Molecule-based force spectroscopy applied to ligand-receptor binding for studying intermolecular recognition and structural unfolding of molecules.

Due its piconewton force sensitivity, AFM has become a widely used technique for investigating several force-related molecular mechanisms, notably using Dynamic Force Spectroscopy (DFS), such as polymer stretching, cellular membrane elasticity, cell adhesion, cell-receptor recognition, protein folding/unfolding, antibody-antigen biorecognition, (more generally biomolecular interactions) and to detect and manipulate single molecules, or cells, providing new insights into their structure-function relationships, even in physiological and pathological contexts. An understanding of the fundamental mechanisms of molecular recognition is central to understanding processes in living organisms.

Biological systems undergoing biorecognition are studied by pulling apart the two partners involved in a complex, once these have been suitably anchored to the AFM and to a substrate, respectively.

One interacting partner is immobilized on the AFM tip and the other on a support, then the functionalized tip is brought into contact with the support and a complex may be formed, provided that the two partners have enough flexibility and orientational freedom. Successively the tip is retracted from the substrate and when the applied external force overcomes the molecular forces, the tip jumps off sharply from contact to a noncontact position and dissociation takes place. Such a jump-off process provides an estimation of the complex unbinding force from which significant kinetics and thermodynamics parameters may be extracted in the framework of suitable phenomenological and molecular models.

Although DFS has demonstrated enormous capabilities to provide detailed information on biological systems, even at the single molecule/cell level, the occurrence of some ambiguous and controversial results has been noted in different experimental and modelling contexts. Therefore, great care should be exercised in both experimental and analysis procedures in order to eliminate possible drawbacks and artefacts which might jeopardize the success of DFS. Careful experiments will eventually allow one to obtain reliable and reproducible information (as normally required in the biomedical context). Within a broad collaborative milieu involving the related expertise of the most active EU AFM laboratories, the WG 2 will aim at developing well established protocols to be adopted in experiments and applications in life science and nanomedicine.

In particular, special attention will be devoted to the immobilization procedures (binding strength and specificity, active site orientation, flexibility, re-orientational freedom, native configuration, and so on) of the biomolecular partners to the AFM tip and substrate that could single out the specific biorecognition events. It is also important to control the number of interacting partners and to reliably take into account for the unbinding forces. An intense coordinated activity will be devoted to better understand the mechanisms involved in the unbinding processes under the application of an external force to develop alternative theoretical approaches which better describe the experimental results (for instance to derive the equilibrium free energy from the mechanical work performed in non equilibrium measurements). Within such a context, reliable, automatic procedures to analyze the large amount of force curves of DFS experiments will be implemented, by promoting, in this way, DFS as a routine approach in the biomedical field. The WG 2 of the Action will also develop new advanced applications of DFS such as the early detection of biomarkers (especially in a pathological context) where very high detection sensitivity is required.

Indeed DFS has demonstrated high potentialities in nano-biodiagnostics, especially in combination with ultrasensitive optical spectroscopies, such as advanced fluorescence.

Finally, the coupling of DFS ability to sense and manipulate single molecules with the detection of mechanical, chemical and electrical effects will be exploited to develop innovative nanodevices such as biosensors, also for application in nanomedicine.