A Study On The Biosensor For Monitoring Organophosphorus Pesticides In Seawater

This work reported that acetylcholinesterase(AChE) from Cwrier(Scomberomorus niphonius) brain was immobilized on CNBr activated sepharose 4B by directly covalent combination, moreover, part of the characters of the immobilized enzyme was identified. Then A flow injection analysis system (FIA) was constructed based on immobilized enzyme and pH electrode.Using seawater as a stream, this work also researched the character of immobilized enzyme in FIA. Furthermore,being found on FIA,a based on immobilized AChE biosenor was made.Malathion and methl-parathion in seawater was used for testing the biosensor. After oxidization treatment of malathion and metyl-parathion, sensitivity of biosensor to Organophosphorus Pesticides (OPs) was enhanced. Finally, automated detecting of OPs in seawater was processed.After AChE was immobilized on CNBr activated sepharose 4B by directly covalent combination, the immobilized enzyme showed its highly activity of catalyzation and activity recovery of 96% after optimum. Stability of Enzyme activity was improved after immobilization ,and the enzyme activity only decreased 13% of original in 3 months. The Michaelis constant(Km) for immobilized enzyme is 0.444mmol/L.The characters of immobilized enzyme in seawater showed that it had a good reproducibility(RSD=2.33%,n=12). when concentration of subsrate(ATCh) ranged from5.2 mmol/L to 41.6 mmol/L, there was a good linearity (r=0.997)between response of biosensor and ATCh, and the optimum concentration and injection volume of ATCh were 41.6 mmol/L and 40UL respectively. The work also showed excessive substrate decreased the activity of immobilized enzyme. The optimum salinity and temperature of immobilized enzyme were 24.9%o and 30癈 respectively in different seawater. but when pH of seawater ranged 7 to 9,the activity ofimmobilized enzyme rised with ascending of pH value.Based on immobilized AChE mentioned above, biosensor of FIA was made, and used for detrmination of OPs in seawater directly. The calibration curves for malathion ,malathion after oxidization treatment ,methl-parathion after oxidization treatment over the concentration range of l-100Hg/L, 0. l-10Hg/L, 50-300ug/L (and 300-lOOOH-g/L) respectively, were obtained. The lower detection limit of malathion ,malathion after oxidization treatment ,parathion after oxidization treatment were 1. 5H-g/L, 0. 05M-g/L, 50ug/L respectively. Treatment of the inhibited enzyme by Ops mentioned above with 0.5mmol/L 2-PAM restored the enzyme to near full activity(99%),allowing repeated use of the biosensor.Finally, we probed on automated detecting of OPs in seawater .There was a good reproducibility of the response of immobilized enzyme to ATCh in every channel,(all of RSD<2%). The concentration of Ops that automated biosensor detected calculated by standard addition method was 90% of theory value.