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A method for measuring superoxide anion free radicals in microorganisms

A technology of superoxide anion and microorganisms, which is applied in the preparation of test samples, color/spectral characteristic measurement, etc., to achieve the effects of good specificity, short time consumption and simple operation

Active Publication Date: 2020-12-15
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective method for determining the concentration of superoxide anion free radicals in microorganisms. Therefore, it is of great significance to develop a method for the determination of superoxide anion free radicals in microorganisms that is easier to operate and has a high degree of test stability.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: the assay method of superoxide anion free radical in the microorganism sample body after phosphine acclimation, concrete operation is as follows:

[0021]Take 20mL of the suspension of microorganisms acclimated by phosphine, in which the biomass is about 5000mg / L, centrifuge at 10000r / min for 8min, take 1g of microbial samples from the bottom of the centrifuge tube, add 3.5mL of carbonic acid buffer solution with a pH of 9.5, and Microbial samples were placed in an ice-water bath for 2 minutes and then centrifuged at 10,000 r / min for 10 minutes, and the supernatant was taken for experimental determination;

[0022] The formulation of the standard curve equation: with NaNO 2 Prepare 8 groups of NO concentrations of 3, 6, 9, 12, 15, 18, 21 and 24 μmol / L 2 - For the standard solution, take 8 test tubes, add 0.5mL carbonic acid buffer solution with a pH of 9.5, and then add 0.1mL hydroxylamine hydrochloride solution with a concentration of 70mmol / L, and then...

Embodiment 2

[0024] Embodiment 2: the assay method of superoxide anion free radical in the microorganism sample body of urban sewage treatment plant, concrete operation is as follows:

[0025] Take 20mL of microbial suspension from urban sewage treatment plants, in which the biomass is about 6800mg / L, centrifuge at 11000r / min for 9min, take 1.5g of microbial samples from the bottom of the centrifuge tube, add 6mL of carbonic acid buffer solution with a pH of 10.00, and Microbial samples were placed in an ice-water bath for 2.5 minutes, and then centrifuged at 11,000 r / min for 10 minutes, and the supernatant was taken for experimental determination.

[0026] The formulation of the standard curve equation: with NaNO 2 Prepare 7 groups of NO concentrations of 4, 8, 12, 16, 20, 24, 28 μmol / L 2 - For the standard solution, take 7 test tubes, add 1.5mL of carbonic acid buffer solution with a pH of 10.0, and then add 0.45mL of a hydroxylamine hydrochloride solution with a concentration of 100mm...

Embodiment 3

[0028] Embodiment 3: the assay method of superoxide anion free radical in the microorganism sample body of coking wastewater treatment plant, concrete operation is as follows:

[0029] Take 20mL of microbial suspension from the coking wastewater treatment plant, in which the biomass is about 4500mg / L, centrifuge at 12000r / min for 10min, take 2g of microbial samples from the bottom of the centrifuge tube, add 8mL of carbonic acid buffer solution with a pH of 10.00, and the microorganisms The sample was placed in an ice-water bath for 2.5 minutes and then centrifuged at 11000r / min for 10 minutes, and the supernatant was taken for experimental determination;

[0030] The formulation of the standard curve equation: with NaNO 2 Prepare NO concentrations of 3, 6, 9, 12, 15, 18, 21 and 24 μmol / L 2 - For the standard solution, take 8 test tubes, add 1mL of carbonic acid buffer solution with a pH of 10.0, and then add 0.3mL of a hydroxylamine hydrochloride solution with a concentrati...

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Abstract

The invention discloses a method for detecting superoxide anion free radicals in microorganisms. The method comprises the following steps: firstly extracting a microorganism sample to formulate a standard curve; then, setting an experimental group, a control group and a blank group to respectively detect the absorbance value of the microorganism sample, and taking difference values of the three groups of absorbance as absorbance values of microorganisms in the experimental group; and thirdly, calculating concentration values of superoxide anion free radicals in the microorganisms by utilizingthe standard curve and formula, and calculating the content of O2<-.> in the microorganism with unit mass according to the formula to complete detection of superoxide anion free radicals in the microorganisms. Compared with a traditionally used phosphate buffer solution having a pH value of 7.8 and a Tris-HCl buffer solution having a pH value of 7.6, a carbonate buffer solution having a pH value of 9.5-10.5 used in the method has the advantage that the sensitivity of a detection system for detecting O2<-.> is increased by about 10 times. The method has good specificity, simple operation and short time consumption, and can be easily popularized.

Description

technical field [0001] The invention relates to a method for measuring superoxide anion free radicals in microorganisms, and belongs to the technical field of biological purification treatment of waste water and waste gas. Background technique [0002] superoxide anion radical (O 2 -. ) belongs to the active oxygen group and has strong oxidizing properties. o 2 -. Ubiquitous in microbial systems, when organisms are stressed by pollutants, the production of active oxygen in the body will greatly increase; when it exceeds the antioxidant defense capacity of organisms, cellular components such as lipids, proteins, and nucleic acids in organisms will be at O 2 -. Irreversible damage occurs under the action of superoxide anion free radicals, which interfere with cell metabolism or lead to cell death. Therefore, superoxide anion radicals are considered to be an important way and mechanism of pollutant toxicity. In addition, studies have shown that reactive oxygen species ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31G01N1/34G01N1/38
CPCG01N1/34G01N1/38G01N21/31
Inventor 刘树根肖瑢杨希宁平
Owner KUNMING UNIV OF SCI & TECH
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