Protein Interactions With Nitrogen-Doped Amorphous Carbon Surfaces

Amorphous carbon is a very promising material for biocompatible devices. It can be made by a variety of plasma-assisted deposition techniques and is readily doped with other elements, such as nitrogen, which allows tuneable mechanical and tribological properties, including high hardness, low coefficient of friction, and high chemical resistance. It has also been applied to polymer surfaces like poly(tetrafluoroethylene) which gives it the potential for coating applications to hemocompatible devices such as vascular grafts. Although advances in biomaterials used in both surgical and biomedical applications have steadily improved over the past 30 years, improvements towards their biocompatibility and longevity are still needed. Proteins immediately adsorb to the biomaterial interface when it is exposed to bodily fluids such as blood, and this protein layer mediates cellular adsorption on the biomaterial, ultimately playing a major role in the overall success of the biomaterial. Despite advances over the past 40 years in understanding protein interactions at biomaterial interface, there is still a lack of understanding on many of the mechanisms and factors affecting protein adsorption. The main objective of the work presented in this thesis is to (1) develop a surface plasmon resonance (SPR) assay to measure the initial binding kinetics of two major serum proteins, human serum albumin (HSA) and fibrinogen (Fib), to amorphous carbon films prepared with different amounts of nitrogen incorporation. The nitrogen incorporation was controlled by adjusting the %N2 plasma discharge gas during plasma enhanced chemical sputtering using a graphite target onto a Au sensor surface. The initial binding kinetics measurements from SPR experiments found the dissociation kinetics (kd) for both Fib and HSA were comparable onto fullerene-like carbon nitride films (FL-CNx). The association kinetics (ka) was determined to be an important factor for protein adsorption, and the ka was an order of magnitude larger for Fib than HSA. In addition, nitrogen incorporation into the FL-CNx initially decreased ka for both Fib and HSA. However, increasing the nitrogen incorporation due to higher %N2 plasma discharge gas ratios during FL-CNx film deposition increased the ka values for both Fib and HSA. Atomic force microscopy, Raman spectroscopy, and sessile contact angle measurements on the FL-CNx films indicated that the surface hydrophobicity, and the film structure played roles in the changes in protein binding kinetics. The FL-CNx films prepared in the original deposition chamber were also found to contain trace amounts of metals, mainly Fe, incorporated into the films during the deposition process. A second objective (2) of the thesis was to characterize the trace Fe in FL-CNx films deposited onto poly(tetrafluoroethylene). X-ray photoelectron spectroscopy, Fe L-edge x-ray absorption near edge spectroscopy, and electron spin resonance spectroscopy were used to elucidate the Fe structure in the FL-CNx films. The Fe was found to exist in different Fe(III)-oxide and Fe(II) oxide forms, and the Fe valency and concentration was dependent on the %N2 plasma discharge gas during film deposition, and differences were observed for the Fe in the surface and bulk regions of the film. A third objective (3) of this thesis was to design a “metal free” plasma deposition chamber. The films generated using this new chamber were amorphous carbon nitride (a-C:N), and the nitrogen incorporation was controlled by changing the %N2 plasma discharge during a-C:N film deposition. The SPR measurements on the a-C:N films found that the kd values were very similar for HSA and Fib, indicating that the protein/surface interaction is very stable and independent of the protein. The ka(Fib) > ka(HSA) by and order of magnitude. The incorporation of nitrogen into a-C:N film initially decreased the ka for both HSA and Fib, but incorporation of nitrogen using higher %N2 plasma discharge gas during a-C:N film formation increased the ka values for both HSA and Fib. Film characterization suggested that changes in the a-C:N film surface wettability, the type of nitrogen functionalization within the film matrix, and the electronic workfunction may play a role in the changes in ka values measured for HSA and Fib. In addition, it was found that Fe-doping (1.3 at.% Fe) into a-C:N film did not change the HSA and Fib binding kinetics compared to the “metal-free” a-C:N film. Overall, a SPR assay was successfully developed and the initial binding kinetics of HSA and Fib onto amorphous carbon surfaces prepared with different amounts of nitrogen incorporation are reported for the first time. The kinetic results show that the major differences in the binding strength between the two different proteins are the differences in the protein’s ka (recognition rate) towards the surface. This fundamental assay can be expanded in future experiments to study specific surface properties and quantitatively measure the effects of protein binding.

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