Malathion, a widely used organophosphate (OP) pesticide, poses serious health risks due to its neurotoxic effects, highlighting the urgent need for rapid and reliable detection in agricultural products. In this study, a simple, low-cost, and portable colorimetric indicator was developed by incorporating copper nanoparticles (CuNPs), acetylcholinesterase (AChE), and acetylthiocholine iodide (ATChI) within an agar matrix. The sensing mechanism is based on the inhibition of AChE by malathion, which suppresses thiocholine generation and prevents CuNP aggregation, thereby inhibiting the expected color change of the film. The system exhibited optimal activity at pH 7.0 using 400 mU/mL AChE and 120 µM ATChI. TEM and FE-SEM analyses revealed dense CuNP aggregation in the absence of malathion, while UV–Vis spectra showed a red shift in the LSPR peak (from 228 to 259 nm) accompanied by reduced absorbance intensity, both consistent with AChE inhibition. FTIR spectra confirmed pesticide interaction through a characteristic P=O stretching band. The solution-based assay achieved a limit of detection (LOD) of 0.013 ppm, whereas the film-based colorimetric sensor exhibited a slightly higher LOD of 0.027 ppm, attributed to diffusion limitations within the agar matrix compared with the homogeneous solution phase. Interestingly, a similar concentration-dependent behavior was observed for both systems, where enzyme inhibition dominated at low malathion levels (≤0.1 ppm), while direct CuNP–pesticide interactions prevailed at higher concentrations. The sensor demonstrated excellent selectivity toward malathion over other pesticides and maintained stability for up to 20 days. When applied to real samples (lettuce, cucumber, and strawberry), recoveries ranged from 85.8 % to 104.3 %. Owing to its high sensitivity, stability, and instrument-free operation, the developed indicator provides a promising platform for on-site and real-time monitoring of OP residues in food and environmental samples.
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