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Fundamentals of Protein Adsorption at the Solid-Liquid Interface over Short Time Periods

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Hall_Brad.pdf (7.07 MB)

Date

2014-12-16

Authors

Hall, Brad

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University of Waterloo

Abstract

Purpose: To directly measure the quantity and degree of denaturation of biomaterial- adsorbed proteins over short time periods. Methods: Contact lenses were used as model biomaterials as they are widely used, readily available, and have a wide variety of material properties. The proteins lysozyme, lactoferrin, and albumin were investigated, as they are major protein components of bodily fluids, notably tears. Time points within the first few minutes of protein-material interactions were concentrated on. • A novel technique to measure the activity of surface adsorbed lysozyme was developed (Chapter 3) • Direct comparison of traditional extraction procedures and the novel surface technique for measuring the activity of adsorbed lysozyme (Chapter 4) • The effect of competitive adsorption between lysozyme and lactoferrin and the effect on lysozyme activity (Chapter 5) • Investigations of using I125 radiolabeling for protein adsorption experiments with contact lenses (Chapter 6) • The effect of competitive adsorption of an artificial tear solution to the deposition of lysozyme, lactoferrin, and albumin (Chapter 7) Results: Using the novel technique, the activity of surface adsorbed lysozyme and any biologically relevant lysozyme can be measured and distinguished within the first few minutes of lysozyme-material interaction. Using the novel technique in conjunction with protein extraction provides detailed activity information about deposited biologically relevant adsorbed lysozyme and lysozyme which is in underlying protein layers or in the bulk of the material. Lactoferrin co-adsorption with lysozyme did not affect the surface activity of lysozyme, but did decrease the activity of desorbed lysozyme. Radiochemical experiments using I125 can provide sensitive measurements of protein adsorption to contact lens materials, but extra steps need to be taken to limit and measure the amount of unbound I125 in solution and to quantify the ‘apparent mass’ adsorbed unbound I125 represents. Lotrafilcon B was the only lens material to show decreased protein adsorption due to competitive adsorption effects. This effect occurred when lysozyme and lactoferrin were competing and when lysozyme, lactoferrin, and albumin were competing with components from the artificial tear solution. Conclusions: This thesis has developed and refined methods to measure biomaterial- adsorbed proteins over short time periods. These techniques can be utilized in the future to measure both the quantity and degree of denaturation of adsorbed proteins to contact lenses and other biomaterials.

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Keywords

protein adsorption, contact lens, protein activity on biomaterials, protein surface activity, lysozyme kinetics, silicone hydrogel, competitive protein adsorption, I125 radiolabeling, free I125, artificial tear solution

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http://hdl.handle.net/10012/8994

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Theses
Optometry and Vision Science