Acridine ester chemiluminescence can study and analyze the DNA damage of environmental pollutants
The rapid development of molecular biology and genetic engineering has provided a large amount of information for the study of human genes, and has brought revolutionary changes to the diagnosis of genetic diseases and the study of pathogenic mechanisms. Establish simple, rapid and sensitive detection of DNA hybridization and inheritance The method of toxic effect is a very meaningful research topic. For the detection of DNA damage by genotoxic substances, the commonly used method is to detect the different signals generated by the interaction between the indicator and the DNA before and after hybridization. Now more indicators are used. The agent is acridine ester.
DNA damage detection methods include gel electrophoresis, ultraviolet spectrophotometry, fluorescence and electrochemistry, and chemiluminescence analysis. Chemiluminescence analysis (CL) has been widely used in the fields of environment and life sciences due to its high sensitivity, wide linear range, convenient operation, fast analysis, and easy automation. Formaldehyde and acetaldehyde are both environmental pollutants. To a certain extent, it can cause DNA damage. It has been reported that the detection methods of formaldehyde and acetaldehyde on DNA damage include ultraviolet spectrophotometry, fluorescence method, capillary electrophoresis fluorescence method, etc. Some of these methods have low sensitivity, and some are cumbersome to operate. In this paper, based on the changes in the amount of acridine esters embedded in DNA before and after DNA damage by formaldehyde and acetaldehyde, the changes in the chemiluminescence intensity of acridine esters are caused to study the damage behavior of formaldehyde and acetaldehyde on DNA, and a simple evaluation of formaldehyde and acetaldehyde Chemiluminescence analysis method of aldehyde damage to DNA.
Principles of Chemiluminescence Detection
Chemiluminescence experiment method
The principle of chemiluminescence analysis to detect the damage of formaldehyde and acetaldehyde to DNA is shown in the figure above. First, the glass luminescent cell used is immersed in a certain amount of concentrated nitric acid for several hours, acidified, and then washed with water. Add 20 μL of 1 mg/mL calf thymus DNA to the luminescent pool, and place it for 1 hour to make the DNA adsorb to the bottom of the luminescent pool, and then wash the unadsorbed calf thymus DNA with secondary water to obtain a luminescent pool with DNA adsorbed.
Add 50μL of 9.6×10-7g/mL acridinium ester to this luminous cell, and the chimerization reaction is 1h, so that AE is embedded in the DNA double-stranded structure, and the non-chimeric AE is washed with secondary water. Finally, press in the luminous cell. Add luminescence initiation reagents in sequence to detect the chemiluminescence generated by AE chimerized in DNA. When detecting the damage of calf thymus DNA by formaldehyde and acetaldehyde, add damage reagent to the DNA after AE chimerization, and use it twice after a certain period of time. The water washes away the AE released after DNA damage, and the luminescence initiation reagent is added to detect the chemiluminescence intensity of the AE chimeric in the DNA.
The research in this article shows that low concentration of formaldehyde causes DNA breakage and damage, high concentration of formaldehyde causes DNA strand cross-linking, and acetaldehyde only causes DNA strand breakage and damage. Acridinium ester can be used as a good chemiluminescence indicator for the structure of DNA double helix. The established chemiluminescence detection method for DNA damage caused by formaldehyde and acetaldehyde is feasible and has the advantages of simplicity and sensitivity. It provides a simple research method for the study of environmental pollutants on DNA damage.
HEPES, as a zwitterionic buffer, increases the osmotic pressure of the cell culture system by increasing the concentration of solution ions, maintaining normal cell morphology and function, and improving cell survival rate. Widely used in cell culture, especially under specific conditions such as tumor cell culture, it is crucial to maintain cell growth and function.