| Organophosphorus compounds (OPs) have been not only used as pesticides and herbicides in current agriculture, but also used as chemical warfare agents, becoming a serious threat to ecological environment, personal and social security. While traditional detecting methods have very high sensitivity, reliability and precision, they require expensive laboratory instrumentation, highly trained personnel and time-consuming analysis. These techniques are unsuitable for rapid warning and field analysis. Therefore, rapid, sensitive, and precise detection of OPs in the environment, public places or workplaces become increasingly important for homeland security and environment monitoring. In this thesis, the study was focused on the preparation and selection of plant-esterase-porphyrin complex. The interaction of the complex with OPs was also investigated to construct a new sensor array system integrating plant-esterase-porphyrin film array finally. Researches were carried out as follows:(1) Purification of plant-esterase in an aqueous two-phase system (ATPS) was investigated. The effect of various process parameters such as the type of ATPS, the phase-forming salt, the molecular weight and concentration of PEG, the system pH, and the types and concentrations of neutral salts on partitioning of plant-esterase were evaluated. Using 27% PEG1000/ 13% NaH2PO4, and 27% PEG1000/ 13% NaH2PO4/ 6.0% (NH4)2SO4, plant-esterase was purified by a two-step extraction. Compared to the results obtained with the conventional salting-out method, this method had a comparable yield (83.16% versus the original yield of 80%), but produced plant-esterase that was 4.8 times as pure (18.46-fold). Integrating dialysis into the aqueous two-phase extraction removed (NH4)2SO4 from the purified plant-esterase. Finally, plant-esterase was freeze-dried to convert the product to powder.(2) Plant-esterase-porphyrin complexes sensitive for dimethyl methylphosphonate (DMMP), a simulant for organophosphorus agents, were selected with UV-Vis spectroscopy. The results showed that plant-esterase-H2TPPS1 had the highest sensitivity of 22380.95. The sensitivity of the plant-esterase-CoTPPCl, EuTPPCl, ZnTPP and FeTPPCl complex was 1318.3, 0.61, 0.17 and 0.05, respectively. Plant-esterase-CuTPP complex has the lowest sensitivity (0.94×10-6). Compared to that got with the porphyrins only, the sensitivity was increased except plant-esterase-CuTPP. Especially for plant-esterase-H2TPPS1, the sensitivity was significantly increased by 3.42×106. Therefore, five complexes, plant-esterase- H2TPPS1, CoTPPCl, EuTPPCl, ZnTPP and FeTPPCl, were selected as sensing material of biosensor for detection of organophosphorus agents.(3) To evaluate the mechanism of detection with plant-esterase-porphyrin complexes, the interaction of porphyrin-plant-esterase-OPs were investigated by the combination of UV-Vis absorption spectroscopy, fluorescence spectroscopy and enzyme kinetics methods. It was found that both H2TPPS1 and DMMP were competitive inhibitors of plant-esterase. They bind to plant-esterase at the active site, just like the substrate. However, DMMP exhibites higher inhibition potency than H2TPPS1 (ki(H2TPPS1) > ki(DMMP)). Therefore, the addition of DMMP to the performed plant-esterase-H2TPPS1 complex resulted in a loss of the 420 nm absorbance band as the porphyrin was displaced from plant-esterase.(4) A novel sensing nano-film immobilized plant-esterase-H2TPPS1 was prepared for the detection of DMMP. The results showed that the interaction of H2TPPS1 with immobilized plant-esterase yielded a characteristic peak at 422 nm. Exposure of the immobilized plant-esterase-H2TPPS1 surface to DMMP resulted in a decrease in absorbance intensity at 422 nm due to the displacement of the porphyrin from the active site by DMMP. The nano-structure of the film had a good effect on its sensitivity to DMMP and lowered the detection limit. The loss in absorbance intensity at 422 nm was linearly dependent on DMMP concentration below 9×10-7 M. DMMP concentration as low as 4.5×10-10 M can be detected by the sensing nano-film. |
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