Post on 04-Apr-2018
7/29/2019 Psi Lbr Electrophysiology
1/4
Paul Scherrer Institut, 5232 Villigen PSI, Schweiz
Telefon +41 56 310 21 11, Telefax +41 56 310 21 99
http://www.psi.ch
Printed from PSI home page
Electrophysiological methods and devicesfor the activity measurement of membrane proteins
Translocation of ions and charged molecules across biological membranes are sensitively measured using
electrophysiological instruments. In principle, an electrical potential is applied across a lipid bilayer membrane and, if
ion channels are open or membrane transporters active, the resulting currents are measured. Membrane resistance in
the Giga-Ohm range of lipid bilayers suspended in micro- or nanopores of the supporting material can be achieved, a
prerequisite for sensitive measurements. Using impedance spectroscopy, the sealing resistance and the total
capacitances of the bilayers and the supporting material are determined. As in patch-clamp measurements (see insert),
voltage pulses can be applied to planar lipid bilayers and opening of ion channels determined as intermediate currents
of some pico Ampres. To measure these low currents, measurements have to be carried out in a Faraday-cage which
shields the measurement cell from outside disturbances.
Instrumentation
Two instruments (Potentiostat Autolab, PG12) are available, equipped with an impedance and a low current
module. Voltage-clamp measurements are carried out using an Axopatch 200 B instrument and a Digidata
1440 A analyser inside a Faraday cage (Warner) .
Rotavap and extruders are used to generate liposomes of uniform sizes.
7/29/2019 Psi Lbr Electrophysiology
2/4
Nanopore arrays in a silicon nitride membrane
Suspended bilayer in a nanopore
Instrumental setup for ion channel recording
Transport of solvents in microfluidic systems is achieved by computer-controlled NEMESYS syringe devices.
Materials
Fabrication, derivatization and analysis of micro- and nanopore materials are carried out in collaboration with PSI
Laboratory for micro- and nanotechnolgy and external partners.
Two kinds of materials have been used for bioanalytical devices: silicon and polymers. Silicon technology requires clean
room laboratories including essentially photolithography or e-beam facilities for structure definition and etching processesfor structure generation. Polymers can be structured by hot embossing and molding techniques and surfaces can be
functionalized using chemical methods. Scanning electron microscopy, contact angle measurements and fluorescence
microscopy are methods to analyse surface properties.
Pore generation
In a 300 nm thin silicon nitride membrane on a silicon chip, pores of 200 nm to 25 micrometers have been made. For
ion channel recordings one pore is sufficient, whereas for transporter protein recordings arrays of pores are required.
Since the unitary translocation rate of transporters is about 1000 times lower than that of ion channels, a sufficient
surface density of transporter molecules is required.
The quality of bilayer sealing depends on the lipid composition, the bilayer formation method, the material and the
surface properties (roughness, contact angle). The capacitance mainly depends on the thickness of the supporting
material. If it is thinner than about 10 micrometers, the capacitance values can be hardly compensated to the low values
necessary for Voltage-clamp experiments.
[1]
7/29/2019 Psi Lbr Electrophysiology
3/4
Chip i ntegrated in a PDMS microfluidic system
(courtesy NTB)
Polymer-based microfluidic channel with a
micropore (courtesy FHNW, Eugen Mller)
Bilayer formation and protein integration
Microfluidic systems
Fluidic transport to the micrometer-sized pores is tricky. Solvents have to be transported, electrodes integrated and
membrane protein activities of interest measured. Presently emphasis is put on making prototype devices of polymers.
Methods
The integration of membrane proteins into lipid bilayer membranes suspended in micro- or nanopores is a critical step.
Three different strategies (A, B or C) have been evaluated. Pore size, lipid composition and the bilayer formation method,
influence the stability of the planar lipid proteobilayers. Electrophysiological methods are needed to monitor bilayer
formation and to determine membrane-sealing resistance and capacitance of the bilayers formed.
Measuring activities: current traces
Single voltage-gated ion channels are activated by changing the potential from about - 150 mV to + 150 mV. Individual
channels remain open for some ms and the opening and closing of channels can be recorded using the Axopatch
7/29/2019 Psi Lbr Electrophysiology
4/4
Current peaks of about 12 pA from single
KvAP ion channels reconstituted in a
DOPG/DOPE bilayer
Membrane proteins having electrogenic
activity
instrument.
Membrane proteins of interest
Using electrophysiological methods, membrane protein mediated translocation of ions across bilayers can be monitored.
Ion channels and transporters are important targets for drugs and thus highly relevant in drug discovery.
For more information Tiefenauer Group Publications and Publications LBR
URLs:
[1] : http://lmn.web.psi.ch/
[2] : http://www.psi.ch/lbr/tiefenauer-group-publications
[3] : http://www.psi.ch/lbr/publications
http://www.psi.ch/lbr/electrophysiology
[2] [3]