On-chip integrated hydrolysis, fluorescent labeling, and electrophoretic separation utilized for acetylcholinesterase assay

Abstract

A sensitive on-chip acetylcholinesterase (AChE) assay that serves as a basis for the development of a fully integrated on-chip AChE-inhibitor detection assay is presented. The sequential steps required for the on-chip analysis process were integrated into a microchip. Transport and mixing of the reagents occurred by a combination of electroosmosis and electrophoresis using computer-controlled electrokinetic transport. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was determined by on-chip reaction of thiocholine with eosinmaleimide, and the resulting thioether was electrophoretically separated and detected by laser-induced fluorescence (LIF). Enzyme-substrate mixing and reaction by confluent flow of reagents was compared with electrophoretically mediated microanalysis (EMMA), based on injection of an enzyme plug, and the utilization of differences in electrophoretic mobility as a driving force for efficient mixing and reaction. Both methods yielded similar results, however the EMMA-plug technique is preferable. The EMMA-plug technique was optimized for length and pushing time of enzyme plug, length of dyes mixture plug, acetylthiocholine concentration, and detector location. Detection of O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and paraoxon, two AChE inhibitors, was demonstrated by off-chip mixing of the inhibitor and AChE, followed by the on-chip AChE assay. Limit of detection of VX for 5.5 min incubation and of paraoxon for 8 min incubation was 4 × 10 −10 and 4 × 10 −7 M, respectively. Utilization of the AChE microchip assay for inhibition kinetics was demonstrated also by evaluation of the inhibitor-enzyme bimolecular reaction constant ( k i ). The evaluated k i values for VX and paraoxon for AChE from the electric eel were 3.5 × 10 7 and 1.7 × 10 5 M −1 min −1, respectively, conforming well to reported values obtained by bulk methods.