Re-selection of DNA aptamers for acetamiprid for light-up fluorescent detection

Acetamiprid is currently the only member of second-generation neonicotinoid insecticides permitted for open-field use within the European Union following the ban of other neonicotinoids. Its potential environmental and health risks underscore the need for reliable detection methods. Although an acetamiprid aptamer was previously reported, its widely adopted 20-nt and 49-nt truncated variants have not been rigorously validated. By tracing their truncation history and evaluating their binding using isothermal titration calorimetry (ITC), neither sequence exhibited measurable affinity for acetamiprid. To address this gap, we isolated a new DNA aptamer, Acet-1, through 12 rounds of capture-SELEX. Acet-1 exhibited a dissociation constant (K_d​) of approximately 10 μM for acetamiprid, as consistently determined by ITC, thioflavin T fluorescence, and DNA strand-displacement assays. The strand-displacement assay based on Acet-1 enabled concentration-dependent detection of acetamiprid with limits of detection of 7.6 μM in buffer and 3.2 μM in spiked lake water. The aptamer displayed good selectivity against structurally unrelated compounds but cross-reacted with the closely related neonicotinoid imidacloprid. These results establish the 42-nt Acet-1 aptamer as a short, experimentally validated recognition element for acetamiprid and provide a robust foundation for the development of aptamer-based analytical methods for monitoring neonicotinoid pesticides.