Sensing Discrete Streptavidin-Biotin Interactions with Atomic Force Microscopy

Abstract

Molecular recognition forms the basis for assembly and regulation in living organisms. We have used the atomic force microscope (AFM) to study the interaction of a model receptor streptavidin with its ligand biotin under physiological conditions. Surfaces functionalized with biotin and streptavidin exhibited adhesive forces 3-8 times greater than the nonspecific interactions observed between blocked streptavidin and biotinylated surfaces. The magnitude and distribution of the observed adhesive forces suggest they result from individual streptavidin-biotin interactions. This technique provides a means to directly study molecular recognition interactions at the molecular level. Nature has developed the unique ability for molecular recognition through the use of multiple noncovalent bonds (Le., van der Waals, hydrogen, ionic, and hydrophobic interactions) which possess a high degree of spatial and orientational specificity. Molecular recognition plays a central role in cellular behavior1 and the immunological response,2 and has also become the basis for a wide range of bioanalytical technique^.^ Although the structure and binding properties of molecular recognition systems can be measured, the forces involved in intermolecular interactions remain largely unknown. Molecular recognition interactions have been characterized through observations of the behavior of cells on which ligands and receptors naturally occur or have been atta~hed.~?~ Although these studies have largely been qualitative, the application of micropipet techniques to the study of cellular interactions5 has provided the meansfor micromanipulation with a force sensitivity of N. The surface force apparatus has also recently been used to characterize quantitatively the surface forces between model molecular recognition systems.6 The control of surface properties and the very small forces and distances involved in intermolecular interactions continue to limit the characterization of discrete molecular recognition interactions. The AFM has several properties that make it an ideal tool for measuring intermolecular forces: theoretical force sensitivity on the order of N, displacement sensitivity of 0.01 nm, contact areas as small as 10 nm2, and ability to operate under physiological condition^.^ In fact, the measurement of interactions as small as asingle hydrogen bond has recently been reported.8 The bond energies for specific molecular interactions fall between those typical * To whom correspondence should be addressed.