| Organophosphates (OPs) are widely used as pesticides and insecticides in China, while the problems of residual OPs on food and in the environment are increasingly serious. OPs can combine with cholinesterase (ChE) rapidly to form phosphoric cholinesterase after entering human body. To prevent and conduct the issues of pollution of the food and environment, it's very important to use fast, sensitive and accurate assay methods. Electrochemical biomonitoring is simple and fast. What's more, the instruments are inexpensive and afford stable and highly sensitive performance as well. This approach is especially suitable for in-situ monitoring and large-scale screening of food. Acetylcholinesterase (AChE) is always used to evaluate the ecotoxicity of OPs for its good sensitivity and high activity.Based on the principle of inhibition of AChE by OPs, this thesis focuses on the research of electrochemical biomonitoring of residual OPs. The contents and results are as following:(1) Electrochemical assessment of parathion-methyl poisoning on the biomarker of ChE from pheretima using 2,6-dimethyl-p-benzoquinone (2,6-DMBQ):A square wave voltammetry (SWV) method was developed for the assessment of OPs impact on ChE of Pheretima with 2,6-DMBQ as a redox indicator. Acetylthiocholine is hydrolyzed by ChE to produce thiocholine which reacts with 2,6-DMBQ to give obvious shift of electrochemical signal. The reduction peak of 2,6-DMBQ is located at around-0.18V, far away from the oxidation potential of possible interference components often present in bio-sample. The decrease rate of reduction current was related with the activity of ChE. The inhibition rate of parathion-methyl on ChE activity increases quickly during the first 10 minutes incubation, after then it approaches to be constant. ChE loses almost 29.3% activity after 10min incubation with 1μg/ml parathion-methyl and 67.5% with 10μg/ml parathion-methyl, while the activity that corresponds to 40μg/ml parathion-methyl was nearly completely inhibited (94.9%). Compared to cyclic voltammetry (CV) and amperometry, SWV method is a high sensitive electroanalysis with fast scan-rate. This method can be employed to assess the inhibition of ChE and investigate OPs impact on environmental animals.(2) Construction of AChE-lecithin biomimetic structure at the cyclohexane/water interface for the electrochemical determination of fenthion:AChE was incorporated into a lecithin-based biomimetic structure at the cyclohexane/water interface for the determination of fenthion using a SWV method. Water insoluble indophenol acetate in cyclohexane phase was hydrolyzed by AChE at the interface to produce water soluble indophenol that was detection by the electrochemical means in the aqueous solution. The electrochemical signals were used to quantify the enzymatic activity of AChE and inhibition percentage caused by fenthion in cyclohexane phase. A relationship between the inhibition percentage and the fenthion concentration was obtained in a concentration range from 1ng/mL to lmg/mL. It was also observed that AChE in biomimetic structure possessed higher enzymatic activity and was more sensitive to fenthion inhibition than freestanding AChE.(3) Electrochemical assay of methyl-parathion in homogeneous reaction using water soluble films with immobilized AChE:Water soluble poly (vinyl alcohol) (PVA) was used to prepare the enzyme membranes of AChE for electrochemical assaying methyl-parathion in water insoluble organic solvent. The immobilized enzyme was separated from electrode for the development of enzyme inhibition-based bioassays. This assay is simple and convenient where enzyme membrane is applicable for single use and the electrode for repeated use. The enzyme membranes can be used handily piece by piece and the released AChE performs catalytic reaction homogeneously. The inhibition percentage of AChE increases with the concentration of methyl-parathion ranging from 0.1 to 1mg/mL. |
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