Low-Micromolar-Pressure Capture-SELEX Yields Fold-Switching DNA Aptamers for Label-Free Quantification of Benzovindiflupyr

Benzovindiflupyr (BZF) is a widely used succinate dehydrogenase inhibitor whose persistence complicates monitoring in food and environmental matrices. We implemented a capture-SELEX campaign that constrained ligand concentrations from low micromolar to submicromolar across 13 rounds, creating a low-pressure evolutionary bottleneck. This approach yielded two high-affinity aptamers: Apt.BZF01 (K D = 67.0 ± 9.7 nM) forming a parallel G-quadruplex and Apt.BZF02 (K D = 25.8 ± 5.8 nM) exhibiting ligand-induced fold-switching. Both aptamers showed no cross-reactivity against 21 pesticides. Leveraging Apt.BZF02’s conformational switching, we developed a label-free Thioflavin T displacement assay achieving 17 nM detection limit that maintains performance in complex matrices, with 93–104% recoveries in grape homogenate. This study provides a sensitive detection platform and offers a generalizable blueprint for coupling low-target-pressure SELEX with fold-switch-enabled readouts for challenging hydrophobic targets.