Application Of Core-shell CdSe/ZnS Quantum Dot In Biological Analysis

Core-shell quantum dot (QD) is a new inorganic semiconductor nanocrystal material. As a fluorescent probe, it has many unique excellent optical characteristics compared with traditional organic dyes. Firstly, as QDs have broad effective excitation spectra, simultaneous excitation of multiple QDs can be accomplished easily with a single excitation light, which can promote multiple applications of QDs. Secondly, it is not necessary to strictly compensate for the measurements owing to the narrow and symmetrical emission of QDs. Thirdly, the fluorescence strength of QDs do not decrease when exposed to light for long periods of time (that is, QDs are photostable), and QDs are less susceptible to metabolic degradation than organic fluorophores. Finally, although QDs with different sizes have different emission wavelengths (different colors), they have similar physical properties (such as dimension and polarity) and biological conjugation ability (namely, that can effectively conjugate to biomolecules and proteins). In recent years, quantum dots have been widely used in optical encoding of microbeads, encoded bead based array technology (including DNA hybridization assay and antibody/antigen immunoassay), molecular and cellular imaging, fluorescence resonance energy transfer (FRET) and capillary electrophoresis. This thesis mainly focuses on the synthesis and control of different size QDs, optical properties, surface chemistry and bioconjugation, and especially its practical application in biological and analytical filed as fluorescent probe. The main contents and results are summarized as follows:1. Different diameter CdSe/ZnS semiconductor nanocrystals (average diameter: from ~3.5 to ~20 nm), quantum dots (QDs) were synthesized by changing the nucleation time, using organometallic reagents. These quantum dots possess narrow and symmetrical fluorescent emissions. The emission wavelengths of these composite dots span most of the visible spectrum from 500 nm through 700 nm. Furthermore, it is found that the quantum dots with an emission at ~590 nm, tend to have a good quantum yield (such asΦ590= 43.5%). While the emission wavelength of prepared CdSe/ZnS QDs shifts toward blue or red from 590 nm, the quantum yield tends to decrease.2. Multiple-color encoded beads were achieved by incorporating two color core-shell quantum dots (CdSe/ZnS) to commercial polystyrene beads. By controlling the molar ratio of the QDs in doping solution, various encoded beads with different discriminable codes could be obtained. For two certain QDs, the relation curve about the intensity ratio of single encoded bead and the molar ratio of the two QDs in doping solution could be achieved firstly. Then the curve could be used to estimate the capacity of encoding based on these two QDs, and guide the quantitative encoding in the coming experiment. The results of antibody conjugation suggest that the encoded beads obtained in this method could be effectively used in biological applications. After that, optical encoding of microbeads with these quantum dots was carried out, and the spectra of encoded beads were identified. The result indicates that, to identify the encoded beads with different emission wavelengths and emission intensities, it is needed to acquire and differentiate the spectra of beads. After immobilized with human IgG, the encoded beads were used to detect the corresponding antibody in solution. The result indicates that the encoded beads can detect the antibody signal effectively. And the effective detection range of the antibody is about 2~15μM.3. A flow cytometric detecting technology based on quantum dots (QDs)-encoded beads has been described. Using this technology, several QD encoded beads with different code were identified effectively, and the target molecule (DNA sequence) in solution was also detected accurately by coupling to its complementary sequence probed on QDs-encoded beads through DNA hybridization assay. The resolution of this technology for encoded beads is resulted from two longer wavelength fluorescence identification signals (yellow and red fluorescent signals of QDs), and the third shorter wavelength fluorescence signal (green reporting signal of fluorescein isothiocyanate (FITC)) for the determination of reaction between probe and target. In experiment, because of QDs'unique optical character, only one excitation light source was needed to excite the QDs and probe dye FITC synchronously comparing with other flow cytometric assay technology. The results show that this technology has present excellent repeatability and good accuracy. It will become a promising multiple assay platform in various application fields after further improvement.4. Quantum dot (QD) solubilization, conjugation with biomolecules, column purification and labeling of human HepG2 cells with transferrin-QD (Tf-QD) conjugates are reported in detail in this paper. Water-soluble QDs (WQDs) were obtained using sodium thiolycolate to replace the surface ligand tri-n-octylphosphine oxide (TOPO) on the surface of oil-soluble QDs, and Tf-QD conjugates were produced by coupling Tf to WQD. The resulting Tf-QDs were characterized by UV and luminescence spectrophotometry, and purified by Sephadex column. The results indicate that Tf has been conjugated to QD successfully. Based on transferrin/transferrin-receptor-mediated delivery system, the Tf-QD conjugates were used to label human HepG2 cells. After a short incubation, the QDs were mainly localized to the membrane of cells. After 12-hour incubation, QDs appear mainly in the cytoplasm portion. However, QDs were not found in the nucleus of the cells. Furthermore, the fluorescence intensity of QDs in the cells reduces gradually over time, and fluorescence cannot be observed after 10 days. However, the growth of the labeled cells was not markedly affected by the toxicity of QDs, and they are alive for 10 days. These results can be used for further application of QDs in bioscience.5. The resonance energy transfer between chemiluminescence donor (luminol-H2 O2 system) and Quantum dots (QDs, emission at 593 nm) acceptors (CRET) was investigated. Concretely, the resonance energy transfer efficiencies were compared while the oil soluble QDs, water soluble QDs (modified with thioglycolate) and QD-HRP conjugates were used as acceptor respectively. The fluorescence of QD acceptor can be observed in the three cases, which suggests that the CRET occurs while the different QD acceptor was used. The highest CRET efficiency (10.7%) was observed in the case of oil soluble QDs. And the lowest CRET efficiency (2.7%) was observed in the QD-HRP conjugates case. This result is coincident with the quantum yields of the acceptors (18.3% and 0.4%). The same result was observed in another similar set of experiment, in which the amphiphilic-polymer modified QDs (emission at 675 nm) were used. It suggests that the quantum yield of the QD in different status is the crucial factor to the CRET efficency. Furthermore, the multiplexed CRET also be observed between luminol donor and three different size QD acceptors simultaneously. This work will offer useful support for improving the study of CRET based on QDs.