Nanopores with Fluid Walls for Characterizing Proteins and Peptides.

[摘要] 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 a nanopore 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 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.

[发布机构] University of Michigan

[效力级别] Coulter Couting

[关键词] Resistive Pulse;Coulter Couting;Nanopore;Protein Characterization;Protein Size Charge Shape Rotational Diffusion Coefficient and Dipole Moment;Biomedical Engineering;Chemistry;Physics;Science (General);Health Sciences;Science;Biomedical Engineering