Investigation of Structure and Function of Thin Films of Nitric Oxide Synthase and Polyethylenimine Formed Using The Layer-by-Layer Deposition Method

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Layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/polyelectrolytes nanostructured films. Application of these films as functional coatings particularly on medical implants is a transformative leap for biocompatible coatings that can be tailored to fulfill various biological functions. In this project, we investigate the layer-by-layer (LBL) deposition of nitric oxide synthase (NOS) as a functional component and polyethylenimine (PEI) as a matrix on pyrolytic graphite and mica surfaces as model surfaces. We have previously shown that these films, when exposed to NOS substrate arginine and other ingredients, can release fluxes of nitric oxide (NO). The latter is a molecule known to counteract platelet aggregation, and thus can stop the thrombosis cascade on the surface of medical devices. Negative and positive charge density in each layer is the driving force that leads to adhesion of film components. In this work we examined if the pH of the medium of each component can modulate the charge achieved in each layer. We used buffered NOS solutions and saline PEI adjusted to desired pHs to investigate charge-driven adhesion of NOS and PEI as well as extent of NO release that results. In addition to measurements of NO release using the Griess assay, we also used the catalytic reduction of nitric oxide as an electrochemical handle to monitor functional aspects of various films built under different pH conditions. In terms of structure, we used atomic force microscopy to characterize the morphology of the outermost NOS layer. Our results show that the charge density of each layer brought about by the pH of film components during the LBL process affects the structure of the film as well as the extent of NO release by the resulting NOS/PEI thin film.