Immobilizing Biological Molecules for Recognition Imaging and Force Spectroscopy Applications

W. Travis Johnson, PhD
MRS Spring 2008

There are many biochemical immobilization and bioconjugation chemistry schemes that have been applied to the investigation of ligand-receptor interactions by AFM (atomic force microscopy). In these studies, biological ligands are typically bound to the tip of an AFM probe while corresponding receptor molecules or whole cells are bound to a flat substrate such as mica, silicon, flat glass, or a metal coated substrate. Ligand molecules for a particular receptor can be attached to the tip of an AFM probe, transforming the probe into a sensitive, chemically selective biosensor for that receptor. Molecular recognition force microscopy (MRFM) is a single molecule AFM-based technique which relies heavily on nanoscale surface chemistry, nanoscale biochemical immobilization chemistry and bioconjugation chemistry. In MRFM, single molecule unbinding interactions between AFM probe-bound ligands and substrate-bound receptor pairs are observed and quantified one by one as the AFM cantilever approaches and then is subsequently withdrawn away from the surface many times. The nanoNewton-scale molecular unbinding events are generally detected by measuring the optical deflection of the flexible AFM cantilever. These force spectroscopy (FS) experiments can give valuable information about the structure and dynamics of molecular unbinding events at the single molecule level. In addition to intermolecular interactions, this technique has also been effectively applied to gain an understanding of the intramolecular forces involved in the dynamics of protein folding and polymer elongation. Topography and recognition (TREC) imaging is another single molecule AFM technique that also utilizes probe-bound ligands and substrate-bound receptor pairs. TREC imaging is a dynamic force microscopy (DFM) technique in which a ligand-coated AFM probe is scanned and oscillated over a biological surface in magnetic AC (MAC) Mode in order to resolve recognition maps of ligand-receptor interactions. Specific interactions between the ligand attached to the AFM probe and receptor molecules on the substrate are resolved during scanning as small changes in the MAC Mode signal. TREC imaging is a powerful technique with many potential applications because it allows a specific type of molecule to be identified in compositionally complex samples, such as biological materials. The lateral positions of functionally active receptors on a cell or other biological surface can be resolved with nanometer resolution. TREC has been used to image, map and analyze the chemical compositions of a variety of samples; including molecular interactions between nucleic acids and proteins, antibodies and antigens, and small ligands and their receptors.

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