From rational engineering of a broad-spectrum aptamer to multi-residue screening: A fluorescent aptasensor for multiplexed sulfonamides.

The increasing prevalence of multiple antibiotic residues in animal-derived foods presents significant challenges for monitoring efforts. Broad-spectrum aptamers offer great promise for enabling multiplex detection of various antibiotic residues. However, aptamers directly screened for this purpose often exhibit low affinity and poor stability, necessitating further structural optimization. In this study, a novel aptamer engineering strategy, termed "pairwise mutations," was developed and combined with stepwise rational truncation, molecular simulation, and bivalent engineering to systematically optimize the previously identified broad-spectrum aptamer, SAs16 (80 nt), from our laboratory. SAs16 was progressively truncated to a minimal active structure of 19 nt, designated SAs16-2. Through pairwise mutations, G13 was mutated to A and G4 was mutated to T, thereby establishing complementary base pairing between these positions and enhancing structural stability. Molecular simulations revealed that the loop region of the aptamer plays an essential role in the molecular recognition of the target antibiotics via hydrogen bonding and hydrophobic interactions. Ultimately, a bivalent broad-spectrum aptamer SAs16-A0 was designed, with enhanced binding affinity. Following these optimizations, a label-free fluorescent aptasensor was developed based on SAs16-A0, enabling multi-residue screening of sulfadiazine (SDZ), sulfamethoxypyridazine (SMP), and sulfadimethoxine (SDM) in real samples. The aptasensor demonstrated a LOD as low as 3.14 ng/mL and a detection range of 4-200 ng/mL. The rational aptamer redesign strategy proposed in this study successfully created a promising molecular probe for identifying sulfonamide antibiotics (SAs) and offers valuable insights for the development of engineered aptamers.