On the detection of single bond ruptures in dynamic force spectroscopy by AFM

Force spectroscopy is a novel tool in physical chemistry and biophysics. This methodology is aimed at providing kinetic parameters of dissociation at a single-molecule level by rupturing molecular bonds subjected to different loading rates. One persistent problem in the implementation of this methodology is a question about the single-bond nature of the rupture events detected in experiments based on atomic force microscopy. Here we address this question by considering the probability that the nearly simultaneous rupture of two molecular bonds might appear as a single bond rupture in the experimental data, complicating the data analysis and contributing to systematic errors in the extracted kinetic parameters. An approximate analytical model predicts that such events might be common in experiments employing soft cantilever force sensors and short tethers to immobilize the interacting molecules. These findings are confirmed by a more elaborate numerical model providing valuable guidelines on performing single-molecule force spectroscopy experiments.