SU940020A1 - Glucose determination method - Google Patents

Description

(54) METHOD FOR DETERMINING GLUCOSE

one

The invention relates to analytical biochemistry and can be used in research and development, the food and microbiological industries, fermentation production, clinical research.

A known method for determining glucose by introducing a sample of an analyte into a mixture containing a buffer solution, glucose oxidase, peroxidase, and an indicator substrate, o-toluidine, followed by measuring the density of the resulting solution 1.

The disadvantage of this method is its complexity, associated with the need to introduce two enzymes, glucose oxidase and peroxidase, which leads to a limitation of the possibility of using the method.

The closest in technical essence and achievable results to the proposed method is the determination of glucose by introducing a sample of the analyte into a mixture containing a buffer solution with pH 4-5, glucose oxidase and an indicator substrate - 2,6-dichlorophenolindophenol, followed by measuring the optical density of the resulting solution 2 .

The disadvantage of this method is its low sensitivity (MB) and accuracy (the error is 20%).

The purpose of the invention is to increase the sensitivity and accuracy of the method.

The goal is achieved in that in the method of determining glucose by introducing a sample of an analyte into a mixture containing a buffer solution, glucose oxidase and indicator substrate, followed by measuring the optical density of the resulting solution, N, N, N, N-tetramethyl-p is used as an indicator substrate -phenylenediamine and the process is carried out at a pH of 7.3-8.

The radical ion salt N, N, N, N - tetra15 of methyl-p-phenylenediamine has a bright blue color (A.max. 606 nm), which disappears when reduced, is characterized by a high molar reduction ratio (1, CM), is stable for long time at pH 4.6.

Example}. When measuring, 2.2 ml of Na-phosphate buffer solution (pH 7.7; 0.2 M), 0.3 ml of the working solution of the radical ion N, N, N, N-tetramethyl-n-phenylenediamine are introduced into the spectrophotometric cuvette. (0.164 mg / ml, pH 4.6), 0.1 ml of glucose oxidase solution (2 mg / ml). After measuring the initial optical density at 606, the reaction is initiated by adding 50 µl of the glucose solution to be examined to the solution. After completion of the reaction (5 min), the total change in optical density is recorded. The glucose concentration is calculated from a calibration plot of absorbance versus glucose concentration. The results of the analysis are shown in table. one.

Table 1 Dilution in the cuvette by 53 times. Example 2. When carrying out measurements, 2.2 ml of Na-phosphate buffer solution (pH 7.6; 0.2 M), 0.3 ml of the working solution of the radical ion N, N, N, N-tetramethyl were introduced into a spectrophotometric cuvette. -p-phenylenediamine (0.164 mg / ml, pH 4.6), OD ml of glucose oxidase solution (2 mg / ml). Photometry of the reaction is carried out at 606 nm. The reaction is initiated by adding to the solution 50 μl of the glucose solution under study. The reaction rate is judged by measuring the tangent of the angle of inclination of the linear portion of the plot of the dependence of optical density on time. The glucose concentration is calculated from the calibration graph of the dependence of the rate of the enzymatic reaction on the glucose concentration. The results of the analysis are presented in table. 2

table 2

Claims (2)

 Dilution in a ditch in 53 times. DETAILED DESCRIPTION OF THE INVENTION Glucose determination method by introducing a sample of an analyte into a mixture, containing buffer solution, glucose oxidase and indicator substrate, followed by measuring the optical density of the solution obtained, characterized in that, in order to increase the sensitivity and accuracy of the method, N , N, NiN-tetramethyl-p-phenylenediamine and the process is carried out at a pH of 7.3-8.0. Sources of information taken into account in the examination 1. US patent Re No. 29498, cl. 195-1035, published. 1977. 2. US patent number 3009862, cl. 195-1035 published 1961 (prototype).