Nanopore-Based Methods for Characterizing Single Proteins.

Abstract

Proteins represent the most diverse class of biomolecules in both structure and function and are involved in nearly every physiological process; their quantification, identification, and biophysical characterization is therefore of fundamental and practical importance. This dissertation introduces two distinct techniques that use nanopores to characterize and identify single unlabeled proteins in a high-throughput manner. The first technique uses femtosecond-laser-fabricated dual-pore glass chips for performing cell-attached single-ion-channel recordings. Existing planar patch-clamp platforms are generally unable to perform these types of recordings due to excess noise arising from low seal resistances and the use of substrates with poor dielectric properties. While these platforms tend to use a single pore (D ~ 1–2 μm) to position a cell by suction and to establish a seal, the dual-pore glass chips employ separate pores optimized for each function, enabling the use of a relatively small patch aperture (D ~ 150–300 nm) that is more suitable for forming high-resistance seals than micropores used currently. Patch-clamp experiments with these chips achieved high seal resistances and the lowest RMS noise ever reported for a planar patch-clamp platform. This platform enables semi-automated single-channel recordings in the cell-attached configuration that are comparable to those obtained by conventional patch-clamp, which is laborious and requires manual control of micropipette position. The second technique uses electrolyte-filled nanopores coated with a lipid bilayer to characterize single lipid-anchored proteins via resistive-pulse sensing. Lipid-coated nanopores have previously been used to determine a protein’s volume, charge, and ligand affinity by measuring the change in ionic current, DI, through the nanopore as a protein travels from one side to the other. Exploiting the dependence of DI on the shape and orientation of a particle in the nanopore, this work extends the capabilities of resistive-pulse sensors by enabling determination of the shape, volume, rotational diffusion coefficient, and dipole moment of individual non-spherical proteins. The techniques introduced here may ultimately reveal insights into conformational protein dynamics, expedite biomarker and drug discovery, enable the characterization of personal proteomes, and improve our understanding of proteins and protein complexes regarding health and disease.