Nanopores with Fluid Walls for Characterizing Proteins and Peptides

Nanopore-based, resistive-pulse sensing is a simple single-molecule technique, is label free, and employs basic electronic recording equipment. This technique shows promise for rapid, multi-parameter characterization of single proteins; however, it is limited by transit times of proteins through nanopores that are too fast to be resolved, non-specific interactions of proteins with the nanopore walls, and poor specificity of nanopores for particular proteins.;This dissertation introduces the concept of nanopores with fluid walls and their applications in sensing and characterization of proteins, disease-relevant aggregates of amyloid-beta peptides, and activity of membrane-active enzymes.;Inspired by lipid-coated nanostructures found in the olfactory sensilla of insect antennae, this work demonstrates that coating nanopores with a fluid lipid bilayer confers unprecedented capabilities to a nanopore such as precise control and dynamic actuation of nanopore diameters with sub-nanometer precision, well-defined control of protein transit times, simultaneous multi-parameter characterization of proteins, and an ability to monitor the enzyme phospholipase D.;Using these bilayer-coated nanopores with lipids presenting a ligand, proteins binding to the ligand were captured, concentrated on the surface, and selectively transported to the nanopore, thereby, conferring specificity to a nanopore. These assays enabled the first combined determination of a protein's volume, shape, charge, and affinity for the ligand using a single molecule technique. For non-spherical proteins, the dipole moment and rotational diffusion coefficient could be determined from a single protein.;Additionally, the fluid, biomimetic surface of a bilayer-coated nanopore was non-fouling and enabled characterization of Alzheimer's disease-related amyloid-beta aggregates. The presented method and analysis fulfills a previously unmet need in the amyloid research field: a method capable of determining the size distributions and concentrations of amyloid-beta aggregates in solution.;The experiments presented here demonstrate that the concept of a nanopore with fluid walls enables new nanopore-based assays. In particular, it demonstrates the benefits of this concept for simultaneous, multi-parameter characterization of proteins with a single-molecule method. This technique may, therefore, be well-suited for identification of proteins directly in complex biological fluids. Based on these findings, the addition of fluid walls to nanopores holds great promise as a tool for simple, portable single molecule assays and protein characterization.