Development of an acetylcholinesterase immobilized flow through amperometric detector based on thiocholine detection at a silver electrode

This paper describes the use of a commercially available thin-layer flow through a detector with the sensing block customized in an original design for acetylcholinesterase (AChE) immobilization and suitable for inhibition studies by flow injection analysis (FI). AChE was chemically linked onto a gold disk substrate adjacent to a silver disk electrode. The downstream positioned silver electrode, poised at 0.08 V vs. Ag/AgCl, KCl 3 M, permitted the sensitive amperometric detection of liberated thiocholine (TCh) using acetylthiocholine (ATCh) as enzyme substrate. A typical Michaelis-Menten curve was obtained for ATCh within the concentration range 1 x 10(-5)-1 x 10(-2) M with a Km(app) of 5.93 x 10(-4) M. ATCh quantification was achieved with a limit of detection (LOD) of 5.3 x 10(-6) M. The utility of the developed FI setup was demonstrated for AChE inhibition studies using neostigmine as model compound. The IC50 for neostigmine was obtained to be 1.45 x 10(-7) M. (C) 2013 Elsevier B.V. All rights reserved.