Dual and ultrasensitive electrochemical aptamer-based antibiotic biosensor via cascaded DNA assembly amplifications

Kanamycin (KAN) and tobramycin (TOB), as aminoglycoside antibiotics, can induce irreversible ototoxicity and dose-dependent nephrotoxicity risks to human health through residual contaminations. Consequently, sensitive monitoring of TOB and KAN residues in animal-derived foods is of critical importance; however, methods that can enable simultaneous detection of these two antibiotics have been rarely reported. This work demonstrates an ultrasensitive and multiplexed electrochemical aptasensor for simultaneous sensing of KAN and TOB via catalytic gold nanoparticles (AuNPs)/reduced graphene (rGO) nanocomposites and cascaded DNA assembly signal amplifications. Upon binding with their corresponding aptamers in duplex probes, KAN and TOB trigger initiator strands liberation to trigger cascade hybridization DNA assemblies, which enrich numerous auxiliary probes labeled with distinct electroactive reporters, methylene blue (MB) and ferrocene (Fc), on AuNPs/rGO-modified electrode surface. Subsequent electro-catalytic oxidation of MB and Fc yields markedly amplified and well-resolved redox currents, leading to highly sensitive aptamer-based assay of KAN and TOB in dynamic range of 0.1-300 nM with detection limits of 42.14 pM (KAN) and 58.42 pM (TOB). Such multiplexed sensor also enables the monitoring of low-level antibiotic residues in milk matrices, thereby providing promising potentials for the construction of sensitive aptasensors for convenient and simultaneous detection of various antibiotic residues.