Hairpin DNA and Linear Single-Stranded DNA Synergy-Engineered Dual-Ratiometric Electrochemical Aptasensor for Simultaneous Malathion and Omethoate Detection with Enhanced Performance

Simultaneously determining multiple pesticide residues in agricultural products is of great practical significance. This study presents a dual-ratiometric electrochemical aptasensor via the synergy of hairpin DNA and linear single-stranded DNA (ssDNA) for the simultaneous determination of two pesticides of malathion (MAL) and omethoate (OMT). MXene@AQ/AuNPs were prepared through a facile room-temperature self-assembly strategy, serving as a dual-functional platform to yield a stable reference signal (<i>I</i>_AQ) and amplify response signals. Hairpin DNA and the methylene blue-labeled OMT aptamer (MB-OMT Apt1) were subsequently coimmobilized, with hairpin DNA providing binding sites for assembling the ferrocene-labeled MAL aptamer (Fc-MAL Apt1). In the presence of MAL and OMT, specific bindings triggered the detachment of Fc-MAL Apt1 (causing a decrease in <i>I</i>_Fc) and the approach of MB-OMT Apt1 toward the electrode (causing an increase in <i>I</i>_MB), with ratios of <i>I</i>_Fc/<i>I</i>_AQ and <i>I</i>_MB/<i>I</i>_AQ used for the quantification of MAL and OMT, respectively. Following this principle, two additional aptasensors based exclusively on hDNA or ssDNA were constructed for performance comparison. The findings indicate that hairpin DNA and linear ssDNA act synergistically through structural and characteristic complementarity, significantly enhancing the assembly and identification efficiency of the aptasensing platform, which endows the hDNA&ssDNA-based aptasensor with remarkable reliability and anti-interference performance for multipesticide analysis.