A supersandwich fluorescent aptasensor leveraging the synergy of mini-nanotetrahedron, high-affinity split aptamer and rolling circle amplification for kanamycin analysis in milk.

Sensitive detection of kanamycin (KAN) is vital for ensuring food safety. In this study, a novel fluorescent aptasensor for KAN detection was developed by synergizing high-affinity split aptamers, a miniaturized DNA nanotetrahedron (mini-NTH) scaffold, and rolling circle amplification (RCA). Contrasting conventional views, the split aptamers exhibited superior affinity to intact ones, a phenomenon elucidated by molecular dynamics (MD) simulations and validated via bio-layer interferometry (BLI). The mini-NTH scaffold ensured precise probe orientation on magnetic beads. Upon KAN binding, a ternary complex formed that consumed RCA primers, triggering a sensitive "signal-off" fluorescence response. The aptasensor achieved a linear range of 100 pM-100 nM with a detection limit of 0.5 nM. It demonstrated excellent stability (96.1% retention after 15 days), high precision (RSD < 5%), and reliable accuracy in milk samples (recoveries: 93.9-105.0%), showing strong agreement with HPLC analysis. This work presents a robust tool for antibiotic monitoring.