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 1×10−5–1×10−2 M with a Kmapp of 5.93×10−4 M. ATCh quantification was achieved with a limit of detection (LOD) of 5.3×10−6 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 1.45×10−7 M.