Quantifying Fibrinogen Adsorption on Metal/Metal Oxide Surfaces by Chemiluminescence

Introduction: Systematic studies of protein adsorption onto metallic biomaterials are lacking, despite the important role these materials play in health care (dental implants, stents). Wavelength dispersive spectroscopy (WDS) has been shown to be a potentially useful tool to rapidly detect adsorbed protein on sputtered combinatorial metallic surfaces having well-defined compositional gradients. However, WDS involves drying surfaces with adsorbed fibrinogen prior to analysis, possibly underestimating protein quantity. Objectives: To develop an antibody-based chemiluminescence approach to assess fibrinogen adsorption on sputtered combinatorial metallic surfaces, and to compare these data with that obtained from WDS. Methods: A direct ELISA technique was used to detect fibrinogen adsorbed to Silicon wafer and to a titanium-aluminum (Ti-Al) binary library obtained using a well-established combinatorial sputtering technique. Samples were attached to “bottomless” 96-well plates by an adhesive prior to protein adsorption (1 mg/mL bovine fibrinogen; 1 hr). Following rinsing and blocking (1% BSA), surfaces were incubated in the presence of an HRP-conjugated anti-fibrinogen antibody (1:20,000 dilution) prior to adding the chemiluminescence substrate. A standard curve obtained using a high-binding plate enabled conversion of these results into concentration. Results: The surface density of adsorbed fibrinogen on a Ti-Al metal/metal oxide library decreased as the composition of aluminum increased (~900 ng/cm2 to ~550 ng/cm2). A value of 804 ± 82 ng/cm2 was obtained on Si wafers. Fibrinogen levels on either sample was comparable to that reported by WDS, provided one adequately accounted for additional protein adsorption to the plate walls. Conclusions: An antibody-mediated chemiluminescence assay can be used as a high-throughput method for evaluating preferential fibrinogen adsorption to combinatorial metal surfaces. However, an alterative method of sample attachment to the multi-well plate is needed to enable plate assembly following adsorption, thus avoiding non-specific protein adsorption to the plate walls. External Funding Source: Medtronic, Inc.