In vitro selection and engineering azithromycin-specific aptamers and construction of a ratiometric fluorescent aptasensor for sensitive detection of azithromycin

Azithromycin can effectively inhibit bacterial protein synthesis and reduce biofilm formation and quorum sensing, and thus has been preferentially used in the treatment of various infections. However, azithromycin is also among the most highly concentrated antibiotics present in wastewater due to the inappropriate use and treatment. In order to sensitively and rapidly monitor the azithromycin residues, aptamers specific to azithromycin were selected through 12 rounds of screening by using magnetic beads-SELEX (MB-SELEX). The screened aptamers were assessed for their affinity and specificity using fluorescent assay. Apt3 emerged as the optimal candidate aptamer with the dissociation constant (Kd) of 235.07 nM. After secondary structure analysis and molecular docking, Apt3 was systematically truncated and subjected to single-base mutations. The optimal aptamer Apt3–27 T was identified, exhibiting the Kd value of 215.84 nM and the improved specificity compared to the original aptamer. Then Apt3–27 T was utilized to construct a ratiometric fluorescent aptasensor, which exhibited the linear dynamic range of 25–400 nM and low detection limit of 9.78 nM. More importantly, the ratiometric fluorescence mechanism further improved the specificity of the biosensor, which finally can discriminate azithromycin from structurally similar erythromycin. Evaluation experiments using actual samples spiked with azithromycin showed the recovery ranging from 97.1% to 106% and the relative standard deviation ranging from 4.29% to 8.61%. The screened aptamer and constructed aptasensor offer the advantages of high affinity, high effectiveness and ease of use in actual sample detection, thereby displaying promising applications in the monitoring of azithromycin residues.