Design Of Functionalized Nucleic Acid Probes And Their Application In Biochemical Detection

Cancer is the representative of malignant tumor,which seriously threaten the human life and health.With the development of biosensor technology,people began to investigate the physiological and pathological mechanisms of cancer from the molecular level,in order to be used for the early diagnosis and treatment of cancer.As an important molecular probes,nucleic acid probes are widely used in gene diagnosis because of their advantages of easy synthesis,easy modification,flexible design,stable properties,and easy functionalization.In addition,nucleic acid probes are offen used to construct various DNA nanostructures for tumor cell imaging and drug delivery,due to their controllable assembly,biocompatibility,and low cytotoxicity.However,there are still many problems in the analysis method,such as low sensitivity,poor selectivity,slow analysis speed,and high experimental background.Based on the above problems,several functional nucleic acid probes have been successfully development in the present dissertation.Though the use of enzyme tools,gold nanoparticles and signal amplification technology,we have developed a variety of novel and highly efficient biological analysis strategies.Compared with other detection methods,the proposed detection methods which show their great application potential on the early detection of tumors.Firstly,a palindromic molecular beacon sensing system based on the character of pallet sequence and isothermal strand displacement amplification was developed for the ultrasensitive detection of K-ras gene.There was a good linear response range of 10 pM-150 nM and the target K-ras gene could be detected down to 10 pM.Furthermore,the wild-type gene against other mutant targets was easily distinguished.Secondly,we developed a novel sensing system based on strand displacement amplification which mainly contained a labelled hairpin probe,a linker strand,a primer and gold nanoparticles as a quencher for the detection of p53 gene.In this sensing system,the fluorescence of hairpin probe was quenched by gold nanoparticles because of fluorescence resonance energy transfer.In the presence of target DNA,hairpin probe could hybridize with target DNA and force fluorescent groups away from the surface of gold nanoparticles,leading to the fluorescence signal recover.This strategy possessed high speed of analysis,low background,good selectivity and high sensitivity,with a detection limit of 5 pM.In addition,the result of real sample was also satisfied.Finally,we proposed a DNA hexahedron nanostructure for the detection of pH values in vitro.The synthesis of the nanostructure was simple,did not require expensive experimental instruments and could delay the degradation of nucleic acids by nucleases.In addition,the fluorescence signal of the I-motif sequence was capable of rapid response at a range of pH values.Therefore,this nanostructure possessed a great potential application in intracellular pH detection and cell imaging.