Alpha-hemolysin is secreted by Staphylococcus aureus, this toxin causes cell death by binding with the outer membrane,
Alpha-hemolysin forms a homo-heptameric β-barrel in biological membranes.
Hemolysins are thought to be responsible for many events in host cells. For example, iron may be a limiting factor in the growth of various pathogenic bacteria. Since free iron may generate damaging free radicals, free iron is typically maintained at low concentrations within the body. Red blood cells are rich in iron-containing heme. Lysis of these cells releases heme into the surroundings, allowing the bacteria to take up the free iron.
Data and Applications
Orbit 16 data and applications:
Data courtesy of Dr. Gerhard Baaken et. al., University of Freiburg / Ionera.
Event-averaged histograms (black) and overlaid current traces (blue) of parallel and simultaneous recordings on a MECA chip of monoPEG-28-mediated blockages of hemolysin nanopore(s). The current traces were recorded with a multichannel amplifier (Tecella Jet 16). Histograms were derived from the mean current levels of at least 2000 visits of blocked stated per cavity (20 kHz sample frequency).
Read the full paper. (Am. Chem. Soc Nano, 5(10), 8080-8088, 2011)
Data were kindly supplied by Prof. Fritz Simmel, Technical University of Munich, Munich, Germany.
Reconstituted Alpha hemolysin channels are constantly open at positive and negative membrane potentials. Gating is observed as a result of the passage of a single stranded DNA molecule through the pore. Recordings were performed on the Port-a-Patch.
Orbit 16 data and applications:
Data courtesy of Dr. Gerhard Baaken et.al, University of Freiburg / Ionera.
Current traces and histograms derived from recordings of αHL pores blocked by monoPEG-28 and polyPEG-1500 on an Ionera MECA chip (AxoPatch 200B, filter freq: 20kHz, digitized at 200 kHz).
Read the full paper: (Am. Chem. Soc Nano, 5(10), 8080-8088, 2011)
Automated formation of membranes from polyoxazoline based triblock popolymers. Screenshot of a recording of Alpha-Hemolysine in a polyoxazoline based triblock copolymer membrane on the Orbit 16.
Alpha-Hemolysin is capable of insertion into triblock copolymer membranes.
(A) Current-voltage relationship of Alpha-Hemolysin pore in Poly(2-methyloxazoline-b-dimethylsiloxane-b-2-methyloxazoline) membrane. Average of two channels. Conditions: 25 mM Tris, 4 M KCl, pH 8.0.
(B+C) Representative recordings of Alpha-Hemolysin with PEG-28 at 40 mV and -40 mM. Conditions: 25 mM Tris, 4 M KCl, pH 8.0. Note different time scale at positive (B) and negative (C) potentials.
Temperature control of the Orbit mini: PEG induced current blockages of a alpha-hemolysin pore
(A) Current traces recorded at 10°C and 40°C illustrating a strong increase of the open pore current as well as the blockage frequency at elevated temperature.
(B) Event averaged histograms of the residual current during blockages. The open pore current scales with the temperature as well as the dwell time of the blockages.
(C) Dependence of the open pore current on the temperature.
(D) Dependence of the frequency of blockages on the temperature.
Screenshot of the recording window showing simultaneous and parallel PEG detection with single aHL-nanopores. Channels 1-5,7,12-14 contain a single aHL-nanopore. Channels 10 and 11 have two and Channel 9 has three aHL-nanopores. In Channels 8 and 14 single aHL-nanopores are assembled as hexamer. Channels 6 and 16 are switched off.
Conditons: 3 M KCl, 20 mM TRIS, pH 8, +40 mV
2019 - A comparison of ion channel current blockades caused by individual poly(ethylene glycol) molecules and polyoxometalate nanoclusters
Orbit 16 publication in The European Physical Journal E (2019)
Wang H., Kasianowicz J.J., Robertson J.W.F., Poster D.L., Ettedgui J.
2018 - Size-dependent interaction of a 3-arm star poly(ethylene glycol) with two biological nanopores
Orbit 16 publication in The European Physical Journal E (2018)
Talarimoghari M., Baaken G., Hanselmann R., Behrends J.C.
2018 - Cell‐free production of pore forming toxins: Functional analysis of thermostable direct hemolysin from Vibrio parahaemolyticus
Orbit 16 publication in Engineering in Life Sciences (2018)
Dondapati S.K., Wüstenhagen D.A., Strauch E., Kubick S.
2016 - Probing driving forces in aerolysin and α-hemolysin biological nanopores: electrophoresis versus electroosmosis
Orbit 16 publication in Nanoscale (2016)
Boukhet M., Piguet F., Ouldali H., Pastoriza-Gallego M., Pelta J., Oukhaled A.
2015 - High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore
Orbit 16 and Vesicle Prep Pro publication in American Chemical Society Nano (2015)
Baaken G., Halimeh I., Bacri, Pelta J., Oukhaled A., Behrends J.C.
2015 - Automated Formation of Lipid Membrane Microarrays for Ionic Single-Molecule Sensing with Protein Nanopores
Orbit 16 publication in Small (2015)
Del Rio Martinez J.M., Zaitseva E., Petersen S., Baaken G., Behrends J.C.
2014 - Generation of chip based microelectrochemical cell arrays for long-term and high-resolution recording of ionic currents through ion channel proteins
Orbit 16 publication in Sensors and Actuators B: Chemical (2014)
Zheng T., Baaken G., Vellinger M., Behrends J.C., Rühe J.
Orbit 16 publication in Science (2012)
Langecker M., ArnautV., Martin T.G., ListJ., RennerS., Mayer M., Dietz H., Simmel F.C.
Port-a-Patch, Patchliner, SyncroPatch 96 ((a predecessor model of SyncroPatch 384PE) and Vesicle Prep Pro publication in Analytical and Bioanalytical Chemistry (2012)
Steller L., Kreir M., Salzer R.
Orbit 16 publication in Journal of the American Chemical Society Nano (2011)
Baaken G., Ankri N., Schuler A.K., Rühe J., Behrends C.
2008 - Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents
Orbit 16 publication in Lab on a Chip (2008)
Baaken G., Sondermann M., Schlemmer C., Rühe J., Behrends J.C.
Orbit 16 and Orbit Mini
This webinar covers the use of the lipid bilayer platforms from Nanion: the Orbit16 and the Orbit mini for characterization of membrane proteins like ion channels, bacterial porins and biological nanopores. Both bilayer systems support high quality low noise recordings, but differ in throughput capabilities and experimental features. The Orbit16, introduced in 2012 is a device for efficient formation of 16 lipid bilayers simultaneously, allowing for parallel bilayer-reconstitution of ion channels and nanopores.