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Colorimetric analysis method for measuring content of glucose and activity of glucose oxidase

A technology of glucose oxidase and glucose, applied in the determination/inspection of microorganisms, biochemical equipment and methods, color/spectral characteristic measurement, etc., which can solve the adverse effects of detection sensitivity, narrow applicable pH range, and repeatable operation Poor performance and other problems, to achieve the effect of less sample consumption, wide application range and rapid response

Inactive Publication Date: 2010-02-24
CHENGDU ORGANIC CHEM CO LTD CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, such a method has great disadvantages: firstly, ferrous ions are easily oxidized slowly by oxygen in the air, thus adversely affecting the sensitivity of detection; pH 4.0 approach to slow down Fe 2+ Oxidation in air, but this leads to a narrow range of applicable pH values, time-consuming and labor-intensive operations, and poor repeatability of operations; in addition, due to the oxidation of Fe by hydrogen peroxide 2+ It often takes tens of minutes to fully react, so the analysis speed is relatively slow, resulting in low test efficiency
[0004] In order to overcome the shortcomings of the existing GOD measurement system, design an easy-to-operate, sensitive, accurate, fast and reliable detection method, and broaden the scope of application of the detection system, it is necessary to readjust the existing evaluation system and change unreasonable factors

Method used

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  • Colorimetric analysis method for measuring content of glucose and activity of glucose oxidase
  • Colorimetric analysis method for measuring content of glucose and activity of glucose oxidase
  • Colorimetric analysis method for measuring content of glucose and activity of glucose oxidase

Examples

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Embodiment 1

[0025]Determination of the content of glucose in the sample to be tested. Prepare the sample to be tested into an aqueous solution with a certain concentration (pH=7.0). Take 1.0mL of the sample solution to be tested, transfer it to a clean test tube, add 4.0mL of GOD standard solution with a concentration of 0.1mg / mL at the same time, and then react in a constant temperature water bath at 25°C for 2.0h under magnetic stirring. Use three clean pipettes to measure 1.0mL 4-aminoantipyrine standard solution, 1.0mL phenol standard solution, and 0.5mL horseradish peroxidase standard solution, and mix them together in another clean test tube. Then pipette 2.5 mL of the above reaction solution of glucose and GOD, and measure the absorbance after stirring for 30 s. Measured with a UV-2010 ultraviolet-visible spectrophotometer, with a 0.1mol / L phosphate buffer solution of pH=7.0 as a reference solution, the wavelength scanning range is 600-400nm, and the absorbance of the measurement ...

Embodiment 2

[0027] The catalytic activity of glucose was determined at pH = 7.0 and 25°C. Take 1.0mL of 0.01mg / mL GOD solution, transfer it into a clean test tube, then add 4.0mL of 50mmol / L β-D-glucose solution, and react at 25°C for 15 minutes under magnetic stirring. Before the end of the reaction, prepare the standard analysis solution 3 minutes in advance, that is, measure 1.0mL 4-aminoantipyrine standard solution, 1.0mL phenol standard solution, and 0.5mL horseradish peroxidase standard solution, and mix together in another clean test tube. When the 14th minute is up, quickly measure 2.5 mL of the glucose-GOD reaction solution and mix it with it. After stirring for 30 s, quickly measure the absorbance of the generated red quinoneimine dye with a UV-2010 ultraviolet-visible spectrophotometer, and record the absorbance value measured at 510 nm. use figure 1 Using the standard curve shown, calculate the concentration of hydrogen peroxide corresponding to a specific absorbance. Fina...

Embodiment 3

[0031] The catalytic activity of glucose was determined at pH = 5.0 and 37°C. First prepare 1000mL of 0.1mol / L phosphate buffer solution with pH=5.0, and then use this buffer as solvent to prepare 50mL of 50mmol / L β-D-glucose solution and 0.1mg / mL glucose oxidase Solution 50mL. Before measuring enzyme activity, get 1.0mL concentration and be the GOD standard solution of 0.1mg / mL in clean test tube, add the β-D-glucose solution of 4.0mL 50mmol / L, operate according to the method described in Example 2, finally That is, the catalytic activity of the enzyme at pH=5.0 and 37°C can be determined.

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Abstract

The invention relates to a colorimetric analysis method for measuring the content of glucose and the activity of glucose oxidase, which is an efficient, quick and sensitive colorimetric method for measuring the content of the glucose or analyzing the activity of the glucose oxidase in a sample. The evaluation system has the characteristics of simple and practical method, mild reaction system, friendly environment, less sample consumption and good analytical repeatability; and in addition, the condition required for analysis is not high, and accurate analysis can be realized only by utilizing the ordinary ultraviolet-visible spectrophotometer in a general laboratory. Compared with the conventional o-diaminobenzene method, the Fitton method and the electrochemical method, the colorimetric analysis method can avoid trivial operation steps effectively, reduce the consumption of time and manual labor, and reduce the influence of environmental factors to the sensitivity of detecting enzymatic activity effectively. Therefore, the novel glucose oxidase analysis method can be widely applied in the fields of food production, brewing, beverages, medical detection and eassay, biochemical research and the like.

Description

technical field [0001] The invention relates to a colorimetric analysis method for measuring glucose content and evaluating the enzymatic activity of glucose oxidase, which belongs to the field of analytical chemistry. Background technique [0002] Glucose Oxidase (Glucose Oxidase, GOD, EC 1.1.3.4) is an important natural enzyme in the field of biomedical and biochemical research. It can catalyze the oxidation reaction of molecular oxygen to β-D-glucose in an aqueous environment. This produces gluconolactone, which is further hydrolyzed to give gluconic acid and hydrogen peroxide. For a long time, glucose oxidase has good commercial value and research value. It can not only be used in food processing and beverage production to remove glucose or oxygen in products to obtain gluconic acid, but also can be used to test D-glucose in various samples. content, such as human and animal blood, urine, and raw material liquids in the fermentation industry. In addition, it can also b...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N21/25C12Q1/54
Inventor 刘白玲侯晓晖罗荣陈华林
Owner CHENGDU ORGANIC CHEM CO LTD CHINESE ACAD OF SCI
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