Antibiotic residues in milk pose public health risks by promoting antimicrobial resistance, necessitating advanced detection methods. This study presents a dual-color "turn-on" aptasensor for simultaneous detection of ampicillin (AMP) and oxytetracycline (OTC). Aptamer-functionalized silica nanoparticles doped with Rhodamine B isothiocyanate (apt-RBITC@SiO) and Alexa Fluor 488 (apt-AF488@SiO) serve as fluorescence probes, while multicore iron oxide nanoparticles (MIONs) act as capture probes. The fluorescence intensities at 519 nm (green) and 573 nm (red) correspond to OTC and AMP concentrations, with limits of detection (LODs) of 0.13 ng/mL and 0.17 ng/mL, respectively. Detection ranges span 0.5-100 ng/mL. The sensor demonstrated recovery rates of 94.16%-103.22 % for OTC and 98.33%-101.62 % for AMP in milk, with low relative standard deviations (RSD). Compared to ELISA, the developed aptasensor exhibits superior sensitivity and lower detection limits, making it a highly effective tool for antibiotic residue detection. This aptasensor provides a sensitive, selective, and efficient method for detecting antibiotic residues in milk, offering significant potential for food safety monitoring.
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