Highly Parallel Transport Recordings on a Membrane-on-Nanopore Chip at Single Molecule Resolution

Abstract

Membrane proteins are prime drug targets as they control the transit of information, ions, and solutes across membranes. Here, we present a membrane-on-nanopore platform to analyze nonelectrogenic channels and transporters that are typically not accessible by electrophysiological methods in a multiplexed manner. The silicon chip contains 250 000 femtoliter cavities, closed by a silicon dioxide top layer with defined nanopores. Lipid vesicles containing membrane proteins of interest are spread onto the nanopore-chip surface. Transport events of ligand-gated channels were recorded at single-molecule resolution by high-parallel fluorescence decoding.

Attolitre-sized lipid bilayer chamber array for rapid detection of single transporters

“…S2a ), which are characteristic optical features of thin lipid bilayers, termed the Plateau–Gibbs border 16 23 24 . Thus, we achieved highly efficient formation of stable and uniform lipid bilayers all over the device, even for the 200-aL chambers (30-nm height), where the aspect ratio of chamber height to lipid orifice size was 30 nm/3 μm = 0.01, which is much smaller than previously reported 9 10 11 12 13 20 . This highly efficient formation is due to the advantage of chloroform as a solvent, as we previously reported.…”

“…A microsystem for arrayed micro-sized reactors sealed with lipid bilayers is another option for membrane transporter analysis; in these systems, transport activity is measured optically based on substrate accumulation or consumption in the chamber 6 9 10 11 12 13 14 . The microsystems enhance the sensitivity and throughput of transporter analysis; however, it remains difficult to measure transport activity of single molecules due to large reactor volumes or low lipid bilayer formation efficiency 15 .…”

Polarization Induced Electro-Functionalization of Pore Walls: A Contactless Technology

“…The lipid bilayer is neither mechanically nor electrically stable [31]. Several approaches have been conducted to overcome this inherent limitation such as the inclusion of polymerizable lipids [32,33], the use of hydrogels and inorganic supports [34,35], reduction of the lateral bilayer size [36], 'droplet interface bilayers' (DIBs) [37,38], and replacement of the lipids by amphiphilic polymers [26]. The protein itself is not very stable and has a relatively short lifetime for detection as a result of the sensitivity of the protein to temperature, voltage, ion concentrations, and solvents [39,40].…”

A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings

“…22 So far, many microfluidic platforms and other chip technologies have been developed, usually lacking the possibility of automated serial formation of lipid bilayers. 18,20,21,[23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] Highly parallelized systems capable of the creation of arrays of lipid bilayers usually rely solely on measurements of fluorescence 32,35,[41][42][43] that does not provide for complete characterization of the function of the pores.…”