title: Development of an acetylcholinesterase immobilized flow through amperometric detector based on thiocholine detection at a silver electrode.
abstract: 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.
authors.affiliation: Université Libre de Bruxelles, Faculty of Pharmacy, Brussels 1050, Belgium.
authors.forename: Cobra
authors.initials: C
authors.lastname: Parsajoo
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Kauffmann
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002138:Calibration; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry*; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods*; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012834:Silver / Q000737:chemistry; D013379:Substrate Specificity; D013499:Surface Properties; D013860:Thiocholine / Q000032:analysis*
chemical_list: D002800:Cholinesterase Inhibitors; D004800:Enzymes, Immobilized; D012834:Silver; D013860:Thiocholine; D000110:Acetylcholinesterase