The development of a multiplexed sensing platform is necessary for highly selective, sensitive, and rapid screening of specific antibiotics. In this study, we designed a novel multiplex aptasensor for antibiotics by fluorescence resonance energy transfer (FRET) strategy using DNase I-assisted cyclic enzymatic signal amplification (CESA) method combined with aptamer/graphene oxide complex. The aptamers specific for sulfadimethoxine, kanamycin, and ampicillin were conjugated with Cyanine 3 (Cy3), 6-Carboxyfluorescein (FAM), and Cyanine 5 (Cy5), respectively, and graphene oxide (GO) was adopted to quench the fluorescence of the three different fluorophores with the efficiencies of 94.36%, 93.94%, and 96.97% for Cy3, FAM, and Cy5, respectively. CESA method was used for sensitive detection, resulting in a 2.1-fold increased signal compared to those of unamplified method. The aptasensor rapidly detected antibiotics in solution with limit of detection of 1.997, 2.664, and 2.337 ng/mL for sulfadimethoxine, kanamycin, and ampicillin, respectively. In addition, antibiotics dissolved in milk were efficiently detected with similar sensitivities. Multiplexed detection test proved that the fluorescently modified aptamers could work separately from each other. The results indicate that the aptasensor offers high specificity for each antibiotic and enables simultaneous and multicolor sensing for rapid screening of multiple antibiotics at the same time.

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