Application Of Quantum Dots In Cellular Imaging Analysis

As a new type of nano-fluorescent material, quantum dots (QDs) have many advantages that traditional dyes do not possess. The fluorescence of QDs is very intrinsic, bright, and prolonged which endows them with new possibilities in long time imaging. Furthermore, QDs have similar physical properties. They can effectively tag different biological molecules such as macromolecular proteins, antigens and nucleic acid to the detection of various biological activities. In recent years, QDs with the tunable optical characteristics have been used in many fields including live cells labeling, macromolecular protein detection, the animals imaging, and have made many significant achievements. It has brought a broader application prospect in the field of life science and biology. This thesis mainly focuses on the optical properties and bioconjugation of the core-shell QDs, especially its practical application in biological as fluorescent probe. The main contents and results are summarized as follows:(1) Four probes were synthesized by chemical coupling and protein denaturation with CdSe/ZnS, CdTe QDs, and transferrin. SDS-PAGE electrophoresis and capillary electrophoresis were used to verify the conjugation of these luminescent probes. The cytotoxicity of these four luminescent probes and the original QDs were evaluated in HeLa cells. The results showed that CdSe/ZnS QDs capped with denatured transferrin had the lowest cytotoxicity. Furthermore, while the probe preparation was very simple, the photoluminescence quantum yield of this probe was 7% higher than the original CdSe/ZnS QDs. This provides a new way for exploiting QD probes with low cytotoxicity, which will expand applications of nanocomposite assembly in biolabeling and imaging.(2) Quantum dots were labeled to transferrin and the endocytosis of transferrin into HeLa cells was observed under normal state, iron overloading and iron deficient state. In three states the fluorescence underwent a gradual process of first dark, then light and finally dark, indicating the endocytosis of transferrin. The fluorescence intensity analysis showed that a platform was emerged when fluorescence changed to a certain degree in each of the three states. Experienced a same period of time after platform, the fluorescence strength of cells was different. Deficiency state was the highest and iron overloading state was the lowest. We also found that the average fluorescence intensity in cells detected by the spectrum in iron deficiency state was almost 7 times than that in high iron state. All these proved that iron overloading would slow down the progress, but iron deficiency would accelerate the endocytosis. This work advances a direct observational method and may contribute to further study the relation between iron and transferrin.(3) Water-soluble CdTe/CdS-QDs were synthesized in aqueous solution with captosuccinic acid as stabilizer. The absorption and fluorescence spectra showed that the QDs had good optical properties. The QDs were used to prepare fluorescent probes. The experiment results showed that the denatured transferrin could conjugate to CdTe/CdS QDs effectively and the HepG2 cells could be recognized successfully. This work is of great significance for the preparation of high-quality QDs and their applications in life science. It has an important reference value for the recognition of cancer cells.(4) A series of water-soluble CdSe/ZnS quantum dots with different electric charges were obtained by coating with amino and carboxyl terminal at different ratios. Spectrum and agarose gel electrophoresis were proved that this kind of quantum dots could be easily associated with plasmid DNA via electrostatic interaction, which was resistant to DNase I digestion. The further experiments found that the transfection efficiency changed with different electric charges in HeLa cells transfected with these nanoparticles. The whole transfection process was real time monitored by this complex. The results showed that the nanoparticles experienced binding, penetrating the cell membrane and going into cytoplasm in the first 6 h. After that, the QDs-DNA complexes had been stranded within the cytoplasm. At 18 h after transfection, the green fluorescence of EGFP began to appear, in the following 6 h, the plasmid DNA fully expressed. Toxicity test showed that the toxicity of these complexes was lower than that of the commercial liposome reagent. It offers one approach for development of new gene vector in this work.