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Abstract
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.
This article was submitted as part of a Special Memorial Issue honoring Prof. George G. Guilbault.
Financial support for this work was given by the EPA, Environmental RTDI Programme 2000–2006 (Grant agreement No. 2006-PhD-RCA-18-M1) and the Marine Institute and Marine RTDI Measure, Productive Sector Operational Programme, National Development Plan 2000–2006 (Grant-aid agreement No. AT-04-01-01) and is gratefully acknowledged.
Notes
Cmin – concentrations which cause significant change in respiration calculated by ANOVA test with confidence limits of >99%.
*Novel metal clad leaky waveguide (MCLW) sensor.
+Biosensor (enzyme-CSSD) BChE-ISE (ion selective electrode).
−Biosensor (the enzyme-FET) BChE-ISFET (ion selective field effect transistor).
x Amperometric detection with organic phase enzyme electrode (OPEE).
Δ Continuous-Flow system based enzyme sensor.
□ Amperometric analyzer-automatic flow-injection system.
⋄ Amperometric screen printed electrode.
(hr) – Horse serum.
(hu) – Human serum.
