A screening assay for the measurement of activity and inhibition of cholinesterase enzymes is described. It employs a coupled bi-enzymatic reaction of butyrylcholinesterase and choline oxidase and quenched-phosphorescence detection of dissolved oxygen with an oxygen-sensitive probe. The assay can operate in 96-well plates or capillary micro-cuvettes with detection on conventional fluorescent plate reader or the LightCycler® platform, respectively. It was demonstrated with the inhibition assays of paraoxon and carbofuran pesticides for which IC50 values were determined and system performance assessed. This methodology provides moderate sensitivity (nM-μM range of enzyme or inhibitor concentrations), simplicity, sample throughput, and convenience.
- Acetylcholine esterase inhibitors
- Choline oxidase
- Enzymatic assays
- Optical oxygen sensing
- Phosphorescence quenching