Research On Fluorescence Detection Of Cancer-related Endogenous MicroRNA

At present,it has become current research hotspots for cancer early diagnosis to screen suitable cancer-related biomarkers and develop efficient as well as convenient detection strategies.Previous studies have shown that obvious abnormal expression levels of non-coding microRNA(miRNA)always occurs in the early stage of cancer cells or tissues,which could be used as the appropriate cancer-related nucleic acid biomarkers.In this dissertation,the fluorescence detection has been combined with the designed deoxyribonucleic acid(DNA)sequence,which construct the identification and imaging system with high sensitivity and specificity for microRNA detection in cancer cells,and could be introduced into practical application.The following studies were carried out:Staudinger reduction has been applied for endogenous miRNA detection,and established a hybridization-mediated Staudinger reduction(HMSR)probe which made target miRNA circulate in the testing system.HMSR probe could recognize the target miRNA by base complementary pairing and lead to Staudinger reaction,which caused the fluorescence recovery of fluorescent molecule.HMSR probe showed good selectivity and high sensitivity with ameliorative detection limit(1.3 × 10-15 M).Meanwhile,HMSR probe could display accurate fluorescence imaging in living cells and tissues under both one-photon and two-photon(785 nm)excitation.Moreover,40 serum samples have been tested for exploring the potential clinical application,and the reliable results in distinguishing pancreatic cancer patients from different morbid stages was obtained.Those consequence could supply a feasible method for endogenous miRNA detection in cancer clinical diagnostics.Telomerase could extend hexamer telomeric repeats using the 3' end of the DNA sequence as the specific primer.Therefore,a telomerase-catalyzed FRET(Fluorescence resonance energy transfer)ratioflare(TCFR)nanoprobe has been developed for accurate sensing of low-abundance cancer-related miRNA.In this work,low false positive signals could be achieved through ratiometric measurements.Besides,TCFR nanoprobe could bind with target miRNA repeatedly for the synergetic work of telomerase and DNA probe,which resulting in high sensitivity with the improved detection limit of 2.3× 10-15 M.Meanwhile,high expression level of endogenous miRNA and telomerase allow screening cancer cells from normal cells reliably.It is reported endogenous glutathione(GSH)could react with disulfide bond.A GSH-cleavable nanoprobe(GCN)has been proposed for monitoring endogenous miRNA with gold nanoparticles as carrier.The synergetic work of miRNA and GSH enabled the self-assembly of DNA sequence with the FRET signal between fluorescent molecule.Meanwhile,each gold nanoparticle could load about 58 double-stranded DNA sequences,and turn into living cells through endocytosis with high efficiency.The covalent bonds between gold and sulfhydryl group could avoid the degradation of DNA probes.Besides,GCN provide obvious ratio fluorescent signal between cancer cells and normal cells.