Studies On New Detection Technique Based On The Separation Of Quantum Dot Bioprobes Using Capillary Electrophoresis

With the development of life science, the complexity of biological sample is increasing, which makes higher request for the detection speed, efficiency, throughout, sensitivity and cost of analytical method. As a new type of fluorescent probes, quantum dots (QDs) have many unique excellent optical characteristics compared with traditional organic dyes and fluorescent proteins, and QD-based fluorescent sensors have been widely used in biological analysis in recent years. Capillary electrophoresis (CE) is one of the most powerful micro-separation techniques with advantages of fast, high sparation resolution, high sensitive, low sample consumption and high throughout, and also has been applied in bioanalysis. Therefore, the combination of QDs probes with CE detection techniques would have broad application prospects in bioanalysis. This thesis mainly focuses on the establishment of a new analytical method based on the combination of QDs probes with CE techniques, and its application in biological analysis and detection. The main contents and results are summarized as follows:(1) A highly efficient method for size determination of water-soluble CdSe/ZnS core-shell QDs was set by CE using polymer additive as sieving medium. The influence of some factors, such as kinds and concentrations of the sieving medium, pH and concentrations of the background electrolyte, and applied voltage, on the separation of QDs were investigated. Under the optimal separation conditions, four different sized QDs were successfully separated, and the relative standard deviation of the migration times for these QDs were 1.01%. In addition, an equation was fit by taking into account the correlation existing between the electrophoretic mobilities and the sizes of a set of QDs. The feasibility of this equation to measure the sizes of other QDs was confirmed by comparison with the sizes obtained by transmission electron microscopy experiment. This work offers a novel method for size determination of QDs, and provides an important reference on our subsequent bioanalysis based on CE and QDs probe. (2) A fluorescence resonance energy transfer (FRET) system was designed, which consisted of water-soluble 532 nm-emitting CdTe QDs donor and 632 nm-emitting CdSe/ZnS QDs acceptor. The QDs donor and acceptor were covalently conjugated with mouse 1gG and goat anti-mouse 1gG, respectively. The bio-affinity between antigen and antibody brought two kinds of QDs close enough to make the FRET happen between them. Then CE with fluorescence detection was used for the analysis of above QD-based FRET system. In the CE experiments, the real-time fluorescence signal of donor and acceptor were simultaneously collected by two signal channel with fixed detecting wavelength, and an effective separation of donor-acceptor immunocomplexes from free donor and acceptor was achieved, avoiding the fluorescent interference of non-FRET signal from those free ones and making the analysis of FRET more accurate. Results showed that FRET efficiency obtained by CE (38.56-69.58%) improved substantially in comparison with that obtained by ensemble measurement (12.77-52.37%). This work offers a novel approach for the FRET study possessing the unique advantages of improved FRET efficiency, high sensitivity, intuitionistic observation and low sample consumption.(3) Two quantum dot-molecular beacon (QD-MB) probes were designed using 585 and 650-nm emitting CdTe QDs which were covalently conjugated to MBs with different DNA oligonucleotide sequences by amide linkage and streptavidin-biotin binding, respectively. The hybridizations of QD-MB probes with different DNA targets were then monitored by CE, and results indicated that the two QD-MB probes specifically hybridized with their complementary DNA sequences, respectively. Furthermore, the simultaneous detection of dual single-base mutations in a given DNA oligonucleotide was successfully achieved in CE using above two QD-MB probes. This work offers a promising approach for simultaneous detection of multiple single-base mutations, and exhibits potential capability in the single nucleotide polymorphism analysis and high-sensitivity DNA detection.(4) Complementary DNA oligonucleotides-modified CdSe/ZnS QDs and gold nanoparticles (Au NPs) were connected together in solution by the hybridization of complementary oligonucleotides, and a model system (QD-Au) for the study of metal-enhanced fluorescence was constructed, in which the distance between QDs and Au NPs was controlled by adjusting the base number of oligonucleotide. The distance dependence of metal-enhanced QDs fluorescence in solution was then studied systematically by CE. In the CE experiments, the metal-enhanced fluorescence of QDs solution was only observed when the distance between QDs and Au NPs ranged from 6.8 to 18.7 nm, and the maximum enhancement by a factor of 2.3 was achieved at 11.9 nm. Furthermore, minimum 19.6 pg (15 nM) target DNA was identified in CE based on its specific competition with the QD-DNA in QD-Au system with the optimal QD-Au distance. This work provides an important reference for future study of metal-enhanced fluorescence in solution, and exhibits potential capability in the nucleic acid hybridization analysis and high-sensitivity DNA detection.