| The micrococcal nuclease (MNase) is an extracellular endo-exonuclease of Staphylococcus aureus (S. aureus). It can digests DNA and RNA at AT or AU-rich regions to yield mononucleotides and oligonucleotides with terminal3’-phosphates. MNase has been used in hydrolysis of nucleic acids, sequencing of RNA, studies of chromatin and protein structure. In addition, the existence of MNase can be the standard to identify S. aureus and the content of MNase can be used to evaluate the pathogenicity of S. aureus.Mercury, widely distributed in the water, air, and soil, is considered to be a highly harmful heavy metal pollutant which exerts adverse effects on the environment. Divalent mercury ion (Hg2+) is the most common and stable form of mercury pollution. Hg2+can be converted to organic mercury by methylation and accumulated in the animal and human body through the food chain, posing a threat to human and animal health. Therefore, a new, sensitive assay for Hg2+in the environment and food industry is highly demanded.As an efficient optical molecular ruler, fluorescence resonance energy transfer (FRET) has broad applications in the nucleic acid detection, immunoassay, enzyme activity analysis and so on. However, owing to the poor photostability, narrow absorption spectra, and broad emission spectra of organic dyes, the application of FRET has been limited. Compared with organic fluorophores, quantum dots (QDs) possess unique optical characteristics. It has expanded the range of FRET and has been considered as new and significant development trend of FRET nowadays.In view of the importance of MNase and Hg2+detection, in this work, we establish the assays for MNase and Hg2+based on the FRET technique and QDs.The main contents are as follows:The first chapter is an introduction part to summarize the FRET principle, researches of fluorescent probes, the properties of QDs and the QDs-FRET applications to biological analysis. The aspects of nucleic acid detection, immunoassay and enzyme activity analysis have been described in detail.In the second chapter, a novel and simple method is presented for MNase detection based on QDs-FRET system through peptide acting as an electrostatic linker. The positively charged peptide serves as a bridge to bring negatively charged QDs and negatively charged ROX-ssDNA into close contact to energy transfer. When the ROX-ssDNA is cut into fragments by MNase, the FRET efficiency decreases, thus providing a sensing platform for MNase. This approach is simple in design. QDs covalent modification and doubly labeled DNA strand are avoided. Under the optimal conditions, the linear range covered4.0×10-3to8.0×10-2U mL-1. The limit of detection achieved2.9×10-3U mL-1.In the third chapter, a novel Hg2+detection based on QDs and nicking endonuclease (NEase) signal amplification technology is studied. The QDs are functionalized with the hairpin-shape probe A, modified with a quencher BHQ-2. The fluorescence of the QDs is quenched by the BHQ-2through FRET quenching. In the absence of Hg2+, probe B could not hybridized with probe A due to the three thymine-thymine (T-T) mismatched base pairs. When the Hg2+is present, probe B hybridizes with the loop of probe A due to the formation of T-Hg2+-T and the hairpin-shape probe A is opened. Then NEase recognizes specific nucleotide sequences and cleaves the probe A. After nicking, the probe A fragments dissociates with the probe B and Hg2+, and the distance between the QDs and BHQ-2increases, which generates a fluorescence increase of the QDs, The released Hg2+and probe B could then hybridize with another probe A to start a new cycle, therefore a remarkable signal amplification is achieved. A liner relationship between the QDs intensity and the Hg2+concentration was obtained over the range of1.0×10-9mol L-1to1.5×10-8mol L-1, with a limit of8.0×10-1mol L-1.The result showed this method had a good selectivity to Hg2+... |
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