Carbamates (CMs) and organophosphates (OPs) are widely used pesticides with known neurotoxicity arising from the inhibition of acetylcholinesterase (AChE). When AChE is active, in vitro, it can hydrolyze certain substrates to colored products while in the presence of an inhibitor this color development is decreased. Based on this principle, an AChE assay for CM and OP compounds was optimized and validated for carbofuran, carbofuran-3-hydroxy and dichlorvos in lettuce and strawberry extracts. The analytical performance of the assay was confirmed by an accredited liquid chromatography tandem mass spectrometry (LC–MS/MS) method. The developed AChE assay achieved low limits of detection (LODs) at the part per billion (ppb) level, depending the analyte inhibitory strength, recovery rates higher than 70% and good repeatability. Moreover, the toxic unit (TU) approach was applied, for extracts containing the validated analytes, and antagonism was noticed in all cases. Overall, the developed method is rapid, simple, cost-effective and may find application as a low-cost pre-screening tool of AChE inhibitors presence. Last but not least, this study can be considered a guide on development, validation and benchmarking of bioassays in food safety, a topic, which is commonly mispresented in the available literature.

Acute intoxication incidents due to neurotoxic organophosphate (OP) insecticides are occasionally reported, related either to suicidal attempts or occupational exposure due to the misuse of protective equipment. Among them, chlorpyrifos is a compound related to great controversy, which is still authorized and easily accessible in many countries around the world. However, to screen for its exposure markers, instrumental methods are commonly applied, which cannot enable rapid monitoring at an early stage of an intoxication. Therefore, in this study, a microfluidic paper-based analytical device (μPAD) able to rapidly screen for chlorpyrifos-oxon, the toxic chlorpyrifos metabolite, in human serum was developed and fully validated. The μPAD combines wax-printed butyrylcholinesterase (BChE) paper sensors, a lab-on-a-chip (LOC) prototype injector and a smartphone as the analytical detector. In principle, the wax-printed strips with adsorbed BChE are embedded into LOC injectors able to deliver samples and reagents on-demand. A smartphone reader was used to monitor the color development on the strips providing binary qualitative results. μPAD method performance characteristics were thoroughly evaluated in terms of specificity, detection capability (CCβ) and ruggedness. The developed analytical platform is rapid (results within 10 min), cost-efficient (0.70 €), potentially applicable at the point-of-need and attained a low CCβ (10 μg L-1 in human serum). Finally, μPAD characteristics were critically compared to well-established methods, namely an in-house BChE microplate assay and liquid chromatography tandem mass spectrometry.