Abstract
Rapid and accurate detection of antibiotic residues in milk is essential for public health and food safety. Here, we report a multiplexed ratiometric fluorescent sensor for the simultaneous detection of kanamycin (KAN) and doxycycline (DOX). The sensing platform combines copper nanoclusters encapsulated within zeolitic imidazolate framework-8 (CuNCs@ZIF-8), which provide a stable blue fluorescence reference, with DNA-templated silver nanoclusters (DNA-AgNCs) serving as a red fluorescence reporter. The CuNCs@ZIF-8 efficiently quench the fluorescence of DNA-AgNCs via fluorescence resonance energy transfer (FRET). Upon the introduction of KAN, the aptamer domain on the DNA-AgNCs specifically binds to KAN, releasing DNA-AgNCs from CuNCs@ZIF-8 and restoring the red fluorescence of DNA-AgNCs in proportion to KAN concentration. In parallel, the intrinsic green fluorescence of DOX is significantly enhanced through confinement within ZIF-8 and suppression of non-radiative transitions. This multiplexed sensor demonstrated a linear correlation between the ratiometric fluorescence intensity ratio and analyte concentrations, enabling quantitative analysis. The sensor achieved detection limits of 7.34 nM for KAN and 8.65 nM for DOX, well below regulatory limits, and exhibited robust performance in spiked milk samples. The approach meets fit-for-purpose standards by providing rapid, simultaneous detection of antibiotics in dairy products, underscoring its potential for practical and reliable antibiotic residue monitoring.