Quantification of primary amine groups available for subsequent biofunctionalization of polymer surfaces

Biocompatible polymers are commonly functionalized with specific moieties such as amino groups to modify their surface properties and/or to attach bioactive compounds. A reliable method is usually required to characterize amino group surface densities. In this study, aminated polyethylene terephthalate (PET) films were generated via an aminolysis reaction involving either ethylenediamine molecules (EtDA), in order to vary easily the amino group density on PET surfaces, or 25 kDa polyvinylamine (PVAm) as an alternative reagent preventing bulk damages resulting from the aminolysis reaction. Among commonly used dyes for amino group quantification, Orange II and Coomassie Brillant Blue (CBB) were selected to quantify the extent of amine grafting resulting from these derivatization procedures. Rapid and convenient colorimetric assays were compared to surface atomic compositions obtained from X-ray photoelectron spectroscopy (XPS) measurements. Orange II was found to be the most appropriate dye for quantifying primary amine groups in a reliable and specific way. Due to its unique negative charge and low steric hindrance compared to CBB, the Orange II dye was very sensitive and provided reliable quantification over a wide range of amino group surface densities (ca. 5 to at least 200 pmol/mm(2)). In order to further validate the use of the Orange II dye for amino group quantification, a heterobifunctional linker reacting with amino groups was then grafted on modified PET surfaces. Interestingly, the good correlation between the densities of adsorbed Orange II and covalently grafted linkers suggests that the Orange II method is a relevant, reliable, easy, and inexpensive method to predict the amount of amino groups available for subsequent functionalization of polymer surfaces.