Construction Of Upconversion Flurescent Probes And Their Applications In Micro Rna Detecion And Cell Imaging

Micro RNA is an endogenous non-coding RNA with a length of about 18-24 nucleotides,which is an important regulator of various physiological processes.The abnormal expression of micro RNA is closely related to the proliferation,angiogenesis,immune escape,migration,invasion and adhesion of tumors.Therefore,the rapid and accurate detection of micro RNA is of great significance for the early diagnosis and pathological analysis of tumors.Compared with the nanoprobes that use fluorescent dyes or quantum dots as signal tags,the micro RNA nanoprobes based on lanthanide-doped upconversion nanomaterials?UCNPs?possess the following advantages:less autofluorescence,good biocompatibility,good photostability,tunable emission spectra and large anti-Stokes shifts.However,the lack of detection sensitivity hinders the application of micro RNA upconversion nanoprobes in biological samples.Therefore,the development of micro RNA upconversion fluorescent nanoprobes with superior sensitivity and selectivity has important implications for the early diagnosis of tumors.This thesis is devoted to improving the detection sensitivity of upconversion nanoprobes and realizing the application of UCNPs in the diagnosis and treatment of tumor.Combining signal amplification strategy and nano-assembly technology,several novel upconversion nanoprobes have been developed for micro RNA detection and intracellular imaging analysis.The main contents are as follows:1.Assembly-disassembly of core-satellite UCNPs-Au NPs nanostructure for the detection of micro RNAMicro RNA responsive nanoprobes were engineered by incorporating upconversion nanoparticles Na YF₄@Na YF₄:Yb,Er@Na YF₄?UCNPs?and Au NPs in a core-satellite format.A lock-like DNA?LLD?constructed by the hybridization between the hairpin H1 and b DNA served as the linker molecule to anchoring Au NPs on the surface of UCNPs,thus quenching the upconversion emission of UCNPs.LLD can specifically recognize the target mi R-21,and allows target-induced circle amplification with the aid of fuel hairpin H2.With the assistance of H2,mi R-21 was able to unlock the LLD structure repeatedly,thereby causing the detachment of Au NPs from the UCNPs and the recovery of upconversion fluorescence.The proposed nanoprobes demonstrated low detection limit of 0.76 f M and could achieve highly sensitive detection of mi R-21.2.Target-induced circle amplification strategy for dual-channel fluorescence imaging of intracellular micro RNAAn upconversion fluorescent nanoprobe featuring target-induced circle amplification and dual-channel fluorescence was designed for accurate imaging of intracellular micro RNA.The lock-like DNA?FAM-LLD-SH?derived from the hybridization between FAM-H1 and HS-b DNA,was assembled onto the surface of PDA-UCNPs.Afterwards,Au NPs were grafted onto the surface of PDA-UCNPs via Au-S bond.In this nanoprobe,the green fluorescence emission of UCNPs and of FAM could be quenched by Au NPs.Target mi R-21 could change the structure of lock-like DNA,resulting in the departure of Au NPs.The hairpin H2 can take place of the target mi R-21,allowing the target-induced circle amplification,and thus enhanced fluorescence of FAM and UCNPs under excitation at 488 nm and 980 nm.The nanoprobes could significantly enhance the signal of intracellular micro RNA imaging,thus indicating excellent capability in distinguishing between normal and tumor cells.3.Ratiometric fluorescent nanoprobes for the sensitive detection of micro RNA and precise photodynamic therapyA multifunctional upconversion theranostic nanoprobe was constructed by combining the near-infrared excitation of UCNPs,the DNA loading and fluorescence quenching ability of Mn O₂nanosheets,and the signal amplification of hybridization chain reaction?HCR?.In this nanoplatform,mesoporous silica?m Si O₂?encapsulated with photosensitizer Ce6 was coated with Mn O₂ nanosheets,on which the hairpins HP1 and HP2 modified with fluorescent dyes were adsorbed.The reductive environment in tumor cells facilitated the dissolvation of Mn O₂,subsequently releasing HP1 and HP2 for the target-initiated hybridization chain reaction.Mi R-21 triggered cascaded assembly of HP1 and HP2 into long ds DNA polymers,which bought fluorophores FAM and TAMRA into close proximity.Due to F?rster resonance energy transfer?FRET?,the emission ratio of TAMRA to FAM would be increased with the mi R-21concentration under excitation at 488 nm.The nanoprobes realized the sensitive detection of micro RNA through ratiometric fluorescence,showing the potentials in fluorescence guided photodynamic therapy.In this research,we are commited to explore signal amplication strategies for the application of UCNPs-based nanoprobes in highly sensitive detection of tumor-associated nucleic acids,including biosensing and bioimaging.