New principle of direct real-time monitoring of the interaction of cholinesterase and its inhibitors by piezolectric biosensor

This paper describes a new method for the sensitive detection of cholinesterase inhibitors based on real-time monitoring using a piezoelectric biosensor. The cholinesterase inhibitor paraoxon was immobilized on the sensing surface via a chelate complex as the recognition element. At first, the conjugate of N-mercaptoundecanoic acid (MUA) with Nalpha,Nalpha-bis (carboxymethyl)-L-lysine (NTA-Lys) was chemisorbed to form a self-assembled monolayer on the surface of the gold electrode of the piczosensor. In the next step, paraoxon-spacer-hexahistidine conjugate was linked to the MUA-Lys-NTA layer via the chelate complex with Ni2+. The paraoxon-modified surface thus obtained was applied for the binding of human butyrylcholinesterase (BChE). Regeneration of the sensing surface was achieved by splitting the chelate complex with EDTA and depositing a fresh layer of Ni2+ followed by addition of the paraoxon-spacer-hexahistidine. In the presence of free inhibitors like diisopropylfluorophosphate (DFP), binding of BChE to the surface-bound paraoxon was decreased. In this way, a competitive affinity assay for organophosphorus compounds was developed. The limit of detection for DFP as a model compound was 10 nmol/l (ca. 2 mug/l). This new concept seems suitable for constructing biosensors for the group-specific detection of cholinesterase-inhibiting substances like insecticides in the field. (C) 2003 Elsevier Science B.V. All rights reserved.