title: The effect of fluorescent labels on protein sorption in polymer hydrogels.
abstract: Hydrogels are an increasingly important class of medical device materials that enable diverse and unique function, but can also be subject to significant biofouling and contamination. Although it is challenging to accurately quantify protein biofouling in hydrogels, spectroscopic detection of fluorescently labeled proteins is one method with the potential to provide direct, sensitive quantitation in transparent materials. Therefore, it is important to understand how fluorophores can affect protein-material interactions in hydrogels. This work uses an independent method, native ultraviolet fluorescence (native UV) of proteins, in conjunction with labeled protein fluorescence and the bicinchoninic acid assay (BCA), to assess the effect of fluorescent labels on protein sorption in polymer hydrogels. Bovine serum albumin (BSA) and lysozyme (LY) were labeled with two common but structurally different fluorophores and used as model biofouling proteins in three contact lens hydrogel materials. Native UV was used to directly measure both labeled and unlabeled protein sorption, while orthogonal measurements were performed with extrinsic fluorescence and BCA assay to compare with the native UV results. Sorption of labeled proteins was found to be <2-fold higher than unlabeled proteins on most protein-material combinations, while differences of up to 10-fold were observed for labeled BSA in more hydrophobic hydrogels. Fluorescence recovery after photobleaching (FRAP) also showed that the fluorescent label chemistry can significantly affect surface adsorption of sorbed proteins on the internal surfaces of hydrogels. This study reveals the complex nature of fluorophore-protein-material interactions and shows the potential of native UV for investigating unlabeled protein biofouling in hydrogels.
authors.affiliation: Department of Biomedical Engineering, The George Washington University, Phillips Hall - Room 617, The Academic Center 801 22nd Street NW, Washington, DC, 20052, USA.
authors.forename: Allan
authors.initials: A
authors.lastname: Guan
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Li
authors.forename: K Scott
authors.initials: KS
authors.lastname: Phillips
mesh_terms: D000327:Adsorption; D000818:Animals; D002417:Cattle; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D015394:Molecular Structure; D009113:Muramidase / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013499:Surface Properties
chemical_list: D005456:Fluorescent Dyes; D020100:Hydrogels; D011108:Polymers; D012710:Serum Albumin, Bovine; D009113:Muramidase