Construction Of Live Cell MicroRNA Sensing Probes Based On Metal Organic-framework Materials

MicroRNAs(miRNAs)play a critical regulatory role in DNA damage,biological metabolism,cells differentiation,apoptosis and cancer.The expression level of miRNAs is closely related to the degree of tumor progression,which means that miRNAs have great potential to be used as markers in diagnostic and cancer treatment.Therefore,in order to further study the function of miRNAs,it is urgent to develop an in situ,rapid,sensitive and specific method for the detection of intracellular miRNAs.In this thesis,two high sensitive,high specific nanoprobes have been developed for in situ miRNA imaging based on the metal-organic frameworks nanomaterials and functional nucleic acid.The detailed description is listed as followed:1.Detection and imaging of miRNAs in living cell based on nucleic acid molecular beacon-metal organic framework nanoassembliesIn this work,nanoassemblies which include a metal organic framework nanomaterials and a molecular beacon(MB),is constructed for detection and imaging miRNA in living cell.The acid-sensitive zinc-imidazole metal organic framework nanomaterials(ZIF-8)are used as the carrier,the nucleic acid molecular beacon are capable of specifically recognizing a target miRNA sequence(miR-21 as a modal molecule).Under neutral conditions,MB can be effectively loaded on ZIF-8(MB@ZIF-8),while in acidic environments,ZIF-8 dissolves and releases loaded MB molecules.The results showed that MB@ZIF-8 can enter the cancer cells through the lysosomal mechanism,release MB in the lysosomal acidic condition,and then bind to the highly expressed miR-21 in the cancer cells to activate the fluorescent signal to achieve the detection and imaging of miRNAs in living cell.Combining metal-organic framework nanomaterials with molecular beacon nucleic acid probes is a major innovation in this experiment,creating a promising prospect for the new material in the direction of biosensor.On the other hand,the MB@ZIF-8 nanoparticles have simple preparation,good biocompatibility and low toxicity,a low background fluorescence signal,low cost.Therefore,the approach demonstrates its potential in biosensing analysis,disease diagnosis and treatment of further research.2.High sensitive detection and imaging of microRNAs in living cell based on metal-organic framework nanomaterials and hybridization chain reactionA high sensitive intracellular miRNA detection method based on metal-organic framework nanomaterials,fluorescence resonance energy transfer(FRET)and hybridization chain reaction(HCR)signal amplification,has been developed.In this design,hairpin probe 1(hpl)is used as the recognition probe and trigger chain,the hairpin probe 2(hp2)is the reporter probe.hpl and hp2 are labeled with a donor fluorophore(FAM)and acceptor fluorophore(TAMRA),respectively.In the presence of the target miRNA,hpl will hybridize with the target and initiate the HCR,realizing signal amplification.Since the hybridization of the hp1 and hp2,the donor FAM and acceptor TAMRA will be close and produce FRET effect,along with the superimposed FRET fluorescence signal.When no target exists,the hp1 hairpin structure will not be opened and thus can not produce the FRET effect.The approach can effectively avoid the production of false positive signals,with high sensitivity,low detection limit,high specificity,a low background fluorescence signal,easy operation.