The Impact of Pesticides - Academy Publish

Acetylcholine + H 2 O AChE Choline + Acetate (1)Choline + O 2ChOBetaine Aldehyde + H 2 O 2 (2)Acetylthiocholine + H 2 O AChE Thiocholine + Acetic Acid (3)In the bienzyme system (equation 1 & 2) AChE catalyzes the hydrolysis of theneurotransmitter acetylcholine into acetate and choline. The choline is subsequentlyconverted by ChO, producing hydrogen peroxide in the presence of oxygen.Hydrogen peroxide can be detected amperometrically with different electrochemicaltransducers. Whereas, in a single enzyme system (eq 3), thiocholine (TCh) ester,acetylthiocholine (ATCh), is preferred as substrate. Acetylthiocholine can beenzymatically hydrolyzed by AChE to TCh, which in turn is oxidized at constantpotential at the electrochemical transducer, producing the initial biosensing response.One approach to improve the performance characteristics of the AChE-basedinhibitor biosensors would be to design and produce appropriate enzymes withcharacteristics more suitable for biosensor applications. Initial biochemical studiesrevealed that Drosophila melanogaster acetylcholinesterase (Dm. AChE) is themost sensitive enzyme toward OPs. The Dm. AChE-based inhibitor biosensorsshow great promise to improve the sensitivity of the biosensor system. Sotiropoulouet al.(2005) reported a Dm. AChE based inhibitor biosensor for the detection ofdichlorvos with a detection limit of 10 -17 M, which is 5 orders of magnitude lowerthan the Electropharus electricus AChE-based biosensor. However, this approachhas been hampered by the fact that AChE from various sources was not easilyavailable because of difficulties in isolation and purification procedures. Anotherpossible approach for improving the performance characteristics of the AChE-basedinhibitor biosensors is to improve the biosensor design and the electrochemicaldetection of the enzymatic product.The immobilization of enzymes on solid electrode is largely influenced by methodof deposition, which could significantly affect the electrocatalytic activity of theelectrode. Various methods including physical adsorption, covalent attachment,layer-by-layer self-assembly, and electropolymerization method were reported forimmobilizing AChE enzyme on electrode surfaces.Physical Adsorption: The physical adsorption is one of the simple procedures toimmobilize the enzyme onto the transducer (Bonnet et al, 2003). AChE wasimmobilized by adsorption on MWCNTs modified electrodes. In this way, few μLof AChE solution were dropped on the MWCNT modified electrode surface andallowed to evaporate at room temperature under a current of air. To remove loslybound enzyme molecules the AChE/MWCNT modified electrode was washedcarefully with buffer solution (Joshi et al, 2005). The draw backs of physicaladsorption are low quantity of adsorbed enzyme, leaching of the enzyme. Some ofAcademyPublish.org - The Impact of Pesticides400