A Tripod Molecular Tip for Single Molecule Ligand−Receptor Force Spectroscopy by AFM

Tripod-shaped molecules were designed for chemical modification of the surface of probes used for atomic force microscopy (AFM). These chemically functionalized tips were used for chemical force spectroscopy (CFS) measurements of the ligand−protein receptor interaction in a biotin−NeutrAvidin model system. We demonstrate that by using this unique tripodal system, we can achieve significantly lower density of ligand on the AFM tip apex, which is optimal for true single molecule measurements. Furthermore, the molecular tripods form highly stable bonds to the AFM probes, leading to more robust and reproducible unbinding force data, thereby addressing one of the challenges in CFS studies. Histogram analysis of the hundreds of collected unbinding forces showed a specific distribution with a peak force maximum at ∼165 pN, in good agreement with the previously reported data of single rupture events of biotin−avidin. We compared these molecular tripod tips with a molecular monopod. The results showed that the mol...