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Determination of Organophosphorus Pesticides Based on Enzyme Inhibition

Author: WangYuHui
Tutor: ZuoJiGen；ZuoZuoQing
School: Zhejiang Normal University
Course: Analytical Chemistry
Keywords: Enzyme inhibition Acetylcholinesterase Merthiolate Poly (vinyl-alcohol) Surfactant Organophosphorus pesticide
CLC: X839.2
Type: Master's thesis
Year: 2011
Downloads: 54
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Abstract

Organophosphorus compounds (Ops) are widely used in modern agricultural, industry as pesticides or insecticides due to their broad-spectrum, easy degradation, high effectiveness and short half life in the environment, which contribute significantly to the economic benefits. Meanwhile, they also lead to a series of problems of environmental pollution and food contamination. A majority of OPs have great threat for the survival of human and animal due to their highly toxic property and the accumulation in living organisms. It’s significant to prevent and deal with the OPs pollution of food, avoid acute comestible poisoning, improve the study of OPs analyses and detect pesticide residue duly and accurately.In recent years, there were some reports about the fast determination of organophosphorus residue. Compared to traditional methods such as spectroscopic method, enzyme inhibition is a usual method of a range of samples and detecting samples, which is simple per-disposal to sample, the examination time short, cheap instrument. The sensitivity and reliability of enzyme inhibition method is closely related to the used enzymes. It mainly adopts acetylcholinesterase (AChE) in enzyme inhibition method to detecting pesticide residue. AChE is extracted from the head of sensitive insects and the electric organs of electric eel which is hard to get. Acetylcholinesterase also has some othe disadvantages as below:complex the process of extraction, low output, and high cost of detection, poor stability and easy losing activity. To meet the demand of the detection of pesticide residues it is very important to study the stability and the conservation of activity of AChE. Hereby, this article was based on the principle of the inhibition of enzyme, using spectrophotometry to study acetylcholinesterase’s immobilization and its application in the detectiondetermine of OPs residue. The content in detail and results are as follows:1. The influence of Merthiolate on the immobilized enzymeImmobilized enzyme was prepared by using AChE from Electrophorus electricus with chitosan as its carriera and Merthiolate as its preservative. The influence of the immobilized conditions on the activity of immobilized enzyme and the properties of the immobilized enzyme were investigated. The optimum pH, temperature of the immobilized enzyme contained Merthiolate were 8.2,38℃, respectively, and the immobilized enzyme without Merthiolate were 8.0,38℃, respectively. The bound enzyme contained Merthiolate showed a high thermal stability at 50℃, while the activity of ordinary immobilized enzyme decreased obviously; the activity of bound enzyme with 0.05% Merthiolate remains 92.3% after 90 d.2. A novel technique of acetylcholinesterase immobilization for assaying carbaryl pesticideA novel technique of enzyme immobilization is introduced where immobilized enzymes are able to perform homogeneous reaction. Water-soluable poly (vinyl alcohol) (PVA) was used to immobilized a model enzyme, AChE and the PVA-AChE enzyme membranes were obtained. When used, a piece of enzyme membrane is put into the solution and dissolved quickly. The released AChE from PVA-AChE membranes is mixed and interacted fully with substrates and carbaryl inhibitors. It shows almost equivalent performance to the catalytical activity of free AChE and the sensitivity of inhibition by inhibitors due to the homogeneous reaction of released enzymes. The values of Michaelis constant (Km) and maximum reaction rate (Vmax) for immobilized AChE are calculated as 0.16 mM and 0.058 A/min respectively which are very close to the Km value 0.14 mM and 0.057 A/min for free AChE. The concentration of PVA and aceton, the time of enzyme reaction, and the time of AChE inhibition by carbaryl pesticide were optimized. The relative Inhibition of AChE activity is increasing with the concentration of carbaryl ranging from 0.1 ng/mL to 100μg/mL and is nearly linearly with log [carbaryl] at the concentration range from 10 ng/mL to 10μg/mL. Compared with that free AChE in solution or solid powder, the prepared PVA-AChE enzyme membranes are easier to store and be used. Also, the procedure of weighting and dispersing AChE can be omitted. The suggested technique of enzyme immobilization is suitable for the widely applications with enzyme catalysed reactions involved for single-use cases.3. The new application of surfactants in determination of malathionThe influence of the three different types of surfactant on immobilized enzyme activity was researched. In order to improve the detection sensitivity andd we must choose appropriate surfactant as sensitizer and the termination reagent of reaction system. After 15 min of incubation, add 1mL the concentration of anionic surfactant sodium lauryl sulfate (10%) and the activity of immobilized enzyme basically did not change in 1 hour that mean the reaction completely ended. When the concentration of anionic surfactant sodium lauryl sulfate (CTAB) was 0.1% in the response system, the sensitivity of Ellman spectrophotometry method increased 27% and the detection limit of malathion to 4 ng/mL.

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