Bienzyme amperometric probes for choline and choline esters assembled with nonconducting electrosynthesized polymers

Different nonconducting polymers have been synthesized on the surface of a platinum (Pt) electrode to assemble fast-response and sensitive amperometric biosensors for choline, butyrylcholine, and acetylcholine, based on choline oxidase (ChOx) and acetylcholinesterase (AchE) or butyrylcholinesterase (BuchE), co-immobilized by crosslinking with bovine serum albumin (BSA) and glutaraldehyde (GLT). Also the electropolymerization conditions as scan rate and time were optimized for each monomer in order to obtain the best permselectivity, the highest interferences rejection and stability. The procedure of immobilization was proven to be fast and simple and resulted in a better longterm stability and in a higher loading of the enzyme than that obtained by the co-electropolymerization. Studies on reproducibility, lifetime of the immobilized enzymes, pH, temperature, interferences, buffers, response time, storage and operational time of the biosensors have been carried out. The optimized biosensors allowed rapid quantitative assay of cholinesterase inhibitor paraoxon, with a detection limit of 0.1 ppb and a response time of less than ten seconds.