A dual-color fluorescent biosensing platform based on amine functionalized 3D copper Prussian blue nanocubes and exonuclease I activity for simultaneous detection of kanamycin and streptomycin

Abstract

A dual fluorescent oligosensor was fabricated to detect kanamycin (KAN) and streptomycin (STR). Complementary DNA oligomers with a carboxyl group (COOH–CDNAs) were attached on amine-functionalized copper Prussian blue nanocubes (NH₂–CuPBNC) through HN-CO bonds. Then, based on complementary base pairing between the FAM labelled KAN oligomer (FAM-ssDNA), TAMRA labelled STR oligomer (TAMRA-ssDNA) and NH₂–CuPBNC/COOH–CDNA system, an FAM-ssDNA/ssDNA-TAMRA/OC–NH–CuPBNC oligosensor was fabricated. The FAM and TAMRA fluorophore emission decreased by NH₂–CuPBNC owing to the fluorescence resonance energy transfer between them. When KAN and STR antibiotics were added, the FAM-ssDNA and TAMRA-ssDNA structure changed into FAM-ssDNA-KAN and TAMRA-ssDNA-STR complexes due to the high binding specificity between KAN, STR and their oligomers, resulting in restoration of FAM and TAMRA fluorescence. The sequential addition of exonuclease I (Exo I) is to trigger and digest the FAM-ssDNA-KAN and TAMRA-ssDNA-STR compounds, resulting in the remarkable recovery of FAM and TAMRA emission. Simultaneously, the freed antibiotics initiate a new cycle of complex formation, culminating in the ultra-trace sensing of both antibiotics. Under the optimized circumstances, the existing oligosensor exhibited great sensitivity toward KAN in the concentration range of 0–50 nM and STR in the concentration range of 0–50 nM with a LOD of 70 and 52 pM. Moreover, the fabricated oligosensor was effectively used to determine KAN and STR with outstanding target discrimination, reproducibility and stability in raw milk and honey, which represents a better platform for antibiotics detection in real food samples.