Detection Of Telomerase Based On Isothermal Amplification Reaction And Detection Of Protein By Terminal Protection Colorimetry

Telomerase,a ribonucleoprotein reverse transcriptase,can prevent the shortening of telomere DNA.In normal cells,telomerase activity is very low and difficultly detected.But it is activated and has higher activity in cancer cells.Telomerase has been recognized as a potential biomarker for cell carcinogenesis.Therefore,the simple,rapid and high-sensitivity detection of telomerase is of great significance for the early diagnosis of cancer and the development of anticancer drugs targeting telomerase.Protein,the material basis of life,is involved in all kinds of human life activities,such as metabolic reactions,immune responses,matter and energy transfer,and so on.Some studies indicate that abnormal protein expression is closely related to the occurrence of some diseases.Therefore,the realization of simple,rapid and high-sensitivity detection of proteins is of great significance in clinical diagnosis and corresponding drug research and development.Based on isothermal exponential amplification reaction(IEXPAR)and small molecule-linked DNA terminal-protection,simple and sensitive methods for the detection of telomerase and streptavidin have been developed in this dissertation.(1)Detection of telomerase activity based on isothermally exponential amplification reaction and magnetic separation.Even in cancer cells,telomerase activity is low and requires a high sensitive assay to detect it.IEXPAR technology can rapidly and efficiently amplify short strand DNA in a short time under constant temperature.Its amplification efficiency in a few minutes is comparable to that of PCR in a few hours.We designed highly sensitive method for the detection of telomerase activity based on IEXPAR coupled with linear amplification by chain displacement.At the same time,we use magnetic microspheres to capture effective signal products.Through magnetic separation,excess reactants are effectively separated after each step of the reaction to avoid their influence on subsequent reactions.telomerase activity in as low as 20 cancer cells could be detected in this method.(2)Colorimetric detection of protein via terminal protection of small-molecule-linked DNA and unmodified gold nanoparticlesA simple and novel colorimetric strategy for protein detection was developed based on terminal protection from target protein and unmodified gold nanoparticles(AuNPs).We designed a hairpin-structured DNA probe with a biotin modification at the 3? terminal.Based on high affinity between biotin and streptavidin(SA),In the presence of the target protein SA,SA specifically binds to the biotin at the 3' terminal of the biton-DNA probe,which prevents the exonuclease III(Exo III)from degrading the biton-DNA probe to form terminal protection.In the absence of the target protein SA,The biton-DNA probe is degraded by Exo III to generate single-stranded DNA.By adding gold nanoparticles,single-stranded DNA can be better adsorbed on the surface of gold nanoparticles,protecting AuNPs from aggregation by salt ions,and the solution color is red;However,the adsorption of gold nanoparticles on biton-DNA probe is weak,which can not prevent salt ions from inducing aggregation of gold nanoparticles,and the solution appears blue.The detection limit of this method for SA was 0.24 pmol.The method is simple and sensitive,has good selectivity,no modification,no enzyme amplification reaction,and low detection cost.