title: Quantification of surface-bound proteins by fluorometric assay: Comparison with quartz crystal microbalance and amido black assay.
abstract: Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only method to give absolute quantities of surface-bound protein. The versatility of the fluorometric assay was also probed by analyzing protein adsorption onto porous superhydrophobic and superhydrophilic surfaces. Results obtained using the assay in conjunction with these surfaces were surface chemistry dependent. Imbibition of water into the superhydrophilic coatings provided greater surface area for protein adsorption, although the protein surface density was less than that found on a comparable flat hydrophilic surface. Superhydrophobic surfaces prevented protein solution penetration. This paper demonstrates the potential of a fluorometric assay to be used as an external calibration for other techniques following protein adsorption processes or as a supplemental method to study protein adsorption. Differences in protein adsorption onto hydrophilic vs superhydrophilic and hydrophobic vs superhydrophobic surfaces are highlighted.
authors.affiliation: Interdisciplinary Biomedical Research Centre, School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton, Nottingham NG11 8NS, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Roach
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Shirtcliffe
authors.forename: David
authors.initials: D
authors.lastname: Farrar
authors.forename: Carole C
authors.initials: CC
authors.lastname: Perry
mesh_terms: D000327:Adsorption; D000580:Amido Black / Q000494:pharmacology*; D000818:Animals; D001703:Biophysics / Q000379:methods*; D002417:Cattle; D002627:Chemistry, Physical / Q000379:methods*; D004396:Coloring Agents / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005470:Fluorometry / Q000295:instrumentation* / Q000379:methods*; D006878:Hydroxides / Q000737:chemistry; D009930:Organic Chemicals / Q000494:pharmacology; D011791:Quartz / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D005456:Fluorescent Dyes; D006878:Hydroxides; C433144:NanoOrange; D009930:Organic Chemicals; D013438:Sulfhydryl Compounds; D014867:Water; D011791:Quartz; D012822:Silicon Dioxide; C031356:hydroxide ion; D000580:Amido Black