Nano-patterning of hydrogenated amorphous carbon (a-C:H) surfaces for control of protein adsorption

Abstract

The thesis presents an investigation of the nano-patterning of hydrogenated amorphous carbon (a-C:H) surfaces to control protein adsorption. The relevant literature is first reviewed, noting the link between protein adsorption and misfolding and its relevance to bio-compatibility and nano-toxicity. It then identifies how nano-topography influences protein adsorption, the debates and conflicts in the literature regarding this effect and the issues associated with controlling nanotopography independently of local surface composition.

The first experimental chapter deals with the preparation and analysis of a-C:H patterns made by focused ion beam (FIB) milling and atomic force microscopy(AFM) nanoindentation. These methods resulted in nano-patterns with 2 nm height amplitude and 60 nm spacing, hence of size commensurate with that of proteins. The challenges associated with the production of such patterns are discussed, particularly the analysis and simulation of the implanted gallium profile in the FIB patterns. Advanced AFM techniques were used to investigate the possible compositional nature of the patterns. The Interleave/Lift method detected an onset of long range interactions between a protein coated tip and the patterned surfaces at a 38 nm tip surface distance for both patterning methods. A hill/valley compositional contrast was also noted, stronger in the case of the FIB pattern. Short range adhesive tip/surface forces were mapped with the digital pulse force method (DPFM). Again, this showed stronger compositional contrast for the FIB pattern. These effects were interpreted as arising from the electrostatic interactions between the negatively charged protein coated tip and the patterned surfaces. Using SRIM modelling and the measured contrast values, a negative charge per adsorbed protein of 1.3-3e was estimated.

The second half of the thesis investigates protein adsorption on a-C:H for four different protein/solvent systems; bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) in de-ionized (DI) water or phosphate buffer saline (PBS)solutions. Fourier transform infrared (FTIR) analysis revealed that there is a significant change in the secondary structure of the proteins once they adsorbed onto a-C:H, corresponding to an increase of t -sheet component, often associated with exposure of the buried hydrophobic groups. This is also consistent with the large surface footprint of the adsorbed proteins, measured by AFM microscopy. Adsorption on FIB-patterned surfaces reveals changed adsorption behaviours, with significant increases in adsorbed foot-prints for all systems expect for the BSA-DI system where this footprint decreases slightly. Finally, adsorption experiments were carried out on patterns made by the FIB and AFM techniques. This comparison indicates that, for the FIB pattern, BSA adsorbs preferentially in the valleys whereas, for the AFM pattern, it resides on the hills. This effect, consistent with the previous analysis, was attributed to the buried charges in the valleys of the FIB pattern. Overall, the work presented in this thesis showed that nano-patterned a-C:H model surfaces are useful to study and control protein adsorption, suggesting that, in the case studied here, nano-topography modifies qualitatively the adsorption process. In addition, the methods developed here can be extended to other patterning techniques and protein systems to study independently the influence of topography and composition on protein adsorption.

Thesis is confidential until 30th April 2015

Date of Award	Apr 2013
Original language	English
Sponsors	Vice Chancellor's Research Scholarship (VCRS)
Supervisor	Patrick Lemoine (Supervisor) & Paul Maguire (Supervisor)