title: Single Molecule Characterization of UV-Activated Antibodies on Gold by Atomic Force Microscopy.
abstract: The interaction between proteins and solid surfaces can influence their conformation and therefore also their activity and affinity. These interactions are highly specific for the respective combination of proteins and solids. Consequently, it is desirable to investigate the conformation of proteins on technical surfaces, ideally at single molecule level, and to correlate the results with their activity. This is in particular true for biosensors where the conformation-dependent target affinity of an immobilized receptor determines the sensitivity of the sensor. Here, we investigate for the first time the immobilization and orientation of antibodies (Abs) photoactivated by a photonic immobilization technique (PIT), which has previously demonstrated to enhance binding capabilities of antibody receptors. The photoactivated immunoglobulins are immobilized on ultrasmooth template stripped gold films and investigated by atomic force microscopy (AFM) at the level of individual molecules. The observed protein orientations are compared with results of nonactivated antibodies adsorbed on similar gold films and mica reference samples. We find that the behavior of Abs is similar for mica and gold when the protein are not treated (physisorption), whereas smaller contact area and larger heights are measured when Abs are treated (PIT). This is explained by assuming that the activated antibodies tend to be more upright compared with nonirradiated ones, thereby providing a better exposure of the binding sites. This finding matches the observed enhancement of Abs binding efficiency when PIT is used to functionalize gold surface of QCM-based biosensors.
authors.affiliation: Department of Physics Ettore Pancini, Università di Napoli Federico II , via Cintia, I-80126 Napoli, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Funari
authors.affiliation: Department of Physics Ettore Pancini, Università di Napoli Federico II , via Cintia, I-80126 Napoli, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Della Ventura
authors.affiliation: Department of Physics Ettore Pancini, Università di Napoli Federico II , via Cintia, I-80126 Napoli, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Altucci
authors.affiliation: Peter Grünberg Institute (PGI-8) and Institute of Complex Systems (ICS-8), Forschungszentrum Jülich GmbH , 52428 Jülich, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Offenhäusser
authors.affiliation: Peter Grünberg Institute (PGI-8) and Institute of Complex Systems (ICS-8), Forschungszentrum Jülich GmbH , 52428 Jülich, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Mayer
authors.affiliation: Department of Physics Ettore Pancini, Università di Napoli Federico II , via Cintia, I-80126 Napoli, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Velotta
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D055668:Photochemical Processes; D014466:Ultraviolet Rays
chemical_list: D055910:Antibodies, Immobilized; D007074:Immunoglobulin G