Understand the blood glucose determination kit method and master the basic principles and methods of blood glucose enzymatic determination.

(Source: Laboratory instruction of biochemistry and molecular biology, Shao Xueling et al, Wuhan University Press, 2003)

Operation method

enzymatic

Principle

The oxidation of β-D glucose in blood glucose to gluconic acid catalyzed by glucose oxidase and the production of hydrogen peroxide, which is catalyzed by peroxidase and oxidizes o-toluidine, the receptor for oxygen, to produce colored compounds. In the presence of sufficient glucose oxidase and peroxidase, the amount of colored compound formed is proportional to the amount of glucose and has a maximum absorption at 625 nm.

Materials and Instruments

Glucose
o-Toluidine Acetic acid Glucose oxidase Horseradish peroxidase
Test tubes Test tube racks Pipettes 722S Spectrophotometer

Move

Solution Preparation:

1. 1% o-toluidine solution: 1 g of o-toluidine was dissolved in 100 ml of anhydrous ethanol and kept in a brown bottle.

2. Acetic acid buffer (pH 5): 300 ml 0.15 mol/L acetic acid (8.7 ml glacial acetic acid to 1 L), 700 ml 0.15 mol/L sodium acetate was mixed and adjusted to pH 5 (with sodium hydroxide or hydrochloric acid).

3. Glucose oxidase, horseradish peroxidase: 10 mg dissolved in 10 ml of water and stored at 4 ℃.

4. o-Toluidine-containing reagent: 150 mlpH5 acetate buffer, 1 ml glucose oxidase solution, 1 ml peroxidase, 1 ml 1% o-Toluidine solution, mixed and stored at 4 ℃, can be stored for 7 weeks.

5. Standard glucose solution: 100 mg of dried glucose dissolved in 100 ml 0.3% benzoic acid solution (benzoic acid 0.3 g dissolved in 100 ml of water) into 1 mg/ml.

Experimental operations:

Take three test tubes and operate them in the following order:

Blank tube distilled water 1 ml
Standard tube standard glucose solution 1 ml
Measuring tube for sample to be measured (protein free hemofiltrate) 1 ml

The sugar content of the sample to be tested was controlled at 400-600 μg/ml. 5 ml of o-toluidine reagent was added to each tube sequentially at half-minute intervals, mixed immediately and reacted accurately for 10 min (the reaction time can be shortened in summer), and the optical density values were read at the wavelength of 625 nm at half-minute intervals, and a blank was used as a control for colorimetry.

Calculate according to the following formula:

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