Atomic force microscopy (AFM), developed in the late eighties to explore atomic details on hard material surfaces, has evolved to an imaging method capable of achieving fine structural details on biological samples. Its particular advantage in biology is that the measurements can be carried out in aqueous and physiological environment, which opens the possibility to study the dynamics of biological processes in vivo. The additional potential of the AFM to measure ultra-low forces at high lateral resolution has paved the way for measuring inter- and intra-molecular forces of bio-molecules 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, NTA nitrilotriacetate—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), respectively. 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 to the ligand—receptor connection thus reducing its lifetime until the interaction bond breaks at a critical force (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 to the receptor—ligand bond reveal details of the molecular dynamics of the recognition process and energy landscapes. Similar experimental strategies were also used for studying intra-molecular force properties of polymers and unfolding—refolding kinetics of filamentous proteins. Recognition imaging, developed by combing dynamic force microscopy with force spectroscopy, allows for localization of receptor sites on surfaces with nanometer positional accuracy.