Covalent attachment and non-specific binding of reactive probe molecules onto surfaces

Optimization of strategies for the covalent attachment of proteins onto polymer surfaces requires the development of analytical methods which can differentiate between proteins that are covalently attached and those that are non-covalently bound (physisorbed). We probed for the surface density of reactive amine, carbonyl, and hydrazide groups using solution phase derivatization reactions to mimic and explore protein immobilization reaction strategies. Labeling compounds investigated were fluorescein derivatives, which were quantified by adsorption spectroscopy, and fluorinated phenyl compounds which were quantified by XPS. Control experiments consisted of performing the same labeling reactions using surfaces without reactive groups, or immersing the polymer surface into the labeling solution after blocking the reactive group of the labeling compound by a covalent reaction in solution. We always found non-negligible contributions arising from physisorption of the derivatization labels. Multiple control surfaces and a novel 'crossover derivatization-XPS' method were studied with the aim of improving compensation for physisorption. Our documentation of surprisingly large physisorption components even for small molecule labels, together with the known propensity of proteins to adsorb onto polymers, suggests caution in quantitative analysis of surface groups by derivatization, and in interpreting covalent protein immobilizations onto polymeric surfaces.