The Analysis Of Telomerase Activity Based On Graphene Oxide And Cationic Conjugated Polymer

Telomerase composed of RNA and proteins is a ribonucleoprotein enzyme,with reverse transcription activity.Telomerase can elongate the telomeric sequences of chromosomes with its own RNA sequence as template and prevent telomere from shortening during cell division.telomerase activity in human normal cells is difficultly detected.It is usually activated in tumor or cancer cells.The activated telomerase is closely related to the immortality of tumor or cancer cells.Telomerase activity detection is great potential for reserach of the properties and functions of telomerase,early diagnosis of human cancers and screening telomerase-target anticancer therapy.In this dissertation,a series of magnetic oxidized graphene(MGO)or PFP-based assays coupled with amplification technologies for the detection of telomerase activity have been established.The main contents are as follows:1.Sensitive detection of telomerase activity based on MGO and isothermal exponetial amplification reaction(IEXPAR).Telomerase elongated product has many repeat sequences-(ggttag)n.A specific DNA probe-(ctaacc)2 which is matched with two repeat sequences is used to hybridize with telomerase elongated product.The excess DNA probes are adsorbed to MGO and removed by magnetic separation.The DNA probes hybridized with telomerase elongated product is rapidly amplified by IEXPAR.SYBR Green I(SG)is utilized for real-time fluorescent detection of IEXPAR products.Under the optimal conditions,The telomerase activity in 50 cancer cells has been obviously detected.Sensitive detection of telomerase activity under the condition of constant temperature is achieved.2.The assay for telomerase activity based on cationic conjugated polymer via fluorescence resonance energy transfer(FRET).A simple and fast assay for telomerase activity based on the FRET from a cationic conjugated polymer-PFP to SG has been developed.In the presence of telomerase,the primer is extended,generating a special DNA with many repeat sequences-(ggttag)n.Then,the telomerase elongated products are fixedly connected with magnetic beads through the specific combination of streptavidin(STV)with biotin.A specific DNA probe-(ctaacc)2 which is matched with two repeat sequences is used to hybridize with telomerase elongated product.After the telomerase elongated products form double strands,a fluorescent dye SG is added to the hybridization solution.SG can inset the double-stranded structure of DNA.PFP is added finally.It can adsorbon dsDNA by electrostatic interaction.FRET from PFP to SG occurs.So,quantitative determination of telomerase activity can be achieved by detection of efficiency of FRET.The telomerase activity in 3×105Hela cells has been obviously detected.In order to improve the detection sensitivity,hybridization chain reaction(HCR)as signal amplification technology has been used to coupled with FRET.The telomerase activity in 6×104Hela cells has been detected.The detection sensitivity is improved higher almost one order of magnitude.But,this result is still unsatisfactory.Further efforts are needed to improve the detection sensitivity of this method.