Attaching Biological Entities to AFM Cantilevers for Molecular Recognition Studies
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1025-B14-03
Attaching Biological Entities to AFM Cantilevers for Molecular Recognition Studies W. Travis Johnson Nanotechnology Measurements Division, Agilent Technologies, 4330 W Chandler Blvd, Chandler, AZ, 85226 ABSTRACT Atomic force microscopy (AFM) is an important tool for high resolution studies in biophysics. A strong suit of AFM is its ability to measure ligand-receptor interactions at the picoN scale. Using AFM, scientists can probe and quantify these interactions in their native, liquid environments at physiological pH or perform dynamic experiments in situ by removing or adding ions, solutes, and reagents to the sample environment. Bioconjugation chemistry and surface chemistry are crucial because a selective ligand must be immobilized on the tip of a cantilever so that the AFM can resolve the mechanical force that is required to separate the ligand and its target. The resulting data can be used to calculate forces of unbinding, derive rate constants, and infer structural information about the binding pocket. Biomolecular recognition experiments with AFM can be greatly enhanced through the use of relatively short (~8-10 nm), heterobifunctional, elastic, polyethylene glycol (PEG) linker to immobilize ligands. Selective bifunctional linkers are used in order to permit their sequential immobilization and bioconjugation, while minimizing undesirable polymerizations or self-conjugation. INTRODUCTION Single molecule interactions can be studied by covalently attaching ligands directly to the tip of an AFM (atomic force microscope) probe [1, 2, 3, 4, 5] or by absorbing molecules on the probe tip [6, 7]. Molecular recognition force microscopy (MRFM) is an AFM based technique that relies heavily on nanoscale surface chemistry, biochemical immobilization chemistry and bioconjugation chemistry [8]. In MRFM, nanoNewton interactions are observed and quantified one by one as an AFM probe that has been modified with ligand molecules approaches and is subsequently withdrawn away from a surface that contains immobilized receptors. MRFM experiments can give valuable information about the intramolecular forces involved in protein folding [9] and the structure and dynamics of intermolecular unbinding events at the single molecule level [10]. Topography and recognition imaging (TREC) is another single molecule AFM technique that also relies on interations between ligands and receptor pairs [11]. TREC is a dynamic force microscopy (DFM or AC mode) AFM method in which ligand-modified AFM probes are scanned and oscillated over biological surfaces in magnetic AC (MAC) Mode. TREC imaging allows specific types of molecules to be identified in compositionally complex samples with nanometer-scale lateral resolution [12, 13, 14, 15, 16, 17, 18]. Compared to ligands attached directly to AFM probes, flexible tethers allow ligands freedom to diffuse relatively unencumbered within defined volumes of space, so the effective concentration of ligand molecules in the experiment can be controlled [13]. Another benefit of immobilizing ligands b
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