Acetylcholinesterase Modified Porous Silicon for Electrochemical Measurement of Total Active Immobilized Enzyme Amount and Effective Malathion Detection

Abstract

An amperometric method to measure the total amount of active and inhibited immobilized Acetylcholinesterase enzyme (AChE) and the quantification of AChE inhibitors was realized. In this approach porous silicon (PSi) surface was used as a matrix for AChE immobilization and Boron doped polycrystalline diamant (BDPD) was used as working electrode for the electrochemical measurements. The covalent immobilization of AChE from electric eel was achieved on amine functionalized PSi surface previously decorated with Au particles. This surface is suitable for a stable attachment of AChE enzyme. The amperometric detection of AChE activity at Boron doped polycrystalline diamond electrode is based on the oxidation of thiocholine, the enzymatic reaction product of immobilized AChE in the presence of the substrate acetylthiocholine chlorid (ATCl) and this without the need to further modify the BDPD surface or the use of other reagents. The concentration of immobilized active AChE enzymes was estimated to Γ≈ 1.8 10 12 AChE cm -2 by means of a calibration curve. Michaelis constant was assessed with a Km of 4.3 10 -4 M. Finally, the electrochemical quantification and detection of Malathion shows a good linear calibration curve where the concentration of Malathion range is from 2 to 6 nM within 6 minutes after inhibitor addition.