Microfluidic assays of acetylcholinesterase inhibitors

A microfabricated device for flow injection analysis and electrophoretic se paration of acetylcholinesterase (AChE) inhibitors is described. Solutions of inhibitor, enzyme, substrate, and derivitizing agent were mixed within t he channels of the microchip using computer-controlled electrokinetic trans port. AChE-catalyzed hydrolysis of acetylthiocholine to thiocholine was mea sured in an onchip reaction of thiocholine with coumarinylphenylmaleimide, and the resulting thioether was detected by laser-induced fluorescence. Inh ibitors reduced the fluorescence signal and produced a negative peak diagno stic for the type of inhibition. A Gaussian peak was observed for competiti ve inhibitors, whereas a broad negative peak was observed for irreversible inhibitors. From a microchip assay for tacrine, an inhibition constant, K-i , of 1.5 +/- 0.2 nM was derived, which compared well with a standard cuvett e assay, A now injection assay of two irreversible inhibitors, carbofuran a nd eserine, was performed. With a Ei-min stopped-flow reaction time, a dete ction limit of 10 nM carbofuran was obtained. As a potential multiplex scre ening device, a mixture of four cationic inhibitors, tacrine, edrophonium, and tetramethyl- and tetraethylammonium chloride, was separated and detecte d within 70 s.