Fouling of contact lenses is often due to tear protein diffusion into and aggregation within the contact lens material. These processes can diminish water and oxygen diffusion and create optical cloudiness of the lens. In order to understand the interactions between proteins and hydrogel contact lens materials a study was designed to measure the diffusivity of two model proteins within hydrogel films of varying composition using fluorescence correlation spectroscopy (FCS). Diffusion of human serum albumin (HSA) and apoferritin (aFER) was examined in a range of ~20 μm thick poly(acrylamide) (pAA) and poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels. Protein diffusivity was measured as a function of depth position within each hydrogel film. The characteristic diffusion time for two proteins in pHEMA hydrogels increased relative to both their diffusivity in solution and in pAA hydrogels, indicating that the protein-pHEMA interaction rather than the degree of hydrogel crosslinking is responsible for the observed effects. The resulting spatial representation of the molecular diffusion of proteins into and interaction with hydrogel materials builds a basis on which to conduct similar studies using commercial contact lens samples.
Integrins are an important and highly conserved class of extracellular matrix binding membrane bound receptors that provide a functional linkage between cells and their environment. The excellent spatial resolution and short observation time of a modern fluorescence correlation spectroscopy (FCS) instrument is uniquely suited to the study of quantitative differences in integrin behavior on different parts of a single cell. We hypothesize that the application of FCS to the study of these integrin dynamics on the membranes of nerve cell growth cones will give previously unavailable quantitative insight into nerve cell guidance. Accordingly, we have characterized the application of two integrin-binding small peptide-based FCS analytes to primary rat dorsal root ganglion cells. It results that neither the RGD-based nor the SIKVAV-based analyte exhibited specific association with primary rat dorsal root ganglion growth cone membranes in a range that is accessible to FCS observation. However, the techniques and instrumentation developed for these experiments will be useful for evaluating additional integrin ligands.
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