In-situ protein adsorption study on biofunctionalized surfaces using spectroscopic ellipsometry

Techniques currently employed to evaluate biomolecular interactions on surfaces require the use of radiolabeled, enzymatic, or fluorescent-tags to record and report the binding event. Ellipsometry has proven to be a powerful tool in understanding the biomolecular interactions on solid substrates and, typically does not require the labeling of the ligand or the receptor. In this present study, the adsorption kinetics of Human Serum Albumin (HSA) on functionalized silicon surfaces were evaluated using in-situ ellipsometry. In-situ ellipsometry was used to estimate the thickness of the adsorbed layers and the adsorption and desorption kinetics of HSA on functionalized surfaces. In this study, dense, self assembled monolayers were fabricated using aminopropyltriethoxysilane (APTES) and mixed silanes using APTES and methyltriethoxysilane at a ratio of 1:10, to serve as a template for protein immobilization on silicon surfaces. The silane derivatized surfaces were further modified using three different ligands/receptors that have been reported to bind HSA, namely: a linear peptide, a polyclonal antibody against human serum albumin, and small synthetic ligand (2, 4, 6-Tris(dimethylaminomethyl)phenol. The amount of HSA adsorbed was observed to increase with time, and with the initial concentration of the HSA solution. The adsorption kinetics of HSA on functionalized surfaces was approximated by a simple model for protein adsorption. A good model fit was obtained for the experimental data, thus enabling the interpretation of the adsorption kinetics of HSA on functionalized silicon surfaces. The effect of different HSA binding ligands on the rate constants affecting protein adsorption and desorption were studied.