Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for Quantitative Determination of In Vitro/In Vitro Superoxide Radical Content Generated by Environmental Stress

A quantitative determination and free radical technology, applied in the field of environmental toxicology, can solve the problems of signal loss, inability to distinguish fluorescence, and inability to directly compare the results, so as to reduce the interference of external environmental factors, and the method is simple and reliable.

Active Publication Date: 2022-07-26
维塔探索(广东)科技有限公司
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1) The results of fluorescence microscope observation are easily affected by factors such as ambient light, the quality and parameters of the camera, the light transmission efficiency of the microscope light path, and the thickness of the sample. The results are difficult to quantify accurately, and the results between different batches cannot be directly carried out. Compare
[0005] 2) The organism itself contains a large amount of autofluorescent substances, such as chlorophyll fluorescence. Under a fluorescence microscope, it is impossible to distinguish whether the fluorescence is from 2,7-dichlorofluorescein or other autofluorescence, so the obtained results are easily interfered by the sample
Substances such as ethanol, xylene, and paraffin are used during section preparation, which dissolve 2,7-dichlorofluorescein, resulting in loss of final signal
Therefore, this method is not suitable for larger animals or tissues

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for Quantitative Determination of In Vitro/In Vitro Superoxide Radical Content Generated by Environmental Stress
  • Method for Quantitative Determination of In Vitro/In Vitro Superoxide Radical Content Generated by Environmental Stress
  • Method for Quantitative Determination of In Vitro/In Vitro Superoxide Radical Content Generated by Environmental Stress

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Validation experiments for the determination of superoxide radical content in vitro by the above method.

[0059] Take 0.05 g of nano-titanium dioxide (nTiO 2 ) or large particles of titanium dioxide were dissolved in 500 mL of EPA medium, and the JP-060ST ultrasonic cleaner was used for 30 min (360 W, 50 kHz) in a water bath at 43 °C under the conditions of good dispersion, moderate thermal motion of particles, and no coagulation. , Jiemeng, Shenzhen, China), formulated with 100 mg / L TiO 2 The mother liquid medium needs to be prepared now to reduce the coagulation of titanium dioxide due to its own thermal motion.

[0060] Under the protection of nitrogen gas, accurately weigh 1 mg of 2,7-dichlorodihydrofluorescein into a 1.5 mL centrifuge tube, add 1 mL of methanol solution, vortex to dissolve, and prepare a 1 g / L solution. After observing that there are no solid particles remaining, filter it with a 0.22 μm needle filter and use it as the 2,7-dichlorodihydrofluores...

Embodiment 2

[0064] A method for quantitatively measuring ROS content in rotifers, specifically including the following experimental contents:

[0065] (1) Preparation of 1 mM working solution:

[0066] Accurately weigh 0.0487g of 2,7-dichlorofluorescein diacetate (H2DCFDA) into a 15mL centrifuge tube, use an appropriate amount of dimethyl sulfoxide to prepare a stock solution with a concentration of 10 mM, and then use EPA buffer to dilute to 1 The working solution with mM concentration was dispensed into 1.5mL centrifuge tubes and stored at -20°C.

[0067] H2DCFDA is a highly efficient bioaffinity fluorescent probe. As a non-polar compound, it can quickly diffuse into cells, be hydrolyzed into dichlorodihydrofluorescein under the action of cellular esterase, and react with ROS in cells to generate fluorescence The substance dichlorofluorescein fully excludes the interference of ROS in vitro.

[0068] 3.3.2 Experimental procedure

[0069] The experimental settings were 1 mg / L, 10 mg / L ...

Embodiment 3

[0076] Validation experiment for measuring the content of superoxide radicals produced in hydrogen peroxide by the method of Example 1.

[0077] Use distilled water to configure hydrogen peroxide to two mass percentage concentrations of 0.1% and 1%, and prepare a distilled water sample as a control.

[0078] Under nitrogen protection, accurately weigh 1 mg of 2,7-dichlorodihydrofluorescein into a 1.5 mL centrifuge tube, add 1 mL of methanol solution, vortex to dissolve, and prepare a 1 g / L solution. After observing under a microscope that no solid particles remain, filter it with a 0.22 μm needle filter and use it as a 2,7-dichlorodihydrofluorescein stock solution. To exclude the interference of oxygen in the air, the 2,7-dichlorodihydrofluorescein solution should avoid exposure to air.

[0079] Take 10 ml of 0.1%, 1% mass percentage of hydrogen peroxide and control group liquid and add them to three beakers respectively, add 50 mL of the above-treated EPA medium to each beak...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
diameteraaaaaaaaaa
lengthaaaaaaaaaa
particle diameteraaaaaaaaaa
Login to View More

Abstract

The invention provides a method for quantitatively measuring the content of superoxide radicals in vivo / external generated by environmental stress, which comprises the following steps: step S1, preparing a test solution: taking a sample to be tested, and preparing a test solution; Step S2, prepare a reference substance solution: take the standard substance 2,7-dichlorofluorescein, dissolve it in water, and prepare a reference substance solution; Step S3, use a high performance liquid chromatograph-fluorescence detector to analyze the test solution and the reference substance solution. In step S4, take the peak area of ​​the reference solution as the ordinate and the concentration of the reference solution as the abscissa, draw a standard curve, and calculate the 2,7-dichlorofluorescein in the test solution according to the standard curve the content of superoxide radicals. By adopting the technical scheme of the present invention, ROS can be quantitatively measured, the interference of external environmental factors is reduced, the comparability between different batches is made, and the method is simple and reliable.

Description

technical field [0001] The invention belongs to the field of environmental toxicology, and in particular relates to a method for quantitatively measuring the content of superoxide radicals in vivo / external generated by environmental stress. Background technique [0002] ROS (reactive oxygen species, reactive oxygen species) is an oxygen-containing by-product produced by biological metabolism, and its content will increase strongly under the influence of oxidative stress response after biological exposure to environmental stress. Whether it is important to respond to environmental stress. There are many enzymes that can eliminate ROS in living organisms, such as superoxide dismutase, peroxidase, catalase. If the organism produces ROS, the activity of these enzymes will increase. Therefore, most studies measure the activity of these enzymes to reflect the content of intracellular ROS or the level of oxidative stress from the side. However, the determination of these indicato...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/74
CPCG01N30/06G01N30/74
Inventor 张根李源真杨柳肖湘华潘璐璐欧阳嘉敏张奎王静邹佳林
Owner 维塔探索(广东)科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com