Ultrasensitive SERS aptasensor using multilayered hollow Au/Ag alloy nanourchin SERS tags for trace kanamycin detection in food

Kanamycin (KAN) residues in food pose a serious risk to human health, necessitating the development of rapid and sensitive detection methods. Herein, a novel surface-enhanced Raman scattering (SERS) aptasensor for ultrasensitive detection of kanamycin, which integrates hollow Au/Ag alloy nanourchins (HAAA-NUs) tags with high-density “hot spots” and exonuclease III (Exo III)-assisted cyclic amplification. In the presence of KAN, its specific binding with the KAN aptamer (KAN-Apt) triggers the release of complementary DNA (cDNA), followed by hybridization with hairpin DNA (HAP) and Exo III-mediated digestion, resulting in cyclic amplification of target DNA. The amplified target DNA serves as a bridging mediator that specifically hybridizes with the DNA1 immobilized on Fe 3 O 4 @Au nanoparticles (NPs) and the DNA2 anchored on PATP-loaded HAAA-NUs, leading to the formation of a Fe 3 O 4 @Au NPs-DNA1/target DNA/DNA2-HAAA-NUsPATP complex, which subsequently produces a strong SERS signal. In addition, the background signal can be effectively reduced through magnetic separation. Under optimal conditions, the SERS aptasensor exhibited a wide detection range for KAN from 1 to 10 5  fg/mL with a good linear correlation (R 2  = 0.9913), and a limit of detection (LOD) as low as 0.12 fg/mL. It also performed satisfactorily in the analysis of real samples (milk and honey), achieving good recoveries ranging from 89.72% to 112.54%. Moreover, the method demonstrated excellent specificity and stability.