The widespread use of organophosphate pesticides poses significant environmental and public health risks, necessitating the development of low-cost, sensitive, and portable detection systems. Here, the development and optimization of a field-deployable paper-based colorimetric biosensor (pesticide paper-based analytical device, PAD) for the detection of acetylcholinesterase inhibitors is reported, using indoxyl acetate (IOA) and acetylthiocholine chloride (ATCh) as chromogenic substrates. Assay conditions were systematically optimized for enzyme and substrate concentrations, and incubation time. Kinetic analysis revealed a lower Km for IOA (1.0 ± 0.2 mM) compared to ATCh (6.6 ± 3.2 mM), indicating stronger substrate affinity, while ATCh exhibited a higher Vmax (123.8 ± 6.1 kat), supporting faster signal generation. Ethyl-paraoxon was used as a model pesticide to establish inhibition-based calibration curves. The IOA-based method demonstrated superior sensitivity (limit of detection 0.09 ppm), whereas ATCh provided a broader detection range (1.56–100 ppm). Spike recovery from produce samples yielded recoveries ranging from 59–86%. To enable field deployment, a novel sandwich method of stabilization was developed to preserve both enzyme and substrate activity on paper for over five months at ambient conditions. This stabilization approach maintained ~90% enzymatic activity and enhanced substrate signal intensity, overcoming a major limitation of paper-based biosensors. Pesticide PAD platform offers a promising tool for rapid, on-site pesticide screening with minimal equipment, supporting food safety and environmental monitoring in low-resource settings.
Pokhrel et al. (Fri,) studied this question.