 0.1 M phosphate citrate buffer (pH 5.2). The solution is fixed on the surface by means of a dialysis membrane fixed on the body of the electrode with a rubber ring. The electrode is immersed in an electrochemical cell containing 25 ml of M phosphate-citrate buffer: pH 5.2, t, set a potential of 0.4 V relative to the Ag / AgCl electrode compare, and hold it for 10 minutes. Thereafter, 0.2 ml of 0.63-3.78 mM (concentration range for plotting the calibration graph) is injected into the cell, hydrogen peroxide solution in the same buffer solution, and electricity is accumulated for 1 or 2 minutes (in the reaction mixture from - for dilution the concentration is 5-30 µM). After the accumulation time has expired, a potential of 0.0 V is switched on with a bipolar key and the amount of accumulated electricity is determined for 2 s with a start delay of 120 ms.

These determinations (pH 5.2; 0.1 M phosphate-citrate buffer solution, t) are given in Table. 6

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Table 6

From the data given it can be seen that, with an accumulation time of 1 or 2 min, the calibration graphs for determining the hydrogen peroxide are linear up to 30. The lower limit of detection is 20 μM. The electrode remains operable for a month at 4 C.

Claims (3)

1. The method of electrochemical determination of glucose, which consists in imposing a potential on an enzyme electrode, characterized in that, in order to increase the sensitivity, the enzyme electrode is kept in the analyzed solution at 7.0 ^ pH έ 7.5 V before applying potential for 1.5-12 minutes, after which measure the amount of electricity proportional to the glucose content. 2. An electrode for the electrochemical determination of glucose, containing a conductive layer connected to a down conductor and an enzyme layer, characterized in that, in order to increase the detection sensitivity, the electrode is further provided with a carbon black paste layer located between the conductive and enzyme layers - § mi. 3. The electrode according to claim 2, characterized in that the conductive layer is made of a complex of N-methylphenazinium and tetracyano-p-quinodimetry.