Localization of Single Avidin–Biotin Interactions Using Simultaneous Topography and Molecular Recognition Imaging

Abstract

Simultaneous topography and recognition imaging (TREC), 2] a recent development in dynamic force microscopy, has proven to be a powerful technique in biophysical research. In contrast to the common force mapping mode, the slow imaging speed and the low lateral resolution are overcome by oscillating a functionalized tip close to its resonance frequency during the scan across the surface. In these studies, the topographical imaging of receptor molecules is combined with molecular recognition by their cognate ligands bound to the atomic force microscope (AFM) tip via a distensible tether. The binding sites are evident from the reduction in the oscillation amplitude, as a result of specific recognition during the lateral scan. The receptors are recognized by the ligand on the scanning tip with a lateral resolution of a few nanometers, yielding a topographic image and a separate map of recognition sites from a single scan. Because this new technique opens a broad range of biological applications, a stable and easy-to-use setup is of key interest for new users. Using well-characterized, high-affinity biological binding partners, such as avidin–biotin, for TREC imaging enables a better understanding of the key factors for optimizing the scanning parameters. The robust and well-described avidin–biotin interaction offers an ideal pair of binding partners. Avidin–biotin is 1) robust and reliable, 2) well-known in terms of binding properties, 3) easy to prepare with commercially available components. Moreover, 4) avidin can simply be adsorbed to mica, and mica is an ideal support for AFM imaging. Finally, 5) the biotin–PEG (poly(ethylene glycol)) tether is commercially available and can be attached to amino-functionalized AFM tips in one step. Single avidin molecules can easily be immobilized onto mica via electrostatic adsorption by virtue of their positive net charge at neutral pH. Using different concentrations of avidin in the adsorption buffer, the surface coverage of avidin on mica can be adjusted easily. In addition, a biotin residue was coupled to the AFM tip via a distensible PEG chain. More specifically, biotin was covalently tethered to the amino-functionalized tip in a single coupling step, using “biotin-PEG-NHS” which consists of a PEG chain with a biotin on one end and an amino-reactive N-hydroxysuccinimide ester function (NHS group) on the other. With this configuration, two independent maps were simultaneously acquired, that is, a topography image of the immobilized avidin molecules and a lateral map of the corresponding recognition sites, both recorded at experimental times comparable to normal AFM imaging. Before being applied to TREC, the tips were examined for a functional biotin residue by force spectroscopy experiments on a mica surface densely covered with avidin molecules. Force spectroscopy with an oscillating tip offers an important tool for adjusting the scanning parameters in TREC microscopy. In simultaneously recorded amplitude–distance and force–distance cycles (Figure 1), the oscillation amplitude and the cantilever bending force, respectively, were investigated.