Resonant-Cantilever-Detected Kinetic/Thermodynamic Parameters for Aptamer–Ligand Binding on a Liquid–Solid Interface

Nucleic acid aptamers have been widely used as recognition elements on various biosensing interfaces, but quantitative kinetic/thermodynamic analysis for revealing the aptamer–ligand binding mechanism, which occurs on a liquid–solid interface, has not been realized due to a lack of usable biophysical tools. Herein we apply a resonant microcantilever sensor to continuously record the frequency shift according to the binding-induced mass change on the liquid–solid interface. The frequency-shift curve is used for tracing the reaction process and is fitted with classic equations to calculate a set of kinetic/thermodynamic parameters, such as rate constants (k_a = 902.95 M^–1 s^–1, k_d = 0.000141 s^–1), equilibrium constants (K_D = 1.55 μM), the Gibbs free energy (ΔG° = −32.57 kJ/mol), and the activation energy (E_a = 38.03 kJ/mol) for the immobilized aptamer and free ATP. This quantitative analysis method is label-free, calibration-free, and highly sensitive. The kinetic/thermodynamic parameter detection method provides new resolution to the in-depth understanding of the ligand–aptamer interaction on the liquid–solid interface for biosensing or lab-on-a-chip applications.