Novel methods for the control and detection of protein interactions on surfaces

Interfacial interactions with biological macromolecules are of importance to many biotechnological applications. This work describes the use of self assembled polymer thin films consisting of functionalized polyacrylamide to inhibit the non-specific adsorption of dilute protein solutions. These polymer thin films are grafted with functional ligands through succinimide ester moieties copolymerized on the polymer backbone. This general functionalization scheme is used to attach 3-methyl-thiopropyl amine to the polymer in order to provide a means for polymer attachment to gold surfaces. Additionally, a low molecular weight poly-ethylene glycol ligand is used to enhance the films resistance to protein adsorption. The resulting grafted polymers spontaneously form highly stable thin films on both gold nanoparticles as well as flat gold surfaces. The particle systems are thoroughly characterized to show a highly controllable series of aggregates depending upon the amount of polymer loading. The analogous flat gold systems consist of extremely hydrophilic polymer films approximately 25A thick. In both cases, these films exhibit protein affinities that are significantly attenuated relative to an unmodified gold surface. This attenuation can be somewhat controlled by varying the functionalizing ligands and graft density. The interactions of proteins (fibrinogen and bovine serum albumin) with these inert modified surfaces are studied using several novel surface analytical detection schemes. First, a protein assay is developed using secondary electron microscopy (SEM) to image microcontact printed protein arrays on gold surfaces. A mechanism for image formation is proposed and quantitative imaging is demonstrated. Second, an assay is developed based on surface plasmon resonance (SPR) to quantify levels of protein mass coverage using a square hole array in gold coupled to an array of gold disks fabricated by nanoimprint lithography. The nanostructured gold thin film is used to effectively couple photons into a variety of plasmonic modes. The resulting transmission spectra of these plasmonic crystals exhibit numerous resonances in the visible and NIR which are sensitive to surface adsorbed analytes in both frequency and intensity. By integrating the spectral changes over all frequencies, the observed plasmonic responses are used to quantify non-specifically bound fibrinogen monolayers in both spectroscopic and imaging modes with unprecedented sensitivities.