Urban M.,Goethe University Frankfurt |
Kleefen A.,Goethe University Frankfurt |
Mukherjee N.,University of Groningen |
Seelheim P.,Nanospot GmbH |
And 4 more authors.
Nano Letters | Year: 2014
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. © 2014 American Chemical Society.
Kolesinska B.,ETH Zurich |
Podwysocka D.J.,ETH Zurich |
Rueping M.A.,ETH Zurich |
Seebach D.,ETH Zurich |
And 8 more authors.
Chemistry and Biodiversity | Year: 2013
After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β3-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β2h- and oligo-β3h-prolines without and with fluorescence labels (1-8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2-4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6-8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9-11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption. Copyright © 2013 Verlag Helvetica Chimica Acta AG, Zürich.
Urban M.,Goethe University Frankfurt |
Vor Der Bruggen M.,Nanospot GmbH |
Tampe R.,Goethe University Frankfurt
Journal of Visualized Experiments | Year: 2016
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 proteinharboring 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 SiO2surfaces, but further functionalization using gold-coated chip surfaces is also possible. © 2016 Journal of Visualized Experiments.
Nanospot Gmbh | Date: 2011-03-10
A microstructured measurement chip (1) for optical measurement of properties of artificial or biological membranes (40), having a lower, translucent support layer (10) and at least one non-translucent main layer (20) disposed on top of the former, which layer has depressions (30) configured as measurement chambers, having an upper opening (25) and one or multiple inner side walls (26). In order to improve the measurement chip (1) in such a manner that biological systems can be measured with greater measurement accuracy and higher throughput, it is proposed that the side wall or the side walls (26) of the measurement chambers (30) have depressions (27) and/or elevations (28). The invention furthermore relates to a holder (200) for the measurement chips (1) as well as to a method for the production of the measurement chips (1) from a silicon wafer (300).