Acryloyl-calix[4]arene and aptamer synergistic interface on magnetic imprinted polymers for trace enrofloxacin enrichment and detection

Enrichment and detection of trace antibiotics in complex matrices are challenged by severe matrix interference and low analyte abundance. Herein, a synergistic recognition interface was engineered on magnetic imprinted polymers by integrating acryloyl-calix[4]arene (AC[4]A) and aptamer (Apt). This dual-receptor interface exploits complementary binding modes to specifically recognize enrofloxacin (ENR). Specifically, AC[4]A captures the hydrophobic moiety of ENR via host-guest inclusion, while the aptamer targets its aromatic/carboxyl groups through hydrogen bonding and π-π stacking. Multimodal analyses ( 1 H NMR, CD, molecular docking, and MD simulations) reveal spatially distinct binding sites. Molecular docking indicates a synergistic enhancement of −2.9 kcal/mol in the ternary complex relative to binary sums, while MD simulations reveal that the cooperativity arises from entropy-driven conformational adaptation and interface complementarity. Moreover, the imprinted framework stabilizes the aptamer, retaining >80% extraction capacity after DNase I treatment. With an imprinting factor (IF) of 8.5 and a synergistic recognition factor (SRF) of 1.5, this dual-receptor interface demonstrates a clear “1 + 1>2” effect and the resulting method offers a wide linear range (2.5-300 μg/L) with a low detection limit (0.42 μg/L), well below regulatory limits. Applied to the enrichment and analysis of animal-derived samples, it significantly reduces matrix interference with recoveries of 83.3-98.2%. This dual-receptor strategy provides a reliable and sensitive platform for trace antibiotic enrichment and detection in complex matrices.