(121c) Diffusing Colloidal Probe Measurements of Protein and Synthetic Macromolecule Interactions

We report a new method employing ensembles of freely diffusing colloidal probes to directly and nonintrusively measure kT and nanometer scale interactions of surface immobilized proteins and synthetic macromolecules. By combining evanescent wave and video microscopy techniques, this new method monitors the three dimensional excursions of protein/macromolecule decorated colloids as they sample positions normal and parallel to homogeneous, heterogeneous, and patterned substrates also bearing proteins and macromolecules. Analyses of equilibrium particle distributions and dynamic particle trajectories are used to sensitively resolve energetic, spatial, temporal, and statistical information concerning specific and non-specific interactions between proteins and macromolecules either physisorbed or covalently attached and oriented on surfaces.

Initial experiments have focused on measuring non-specific interactions of Bovine serum albumin (BSA) and PEO-PPO-PEO copolymer adsorbed to chemically modified silica particles and planar silica, gold, and polystyrene surfaces. Measurements of all configurations of BSA and PEO-PPO-PEO on particle and planar surfaces demonstrate (1) the role of substrate surface chemistry on macromolecular architecture, (2) the differences in non-specific interactions of BSA-BSA, BSA-PEO, and PEO-PEO, and (3) the importance of heterogeneities on particles, surfaces, and in adsorbed layers for interpreting measured potential energy profiles and probe dynamics. Ongoing work is focused on adapting and extending the use of diffusing colloidal probes to measure both specific and non-specific interactions between extra cellular cadherin fragments that control calcium dependent cell adhesion and sorting processes. These measurements are being conducted as a function of calcium ion concentration between wild type and mutant cadherin fragments covalently attached and oriented on colloids and surfaces.