Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Abstract

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed. The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates. High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results. The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip

“…LUVs composed of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoglycerol (POPG) were purchased from Avanti Polar Lipids Inc. (Hamburg, Germany) and mixed in molar ratios of 4:3:3 to a concentration of 5 mg/mL and subsequently resolved in HEPES buffer (20 mM HEPES/NaOH, 150 mM NaCl, pH 7.4). LUV formation was conducted as described. , Liposomes were extruded 21 times through 100 nm polycarbonate membranes at the LiposoFast-Basic extruder (AVESTIN, Mannheim, Germany). Vesicle sizes were analyzed using the nanoparticle tracking system NanoSight LM10 (Malvern, Herrenberg, Germany).…”

Electrophysiology of Epithelial Sodium Channel (ENaC) Embedded in Supported Lipid Bilayer Using a Single Nanopore Chip

“…Nanospot GmbH′ developed an array of nanopores utilizing reactive ion etching techniques to investigate fusion process and membrane transport . In another approach, an array of nanopores was fabricated in prepattern arranged silicon cavities. − The hybrid biological solid-state nanopore has tremendous applications to study sequencing of DNA, and bacteriophage phi29 connector channel fused in bilayer has revealed the dynamics of ds DNA translocation. , …”