Jonathan Teichroeb

Size-dependent denaturing kinetics of bovine serum albumin adsorbed onto gold nanospheres

We have used localized surface plasmon resonance (LSPR) to monitor the kinetics of thermal denaturing of bovine serum albumin (BSA) adsorbed onto gold nanospheres of size 5 nm-100 nm. The effect of the protein on the LSPR was monitored by visible extinction spectroscopy. The wavelength of the peak extinction (resonance) is affected by the conformation of the adsorbed protein layer, and as such can be used as a very sensitive probe of thermal denaturing that is specific to the adsorbed (as opposed to free) protein. The time dependence of the denaturing is measured in the temperature range 60 °C–70 °C, and the lifetimes are used to calculate an activation barrier for thermal denaturing. The results show that thermally activated denaturing of proteins adsorbed onto nanoparticles has a nanoparticle-size-dependent activation barrier, and this barrier increases for decreasing particle size. This may have important implications for other protein-nanoparticle interactions.

Imaging protein deposits on contact lens materials.

Purpose. The majority of studies investigating protein deposition on contact lens materials require that the deposit of interest be removed, potentially resulting in erroneous results if some proteins are not removed adequately. The purpose of this study was to investigate the use of in situ imaging methods to examine protein deposition on conventional poly(2-hydroxyethyl methacrylate) (polyHEMA)-based and silicone hydrogel contact lens materials. Methods. Six silicone hydrogel and five polyHEMA-based hydrogel contact lens materials were examined by Atomic Force Microscopy (AFM) and/or Scanning Electron Microscopy (SEM) techniques, after being deposited with proteins in an in vitro model. AFM studies examined lenses deposited solely with lysozyme at approximate physiological concentrations and SEM studies were conducted on lenses exposed to a dilute mixture of lysozyme and albumin-conjugated gold spheres. Results. AFM studies demonstrated that the lens materials had markedly differing surface topographies. SEM results showed that galyfilcon A and balafilcon A lenses deposited both lysozyme and albumin in relatively large aggregates, as compared with lotrafilcon A and B, in which the proteins were deposited in a more evenly spread, monolayer formation. Polymacon lenses deposited more protein than any of the silicone hydrogel materials and much of the protein was aggregated together. AFM data indicated that balafilcon A, lotrafilcon A and polymacon deposited lysozyme in a similar manner, with very little lysozyme being deposited in discrete areas. Galyfilcon A behaved very differently, with the lysozyme exhibiting both aggregates as well as string-like formations over the lens surface. Conclusions. Imaging techniques that allow proteins to be examined in situ show much promise for determining the extent and physical characterization of protein on contact lens materials. These techniques indicate that the pattern of deposition of proteins onto silicone hydrogel contact lens materials differs between materials, depending upon their bulk and surface composition.

Quartz crystal microbalance study of protein adsorption kinetics on poly(2-hydroxyethyl methacrylate).

The interaction of macromolecules with artificial biomaterials may lead to potentially serious complications upon implantation into a biological environment. The interaction of one of the most widely used biomaterials, polyHEMA, with lysozyme, bovine serum albumin (BSA), and lactoferrin was investigated using quartz crystal microbalance (QCM). The concentration dependence of adsorption was measured for the aforementioned proteins individually as well as for lysozyme-BSA, and lysozyme-lactoferrin combinations. An extension of Voinovas viscoelastic model to n layers was used to create thickness-time graphs for adsorption. For each of lactoferrin and lysozyme, two distinctly different timescales of adsorption could be differentiated. However, the mechanisms of adsorption appeared to differ between the two. Negative dissipation shifts were measured for low concentrations of lysozyme, trending to positive dissipation at higher concentrations. This suggested that lysozyme was adsorbed initially into the matrix, stiffening the hydrogel, and later onto the surface of polyHEMA. Additionally, trials with commercial no-rub cleaning solutions indicated little added effectiveness over buffer solutions. Mixtures of proteins showed behaviour which differed in some cases from the simple combination of single protein adsorption experiments.

Protein deposition on a lathe-cut silicone hydrogel contact lens material.

Purpose. To determine the quantity of total protein, total lysozyme, and the conformational state of lysozyme deposited on a novel, lathe-cut silicone hydrogel (SiHy) contact lens material (sifilcon A) after 3 months of wear. Methods. Twenty-four subjects completed a prospective, bilateral, daily-wear, 9-month clinical evaluation in which the subjects were fitted with a novel, custom-made, lathe-cut SiHy lens material. The lenses were worn for three consecutive 3-month periods, with lenses being replaced after each period of wear. After 3 months of wear, the lenses from the left eye were collected and assessed for protein analysis. The total protein deposited on the lenses was determined by a modified Bradford assay, total lysozyme using Western blotting and the lysozyme activity was determined using a modified micrococcal assay. Results. The total protein recovered from the custom-made lenses was 5.3 ± 2.3 μg/lens and the total lysozyme was 2.4 ± 1.2 μg/lens. The denatured lysozyme found on the lenses was 1.9 ± 1.0 μg/lens and the percentage of lysozyme denatured was 80 ± 10%. Conclusions. Even after 3 months of wear, the quantity of protein and the conformational state of lysozyme deposited on these novel lens materials was very similar to that found on similar surface-coated SiHy lenses after 2 to 4 weeks of wear. These results indicate that extended use of the sifilcon A material is not deleterious in terms of the quantity and quality of protein deposited on the lens.