AVS 52nd International Symposium
    Biomaterial Interfaces Friday Sessions
       Session BI+SS-FrM

Paper BI+SS-FrM11
Developments of Flexible Tethers to Measure Antibody-Antigen Interactions using AFM

Friday, November 4, 2005, 11:40 am, Room 311

Session: Biomaterials Surface Characterization
Presenter: Z. Suo, Montana State University
Authors: Z. Suo, Montana State University
F. Terán Arce, Montana State University
R. Avci, Montana State University
E. Smith, Montana State University
K. Thiltges, Montana State University
B. Spangler, Montana State University
Correspondent: Click to Email
Functionalization of an AFM tip surface with covalently bound flexible tether molecules is of special interest because such a flexibility is necessary to measure, in a controllable fashion, the receptor-ligand binding events in the physiological environment of the biomolecules. However, the interpretation of the experimental data is often obscured by and confused with the nonspecific binding events between the substrate surface and the so functionalized AFM tip. Effective methods must be developed to eliminate and/or to identify these nonspecific binding events. To achieve these objectives we employed low densities of varying-length poly(ethylene glycol) (PEG) units grafted onto gold-coated AFM tip surfaces. These tethers were covalently linked to the antibodies of interest, in this case anti-cytochrome c. It was necessary to pacify the uncovered portions of the AFM tip in order to block the nonspecific tip-surface interactions. This was achieved by using the flowers-in-the-meadow concept: by mixing a self-assembled monolayers of small molecular size hydroxyl-terminated PEG unit (meadow) with the specifically terminated larger molecular size PEG unit (flower) as described above. AFM force-extension measurements using such a tip conducted on mica substrate covered with cytochrome c resulted in force and length distributions which are consistent with the tether lengths employed. The pH value and the ionic strength of the buffer have considerable influence on the binding events between the AFM tip and the surface covered with cytochrome c. We will present results covering these topics as well as the role of the coupling chemistry between the end terminals of a PEG molecule and the anti-cytochrome c and the AFM tip on the efficiency of antibody-antigen recognition events.