Improving pesticide residue detection: Immobilized enzyme microreactor embedded in microfluidic paper-based analytical devices

Orientationally immobilized enzyme microreactors (OIMERs), embedded in microfluidic paper-based analytical devices (μPADs) were developed for improved detection of pesticide residues in food. Acetylcholinesterase (AChE) was orientationally immobilized on the reusable Part I of the μPADs, using the specific affinity binding of concanavalin A (Con A) to a glycosyl group on AChE. Using the disposable Part II, facile colorimetric quantification was performed with a smartphone and software, or qualitative detection by a naked-eye visual test. The AChE immobilized in OIMERs not only had improved activity and stability, but also high sensitivity, with a limit of detection as low as (0.007 ± 0.003) μg/mL. The method was used to detect pesticides residues in real vegetable samples; the recovery (88.6–102.7%) showed high reliability for pesticide residues detection in foods. A molecular docking study and an enzyme kinetic analysis were conducted to characterize the mechanism of action of the OIMERs.