Detection and localization of single molecular recognition events using atomic force microscopy

Peter Hinterdorfer, Yves F Dufrêne
2006 Nature Methods  
Because of its piconewton force sensitivity and nanometer positional accuracy, the atomic force microscope (AFM) has emerged as a powerful tool for exploring the forces and the dynamics of the interaction between individual ligands and receptors, either on isolated molecules or on cellular surfaces. These studies require attaching specific biomolecules or cells on AFM tips and on solid supports and measuring the unbinding forces between the modified surfaces using AFM force spectroscopy. In
more » ... review, we describe the current methodology for molecular recognition studies using the AFM, with an emphasis on strategies available for preparing AFM tips and samples, and on procedures for detecting and localizing single molecular recognition events. Molecular recognition between receptors and their cognate ligands is important in life sciences. Such specific interactions include those between complementary strands of DNA, enzyme and substrate, antigen and antibody, lectin and carbohydrate, ligands and cellsurface receptors as well as between cell adhesion proteins. These interactions are involved in many important biological processes, including genome replication and transcription, enzymatic activity, immune response, initiation of infection, and many other cellular functions. Furthermore, their selectivity and specificity are widely exploited in nanobiotechnology for developing bioanalytical and biomedical devices such as biosensors 1 . Despite the vast body of available literature on the structure and function of receptor-ligand complexes, information about the molecular dynamics within the complexes during the association and dissociation process is usually lacking. Moreover, until recently, mapping the spatial distribution of individual binding sites on model or cellular surfaces was not accessible because of a lack of appropriate imaging techniques. Consequently, there is clearly a need to develop and exploit single molecule tools for sensing and mapping molecular recognition interactions on biosurfaces. Owing to its capacity to allow observation and manipulation of biosurfaces under physiological conditions, the AFM 2 has revolutionized the way in which researchers now explore biological structures at the singlemolecule level 3 . Although AFM imaging provides threedimensional views of specimens with unprecedented resolution and with minimal sample preparation 4 , AFM force spectroscopy allows measurement of piconetwon (10 −12 N) forces associated with single molecules 5,6 thereby providing fundamental insights into the molecular basis of biological phenomena and properties as diverse as molecular recognition 7-9 , protein folding and unfolding 10,11 , DNA mechanics 12 and cell adhesion 13 . The main parts of the AFM are the cantilever, the tip, the sample stage and the optical deflection system consisting of a laser diode and a photodetector (Fig. 1) . AFM images are created by scanning (in the x and y directions) a sharp tip, mounted to a soft cantilever spring, over the surface of a sample and by using the interaction force between the tip and the sample to probe the topography of the surface. Force spectroscopy relies on measuring this force with piconewton sensitivity as the tip is pushed toward the sample and retracts from it in the z direction. The sample is mounted on a piezoelectric
doi:10.1038/nmeth871 pmid:16628204 fatcat:glvzw7g6pbhyrhcqhvjqq4fwum