Fluorescent Nanoprobe For Telomerase Activity Analysis

Telomerase is a ribonucleoprotein reverse transcriptase responsible for telomere maintenance by adding the telomeric repeats(TTAGGG)_n onto the 3'-end of human chromosomes.In human somatic cells,telomerase activity is highly regulated,but reactivated or over-expressed in tumors to maintain the indefinite proliferation of cancer cells.Telomerase is considered to be a valuable biomarker and a target for anticancer therapy due to its strong association with cellular immortality and tumorigenesis.The determination of telomerase activity is of great significance for the early diagnosis of human cancers,the understanding of tumor development and the screening of anticancer drugs.The significance of biomarkers detection drives the demand for developing sensitive,specific,simple and cost-effective biosensors.Fluorescent biosensors convert the recognition process of molecules into fluorescent signals,which are simple,sensitive,and rapid.They are widely used in the detection of biomolecules.With the rapid development of nanotechnology,nanomaterials with unique optical properties are gradually applied to fluorescent biosensor technology to improve the performance of sensors and promote the development of sensors.Nano-fluorescent biosensors have thus emerged as a new direction for the development of bio-molecular detection technologies.In this study,two fluorescent biosensors based on graphene oxide and cobalt phosphide nanomaterials were developed for telomerase activity analysis.The nanomaterials were used as quenchers and detection platform due to their large surface area,different binding affinity to different structure of nucleic acids,high fluorescence quenching efficiency and good biocompatibility.Thus,fluorescence detection of telomerase activity from cell extracts and in situ analysis of intracellular telomerase can be realized.The main contents are as follows:In the chapter 2,a fluorescence-labeled single-stranded DNA was used as the probe,graphene oxide was used as a fluorescence quencher and a sensing platform.A fluorescence biosensor was established to rapidly detect telomerase activity.Additionally,graphene oxide can also be used as a carrier for fluorescent probes to analyze intracellular telomerase activity.In the absence of telomerase,the fluorescent probe is adsorbed on the surface of the graphene oxide and the fluorescence is efficiently quenched.While telomerase could initiate the elongation of telomere-primer,leading to the hybridization with fluorescent probe to form DNA duplex,followed by the release of the probe from graphene oxide surface,and thus allowing the fluorescence signal to recover.Quantitative detection of telomerase activity in cell extracts was based on changes in fluorescence signal intensity,and the telomerase activity in living cells was monitored by confocal imaging.In chapter 3,two hairpin fluorescent probes with special sequence were designed.Combined with the hybridization chain reaction technology and the selective binding ability and fluorescence quenching effect of cobalt phosphide,a fluorescent biosensor was established for telomerase activity detection.The fluorescence labeled hairpin probes HP1 and HP2 are first adsorbed on the cobalt phosphide nanowires.In the presence of telomerase,the substrate extends out of a single stranded DNA,which can hybridize with probe HP1 to open it,the generated single-stranded portion of HP1 then hybridizes with HP2 probe,triggering a hybridization chain reaction,while the fluorophores adsorbed on the nanowires are released,resulting in numerous fluorescent signals after multiple hybridization chain reactions.Without telomerase,the hairpin structure of HP1 cannot be opened,the hybridization chain reaction cannot proceed,the fluorescence of the probe is effectively quenched by the nanowire through PET.Moreover,it is demonstrated that the cobalt phosphide nanowires-based nanosensor can be used for in situ and real-time analysis of telomerase activity in living cells.