Preparation And Characterization Of Florescent Ouantum Dots-Based Nanomaterials And Their Application For Biomedical Detection

Fluorescent probe is an important tool in biomedical fields for the study of the interaction between biomolecules, such as antigen-antibody, DN A strands, enzyme and substrate. Quantum dots (QDs), as a new class of fluorphores, have unique optical properties including high quantum yields, large molar extinction coefficience, resistance to photobleaching, tunable fluorescent emission and broad excitation wavelength. Thus QDs have many applications in the biological analysis and detection areas. In this dissertation, the following studies were conducted:(1) High quality CdSe quantum dots were synthesized in liquid paraffin, and coated with ZnSe/ZnS shell on the CdSe cores. Firstly, CdSe cores were prepared via high temperature injection method in liquid paraffin with octadecylamine (ODA) and trioctylphosphine oxide (TOPO) as surfactants. The influence of synthesis conditions, including surfactants, temperature and time, on the growth rates and optical properties of QDs were investigated. Further, the optical properies of obtained CdSe QDs were compared with oleic acid stabilized CdSe. The obtained CdSe QDs have high quantum yields over60%, full width at half maximum less than30nm, and emission wavelength coverage at490-650nm. CdSe cores were coated with ZnSe/ZnS shell to improve the quantum yields and stability. The reaction conditions, such as shell thickness, precursors and temperature, were investigated to optimize the optical properties of core/shell QDs. Transmission electronic microscope (TEM), UV-vis and PL spectrometer were used to confirmed that ZnSe/ZnS shell were coated on CdSe cores. The core/shell QDs displayed higher quantum yields and stability than CdSe cores, and can be used for further application.(2) Oil-soluble QDs were transferred to water-soluble by ligand exchange with glutathione molecules, and the surfactants were further crosslinked with bifunctional PEG to achieve compact PEGylated polymer-caged QDs. Dynamic light scattering (DLS), gel elctrophoresis, Fourier transform infrared spectroscopy (FTIR),1H-NMR and thermal gravimetric analysis (TGA) were used to confirm oil-soluble QDs ligand exchange with glutathiones and PEG covalently linked on the surface of QDs. The PEGylation process was optimized to obtain compact monodispersed PEGylated QDs. The pH and chemical stability of QDs in buffer, nonspecific absorption to cells and cytotoxicity of QDs were investigated. It was found the compact PEGylated QDs were more stable in a wide range of pH environment, and in various chemical environments. The cytotoxity and non-specific absorption of QDs to cells can be reduced by the PEGylation of QDs.(3) Immunofluorescent QDs were prepared by covalent conjugation of antibodies on the surface of carboxyl groups capped QDs. EDC/sulfo-NHS coupling agents were used to crosslink the amino groups in proteins with carboxyl group on the surface of QDs, and the bioconjugates were isolated from the free antibodies via ultracentrifugation. The isolation process was monitored with the UV-vis absorption, and the successful conjugation of antibodies with QDs was confirmed with both agarose gel electrophoesis and SDS-PAGE. Immuno activities of obtained bioconjugtes were investigated by dot blot immunoassays, and the influence of blocking agents on the immnuo activities was also investigated. It was shown that PEG-NH2blocked QD-Ab bioconjugates have lower detection limit, and the detection limit for HBsAg can be as low as1.6ng.(4) Monodispersed QDs-encoded polymer microbeads were generated using a simple capillary fluidic device (CFD). The polymer and QDs solution was emulsified into monodispersed microdroplets by the CFD and solidified via solvent evaporation. Polymer microbeads can be fabricated in a range of different size through changing the flow rates of the two immiscible phases, and have a highly narrow size distribution and uniform shape. QDs-encoding capacity of the microbeads was investigated through adjusting the concentrations and ratios of QDs in the polymer solution. Mono-color encoded microbeads with5intensities and a dual-color QDs-encoded5×5microbeads array were obtained, and the spectral profiles of the microbeads were examined by a fluorescent microscope coupled with a spectral imaging system. QDs-tagged microbeads prepared with this method were more stable than the porous beads swollen with QDs in the buffer with various pH and crosslinking chemicals. Finally, the application of such microbeads for biomolecule detection was demonstrated by conjugation of rabbit IgG on the surface of the microbeads via carboxyl groups, which were then detected by fluorophores-labeled goat-anti-rabbit IgG antibodies.