Metal and pH-Dependent Aptamer Binding of Tetracyclines Enabling Highly Sensitive Fluorescence Sensing

Tetracyclines are a widely used group of antibiotics, many of which are currently only used in veterinary medicine and animal husbandry due to their adverse side effects. For the detection of tetracyclines, we previously reported a DNA aptamer named OTC5 that binds to tetracycline, oxytetracycline, and doxycycline with similar K_D's of ~100 nM. Tetracyclines have an intrinsic fluorescence that is enhanced upon binding to OTC5, which can be used as a label-free and dye-free sensor. In this work, the effect of pH and metal ions on the sensor was studied. Mg²⁺ ions are required for the binding of OTC5 to its target with an optimal concentration of 2 mM. Other metal ions including Ca²⁺ and Zn²⁺ can also support aptamer binding. Although Mn²⁺ barely supported binding, the binding can be rescued by Mg²⁺. ITC studies confirmed that OTC5 had a K_D of 0.2 μM at a pH of 6.0 and 0.03 μM at a pH of 8.3. Lower pH (pH 6) showed better fluorescence enhancement than higher pH (pH 8.3), although a pH of 6.0 had slightly higher K_D values. Under optimized sensing conditions, sensors with limit of detections (LODs) of 0.1-0.7 nM were achieved for tetracycline, oxytetracycline, and doxycycline, which are up to 50-fold lower than previously reported. Milk samples were also tested yielding an LOD of 16 nM oxytetracycline at a pH of 6.0.