A New Biosensor Based On Molecular Beacon And Bio-Barcodeand Application In Assay Of Telomerase And Tumor

The accurate and sensitive recognition and detection of trace tumor biomarkersand cancer cells, one of the most threatening diseases of the human being, is theessential need for the understanding of the processes and mechanisms of the diseasesand could be vital for disease diagnosis, prevention, and treatment. This thesisconstructs new biosensors with these characteristics of molecular beacon andnanoparticles, including the amplification of Au nanoparticles, bio-barcode andisothermal circular strand-displacement polymerization reaction. It with highsensitivity, good selectivity as well as being easy to miniaturization and automate hasbeen appliedinthedetectionoftelomeraseactivityandhumor cells.Thethesis mainlyconsistsofthefollowingissues:1 Based on the characteristics of molecular beacon, we designed a novel andPCR-free telomerase activity assay without the radioactive materials and highlypurified telomerase samples. The hairpin fluorescence probe was used as a template ofTS primer extension reaction and fluorescence signal carrier. Upon recognition andhybridization with the telomerase reaction product, the stems of the hairpin probesopened and restored the fluorescence signal, the fluorescence signals increased withthe increasingof the concentrations of the commercial telomerase. With the process ofelongationstep,2.5×10-15gmL-1telomeraseactivitycouldbedetected.Thebiosensorhas high sensitivity and excellent selectivity, the present fluorescence strategy holdsgreatpromiseinthebiochemicalassayfortheearlydiagnosisofcancers.2 Based on the characteristics of molecular beacon and the amplification of circular strand-displacement polymerization reaction, a PCR-free strategy to convertthe telomerase activityinto fluorescence signals employing hairpin fluorescence probeand two primers based on TS primer elongation and polymerase-induced isothermalstrand-displacement polymerization reaction was proposed in this paper, the assaycould be easily used to detect telomerase activity with a high sensitivity. ThetelomeraseactivityintheHeLaextractsequivalentto4-1000cellscouldbedetectedinthe present method. In conclusion, with its simplicity, selectivity and sensitivity, thisstrategy as a molecular tool holds great promise in the biochemical assay not only forthe telomerase activity and the amount of the cancer cells, but also for otherbiomarkersinearlydiagnosisforcancers.3 A novel electrochemical biosensor based on 6-ferrocenylhexanethiol and Aunanoparticles provided a direct method for quantitative detection of HeLa cells.Capture DNA and signal DNA were respectively modified on the surface of goldelectrode and gold nanoparticles. RNA extracted from HeLa cells played as a bridge,then Gold electrode, RNA and Au NPs could fabricate a"sandwich-type"assaystructure. The electrochemical signal of system was found to be dependent on theamount of HeLa cell, producing a highly sensitive sensor for HeLa cells. Wedemonstrated a linear dose response in the range from ranging from 1.0×10-15-1.0×10-13 M, 100-2000 cells, with a limit of detection of 4.2×10-16M, 76 cells. Thebiosensor has high sensitivityand excellent selectivity, the present strategyholds greatpromiseinthebiochemicalassayfortheearlydiagnosisofcancers.