Confirming Silent Translocation through Nanopores with Simultaneous Single-Molecule Fluorescence and Single-Channel Electrical Recordings.

Most of what is known concerning the luminal passage of materials through nanopores arises from electrical measurements. Whether nanopores are biological, solid-state, synthetic, hybrid, glass-capillary-based, or protein ion channels in cells and tissues, characteristic signatures embedded in the flow of ionic current are foundational to understanding functional behavior. In contrast, this work describes passage through a nanopore that occurs without producing an electrical signature. We refer to the phenomenon as "silent translocation." By definition, silent translocations are invisible to the standard tools of electrophysiology and fundamentally require a simultaneous ancillary measurement technique for positive identification. As a result, this phenomenon has been largely unexplored in the literature. Here, we report on a derivative of Cyanine 5 (sCy5a) that passes through the α-hemolysin (αHL) nanopore silently. Simultaneously acquired single-molecule fluorescence and single-channel electrical recordings from bilayers formed over a closed microcavity demonstrate that translocation does indeed take place, albeit infrequently. We report observations of silent translocation as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurement of the translocation rate as a function of applied potential permits estimation of an effective energy barrier for transport through the pore as well as the effective charge on the dye, all in the absence of an information-containing electrical signature.

Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer Applications.

Artificial lipid bilayers have revolutionized biochemical and biophysical research by providing a versatile interface to study aspects of cell membranes and membrane-bound processes in a controlled environment. Artificial bilayers also play a central role in numerous biosensing applications, form the foundational interface for liposomal drug delivery, and provide a vital structure for the development of synthetic cells. But unlike the envelope in many living cells, artificial bilayers can be mechanically fragile. Here, we develop prototype scaffolds for artificial bilayers made from multiple chemically linked tiers of actin filaments that can be bonded to lipid headgroups. We call the interlinked and layered assembly a multiple minimal actin cortex (multi-MAC). Construction of multi-MACs has the potential to significantly increase the bilayer's resistance to applied stress while retaining many desirable physical and chemical properties that are characteristic of lipid bilayers. Furthermore, the linking chemistry of multi-MACs is generalizable and can be applied almost anywhere lipid bilayers are important. This work describes a filament-by-filament approach to multi-MAC assembly that produces distinct 2D and 3D architectures. The nature of the structure depends on a combination of the underlying chemical conditions. Using fluorescence imaging techniques in model planar bilayers, we explore how multi-MACs vary with electrostatic charge, assembly time, ionic strength, and type of chemical linker. We also assess how the presence of a multi-MAC alters the underlying lateral diffusion of lipids and investigate the ability of multi-MACs to withstand exposure to shear stress.