State Key Lab of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, 865 Changning Road, Shanghai 200050, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2020 Aug 18;92(16):11127-11134. doi: 10.1021/acs.analchem.0c01142. Epub 2020 Jul 1.
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 ( = 902.95 M s, = 0.000141 s), equilibrium constants ( = 1.55 μM), the Gibbs free energy ( = -32.57 kJ/mol), and the activation energy ( = 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.
核酸适体已广泛用作各种生物传感界面上的识别元件，但由于缺乏可用的生物物理工具，尚未实现用于揭示发生在液-固界面上的适体-配体结合机制的定量动力学/热力学分析。在此，我们应用共振微悬臂梁传感器根据液-固界面上的结合诱导质量变化连续记录频率位移。频率位移曲线用于跟踪反应过程，并通过经典方程进行拟合，以计算一组动力学/热力学参数，例如速率常数（ = 902.95 M s ， = 0.000141 s）、平衡常数（ = 1.55 μM）、吉布斯自由能（ = -32.57 kJ/mol）和固定化适体和游离 ATP 的活化能（ = 38.03 kJ/mol）。这种定量分析方法是无标记、无校准且高灵敏度的。动力学/热力学参数检测方法为深入了解生物传感或芯片实验室应用中液-固界面上的配体-适体相互作用提供了新的分辨率。
