Synthesis Of CdSe/CdS Quantum Dots And Enzyme Bioconjugate And Its Application On The Analysis Of Organophosphorus Pesticide

In recent years, quantum dots (QDs) have been investigated for their uses as biological fluorescent probes in replace of organic dyes because of their better spectral features and inherent functional advantages. In the dissertation, a new method for the determination of the parathion in the environment sample is developed based on the aqueous soluble CdSe/CdS core-shell type QDs and methyl parathion hydrolase (MPH) bioconjugate.The dissertation is divided into three sections.In chapter 1, studies of quantum dots are reviewed with the emphasis on its properties and applications on bioassays and in ultrasensitive optical detection and imaging.In chapter 2, CdSe/CdS quantum dots are synthesized in aqueous solution and in non-TOP- based organic solution. The size of CdSe-core is estimated by absorption edge of UV-Vis absorption. The obtained quantum dot fractions are characterized with transmission electron microscopy (TEM), spectrophotometry, and spectrofluorimetry. The synthesized QDs are modified by 2-mercaptoacetic acid to achieve better aqueous solubility in basic conditions with preservation of high quantum yields (QYs), which are promising in their use as biological labels.In chapter 3, methyl parathion hydrolase (MPH) is obtained through a serious of purification steps including ammonium sulfate precipitation and anion exchange chromatography. The purified MPH is then coupled to CdSe/CdS core-shell QDs, forming the MPH/QDs bioconjugates through electrostatic interaction between negatively charged QDs surfaces and the positively charged protein side chain and ending groups (-NH3+). Photoluminescence (PL) spectroscopic study shows that the PL intensity of the MPH/QDs bioconjugate is quenched in the presence of methyl parathion. The overall quenching percentage as a function of methyl parathion concentration matches very well with the Michaelis-Menten Equation. Meanwhile, FL intensity of MPH/QDs are detected when p-nitrophenol, phenol, NO₂^-, NO₃^- are added into the solution. This result indicates that the quenching of PL intensity is caused by the structure change in the enzyme. The detection limit of methyl parathion concentration using MPH/QDs bioconjugate is about 2.1×10^-8 mol/L. In the range of 2.1×10^-8 mol/L to 3.0×10^-6 mol/L, the quenching percentage as a function of methyl parathion concentration matches very well with Y=1.5816X/(1.8697×10^-6+X). The calibration graph is linear in range of 2.1×10⁽-8 mol/L to 3.0×10⁽-6 mol/L methyl parathion with the present method. The relative standard deviation is 0.39 % for the determination of 0.5μmol/L methyl parathion (n=11). These properties make the MPH/QDs bioconjugate a promising biosensor for the detection of methyl parathion. The proposed method has been applied to the analyses of several samples, including soil, grass in field, cole, and Chinese chive with satisfactory results.