AFM Imaging of Immobilized Fibronectin:  Does the Surface Conjugation Scheme Affect the Protein Orientation/Conformation?

Abstract

Covalent grafting of biomolecules could potentially improve the biocompatibility of materials. However, these molecules have to be grafted in an active conformation to play their biological roles. The present work aims at verifying if the surface conjugation scheme of fibronectin (FN) affects the protein orientation/conformation and activity. FN was grafted onto plasma-treated fused silica using two different crosslinkers, glutaric anhydride (GA) or sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Fused silica was chosen as a model surface material because it presents a roughness well below the dimensions of FN, therefore allowing AFM analyses with appropriate depth resolution. Cell adhesion assays were performed to evaluate the bioactivity of grafted FN. Cell adhesion was found to be higher on GA-FN than on SMPB-FN. Since FN-radiolabeling assays allowed us to rule out a surface concentration effect (approximately 80 ng/cm2 of FN on both crosslinkers), it was hypothesized that FN adopted a more active conformation when grafted via GA. In this context, the FN conformation on both crosslinkers was investigated through AFM and contact angle analyses. Before FN grafting, GA- and SMPB-modified surfaces had a similar water contact angle, topography, and roughness. However, water contact angles of GA-FN and SMPB-FN surfaces clearly show differences in surface hydrophilicity, therefore indicating a dependence of protein organization toward the conjugation strategy. Furthermore, AFM results demonstrated that surface topography and roughness of both FN-conjugated surfaces were significantly different. Distribution analysis of FN height and diameter confirmed this observation as the protein dimensions were significantly larger on GA than SMPB. This study confirmed that the covalent immobilization scheme of biomolecules influences their conformation and, hence, their activity. Consequently, selecting the appropriate conjugation strategy is of paramount importance in retaining molecule bioactivity.

Anti-Fog Layer Deposition onto Polymer Materials: A Multi-Step Approach

“…As aforementioned, no nitrogen was detected on the inorganic layer, thus signifying that the surface was devoid of amino groups which are mandatory to subsequent covalent conjugation (Table 1). In this context, a N 2 -H 2 filamentary atmospheric plasma discharge was performed to functionalize the surface, as past experience has taught us that similar plasma conditions enable amino group formation on fused silica [41]. Unfortunately, this treatment only allowed for the surface incorporation of 2.7% of nitrogen, with no amino functionality (results not shown).…”

Thermochemical Nanolithography of Multifunctional Nanotemplates for Assembling Nano‐Objects

“…The former value compares well with previous measurements of single dry fibronectin molecules. [35] Comparing the topographical image of the active sites before and after fibronectin functionalization and considering the geometrical convolution with a spherical AFM tip (radius about 15 nm), we conclude that roughly 1-2 fibronectin molecules are exposed at the surface, whereas more fibronectins could fit inside the hole created during TCNL writing. We cannot give more detailed information on the exact numbers of proteins per spot because we do not know the conformation of the proteins inside the holes.…”

Bridging the Gap Between Physicochemistry and Interpretation Prevalent in Cell−Surface Interactions

“…Another type of chemisorption is employing a free sulfur atom in the biomolecule for interaction with a maleimide functional surface, thereby forming maleimide chemisorption. 73,74,83,84 It must be taken into account whether the chemical moieties are available in the biomolecule to be chemisorbed. For example, Fn has ∼75 lysine residues with available NH 2 functionalities for chemisorption.…”