Studies On DNA Fluorescent Biosensor And Water-soluble Quantum Dots In Application Of DNA Analysis

Deoxyribonucleic acid is an important hereditary material, and the base variation of DNA is tightly related to genetic diseases. So it is important to detect DNA sequence for disease diagnosis and clinical applications. Compared with the traditional organic fluorescent dye, the quantum dots exhibit remarkably important advantages, including broad excitation spectrum, the tunable and narrow emission spectrum, large stokes displacement, high photobleaching threshold and excellent photostability. Quantum dots is becoming an increasingly promising method for biomedical analysis. This masteral dissertation constitutes by four parts besides the preface:(1) A novel DNA fluorescent biosensor based on enzyme-enhanced fluorescence detection on etched optical fibers was developed. The hybridization complex of DNA probe and biotinylated target was formed on the etched optical fiber, and was then bound with streptavidin labeled horseradish peroxidase (streptavidin-HRP). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacet- ic acid (DBDA) generated from the substrate 4-hydroxyphenylaceticacid (p-HPA) under the catalysis of HRP, with a detection limit of 1 pM and a linear range from 1.69 pM to 169 pM. It was discovered that the sensor can retain 70 % of its original activity after three detection-regeneration cycles.(2) CdSe and CdSe/ZnS quantum dots was directly synthesized in aqueous phase. In order to overcome the problems of time consuming, harsh reaction conditions and non water-solubility of quantum dots synthesis in organic solvent phase, we synthesize quantum dots directly under non-oxygen, stirring, and using the NaHSe as precursor and mercaptoacetic acid as stabilizer. We also study the effects of temperature, reaction time, pH, stabilizer and its usage to the quantum dots fluorescent. In addition, TEM, fluorescence and UV/Vis spectrum were used to characterize the prepared nanoparticles. These results clearly showed that this method could synthetize the good and stable fluorescence CdSe and CdSe/ZnS quantum dots quickly under the simple condition. We synthesize CdTe quantum dots directly under non-oxygen, stirring, and using the NaHSe as precursor and mercaptoacetic acid as stabilizer. As-prepared CdTe QDs were shown to have narrower full wave at half maximum(FWHM), higher photoluminescence quantum yields(PL QY),and symmetric fluorescent emission peak. The PLQY of CdTe QDs reaches 45% at room temperature after placing three months. To our best knowledge, the simple experimental conditions, low cost and good repeatability, make it become one of the most successful methods which can directly prepare CdTe QDs in aqueous solution with high quality. In addition, it showed feasibility to synthesize CdTe QDs with near infrared fluorescence that make it be able to apply widely to biochemical analysis in the near future.(3) A novel ultrasensitive fluorescence detection method for DNA based on NEase and target recycles assisted QDs and MBs has been developed. The novel method can discriminate complementary target from single mismatch target accurately. The linear relationship between the analytical signal and the concentration of target is in the range from to 5.0 fM to 500 fM. Aside from high sensitivity and selectivity, this method, can be readily coupled to convention analytical techniques and extended useful for various applications involving the nucleic acids, aptamer-binding small molecules, RNA, metal ions. This system can also be used for SNP of a large number of genes and multiplexed analysis, for example, using different QDs to detect a series of DNA and proteins at the same time.(4) Based on binding of Hg2+ to promote these T-T mismatches to form stable T-Hg2+-T base pairs and the binding rate of DNA and the nanotube was lower than DNA hybridization, we have developed a convenient, fast, sensitive method of fluorescence detection of Hg2+. Under the optimal assembling and detection conditions, a good linearity for simultaneous detection was obtained in the range from 4.52×10-8 to 7.21×10-7 M, and the detection limit was estimated to be 0.1 nM. The presence of other metal ions did not interfere the detection of Hg2+.