Measurement of surface-protein interactions on novel surfaces

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Roach, P., 2005. Measurement of surface-protein interactions on novel surfaces. PhD, Nottingham Trent University.

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Abstract

This thesis is concerned with the fundamental principles affecting protein adsorption. The effects of surface chemistry and topography on protein adsorption characteristics have been identified and quantified. Particular attention has been made to understand how the conformation of surface-bound proteins was affected by the surface onto which they adsorbed.

Quartz crystal microbalance (QCM), UV-Vis spectroscopy and fluorometry were used to assess protein-surface affinity and amounts of protein adsorbed at surface saturation levels. Infrared spectroscopy was used to quantify protein conformational changes incurred upon adsorption. A fluorescent assay protocol was developed for use as an external calibration method for the quantification of adsorbed protein an d the results obtained were compared with QCM and an amido black protein assay of the same systems. Model experiments were performed using bovine fibrinogen (an elongated molecule) and albumin (a globular molecule) adsorbing onto flat hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces in the first instance, but later superhydrophilic and superhydrophobic surfaces were also studied. Surface curvature on the nano-scale was used to model topography, wherein protein molecules adsorbed onto spherical substrates (15-165 nm diameter) having chemically defined surfaces.

Results obtained indicate that both proteins exhibit a less organised secondary structure upon adsorption onto hydrophobic compared to hydrophilic surfaces, with this effect being greatest for albumin. Adsorption rates and binding affinities were found to be higher on hydrophobic surfaces although the amounts adsorbed at saturation were lower. Supporting spectroscopic data suggests that proteins undergo surface induced deformation upon adsorption. Topography was shown to compound the effects of surface chemistry, with fibrinogen being more denatured on surfaces presenting high surface curvature whereas albumin was more denatured on larger substrates. These effects are most probably due to the differing size and shape of the proteins investigated.

This study highlights the possibility of using tailor-made surfaces to influence binding rates and the conformation of bound proteins through protein-surface interactions. The data presented in this thesis demonstrates our ability to control protein adsorption characteristics through careful consideration of the underlying surface, which may facilitate the development and fabrication of materials / surface coatings with tailored bioactivity.

Item Type:

Thesis

Creators:

Roach, P.

Date:

2005

ISBN:

9781369316520

Identifiers: