Application Of Potentiometric Biosensor Based On Degrading Bacteria In Determination Of Organophosphorus Pesticides

Ion-selective electrodes (ISEs) are a kind of electrochemical sensors based on phase boundary potential to indicate the activities of analytes. So far, they have been widely used in analysis of biochemical compounds, food safety and environmental monitoring. Organophosphorus pesticides (OPs) are widely used in agriculture, the residual of OPs in the environment have attracted great attention in China. In this thesis, Bacterial strains capable of degrade methyl parathion (MP) to produce p-nitrophenol were isolated in the environment. The highest degrading efficiency strain was identified as Klebsiella sp. and named as MP-6. Klebsiella sp. MP-6 was used as a recognition receptor and a polymeric membrane anion-sensitive electrode as a transducer to construct potentiometric biosensing system. Coupled with molecularly imprinted solid-phase extraction (MISPE), the system is used for the dectection of MP in the environment. The contents are as follows:1. Isolation of MP-degrading bacteria and characterization of its degrading mechanism:Four novel isolated bacterial strains capable of utilizing MP as the sole carbon source were isolated from pesticide contaminated soil. These four isolates were identified based on morphological characteristics and molecularly biology analysis, and their capability of degrade MP were investigated by high performance liquid chromatography. The highest degrading efficiency strain was selected for further study of degrading mechanism. The results indicate that strain MP-6 can degrade MP thoroughly after 7 days cultivation. Strain MP-6 was identified as Klebsiella sp. and named as MP-6, p-nitrophenol is the main metabolite.2. Potentiometric biosensor based on Klebsiella sp. MP-6 for determination of MP:Using Klebsiella sp. MP-6 as a recognition element, a polymeric membrane anion-sensitive electrode as a transducer and a rotating disc electrode as detector, we construct a potentiometric biosensor for the detection of MP. In the optimized conditions, the biosensor shows good performance towards MP. Besides, the biosensor shows excellent selectivity towards chlorpyrifos, fenthion, phoxim, trichlorfon and dimethoate.3. MISPE coupled with potentiometry for the detection of MPDue to the low amount of MP in the environment, a separation and enrichment pretreatment process is required before detection. Hence, the MP molecularly imprinted polymers (MIPs) are synthesized and used as sorbents for solid-phase extraction (SPE). By optimizing the extraction conditions, the efficient separation and enrichment of MP in the environment could be obtained by using the prepared MISPE column. Coupled MISPE with the potentiometric biosensor as illustrated in section 2, under the optimized conditions, the potential response of the present ISE was linear with MP concentration within 5-100 nM, the detection limit was calculated to be 1 nM (3?).