ORIGINAL PAPER The Effect of Fluorescent Labels on Protein Sorption in Polymer Hydrogels Allan Guan & Zhenyu Li & K. Scott Phillips Received: 16 May 2014 /Accepted: 21 August 2014 # Springer Science+Business Media New York (outside the USA) 2014 Abstract Hydrogels are an increasingly important class of medical device materials that enable diverse and unique func- tion, but can also be subject to significant biofouling and contamination. Although it is challenging to accurately quan- tify protein biofouling in hydrogels, spectroscopic detection of fluorescently labeled proteins is one method with the potential to provide direct, sensitive quantitation in transparent mate- rials. 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 direct- ly measure both labeled and unlabeled protein sorption, while orthogonal measurements were performed with extrinsic fluo- rescence 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 ob- served 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. Keywords Hydrogel . Fluorophore . Fluorescent label . Sorption . Biofouling . Protein . Labeling . Interference . Contact lens . Medical device . Adsorption . Absorption . Fluorescence . Fluorescence recovery after photobleaching (FRAP) . Photobleaching . Intrinsic fluorescence . UV fluorescence . Residues . Microplate . Structure . Lysozyme . Ionic . Hydrohobic . BCA assay . Calibration . Response . Transparent . (Hydroxyethyl) methacrylate (HEMA) . Silicone . Bicinchoninic acid . Polymer Introduction The porous water-filled structure of polymer hydrogels en- ables their use in diverse applications for medical devices such as scaffolds, delivery vehicles, barriers,contact lenses, fillers, and bulking agents [1–3]. Although hydrogels are hydrophilic by design, proteins and other biomolecules with hydrophobic functionalities can bind to internal domains by coulombic (charge-charge) interactions [4]. Similar to non-porous mate- rials, hydrogels can undergo protein adsorption and biofoul- ing, resulting in tissue inflammation [5,6], microbial coloni- zation [5], spread of infectious or allergenic agents [7], and performance degradation [8]. Moreover, new hydrogel mate- rials being used or currently being developed for medical devices often contain complex hydrophobic domains to Electronic supplementary material The online version of this article (doi: 10.1007/s10895-014-1450-8) contains supplementary material, which is available to authorized users. A. Guan : Z. Li Department of Biomedical Engineering, The George Washington University, Phillips Hall - Room 617, The Academic Center 801 22nd Street NW, Washington, DC 20052, USA A. Guan : K. S. Phillips (*) Office of Medical Products and Tobacco, Center for Devices and Radiological Health, Office of Science and Engineering Laboratories, Division of Chemistry and Materials Science, United States Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD 20993, USA e-mail: kenneth.phillips@fda.hhs.gov J Fluoresc DOI 10.1007/s10895-014-1450-8