Paper BI-TuP17
Screening Protein-Surface Interactions with Surface Gradients and TIRF

Tuesday, October 16, 2007, 6:00 pm, Room 4C

Macroscopic surface gradients with negative to neutral surface charges were created by reacting 3-mercaptopropyltrimethoxysilane (MTS) with fused silica and selectively oxidizing surface-bound MTS by controlled UV exposure. XPS analysis and contact angle titrations of the MTS gradients showed that UV oxidation of the MTS sulfhydryl group converts it into a charged sulfonate-like moiety. The MTS gradients were also characterized by AFM in topography, adhesion, and friction modes. The reactivity of unoxidized sulfhydryl groups in the gradient region was assessed by quantitative fluorescence microscopy. The MTS gradients were then used to screen protein interactions with model surfaces. Adsorption kinetics of three human blood proteins, albumin, IgG and fibrinogen, were measured at 1% of their plasma concentrations using a dual channel total internal reflection fluorescence (TIRF) technique. For each protein, two binding experiments were carried out using the same surface gradient sample. In one TIRF channel the solitary binding of one of the fluorescently labeled plasma proteins was observed, and in the other the adsorption of the same was observed from a mixture with the other two unlabeled proteins. The TIRF experiments were later quantified using autoradiography. The adsorption behavior of the three proteins along the sulfhydryl-sulfonate surface gradient was analyzed by taking into account the convective/diffusive transport processes. The on- and off-binding rate constants were obtained as a function of the gradient position by fitting the experimental data to a simple model. Because the unoxidized MTS sulfhydryl can be further derivatized, these techniques have potential to be used as screening tools to study protein-surface interactions on a wide variety of gradient surface chemistries.