Surface modification and conjugation strategies for bioassay/biomaterial applications

The aims of this research were to develop novel surface modification strategies that can be used on a range of solid supports including polymeric and metallic matrices, as these could have a significant impact on the performance of bio/sensors employing these surfaces. These approaches were used to develop methods for immobilizing biomolecular recognition elements, such as antibodies, on modified matrices, and to exploit these approaches for the generation of high sensitivity bio-assays. Human fetuin A, mouse immunoglobulin G, and horseradish peroxidase were employed as model analytes. A silane-based surface modification strategy was designed and optimized for planar or flat surfaces such as polymeric sheets, chips or microtitre plates. These polymeric surfaces were activated prior to silane-functionalization using potassium hydroxide (aq.)-mediated mild oxidation (wet method) and oxygen-plasma etching. This novel surface activation strategy was further optimized in combination with surface functionalization and covalent immobilization of antibodies, for enhancing immunoassay sensitivities. Sensitivities obtained for immunoassays using antibodies immobilized with the developed and adsorption-based conventional strategies were 39 and 625 pg/mL, respectively, for human fetuin A. The strategy was demonstrated to be generic in nature and could be employed to activate a wide range of polymeric and metallic surfaces. In addition, highly sensitive detection of human fetuin A was achieved with antibodies captured in an oriented manner on covalently immobilized protein A (EC50 3.7 ng/mL) in comparison to randomly captured antibodies (EC50 5.8 ng/mL). High-brightness NIR664 dye-doped silica nanoparticles were employed to probe various activation states of platelets. These NPs were functionalized with silanes (viz. amine and carboxy-terminal) followed by conjugation to a platelet surface biomarker-specific antibody (anti-CD41) and successfully employed for probing platelet activation. The antibody-NP conjugates were found to be highly sensitive (>95%) and specific (≈100%). In addition, aggregation of NPs was minimized by controlling their surrounding chemical environment and their stability after antibody conjugation