Characterization and optimization of a quantitative colorimetric acetylcholine esterase inhibition assay for biochip integration demonstrated by neurotoxicity evaluation of malathion

Organophosphates (OPs) and carbamates as insecticides, nematicides, fungicides, and herbicides are constantly increasing. Their neurotoxic nature requires careful usage, and misuse can lead to fatalities. OPs classified as 'Class 1′ toxic compounds irreversibly inhibit cholinesterases due to their molecular structure resembling the natural acetylcholine substrate, leading to toxic events in the human brain. Monitoring such chemicals is relevant for agricultural applications and essential for the military sector to ensure the safety of personnel and civilian populations. State-of-the-art analytical detection methods require time-consuming pre-treatments and costly reagents and face challenges associated with pesticide properties like thermal lability, low volatility, and high polarity, which can compromise analysis performance. Advanced systems like electrophoresis or liquid chromatography are used to address these, but these are not well suited for field analysis. Miniaturized colorimetric assays are becoming more popular for various portable devices and kits (i.e., metabolic or blood cell assays) due to their ease of use and practicality. Here, we aimed to establish and optimize a straight-forward paper-based microfluidic acetylcholine esterase inhibition assay for mobile organophosphate detection, laying the groundwork for future microdevice modules to be used in environmental monitoring, public health, and CBRN applications.