The desorption of ribonuclease A from charge density gradient surfaces studied by spatially-resolved total internal reflection fluorescence.

A quaternary amine surface gradient was prepared on fused silica by a three-step surface modification process. The gradient surface displayed a transition of surface charges along the gradient dimension from a net negative surface charge of silica to a net positive surface charge at the quaternary amine end. The gradient surface was characterized by X-ray photoelectron spectroscopy, ellipsometry, colloidal gold decoration, and dynamic contact angle measurements. It displayed an increased adhesion of negatively charged gold particles towards the quaternary amine end. The water contact angles also increased with the increased surface density of aminopropylsilyl groups. The desorption of ribonuclease A labeled with fluorescein-5-isothiocyanate (FITC-RNase) from the quaternary amine gradient surface was measured using spatially resolved total internal reflection fluorescence (TIRF) spectroscopy. The experimental FITC-RNase desorption results fitted exceptionally well to a two adsorbed protein populations model. A tentative assignment of the two adsorbed protein populations is proposed based on the effect of the ionic strength of the desorbing buffer. The faster desorption population interacted primarily with the quaternary amine gradient surface sites through electrostatic interactions. The slower desorption population interacted with the surface sites via hydrophobic and possibly some electrostatic interactions.