The Construction Of Molecular Beacon With ZnS Quantum Dots For The Detection Of Telomerase And Its Performance Studies

Cancer is one of the malignant diseases. The radiologic techniques such as magneticresonance imaging (MRI) and X-ray computized tomography (CT) have primarily providedconventional tumor staging information (e.g., tumor size and lymph node status), which donot facilitate individualization of treatment decision making because cancer is a markedlymolecularly heterogeneous disease. Thus, pathophysiologic characterization of cancer usingmolecular biomarkers is likely the key to advancing early diagnosis and treatment.Telomerase activity is detectable in almost all (≈90%) human tumors, in particularly, thehuman telomerase gene (hTERT) is over-expression in malignant cell but is present at low orundetectable levels in the corresponding normal tissues. So, in this study，a novel molecularbeacon (MB) was developed for detecting hTERT-mRNA using ZnS quantum dots (QDs) asthe energy donors. It mainly contains3parts as follows:1) The synthesis of ZnS QDs and its optical properties.In this study, ZnS QDs synthesized respectively at aqueous phase and benzene phase.Further, ZnS QDs modified by thioglycolic acid.(1) ZnS:Mn2+QDs were synthesized byaqueous-phase chemical precipitation method at different pH by adjusting sodiumtripolyphosphate (STPP) concentration. ZnS:Mn2+QDs synthesized at pH9.5are uniformlydispersed with9.3nm in diameter, and the peak centers at580nm only when under330nmexcitation;(2) ZnS QDs synthesized at [Zn2+]/[S2-]﹦1:10have orange red emission at580nmonly under330nm excitation in benzene solution. QDs present obvious nanorod structurewith8.2nm in diameter,(3) TGA-ZnS:Mn2+QDs were synthesized directly in aqueous phase,which modified by thioglycolic acid. The emitting phosphors is pink in[TGA]/[Zn2+]/[S2-]/[Mn2+]=2.5：1：1.5：0.5when the aging temperature is80℃.2) Screening of the quenchers for molecular beaconQDsZnS-425and QDsZnS-580synthesized through the study of the synthesis of ZnS quantumdots, On this basis, this paper has used bovine serum albumin (BSA) as a linker to connect theQDs with riboflavin, PYCN, HOCY, rhodamine B, DyeA, DyeB. Further the opticalproperties of this FRET system were researched between these quenchers and ZnS QDs.Theresults showed that adding BSA effectively shortens the distance between the donor andreceptor. These quenchers happened FRET effect with ZnS QDs, and had obvious quenchingphenomenon, had the potential to be the ideal fluorescent quenchers of QDs.3)The construction of the wavelength-shifting MBThe study of screening of the quenchers found that riboflavin and PyCN is the mostsuitable quenchers to construct MB. Firstly,16nucleic acid sequences were chosen throughDNA mfold analysis of hTERT gene. DNA modified with sulfydryl and QDs modified withcarboxyl were covalently coupled by cross-linking agent EMCS. Two kinds of the wavelength-shifting MB were successfully constructed when TGA-ZnS:Mn2+QDs asfluorophore and riboflavin, PyCN respectively as quenchers. When there is a targetedsequence, the loop sequence of molecular beacon combined specificity with targeted sequence,fluorophore and quencher were separated, fluorescent signal respectively shifted from518nm,542nm to425nm. Telomerase activity can be detected qualitatively and quantitatively bydetecting the ratio of fluorescence signal.In conclusion, the wavelength-shifting MB with ZnS QDs can detect targeted DNAsensitively, and is hopeful to become the cell staining agents and fluorescence imaging agents.The position of MB can be tracked by detecting fluorescence signal, and it can also be usedfor tumor cell imaging.