Designing a Metal Nanocluster‐Based Fluorescence Assay for the Differential Detection of Nitroimidazole and Nitrofuran Antibiotics In Environmental and Food Samples

The extensive use and improper disposal of nitro‐antibiotics in veterinary medicine pose significant environmental and health risks, necessitating sensitive and selective detection methods. Furthermore, distinguishing between nitroimidazoles and nitrofurans remains challenging. Here, an amino acid‐scaffolded metal nanocluster‐based differential nitro‐antibiotic detection strategy leveraging the inner filter effect (IFE) is presented. Nanoclusters are engineered to align with the distinct absorption maxima of nitroimidazoles ( λ abs max = 320 nm) and nitrofurans ( λ abs max = 370 nm). L‐tyrosine‐capped silver nanoclusters (Tyr‐Ag NCs) (excitation/emission: 320/410 nm) showed significant photoluminescence (PL) quenching in response to both nitro‐antibiotics classes, enabling a turn‐off‐based detection method. In contrast, L‐tryptophan‐ and L‐cysteine‐capped copper nanoclusters (Trp‐Cu and Cys‐Cu NCs), with excitation/emission around 380/500 nm, overlapped spectrally only with nitrofurans, enabling selective quenching and simple visual detection without instrumentation. All three NCs demonstrated nanomolar sensitivity, high selectivity, and minimal interference from non‐target species, with their detection mechanisms elucidated in detail. The practicality of the assay is validated through the successful detection of nitro‐antibiotics in cow milk and groundwater, demonstrating its reliability in real‐world samples. Overall, this study establishes a strategic sensing platform that intentionally leverages the IFE—traditionally considered an experimental artifact—as a powerful and selective tool for antibiotic detection.