Nanopore-Based, Multi-Parametric Characterization Of Single, Unlabeled Proteins In Solution

With large diversity in structure and function, as well as clinical relevance, proteins represent an important target for biophysical characterization. Resistive pulse-based nanopore sensing is a compelling platform for this task, as it provides information about individual proteins during their translocation through the zeptoliter sensing volume inside of a nanopore. Previous work in our group used lipid-coated synthetic nanopores to extract five distinct protein descriptors by analyzing modulations in ionic current, ΔI, resulting from the translocation and rotation of individual lipid-tethered proteins [1]. However, while coating of nanopore walls with fluid lipid bilayers and tethering proteins with lipid anchors is useful for preventing non-specific protein adsorption, extending translocation times, and increasing specificity, it is technically demanding and its success depends on nanopore diameter, geometry, and surface chemistry. This work explores the possibility of using the detergent Tween-20 for nanopore surface coating [2] combined with high bandwidth recording electronics to characterize freely translocating, untethered proteins on a single molecule level. Here, we utilize the dependence of ΔI on the orientation of non-spherical proteins transiting a nanopore to determine their intrinsic shapes, volumes, and dipole moments in solution. The ability to thoroughly examine unlabeled, natively-folded proteins in an aqueous sample on a single molecule level signifies an important step toward the use of nanopores for proteomic and diagnostic applications.[1] Yusko, E.C., et al., Real-time shape approximation and 5-D fingerprinting of single proteins. arXiv preprint arXiv:1510.01935, 2015.[2] Hu, Rui, et al. Intrinsic and membrane-facilitated α-synuclein oligomerization revealed by label-free detection through solid-state nanopores. Scientific reports 6, 2016.