Xiaohan Zhang, Emily Hoi Pui Chao, Qiushi Liu, Xinyuan Mu, Boya Zhao, Ka-Ying Wong, Philip E. Johnson, Juewen Liu
Tobramycin is a frontline aminoglycoside antibiotic with a narrow therapeutic window. Here, we report the selection of high-affinity DNA aptamers using a library-immobilization based SELEX strategy. The lead aptamer, TOB-2, binds tobramycin with a dissociation constant of 420 nM as determined by isothermal titration calorimetry, while exhibiting over 1000-fold weaker affinity for kanamycin. Structural characterization by mutation analysis, NMR spectroscopy, and circular dichroism spectroscopy reveals a non-G-quadruplex core sequence adopting a stem-loop architecture. Comparative evaluation against two previously reported aptamers highlights the superior binding performance of TOB-2. Leveraging this aptamer, a label-free fluorescent biosensor based on thioflavin T was developed, achieving a limit of detection of 29 nM and enabling accurate quantification of tobramycin in commercial TOBREX eye drops. Integration of this aptamer with electrochemical or other advanced sensing platforms may further enable real-time monitoring of this clinically important antibiotic.