Preparation Of Quantum Dots And Their Applications In Biological Analysis

The key skill for developing fluorescent probes is to explore fine fluorescence materials and reagents with high sensitivity, good stability and diversification. Although with characteristics of high fluorescence yields, conventional organic dyes show some shortcomings such as narrower excitation, broader emission spectra, lower sensitivity and poor stability, which lead to much difficulty to distinguish them from self fluorescence of bio-molecules in applications. Quantum dots (QDs) just come to overcome these limitations of organic dyes and attract much attention of researchers in analytical and biological fields.For any fluorescent materials, facile synthesis method should be developed before they are used widely. Now, quantum dots are mostly prepared using TOP/TOPO system in which reagents are injected into a hot coordinating solvent tri-n-octylphosphine oxide (TOPO) at high temperature (about 300℃). This method would not be popular because the synthesis and storage procedures need to be operated under rigorous conditions such as full of nitrogen, without water and oxygen etc, even though the as prepared QDs possess good optical properties and high quantum yields (QYs). Furthermore, overlooking the markets at home and abroad, QDs, as a type of fluorescence reagents, are very expensive. Therefore, it is very important to search greener and effective routes to fulfil the demands in different fields. Among them, synthesis methods in aqueous solution and green organic ligands attracted much more attention. Unfortunately, QDs such as CdS, CdSe, ZnS obtained in aqueous solution usually show low QYs, although they have good biological compatibility. Based on above states, the aim of the present work is to synthesize high-quality QDs in a new way and then use them in macro-molecules probes, as well as immuno-imagings of tumor cells.For the preparation of QDs, as described in the second and third part of this thesis, a convenient and environment friendlier strategy, in which paraffin was used as solvent and reductant while oleic acid acted as stabilizer, was applied to prepare CdS, CdSe/CdS and Se doped CdS QDs. Optical and structure properties indicated that the as prepared QDs showed excellent fluorescence spectra with narrow FWHM (the full width at the half maximum), good dispersibility and desirable fluorescence yields. TEM images indicated that QDs dispersed well in aquous solution and the shape was approximately spherical. After modification with 2-mercaptoacetic acid, QDs were transferred into aqueous solution and got water-solublity for further applications in biological system.For the applications of QDs in analytical and biological fields, a study about the conjugation between CdSe QDs and macromolecules was carried out, in which the interaction rules between QDs and biological macromolecules such as proteins and enzymes were obtained. In this work described in the fourth part of this thesis, four kinds of proteins were chosen, they were normal proteins-bovine serum albumin (BSA) and chymotrpsin, as well as metalloproteinase-Cu/Zn SOD and bovine hemoglobin. Experimental results indicated that normal proteins could be covalently linked to QDs and the fluorescence intensity of QDs enhanced markedly after conjugating with BSA and chymotrypsin due to their passivation on the defects of QDs. However, for the conjugation of QDs with metalloprotein, the presence of metal ions made the conjugation be complicated and lead to the fluorescence spectra showing two kinds of results:the normal proteins could enhance its intensity and the matal ions in metalloprotein could quench it.With the similar linking method as described above, CdSe QDs were conjugated with rabbit anti-CEA8 antibody and goat anti-rabbit IgG through the interaction of carboxylic groups on the surface of QDs and amino groups on antibody. By the reaction between antibody and antigen, both QDs-antibody and QDs-IgG probes were successfully used to label HeLa cells, as described in the fifth part. Experimental results of fluorescence imaging indicated that the non-specific adsorption could be eliminated through the indirect labeling method. Tracking the signal of imaging for 24 h, QDs still exhibited high and stable fluorescence.In the sixth part, the interaction between gold nanoparticles (NCs) and CdTe QDs capped with mercaptopropionic acid or cysteamine were studied. Proteins can be not only adsorbed on the surface of gold NCs, but also linked to QDs in appropriate solution atmosphere. Based on that, fluorescence resonance energy transfers (FRET) assemble between QDs and gold NCs with chymotrypsin as the binding bridge is developed. This work provided some valuable information for the further study on the interaction mechanism among nanoparticles.In conclusion, this work began with seeking for a new environment friendly and simple route to synthesize high-quality QDs, then studied the conjugation ahout QDs with macromolecules. After linking with primary or second antibody, the QDs probes were used to label HeLa cells. At last, a deeper study on interaction between QDs and gold NCs was carried out. All experiments indicated that there are great potential in the multifunction and specific detection of QDs probes, which would turn to one of most promising research branches in this field.