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Extraction versus in situ techniques for measuring surface-adsorbed lysozyme.
Brad Hall1, Chau-Minh Phan, Lakshman Subbaraman
1*BSc †MSc ‡PhD, BSOptom, FAAO §PhD, FCOptom, FAAO ∥PhD Centre for Contact Lens Research, School of Optometry and Vision Science (all authors), Department of Physics and Astronomy and Guelph-Waterloo Physics Institute (LWJ, JF), University of Waterloo, Waterloo, Ontario, Canada.
Two methods for measuring lysozyme activity on contact lenses were compared. The in situ technique measures biologically relevant surface lysozyme, unlike the extraction method which includes underlying layers.
Area of Science:
- Biomaterials Science
- Ophthalmology
- Surface Chemistry
Background:
- Lysozyme is a key enzyme in tear fluid, crucial for innate immunity against ocular pathogens.
- Understanding lysozyme's activity on contact lens surfaces is vital for preventing microbial adhesion and infection.
- Hydrogel contact lenses vary in their material properties, influencing protein deposition and enzyme activity.
Purpose of the Study:
- To compare protein extraction and in situ techniques for measuring lysozyme activity on hydrogel contact lenses.
- To quantify the time-dependent activity of adsorbed lysozyme on different contact lens materials.
- To visualize the interaction of Micrococcus lysodeikticus with lysozyme-coated contact lenses.
Main Methods:
- Adsorbed lysozyme activity was measured using protein extraction and in situ techniques over one week.
- Total, in situ, and surface-adsorbed lysozyme activity were quantified for six contact lens materials.
- Confocal microscopy was used to image bacterial adhesion to surfaces with adsorbed lysozyme.
Main Results:
- Significant material-specific differences in lysozyme activity were observed between the two techniques.
- Extraction methods generally yielded higher activity values than in situ measurements.
- Bacterial adhesion correlated with contact lens surface properties (e.g., contact angle) and lysozyme surface activity.
Conclusions:
- The in situ technique provides a measure of biologically relevant surface lysozyme activity.
- The protein extraction technique quantifies lysozyme within the material matrix and deeper layers.
- Material-specific differences in lysozyme activity and bacterial adhesion highlight the importance of material selection for contact lenses.
