Structure-optimized truncated aptamers coupled with copper tungstate–reduced graphene oxide for multiplexed electrochemical biosensing of chlorpyrifos, diazinon, and malathion

Organophosphorus pesticides such as chlorpyrifos, diazinon, and malathion are major environmental contaminants that require continuous monitoring. In this work, a multiplex electrochemical aptasensor was developed for the simultaneous detection of these pesticides using structure-optimized truncated aptamers. Truncation designs of the aptamers were rationally developed based on Mfold-predicted secondary structures to enhance binding performance. The designed truncations were experimentally evaluated through electrochemical binding studies and further validated using molecular docking to identify the optimum binding aptamer sequences. A copper tungstate (CuWO₄)/reduced graphene oxide (rGO) nanocomposite was synthesized and integrated into the multiplexed electrochemical aptasensor platform. Three CuWO₄ synthesis methods were investigated and optimized with different CuWO₄-to-rGO ratios using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The citric acid-assisted hydrothermal CuWO₄ showed the highest electrochemical performance, particularly at a CuWO₄-to-rGO ratio of 2:1, due to the synergistic enhancement of electrochemical performance compared to rGO alone. The developed aptasensor exhibited excellent analytical performance, with linear detection ranges of 10 pM–100 nM for Chlor-T4 and Mala-T2, and 10 pM–10 µM for Diaz-T1. The platform demonstrated excellent selectivity and high sensitivity, achieving low limits of detection (LODs) of 60.5 pM, 23.9 pM, and 73.0 pM for chlorpyrifos, diazinon, and malathion, respectively. Validation using spiked tomato, strawberry, and water samples confirmed the practical applicability of the developed platform for real-time and on-site detection. This work highlights the effectiveness of structure-optimized truncated aptamers integrated with CuWO₄/rGO-based nanocomposites for advanced multiplexed detection of environmental pollutants and contributes to the development of next-generation biosensing technologies for environmental monitoring.