Flow-through enzyme immobilized amperometric detector for the rapid screening of acetylcholinesterase inhibitors by flow injection analysis.

A commercially available thin-layer flow-through amperometric detector, with the sensing block customized in an original design, was applied to the screening of drug compounds known as acetylcholinesterase (AChE) inhibitors. AChE from electric eel was covalently immobilized onto a cysteamine modified gold disk adjacent to a silver disk working electrode. On-line studies were performed by flow injection analysis (FIA) in PBS buffer pH 7.4. Seven commercially available AChE inhibitors used in the medical field, namely neostigmine, eserine, tacrine, donepezil, rivastigmine, pyridostigmine and galantamine as well as two natural compounds, quercetin and berberine, were investigated. The same trend of inhibitory potency as described in the literature was observed. Of particular interest and in addition to the determination of the IC50 values, this flow-through system allowed the study of both, the stability of the enzyme-inhibitor complex and the kinetic of the enzyme activity recovery.

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 3M, 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.