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

• Published in: Talanta (Oxford) 2013-05, Vol.109, p.116-120
• Main Authors: Parsajoo, Cobra, Kauffmann, Jean-Michel
• Format: Article
• Language: English
• Subjects: Acetylcholinesterase; Acetylcholinesterase - chemistry; Acetylcholinesterase - metabolism; Biosensing Techniques - instrumentation; Biosensing Techniques - methods; Calibration; Cholinesterase Inhibitors - pharmacology; detection limit; Electrochemical Techniques - instrumentation; Electrochemical Techniques - methods; Electrodes; enzyme substrates; Enzymes, Immobilized - chemistry; flow injection analysis; Flow Injection Analysis - instrumentation; Flow Injection Analysis - methods; gold; inhibitory concentration 50; Microfluidics - instrumentation; Microfluidics - methods; Neostigmine; potassium chloride; Reproducibility of Results; silver; Silver - chemistry; silver chloride; Silver electrode; Substrate Specificity; Surface Properties; Thiocholine; Thiocholine - analysis
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2013.01.062

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 0.08V 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 1×10−5–1×10−2M with a Kmapp of 5.93×10−4M. ATCh quantification was achieved with a limit of detection (LOD) of 5.3×10−6M. 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 1.45×10−7M. ► A new flow through design with amperometric detection of thiocholine at a silver electrode is proposed. ► AChE was immobilized on the surface of a gold disk close to a silver disk electrode in the flow injection system. ► Typical calibration curves for acetylthiocholine were obtained and kinetic parameters of enzymatic reaction were calculated. ► Inhibition test for neostigmine as a model inhibitor was done, IC50 and inhibition calibration curves were obtained.