title: Influence of Protein Surface Coverage on Anomalously Strong Adsorption Sites.
abstract: Serum albumin is commonly used as a blocking agent to reduce nonspecific protein adsorption in bioassays and biodevices; however, the details of this process remain poorly understood. Using single molecule techniques, we investigated the dynamics of human serum albumin (HSA) on four model surfaces as a function of protein concentration. By constructing super-resolution maps, identifying anomalously strong adsorption sites, and quantifying surface heterogeneity, we found that the concentration required for site blocking varied dramatically with surface chemistry. When expressed in terms of protein surface coverage, however, a more consistent picture emerged, where a significant fraction of strong sites were passivated at a fractional coverage of 10(-4). On fused silica (FS), "non-fouling" oligo (ethylene glycol) functionalized FS, and hydrophobically modified FS, a modest additional site blocking effect continued at higher coverage. However, on amine-functionalized surfaces, the surface heterogeneity exhibited a minimum at a coverage of ∼10(-4). Using intermolecular Förster resonance energy transfer (FRET), we determined that new anomalous strong sites were created at higher coverage on amine surfaces and that adsorption to these sites was associated with protein-protein interactions, i.e., surface-induced aggregation.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder , 596 UCB, Boulder, Colorado 80309-0596, United States.
authors.forename: Yu
authors.initials: Y
authors.lastname: Cai
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder , 596 UCB, Boulder, Colorado 80309-0596, United States.
authors.forename: Daniel K
authors.initials: DK
authors.lastname: Schwartz
mesh_terms: D000327:Adsorption; D019855:Ethylene Glycol / Q000737:chemistry; D006801:Humans; D011336:Probability; D012709:Serum Albumin / Q000737:chemistry*; D012821:Silanes / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D013499:Surface Properties; D013696:Temperature; D013997:Time Factors
chemical_list: D012709:Serum Albumin; D012821:Silanes; D012822:Silicon Dioxide; D019855:Ethylene Glycol
keywords: blocking agent; heterogeneity; protein adsorption; single molecule; surface coverage; surface fouling