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.
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Dr. Kim Sapsford (FDA) and Center for Devices and Radiological Health.
The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.
Fernández-Cossío S, Castaño-Oreja MT (2006) Biocompatibility of two novel dermal fillers: histological evaluation of implants of a hyaluronic acid filler and a polyacrylamide filler. Plast Reconstr Surg 117:1789–1796PubMedCrossRefGoogle Scholar
Nguyen KT, West JL (2002) Photopolymerizable hydrogels for tissue engineering applications. Biomaterials 23:4307–4314PubMedCrossRefGoogle Scholar
Li L, Chen S, Zheng J, Ratner BD, Jiang S (2005) Protein adsorption on Oligo(ethylene glycol)-terminated alkanethiolate self-assembled monolayers: the molecular basis for nonfouling behavior. J Phys Chem B 109:2934–2941PubMedCrossRefGoogle Scholar
Szott LM, Horbett TA (2011) Protein interactions with surfaces: cellular responses, complement activation, and newer methods. Curr Opin Chem Biol 15:677–682PubMedCrossRefGoogle Scholar
Phillips K, Cheng Q (2007) Recent advances in surface plasmon resonance based techniques for bioanalysis. Anal Bioanal Chem 387:1831–1840PubMedCrossRefGoogle Scholar
Keith D, Hong B, Christensen M (1997) A novel procedure for the extraction of protein deposits from soft hydrophilic contact lenses for analysis. Curr Eye Res 16:503–510PubMedCrossRefGoogle Scholar
Zhao Z et al (2009) Care regimen and lens material influence on silicone hydrogel contact lens deposition. Optom Vis Sci 86:251–259PubMedCrossRefGoogle Scholar
Brynda E, Drobník J, Vacík J, Kálal J (1978) Protein sorption on polymer surfaces measured by fluorescence labels. J Biomed Mater Res 12:55–65PubMedCrossRefGoogle Scholar
Casiano-Maldonado M et al (2013) Protein adsorption on thermoplastic elastomeric surfaces: a quantitative mass spectrometry study. Int J Mass Spectrom 354–355:391–397CrossRefGoogle Scholar
Lipscomb IP, Sihota AK, Botham M, Harris KL, Keevil CW (2006) Rapid method for the sensitive detection of protein contamination on surgical instruments. J Hosp Infect 62:141–148PubMedCrossRefGoogle Scholar
Tworkoski E, Dorris E, Shin D & Phillips KS (2014) A high-throughput method for testing biofouling and cleaning of polymer hydrogel materials used in medical devices. Analytical Methods 6:4521–4529Google Scholar
Bingaman S, Huxley VH, Rumbaut RE (2003) Fluorescent dyes modify properties of proteins used in microvascular research. Microcirculation 10:221–231PubMedCrossRefGoogle Scholar
Teske CA, Schroeder M, Simon R, Hubbuch J (2005) Protein-labeling effects in confocal laser scanning microscopy. J Phys Chem B 109:13811–13817PubMedCrossRefGoogle Scholar
Timperman AT, Oldenburg KE, Sweedler JV (1995) Native fluorescence detection and spectral differentiation of peptides containing tryptophan and tyrosine in capillary electrophoresis. Anal Chem 67:3421–3426PubMedCrossRefGoogle Scholar
Chen S, Lillard SJ (2001) Continuous cell introduction for the analysis of individual cells by capillary electrophoresis. Anal Chem 73:111–118PubMedCrossRefGoogle Scholar
Roegener J et al (2003) Ultrasensitive detection of unstained proteins in acrylamide gels by native UV fluorescence. Anal Chem 75:157–159PubMedCrossRefGoogle Scholar
Zhang H, Yeung ES (2006) Ultrasensitive native fluorescence detection of proteins with miniaturized polyacrylamide gel electrophoresis by laser side-entry excitation. Electrophoresis 27:3609–3618PubMedCrossRefGoogle Scholar
Jing P, Kaneta T, Imasaka T (2002) Determination of dye/protein ratios in a labeling reaction between a cyanine dye and bovine serum albumin by micellar electrokinetic chromatography using a diode laser-induced fluorescence detection. Electrophor 23:2465–2470CrossRefGoogle Scholar
Luensmann D, Heynen M, Liu L, Sheardown H, Jones L (2010) The efficiency of contact lens care regimens on protein removal from hydrogel and silicone hydrogel lenses. Mol Vis 16:79–92PubMedCentralPubMedGoogle Scholar
Olson BJSC, Markwell J (2007) Assays for determination of protein concentration. Curr Protoc Protein SciChapter 3, Unit 3.4Google Scholar
Kohnlein J, Glasmacher R, Heide V (2008) Multicentre trial on standardisation of a test soil of practical relevance for comparative and quantitative evaluation of cleaning pursuant to EN ISO 15883. Zentralsterilisation 16:424–435Google Scholar
Kohnlein J, Glasmacher R, Heide V (2009) Multicentre trial on standardisation of a test soil of practical relevance for comparative and quantitative evaluation of cleaning pursuant to EN ISO 15883 description of test procedure. Zentralsterilisation 17:410–415Google Scholar
Johnson AE (2005) Fluorescence approaches for determining protein conformations, Interactions and Mechanisms at Membranes. Traffic 6:1078–1092PubMedCrossRefGoogle Scholar
Green JA*, Phillips KS*, et al (2012) Material properties that predict preservative uptake for silicone hydrogel contact lenses. Eye & Contact Lens: Science & Clinical Practice November 201238, 350–357 * Equal ContributionsGoogle Scholar
1.Department of Biomedical EngineeringThe George Washington UniversityWashingtonUSA
2.Office of Medical Products and Tobacco, Center for Devices and Radiological Health, Office of Science and Engineering Laboratories, Division of Chemistry and Materials ScienceUnited States Food and Drug AdministrationSilver SpringUSA