Atomic force microscopy (AFM), atomic force microscopy (AFM) developed in the late 1980s to explore atomic details on hard material surfaces, has evolved into a method capable of imaging fine structural details of biological samples. Its particular advantage in biology is that measurements can be carried out in aqueous and physiological environments, which opens the possibility to study the dynamics of biological processes in vivo. The additional potential of the AFM to measure ultralow forces at high lateral resolution has paved the way for measuring inter- and intramolecular forces of biomolecules on the single-molecule level. Molecular recognition studies using AFM open the possibility to detect specific ligand–receptor interaction forces and to observe molecular recognition of a single ligand–receptor pair. Applications include biotin–avidin, antibody–antigen, nitrilotriacetate (NTA)–hexahistidine 6, and cellular proteins, either isolated or in cell membranes.
The general strategy is to bind ligands to AFM tips and receptors to probe surfaces (or vice versa). In a force–distance cycle, the tip is first approached towards the surface, whereupon a single receptor–ligand complex is formed due to the specific ligand receptor recognition. During subsequent tip–surface retraction a temporarily increasing force is exerted on the ligand–receptor connection, thus reducing its lifetime until the interaction bond breaks at a critical (unbinding) force. Such experiments allow for estimation of affinity, rate constants, and structural data of the binding pocket. Comparing them with values obtained from ensemble-average techniques and binding energies is of particular interest. The dependences of unbinding force on the rate of load increase exerted on the receptor–ligand bond receptor–ligand bond reveal details of the molecular dynamics of the recognition process and energy landscapes. Similar experimental strategies have also been used for studying intramolecular force properties of polymers and unfolding–refolding kinetics of filamentous proteins. Recognition recognition imaging imaging, developed by combing dynamic force microscopy force microscopy with force spectroscopy, allows for localization of receptor sites on surfaces with nanometer positional accuracy.