Study of binding mechanism of aptamer to kanamycin and the development of fluorescent aptasensor in milk detection

Aptasensors being versatile sensing platforms presented higher sensitivity toward target detection. However, lacking theoretical basis of recognition between most targets and their corresponding aptamers has impeded their applications. Herein, we conducted a study to explore the binding mechanism of aptamer to kanamycin (Kana) and developed rapid fluorescent aptasensing methods. Based on the fluorescence polarization results, base mutations were performed at different sites of the aptamer. The key binding nucleotides of Kana was identified as T7, T8, C13 and A15 by using isothermal titration calorimetry (ITC). The Kmut3 (2.18 μM) with lower dissociation constants (Kd), one-third of the native aptamer (6.91 μM), was also obtained. In addition, the lower K⁺ concentration and temperature were found to be conducive to Kana binding. Circular dichroism (CD) results revealed that the binding of Kana can trigger the change of base stacking force and helix force. On the aforementioned basis, a fluorescent sensor was designed with the native aptamer and Kmut3 as recognition elements. The comparison results proved that the Kmut3 presented a 3 times lower limit of detection of 59 nM compared to the native aptamer (148 nM). Notably, this developed aptasensor can be finished in 45 min and was convenient to operate.