Serum Glucose Assay

Summary

Glucose oxidase catalyzes the oxidation of glucose to glucuronic acid and produces hydrogen peroxide: glucose ten 2H2O + O2 = gluconic acid ten 2H2O2. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for laboratory tests.

Operation method

Serum Glucose Assay

Principle

Glucose oxidase catalyzes the oxidation of glucose to glucuronic acid and the production of hydrogen peroxide: glucose ten 2H2O + O2 = gluconic acid ten 2H2O2 In the presence of chromogenic oxygen receptors (e.g., bicuculline, 4-aminoantipyrine coupled with phenol), peroxidase catalyzes the oxidation of hydrogen peroxide, which oxidizes pigmentation and produces colored compounds.

Move

I. Experimental reagents:

l, lmol / L phosphate buffer (PH7.0): dissolve anhydrous disodium hydrogen phosphate 8.67 g and anhydrous potassium dihydrogen phosphate 5.3 g in 800 ml of distilled water, with lmoL / L sodium hydroxide or hydrochloric acid to adjust the pH value to 70, and then diluted to 1 L with distilled water.

2, enzyme reagent: take glucose oxidase 1200W peroxidase 1200W, 4-aminoantipyrine 10mg, sodium azide 10Omg, plus phosphate buffer to 80ml or so, adjust pH7.0 plus phosphate buffer to 100ml, stored in the refrigerator, at least three months of stability.

3, phenol solution: redistilled phenol 100mg dissolved in 100ml distilled water, stored in a brown bottle.

4, enzyme phenol mixed reagent; enzyme reagent and phenol solution mixed in equal amounts, in the refrigerator can be stored for one month.

5、Glucose standard application solution (5.6mmol / L)

II. operation:

Take several 16mmm x 100mm test tubes and operate according to the table below.



Mix well, placed in a 37 ℃ water bath, insulation 15min, with a spectrophotometer, wavelength 505nm, colorimetric cup optical diameter of 1.0cm, with a blank tube to adjust the zero, respectively, to read the standard tube and determine the absorbance of the tube.

III. Calculation:

Serum glucose mmol/L = absorbance of measuring tube / absorbance of standard tube

Reference value: fasting serum glucose is 3.89-6.11mmol/L

Caveat

1, the newly prepared glucose standard solution is mainly α-type, so it needs to be placed for more than 2h (preferably overnight), to wait for the right balance before application.

2， glucose oxidase catalyzed reaction of the optimal PH range (6.5-8.0), but when the PH is as low as 6.

6， the absorbance of the reaction endpoints slightly decreased, the choice of PH7.0 earth 0.1.

3、Because the temperature has a big influence on this method, so the reagent is taken out from the refrigerator to reach room temperature, and then measured.

4、Determination of the sample to potassium oxalate - sodium fluoride as anticoagulant plasma is good.

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