A Novel Method For The Sensitive Detection Of Telomerase Activity At Single Cell Level

Telomerase is a ribonucleoprotein,and catalyze the synthesis of telomeric repeat sequences at the end of chromosome to compensate for the deletion of telomere in cell division.A large amount of datas show that telomerase activity is positive in most tumor cells,so researchers regarded it as a tumor marker,and carried out extensive research.At present,a variety of methods have been developed to detect telomerase activity,but we still face the problem of complex operation and low sensitivity.Therefore,the development of methods for simple,fast,reliable and sensitive detection of telomerase activity is very necessary.In this thesis,new methods are proposed to simply,sensitively and reliably detect telomerase activity.1.Homogeneous and ultrasensitive detection of telomerase activity via gold nanorods-based fluorescence reasonance energy transferAs a universal biomarker,telomerase is one of the promising targets for cancer diagnosis and therapy.Therefore,it is meaningful to develop facile,robust and sensitive methods for evaluation of telomerase activity.Herein,combined with fluorescence resonance energy transfer(FRET),we creatively designed a gold nanorod(GNRs)-based FRET method to detect telomerase activity from cell extracts.As the signal probe,carboxyfluorescein-modified DNA probes(F-DNA)hold negative charge.The electrostatic interaction between F-DNA and positively charged GNRs makes F-DNA close to GNRs,which leads to weak FRET between F-DNA and GNRs.In the presence of telomerase,telomerase substrate(TS)was elongated to form a long single-stranded DNA,which could hybridize with numerous of F-DNA to form long dsDNAs.The strengthened electrostatic interaction leads to a more efficient FRET between GNRs and dsDNA.Therefore,the amplified fluorescent quenching efficiency can greatly improve the sensitivity.The telomerase activity in the HeLa extracts equivalent to 1 cell was detected sensitively without the polymerase chain reaction(PCR)amplification and enzyme-aided signal amplification.Moreover,this facile protocol can be used to distinguish tumor cells from normal cells and study inhibition effect of telomerase inhibitors,which shows its potential application value in clinical diagnosis and drug screening.2.Point-of-care assay of telomerase activity at single-cell level via gas pressure readoutThe point-of-care testing(POCT)for simple,fast,reliable and sensitive detection of telomerase is urgent need in clinical diagnosis.Herein,we describe a facile and reliable(POCT)strategy for detection of telomerase activity via a portable pressure meter.Telomerase substrate(TS)was immobilized onto the surface of magnetic beads(MBs),and then was elongated to a long single-stranded DNA by telomerase.The elongated(TTAGGG)n repeat units hybridized with several short PtNP-functionalized complementary DNA(PtNPs/cDNA),which specifically enriched PtNPs onto the surfaces of MBs,which were separated using a magnet.Then,nanoparticle-catalyzed gas-generation reaction converted telomerase activity into significant change in gas pressure.Due to the self-amplification of telomerase and enrichment by magnetic separation,the telomerase activity of single HeLa cell extract was facilely detected.The difference in relative activity between different kinds of cancer cells was easily and sensitively studied.And the POCT strategy can be used to screen the telomerase inhibitors.The method is simple,fast,reliable and ultra-sensitive,which will make a useful exploration for the development of more convenient and practical POCT method.3.A facile and sensitive fluorescent method for detecting the activity of uracil-DNA glycosylase based on photoinduced electron transferHerein,a novel fluorescence strategy for the detection of UDG activity has been firstly established based on photoinduced electron transfer(PIET).A hairpin DNA probe(F-hpDNA)was designed as the substrate of UDG,which was labeled with carboxyfluorescein(FAM)at 5' end and has three deoxyguanosines at 3' end.So,the fluorescence of FAM was effectively quenched by deoxyguanosines.In the presence of UDG,the deficiency of uracil induced by UDG significantly decreased the stability of the F-hpDNA,which decreases the efficiency of photoinduced electron transfer and leads to the recovery of F-hpDNADNA fluorescence.UDG enzymes can be detected as low as 0.0005 U mL-1,and UDG enzyme activity in cell extracts can be also detected by the proposed method.Therefore,this is a simple,rapid and sensitive method for detecting the activity of UDG enzymes,which provides a new way for the detection of other base repair enzymes.