Force spectroscopy 101: how to design, perform, and analyze an AFM-based single molecule force spectroscopy experiment

Single molecule force spectroscopy presents a deceptively simple approach to probing interaction between molecules and molecular assemblies on the nanoscale by measuring forces that it takes to pull the molecules apart. Yet, a more detailed analysis reveals a wealth of different behaviors and interesting physics. This article aims to explore basic physical concepts behind these experiments from a strictly practical point of using these data to extract meaningful information about the interactions. It also focuses on different loading regimes in these experiments, different kinetics that they cause, and different data interpretation that is required for measurements in those regimes.

► Force spectroscopy is a powerful way to characterize kinetics and thermodynamics of intermolecular interactions on a single molecule level.
► Bond rupture can happen in several distinct regimes, and extracting meaningful information from the data requires careful experiment planning and analysis.
► Rupture forces values are meaningless outside of the context of the applied loading rate and the stiffness of the probe used for the measurement.
► Measurements in the slower near-equilibrium rupture regime can determine equilibrium free energy of the interaction.