This proposal is concerned with the application of a conceptually new method, Diffusing Colloidal Probe Microscopy (DCPM), to directly and nonintrusively measure kT interactions of surface immobilized proteins and synthetic macromolecules. Ensembles of freely diffusing colloids will be employed as ultra-sensitive probes to measure energy vs. separation dependent potentials between proteins covalently attached to colloids and planar surfaces. By combining evanescent wave and video microscopy techniques, DCPM will monitor three dimensional Brownian excursions of protein decorated colloids as they sample positions normal and parallel to homogeneous, heterogeneous, and patterned substrates also bearing covalently attached and oriented proteins. Because diffusing probes sample spatial positions according to their relative energies, time and ensemble averaged excursions of probes can be interpreted using statistical mechanical analyses in terms of net potentials of superimposable non-specific (colloidal, macromolecular) and specific (residues, geometry) contributions. Consistent with the nanotechnology paradigm, DCPM will interrogate protein and synthetic macromolecule interactions by exploiting natural gauges for time (a2/D), energy (kT), force (fN), and length (nm) associated with diffusing colloidal probes. The broad aim of the proposed research is to gain fundamental insights into protein-protein and protein-synthetic macromolecule interactions important to non-specific adsorption of proteins on synthetic surfaces and for interpreting specific binding events measured on protein arrays. Specific experimental and modeling objectives are focused on using DCPM to measure interactions of proteins attached and oriented on sub-micron colloids and micron scale surface patterns. The first task of the proposed work is to covalently attach/orient proteins onto gold colloids and either gold or silica surfaces (purified and characterized proteins provided by collaborator). Building on preliminary DCPM results for physisorbed proteins and synthetic macromolecules, the second task is to measure interactions of covalently attached/oriented proteins with various mutations and conformations to allow for deconvolution of non-specific and specific contributions to net potentials. The last task is to measure non-specific and specific interactions on a model array consisting of patterned regions with covalently attached/oriented proteins and regions of physisorbed synthetic macromolecules. Successful completion of proposed objectives will provide new fundamental insights into applications including biofouling and protein array diagnostics, and in the process, will demonstrate a new technology (DCPM) to directly and sensitively quantify weak interactions controlling non-covalent, equilibrium binding of proteins and their non-specific interactions with synthetic macromolecules. The intellectual merit of the proposed research is broadly related to the fundamental knowledge that will be gained in integrating nanoscale synthetic systems and materials of biological origin. Experimental objectives will produce unique, direct measurements of non-specific and specific protein-protein and protein-synthetic macromolecule interactions on energy (kT) and length (nm) scales relevant to equilibrium binding and adsorption. Modeling objectives will interpret/predict multi-scale non-specific and specific contributions to the net interaction of protein conjugated diffusing colloidal probes and surfaces bearing proteins and synthetic macromolecules. Educational objectives will involve using visual content generated in the proposed research in courses and outreach activities. In terms of classroom teaching, research images and videos will be incorporated into undergraduate and graduate colloid/polymer elective courses and an undergraduate thermodynamics core course. In terms of outreach, content involving optical microscopy of colloids will be adapted for use in programs for 7-12 grade teachers and students primarily through the PI's participation in an existing NSF center for learning and teaching. The broader impacts of this proposal involve exploiting visual research content to provide immersive educational experiences for students at all levels that minimizes boundaries between authentic and guided inquiry.
