Dual-signal-amplified nanopore sensor based on a DNA Walker enables simultaneous and ultrasensitive detection of kanamycin and tobramycin

The widespread use of aminoglycoside antibiotics has raised serious concerns regarding food safety and environmental health, necessitating advanced methods for their sensitive and multiplexed detection. However, conventional techniques struggle to directly quantify these small molecules with sufficient sensitivity and specificity. Here, we present an ultrasensitive and simultaneous sensing platform for kanamycin (KAN) and tobramycin (TOB) that synergistically merges a target-triggered DNA walker with a biological α-hemolysin (α-HL) nanopore. In this design, aptamer recognition of the antibiotic activates a DNAzyme-based walker immobilized on magnetic beads, which then processively cleaves a track strand to release hundreds of DNA reporter probes per binding event. This dual-amplification strategy of catalytic chemical amplification coupled with single-molecule physical readout enables exceptional sensitivity, achieving detection limits as low as 3.88 fM for KAN and 4.62 fM for TOB. The sensing platform exhibits excellent selectivity and successfully accomplishes the simultaneous quantification of both antibiotics in a mixture. When applied to spiked environmental water samples, the method demonstrates high accuracy and reliability, yielding satisfactory recovery rates. This work establishes a generalizable DNA walker–nanopore paradigm, offering a versatile blueprint for the development of highly sensitive, multiplexed, and rapid detection systems for a wide range of small-molecule contaminants.