Modification And Bioanalytication Application Of Quantum Dots

Quantum dots (QDs) have been achieved more and more applications in nanotechnology and nanomedicine due to their several remarkable, attractive optoelectronic characteristics, such as high quantum yield, high molar extinction coefficients, broad absorption with narrow, symmetric photoluminescence (PL) spectra, high resistance to photobleaching and exceptional resistance to photo-and chemical degradation. One of the fastest developing and most exciting interfaces of nanotechnology is the analytical applications of quantum dots in biochemistry. Hydrophilic solubilization of the high quality hydrophobic QDs synthesized in organic phase plays the crucial rule in their application in analytical biochemistry. And the biological effect of water-soluble QDs needs to be taken in account before its application to living cell analysis. According to the developing trend, the preparation of simple and universal QDs probe becomes an important aspect of QDs-based biosensors.The main contents of this thesis are described as follows:(1) Since proteins are macromolecules with amphiphilic, the non-covalent interaction between proteins and QDs is inevitable during the preparation of protein modified QDs. In order to control the preparation of protein related QDs probes, the nonspecific interaction between water-soluble mercaptoacetic acid capped CdSe/ZnS quantum dots (MAA-QDs) with six different proteins have been investigated. It was found that bovine serum albumin, ovalbumin and immunoglobulin G, which are not metalloprotein, will enhance the fluorescence intensity of CdSe/ZnS QDs, but the small heme protein cytochtome c with high isoelectric point (PI) quenches the fluorescence of QDs. Although hemoglobin is also metalloprotein, the fluorescence intensity of QDs still enhances due to the hydrophobicity of hemoglobin with lower PI and large size.(2) An alternative approach to obtain the QDs-DNA complexes can be realized by direct electrostatic interaction between positively charged QDs and dye-labeled negatively charged DNA. In the present work, we utilized lysozyme (Lyz), a protein with a high Isoelectric Point (PI 10.7～11.0), as surface ligand to prepare positive charged QDs. The UV-visible absorption spectroscopy, fluorescent spectroscopy and stability of Lysozyme coupled QDs (Lyz-QDs) have been investigated. And the transmission electron microscopy (TEM) demonstrates the size and dispersity of Lyz-QDs. A novel Hepatitis B Virus (HBV) DNA sensing strategy is proposed using the fluorescence resonance energy transfer (FRET) between Lyz-QDs and dye that labeled on the ssDNA (dye-ssDNA). Based on the configuration of dye-ssDNA before and after the hybridization reaction, the linear with HBV DNA concentration over the range of 3-65 nmol/L is achieved, with a limit of detection of 0.4 nM.(3) A new strategy for quantitatively detecting micrococcal nuclease (MNase) is proposed using electrostatic interaction-based FRET between positively charged QDs and negatively charged dye-ssDNA. The dye-ssDNA is absorbed to the surface of QDs through electrostatic interaction, which results in resonance energy transfer between QDs and the dye. In the presence of MNase which cleaves the dye-ssDNA into small fragments, the weakened interaction between QDs and the shortened ssDNA causes the decrease of FRET efficiency. At given amounts of donor and acceptor, the ratio of fluorescence intensity of QDs to ROX changes in a MNase concentration-dependent manner. Under optimized conditions, the ratio is linear with MNase concentration over the range of 8×10-3-9.0×10-2 unit·mL-1, with a limit of detection of 1.6×10-3 unit-mL-1. This new detection strategy features straightforward design and easy operation, which is capable of expanding the application of the positively charged QDs-based FRET in DNA-related bioassays.(4) Recently, mercapto-compounds capping on the surface of QDs have been shown to be deleterious to biological systems during the application of QDs. Adopting natural biomolecule nucleotide that have good water-solubility and excellent biocompatibility as raw modifying reagents, water-soluble QDs were successfully prepared through a very simple mechanic grinding method. Among all of the four common nucleotides, adenosine 5’-monophosphate (AMP) showed stronger interaction with QDs than the others.1HNMR and FTIR spectra were used to investigate the interaction mechanism between AMP and QDs. TEM images demonstrated the size and dispersity of AMP-QDs. Compared with MAA-QDs, the biological effect of AMP-QDs was investigated by biological microcalorimetry with Tetrahymena thermophila BF5 as model. The results indicate that AMP-QDs stimulate the growth of Tetrahymena thermophila while MAA-QDs inhibit its growth seriously. At the same time, the population density determination and fluorescence imaging of Tetrahymena thermophila BF5 also confirmed the good biocompatibility of AMP-QDs, which also testify the results obtained from biological microcalorimetry.