Preparation Of Apurinic/apyrimidinic Endonuclease 1 Fluorescent Probe And Study On Tumor Diagnosis

Apurinic/apyrimidinic endonuclease 1（APE1）is a protease expressed in different subcellular fractions and plays a vital role in different physiological functions.The abnormal expression and subcellular disfunction of APE1 are related to the occurrence of various tumor diseases.Therefore,APE1 can be used as a tumor biomarker to diagnose cancer.Consequently,it is particularly important to develop a rapid and sensitive detection method for APE1.In this thesis,we constructed a rapid and sensitive nano-fluorescence probe（TA@PR-ds DNA）for the detection of APE1 using arginine and double-stranded DNA（ds DNA）modified dendrimer loaded with1,6,7,12-tetrachloroperylene tetracarboxylic acid dianhydride（TCPBA（TA））,and it was applied in vitro detection.The concrete study contents and conclusions were shown below:1.Preparation of water-soluble TA nano-fluorescent dyes.In order to improve the detection speed,arginine（R）was first modified to the surface（PR）of polyamide-amine（PAMAM）to obtain arginine-modified polyamide-amine（PR）.By adjusting the molar ratio of arginine and PAMAM,three kind of PR polymers with different grafting ratios were obtained,which were 13%（PR13）,33%（PR33）,68%（PR68）,and these three polymers were selected to encapsulate TA dyes respectively.By optimizing the concentration of PR and TA,TA@PR13,TA@PR33 and TA@PR68 particles with good stability were obtained,and the particle sizes were 74,68 and 98 nm,respectively.Using PAMAM to encapsulate TA adjusted theπ-πstacking effect between dyes,and improves the dispersion and fluorescence intensity in the water phase.Meanwhile,the three nano-fluorescent dyes showed good photostability,and the fluorescence emission wavelength was about 600 nm,which can avoid interference of background signals such as intracellular autofluorescence.2.Specific ds DNA modification of TA@PR.At the cellular level,TA@PR68 had good biocompatibility and fast cell uptake rate,which can achieve rapid detection of APE1.Therefore,ds DNA with AP site and quench group BHQ2 was modified by TA@PR68 to obtain TA@PR-ds DNA.After adjusting the concentration of TA@PR and ds DNA,the optimized TA@PR-ds DNA particle size was about 122 nm,and surface potential of about 7.23 m V.The fluorescence wavelength and intensity did not change significantly.3.In vitro detection of APE1.TA@PR-ds DNA has high sensitivity,the detection range of APE1 is 1.2-25 U m L^-1,and the detection limit is 1.1 U m L^-1.In conclusion,we have constructed a rapid and specific nano-fluorescent probe for APE1 detection,which has good biocompatibility and rapid cell uptake ability.It can achieve the rapid and sensitive detection of various cancer cells’APE1,which opens up a new idea and way for the initial diagnosis of tumors.