Parallel and automated formation of lipid bilayers on microstructured chips
for ion channel and nanopore recordings
Ekaterina Zaitseva1, Sönke Petersen1, Juan Del Rio Martinez2, Ibrahim Halimeh2, Jan C. Behrends2 and Gerhard Baaken1
1Ionera Technologies, Freiburg, Germany, 2Institute of Physiology, University of Freiburg, Germany

Lipidbilayer

Introduction

Ionic current
MembraneproteinIndividually
addressed Picoliter
cavityAg/AgClelectrode
Glass substrate

SU-8 photoresist
Ti adhesion Au galvanic startlayer
layer

Idea

The microelectrode cavity array (MECA) Chip (Pikt. above) contains a 4 x 4 array of circular microcavities (MECs, diam. 10-50 µm) in ahighly inert polymer. Each MEC accommodates an individual integrated Ag/AgCl microelectrode. A bilayer roofing the electrolyte-filledcavity is automatically formed by remotely actuated painting from a lipid solution (Ionera-SPREAD). Membrane channel proteins, e.g. asingle α-Hemolysin nanopore, are reconstituted in the bilayer. Analytes e.g. PEG or DNA interacting with the pore can be detected via
resistive pulses. The MECA-chip has been validated with a number of different protein pores and ion channels including gramicidin,alamethicin, α-Hemolysin, OmpF, MspA, Aerolysin, KcsA etc.

3: Reconstitution and parallel recordings of ion channels and protein pores on the MECA

Bilayer recording is a well-established technique for in-depth studies ofbiophysical properties of ion channels and is particularly suited for functional
studies on proteins residing in intracellular membranes. Moreover, thistechnique supports a host of powerful emerging analytical methods which
employ biological nanopores as molecular sensors.
Despite its proven value, bilayer recording can be very frustrating due to thecapricious nature of lipid bilayers, which have to be formed manually one by one
and which often lack stability. We here show an approach allowing for rapid andautomated generation of planar arrays of lipid bilayers.
The present format allows for 16 parallel recordings, thereby enabling efficient
data generation, as well as the high resolution measurement from the single
selected bilayer. We here show the validation of the technique throughrecordings of a variety of channel proteins and nanopore-based assays, includingthe detection and characterization of polynucleotides and neutral polymer
mixtures.

1: Bilayer formation: Remotely actuated painting on
Microelectrode Cavity Arrays (MECA)

2: Electrical stability of automaticallypainted bilayers

1.) Drop of lipid solution

2.) Remotely controlled effector3.) Lipid bilayer on MECs
Bilayers from various phospholipids (DPhPC, POPC, POPE/ POPG) and solvents (octane ordecane) can be formed and reformed automatically on the MECA chip surface. For
longer chain solvents like hexadecene, the method can be adapted by pretreating theMECA chip surface with phospholipid in pentane.
Automatically painted bilayers are stable up to a Critical Voltage Amplitude (CVA) of atleast 300 mV and allow for reconstitution of membrane protein channels.

4: Parallel nanopore analytics on the MECA

The stability of bilayers generated in a parallel manner
was examined with voltage pulses of different lengthand different electrolyte concentrations Histograms ofbreakdown voltage related to time, n= 237, 1566, 256,
1570, 243 and 1578 lipid bilayers for 1, 5, 10, 20, 50 and
100 ms respectively. Cumulative curve for 150 mM, 500
mM and 3 M KCl.

5: High resolution Measurements: Single-molecule polymer sizing and DNA-detection with aHL

A: Self-insertion,soluble peptide antibiotika andbacterial toxins
e.g. Alameticin, Gramicidin,
α-Hemolysin, Aerolysin.

Screenshots of a recording windows of a typicalchannel forming activity assays: Gramicidin (above),Aerolysin (on the right)

Screenshot of the recording window showing simultaneous and parallel PEG
detection with single nanopores. Recordings are done only from bilayers
containing solitary, well-oriented, bona-fide αHL-nanopores.

The recording for gramicidin was done in symmetricalsolution of 100 mM HCl; and the recording for Aerolysin
was done in symmetrical solution of 1 M KCl, 10 mM
HEPES, pH 7.0.

B: Membrane Protein Pores reconstituted via detergent dilution
e.g. OmpF, MspA

C: Ion Channels reconstitution via proteoliposomes fusion.
KcsA potassium channel

Detection of single PEG and DNA molecules with a single nanopore. On the left: Superimposed PEG-induced blocking events of one single
pore with a RMS noise of ~ 2 pA @ 10 kHz. Event averaged histograms of residual conductances obtained from the parallel recording
shown before (P4). Note the congruent maxima corresponding to individual PEG species. On the right: Resistive pulses induced by DNA
blockages of a single HL-pore and the corresponding event averaged histogram lower panel recorded at DC-20 kHz.

A modular device for automated formation and parallel recording of bilayer arrays:
the Orbit-16
In order to facilitate use of automated bilayer array formation in the wider
community, Nanion Technologies, Munich, has incorporated all necessary elements
into one versatile device, called the Orbit-16. It allows for both parallel recording of
all 16 channels using a multichannel patch-clamp amplifier (Tecella, San Diego,USA) as well as high-resolution recordings from selected channels using a lownoise, high-bandwidth amplifier such as the EPC-10 (HEKA, Lambrecht, Germany)or the Axopatch (Axon Instruments/Molecular Devices, Sunnyvale, CA, USA).
In summary, the MECA chip in conjunction with the automated bilayer formation
as realized in the Orbit-16 promises to become a new generic tool enabling
faster, easier and more efficient data collection both in protein nanopore-basedanalytics and membrane protein research.

On the left: All-points histogram showing multiple insertions of OmpF on 7 MECs
On the right: Single-channel conductance of recombinant MspA porin. Data from one parallel recording.

References

Single channel currents with corresponding amplitude histograms of KcsA E71A recorded from 6 bilayers
in parallel with Orbit-16. Current traces were recorded under steady-state conditions at pH 4.0 in 150 mM
KCl symmetric solutions with membrane potential held at +150 mV.

G. Baaken, J. C. Behrends, Hochauflösende Einzelmolekülanalyse mit Nanoporen-Arrays. BIOspektrum, 2011, 17, 769-772
G. Baaken, N. Ankri, A.-K. Schuler, J. Rühe and J. C. Behrends, Nanopore-Based Single-Molecule Mass Spectrometry on a Lipid Membrane Microarray. ACS Nano, 2011, 5 (10), pp 8080–8088
G. Baaken, M. Sondermann, C. Schlemmer, J. Rühe, J.C. Behrends, Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents. Lab on a Chip 8 (6), 2008, 938-944

Acknowledgements:
Project Ionera is funded by the German Federal Ministry of Economy and Technology and the European Union (Exist: FK 03EFT9BW44