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Antibiotic residues, generated by the irrational use of drugs and environmental pollution, have always been a great challenge to aquaculture safety. Therefore, a quick, convenient, and performance-excellent way to detect antibiotic residues in aquaculture fish is urgently required. In this study, a multiplex immunochromatographic strip biosensor based on gold nanoparticles was developed for the simultaneous detection of five classes of antibiotic residues (24 β-lactam antibiotics, 26 sulfonamides, five tetracyclines, 24 quinolones, and four amphenicols) in aquaculture fish within 10 min. The detection ranges of five representative antibiotics, penicillin G, sulfamethazine, tetracycline, enrofloxacin, and chloramphenicol, were 2.33–38.4, 0.688–17.1, 1.4–48.1, 1.45–32.9, and 0.537–9.06 µg/kg, respectively. The accuracy and stability of these measurements were demonstrated by analyzing spiked fish samples, with recovery rates of 87.5%–115.2% and a coefficient of variation < 9.5%. The cross-reaction rates, based on the five representative antibiotics, were 3.77%–202% for β-lactams, 3.95%–137% for sulfonamides, 9.19%–100% for tetracyclines, 4.9%–145% for quinolones, and 3.2%–100% for amphenicols. The excellent testing performance of the biosensor strip to most of antibiotic residues in aquaculture fish ensures they meet the maximum residue limits required by countries or organizations. Therefore, this multiplex immunochromatographic strip biosensor is potentially applicable to the rapidly on-site determination of antibiotic residues in aquaculture fish.
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