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Force spectroscopy is a new and valuable tool in physical chemistry and biophysics. However, data analysis has yet to be standardized, hindering the advancement of the technique. In this article, treatment of the rupture forces is described in the framework of the Bell−Evans model, and the systematic errors associated with the tether effect for approaches that utilize the most probable, the median, and the mean rupture forces are compared. It is shown that significant systematic errors in the dissociation rate can result from nonlinear loading with polymeric tethers even if the apparent loading rate is used in the analysis. Analytical expressions for the systematic errors are provided for the most probable and median forces. The use of these expressions to correct the associated systematic errors is illustrated by the analysis of the measured rupture forces between single hexadecane molecules in water. It is noted that the measured distributions of rupture forces often contain high forces that are unaccounted for by theoretical models. Experimental data indicate that the most significant effect of the high forces “tail” is on the dissociation rate obtained from the median force analysis whereas the barrier width appears to be unaffected. [ABSTRACT FROM AUTHOR]

Single-molecule force spectroscopy has become a valuable tool for the investigation of intermolecular energy landscapes for a wide range of molecular associations. Atomic force microscopy (AFM) is often used as an experimental technique in these measurements, and the Bell−Evans model is commonly used in the statistical analysis of rupture forces. Most applications of the Bell−Evans model consider a constant loading rate of force applied to the intermolecular bond. The data analysis is often inconsistent because either the probe velocity or the apparent loading rate is being used as an independent parameter. These approaches provide different results when used in AFM-based experiments. Significant variations in results arise from the relative stiffness of the AFM force sensor in comparison with the stiffness of polymeric tethers that link the molecules under study to the solid surfaces. An analytical model presented here accounts for the systematic errors in force-spectroscopy parameters arising from the nonlinear loading induced by polymer tethers. The presented analytical model is based on the Bell−Evans model of the kinetics of forced dissociation and on the asymptotic models of tether stretching. The two most common data reduction procedures are analyzed, and analytical expressions for the systematic errors are provided. The model shows that the barrier width is underestimated and that the dissociation rate is significantly overestimated when force-spectroscopy data are analyzed without taking into account the elasticity of the polymeric tether. Systematic error estimates for asymptotic freely jointed chain and wormlike chain polymer models are given for comparison. The analytical model based on the asymptotic freely jointed chain stretching is employed to analyze and correct the results of the double-tether force-spectroscopy experiments of disjoining “hydrophobic bonds” between individual hexadecane molecules that are covalently tethered via poly(ethylene glycol) linkers of different lengths to the substrates and to the AFM probes. Application of the correction algorithm decreases the spread of the data from the mean value, which is particularly important for measurements of the dissociation rate, and increases the barrier width to 0.43 nm, which might be indicative of the theoretically predicted hydrophobic dewetting. [ABSTRACT FROM AUTHOR]

The hydrophobic effect is important for many biological and technological processes. Despite progress in theory, experimental data clarifying water structure and the interaction between hydrophobic solutes at the nanometer scale are scarce due to the very low solubility of hydrophobic species. This article describes an AFM single molecule force spectroscopy method to probe the interaction between molecules with low solubility and reports measurements of the strength and the length scale of the “hydrophobic bond” between hexadecane molecules. Hexadecane molecules are tethered by flexible poly(ethylene glycol) linkers to AFM probes and substrates, removing the aggregation state uncertainty of solution-based approaches as well as spurious surface effects. A shorter hydrophilic polymer layer is added to increase the accessibility of hydrophobic molecules for the force spectroscopy measurements. Statistical analysis of the rupture forces yields a barrier width of 0.24 nm, and a dissociation rate of 1.1 s-1. The results of single molecule measurements are related to the theoretical predictions of the free energy of cavitation in water and to the empirical model of micellization of nonionic surfactants. It is estimated that approximately one-quarter of each molecule's surface is hydrated during forced dissociation, consistent with an extended (nonglobular) conformation of the hexadecane molecules in the dimer. [ABSTRACT FROM AUTHOR]