Synthesis Of Doped Quantum Dots And Its Biosensing Application

Quantum dots?QDs?have been widely used in biomedical research due to their unique properties such as high fluorescence quantum yield,broad-band absorption,good photobleaching resistance,easy charge/energy transfer and facile surface functionalization.Doped QDs can emit double light from a single quantum dot by introducing doped ions.Typical doped QDs include manganese doped zinc sulfide?Mn:ZnS?and copper doped indium phosphide?Cu:InP?,which have the advantages of long dopant emission lifetime and low biological toxicity,and are especially suitable for biosensing and bioimaging applications.In this thesis,we designed and prepared a charge transfer complex of Cu:InP@ligand-PEG as ratiometric fluorescent probe via surface functionalization.Combined with nucleic acid isothermal amplification,we realized the visualized microplate analysis of circulating microRNAs in serum,and further realized the ratiometric fluorescence imaging of microRNAs in living cells.The main contents are as follows:1.Dual-emission Cu:InP QDs?peaks at 530 and 650 nm,respectively?were synthesized by a modified"sequential ion layer adsorption reaction".The green/red fluorescence intensity ratio of the QDs can be controlled by adjusting the Cu²⁺concentration or varying the shell-formation time.Based on the QDs with green/red fluorescence intensity ratio of 1:2,Cu:InP@ligand-PEG nanocomplexes were prepared by surface PEGlyzation and ultrasound-assisted assembly of ligands such as ZnPPIX,TMPyP or hemin.The photophysical properties of the nanocomplexes including steady-state and transient fluorescence were characterized.The results show that the fluorescence quenching of Cu:InP QDs is related to the type and number of ligands.The quenching mechanism is assigned to a charge transfer process.2.Relying on the specific ligand/DNA interaction,the Cu:InP@TMTyP-PEG nanocomplex with QD/ligand ratio of 1:3 was developed as a specific double-stranded DNA probe.The fluorescence titration experiments show that the probe is a ratiometric fluorescence probe,displaying a"green-yellow-red"traffic light-type fluorescence-color change,facilitating visual detection.The results of particle size analysis,Zeta potential measurement and cytotoxicity experiments show that the probe has the advantages of small size,electrical neutrality,chemical stability and good biocompatibility,and is suitable for molecular imaging at the cellular level.3.The Cu:InP@TMTyP-PEG probe was used for sensitive,visual detection of circulating microRNAs in serum via microplate analysis.Total RNA was first extracted from the serum of patients with non-small cell lung cancer?NSCLC?;target microRNA in Total RNA triggered hybridization chain reaction?HCR?of hairpins,producing long double-stranded DNA;the Cu:InP@TMTyP-PEG probe specifically identified the HCR product,displaying abundance-dependent traffic light-type fluorescent-color change.The detection limit of circulating microRNAs is calculated to be 1200copies/?L,and the linear detection range is defined to be 4 logs.Further,multiplexed detection of circulating microRNAs in sera of normal person/NSCLC patient was realized.4.The Cu:InP@TMTyP-PEG probe was used for ratiometric fluorescence imaging of intracellular microRNAs.DNA hairpins were first transfected into cells;HCR was triggered by target microRNA in cytoplasm;the Cu:InP@TMTyP-PEG probe entered cytoplasm through endocytosis and identified the HCR product by displaying traffic light-type fluorescent-color change.This ratiometric fluorescence imaging method is advantageous in terms of high resolution.Three target microRNAs in normal lung cells/NSCLC cells were detected by step-by-step fluorescence imaging,and the results were consistent with those of microplate analysis.