As a weak non-covalent interaction, hydrogen bond (H-bond) is highly susceptible to the environmental interference. However, the direct quantification of a single H-bond under an interference-free condition is still a challenge. Herein, the intramolecular H-bond in a model system, poly(N-isopropylacrylamide), is studied in high vacuum by single-molecule atomic force microscopy and steered molecular dynamics simulations, which allows the precise quantification of H-bond strength in an interference-free state. Control experiments show that the H-bond is significantly weakened in nonpolar solvent, even if the dielectric constant is very close to vacuum. If a polar solvent is used as the environment, the H-bond will be further weaker or even broken. These results imply that for experiments in any liquid environment, the H-bond strength (ΔG) will be only ~ 50% or even less of that measured in vacuum. Further analysis shows that in liquid environments, ΔG decays in a quasi-linear way with the increase of the dielectric constant (ε). For H-bond studies in future, the result measured in vacuum can be set as the standard value, namely, the inherent strength. This approach will provide fundamental insights into the H-bond participated nano-structures and materials in different environments.

Ubiquitous van der Waals (vdW) forces are very important for nanostructures. Although the vdW forces between two surfaces (or two layers) have been measured for several decades, a direct detection at the single-molecule level is still difficult. Herein, we report a novel method to solve this problem in high vacuum by means of AFM-based single-molecule force spectroscopy (SMFS). Solvent molecules and surface adsorbed water are removed thoroughly under high vacuum so that the situation is greatly simplified. A constant force plateau can be observed when a polymer chain is peeled off from a substrate in high vacuum. Accordingly, the vdW forces between one polymer repeating unit and the substrates can be obtained. The experimental results show that the vdW forces (typical range: 21–54 pN) are dependent on the species of substrates and the size of polymer repeating unit, which is in good accordance with the theoretical results. It is expected that this novel method can be applied to detect other non-covalent interactions (such as hydrogen bond and π–π stacking) at the single-molecule level in the future.