(639f) Using QCM-D and Ellipsometry to Determine the Orientation and State of Hydration of Antibodies Adsorbed On a Hydrophobic Surface

Understanding and controlling the structure and orientation of proteins at interfaces is critical in designing biosensors where the capture of an analyte is dependent on the correct orientation and conformation of a surface-tethered protein.  Quartz Crystal Microbalance with Dissipation (QCM-D) is a technique often used to monitor protein adsorption due to its excellent sensitivity, ease of experimental design, and label-free detection method.  One important and often underexploited feature of QCM-D in the liquid phase is that it measures a hydrated protein mass, including the mass of mechanically coupled water in the measurement output.  In practice, this means that either viscoelastic modeling or in-situ combination with a complementary measurement technique is needed to understand the effects of water content on the QCM-D response and to separate dry from hydrated protein mass in a quantitative manner.   

In this study, we demonstrate the utility of QCM-D in quantifying adsorbed protein amount as well as characterizing orientation by examining the case of a human monoclonal antibody adsorbed onto a model hydrophobic, CH₃-terminated surface.  An analysis of the protein-binding ability of the antibody combined with an examination of its mechanical coupling to the surface suggests that the antibodies initially adsorb in a 'flat-on' orientation, unable to capture their antigen, until they reach a surface coverage at which they no longer fit 'flat-on' and begin to adopt a mixture of 'end-on' orientations, with both Fab and Fc portions of the antibody accessible on the surface at monolayer coverage.  By combining QCM-D with simultaneous ellipsometry measurements and by varying the density of the surrounding buffer, we demonstrate that the antibody is less hydrated at low surface coverages, while at high surface coverage the mass of hydration is quite significant, comprising more than 50% of the total mass measured by QCM-D alone.  Based on this example, we detail strategies for how to use QCM-D to achieve quantitative measures of protein surface excess as well as qualitative orientational information, with and without the added benefit of a complementary measurement technique.