Capture-SELEX derived aptamers for light-up fluorescent detection of carbendazim

Carbendazim (CBZ) is a widely used benzimidazole fungicide with an environmental half-life of a few months, necessitating rapid, selective and on-site detection methods. Here, we report the identification of high-affinity DNA aptamers for CBZ using a library-immobilization based capture-SELEX strategy. After 15 rounds of selection and deep sequencing, sequence alignment and subsequent truncation and mutational analysis yielded an optimized sequence, CBZ-10s, which was predicted to adopt a stem-loop structure. CBZ-10s exhibited strong binding to CBZ with dissociation constants (K_d) of 104 nM and 162 nM as determined by ThT fluorescence and isothermal titration calorimetry, respectively. This aptamer has similar binding from pH 6 to 8, whereas Mg²⁺ was required for binding. Integration of CBZ-10s into a strand-displacement fluorescence assay enabled rapid, concentration-dependent light-up detection with high selectivity and a limit of detection of 70 nM. The sensor remained functional in lake water samples, demonstrating robustness in complex matrices. A previously reported CBZ aptamer selected by forming a CBZ-protein conjugate showed no measurable binding under comparable conditions. This work establishes CBZ-10s as a well-defined and reliable recognition element and highlights capture-SELEX as an effective strategy for developing aptamers for small molecules.