SOLVENT-FREE BILAYER LIPID DOME DEVICE
FOR CHANNEL PROTEIN RECORDINGS
T. Osaki,1,* R. Kawano,1 K. Kuribayashi-Shigetomi,2 H. Sasaki,1 and S. Takeuchi1,2
1
2
Kanagawa Academy of Science and Technology, JAPAN, and
Institute of Industrial Science, The University of Tokyo, JAPAN
ABSTRACT
This paper describes a device that enables to form solvent-free bilayer lipid membranes for electrical ion-channel recordings. We designed arrayed apertures surrounded by SU8 fences on an Al-coated polymer film for the matrix of the
bilayer. An electrospray method allows a solvent-free, dried lipid coating at Al-exposed surface of the film, and domeshaped bilayer membranes are formed by electroformation/gentle-hydration method. The SU8 fence plays a role positioning the membrane on top of the aperture, allowing electrical monitoring across the membrane. Since avoiding the
drawback of the organic solvent, we believe that this system will be very useful for channel protein study.
KEYWORDS: Solvent-free Lipid Bilayer, Electroformation, Electrospray Deposition, Electrical Recording Device
INTRODUCTION
Ion-channel protein is a membrane protein species that selectively carries ions across biological membranes in response to the external stimulations such as electrical polarities and/or specific substrates [1]. The functionalities of the
ion-channels are for instance signal transduction and neurotransmission, and malfunctions of the proteins cause various
disease. On this account, the ion-channel proteins have become the extremely important targets for drug discovery
whereas most of their details have not yet clarified likewise the other membrane proteins. The major problem is that
the ion-channels must be reconstituted in a lipid membrane to preserve the activity, and that prevented to produce a
functional ion-channel protein chip for longtime.
With continuous technical advances, artificial bilayer-membrane platforms have been finally developed by MEMS
fabrication techniques for electrophysiological analysis of ion-channel proteins. The platform, for instance, consists of
a microaperture on a plastic/semiconductor film that is to suspend a bilayer lipid membrane [2] or aqueous droplets that
are contacted and formed a membrane at the interface [3]. These devices have significant potential on data-throughput
and reproducibility in comparison to the common patchclamp technique using cultured cells. Yet, the bilayer
membranes formed at those devices are inevitable to contain organic solvents due to the formation process, and
one considers that the solvent remaining at the bilayer interface badly affects the reconstitution probability and the
activity of reconstituted proteins [1].
In this work, we focused on the use of solvent-free
giant liposomes to solve the problem [4]. We previously
succeeded in arraying liposome domes by patterning of
dried lipids on Au-electrodes [5]. Following the basic
idea, here we developed a solvent-free bilayer membrane
device for electrical recordings with the integration of
electrodes. The conceptual diagram is shown in Fig. 1:
the deposited lipid films inside a ring fence delaminate by
Fig. 1 Formation of dome-shaped, solvent-free bilayer
lipid membranes by electroformation (left to right line).
The fence surrounding the aperture secures the location of lipidic-dome growth.
978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
Fig. 2 (a) Fabrication process of a parylene-SU8 composite film and a birds-eye view photo of the film. (b)
Diagram of a electrospray deposition method for a lipid
coating. (c) Image of illuminated spray. (d) An example
of a deposited lipid film.
540
14th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
3 - 7 October 2010, Groningen, The Netherlands
Fig. 3 Schematic design of the solvent-free bilayer lipid
membrane device. There are two pairs of electrodes.
One is for electroformation (Al on the composite film
and ITO), and another is for electrical recordings
(Ag/AgCl).
Fig. 4 (a) The assembled device, consisting of 16
chambers for parallel experiments. (b) Enlarged image
of the upper chambers. (c) A composite film laminated
on a PDMS spacer, to be mounted. (electrosprayed lipid at the center.)
hydration, gradually grow and fuse one another, and finally form a dome-shaped bilayer lipid membrane. The use of
the hydration process is technically important in this work since the method does not leave any organic solvent within
the formed bilayer membrane. Another key is the positioning of the membrane. We newly designed the ring fence to
control the position of the membranes on top of the microapertures. The concept is based on restricting the membrane
formation, instead of patterning the lipid film previously reported [5]. The microaperture fabricated on the matrix film
allows electrical access to the backside of the formed membrane.
EXPERIMENTAL
Fig. 2a shows the fabrication process of the composite matrix film. Shortly, 5 to 10 μm of poly(chloro-p-xylylene)
(parylene) film was deposited on Si-wafer by a chemical vapor deposition method, and patterned microapertures with a
common UV-lithography process. Followed by a thin Al-coating, SU8 photoresist was spincoated and the aligned ring
fences were obtained by another lithography step. A microscopic image was shown in Fig. 2a (bottom). Lipid deposition was then performed by electrospray method (Fig. 2b, c). A lipid solution (2 mg/mL of DOPC mixed with 1 wt% of
a fluorescent lipid in chloroform / acetonitrile = 95 / 5) was filled in a glass capillary and sprayed to the composite film
(2 kV/cm applied, 1 min of spray). By the spray method, the lipid is mostly deposited at the conductive Al-surface but
not at the insulating parts (Fig. 2d). The solvent evaporates immediately after the deposition.
The composite film was assembled into PMMA frames with spacers and electrodes illustrated in Fig. 3. The pair of
Ag/AgCl electrodes located at both upper and bottom sides of the film is for the electrical measurements by a patchclamp amplifier after the lipid membrane formation. On the other hand, the ITO-glass and the Al-coating on the composite film are used for electroformation of the lipid membrane, connected to a function generator equipment. The
PMMA frames form chambers and a channel at the top and the bottom of the film, to fulfill aqueous solutions. After
assembling the device, the composite film surface was observed by an inverted confocal microscopy through the ITOglass. The device images are shown in Fig. 4.
RESULTS AND DISCUSSION
In this study, deionized water was filled at the upper chambers (15 μL) and the bottom channel (ca. 100 μL) for the
bilayer membrane formation. Fig. 5 shows confocal xy-plane images (left) and cross sectional xz-plane images (right)
of the formed lipid membranes (see the schematic illustrations in Fig. 5a). We found that the formation spontaneously
started with the injection of deionized water, and the gentle hydration process can form a giant lipidic-dome on top of
the ring fence as shown in Fig. 5b. In most cases, however, the bilayer dome does not become large enough to cover the
aperture, and the domes usually contain small liposomes.
We, therefore, examined the electroformation method instead. Applying an AC voltage of 0.5 to 1.0 Vp at 1 to 10
Hz between the ITO-glass and the Al on the composite film for several minutes, we observed the lipid membranes grew
inside the fences, fused each other, and successfully covered the aperture (Fig. 5c). Since the solvents prepared for the
electrospray evaporate quickly, the formed bilayer lipid domes would not contain such organic solvents. Although the
reproducibility of the dome formation was still not sufficient, the ring fence was able to control the position of the
domes, as we designed, and we believe that the system can be systematically optimized.
After the electroformation we characterized the resistance and the capacitance of the dome membranes. As illustrated in the inset of Fig. 6, the Ag/AgCl electrodes were connected to a patch-clamp amplifier, and the properties
were investigated. The averaged resistance was ca. 2 GΩ and the capacitance was ca. 20 pF, respectively (n=10), indicating the sealing for the electrical recordings was acceptable. Finally, we observed current increase that would correspond to the signal from transmembrane nanopores (α-hemolysin) by addition of the protein solution at the upper chamber (Fig. 6).
541
CONCLUSION
In this work, we developed a solvent-free bilayer lipid
membrane device that endured electrical measurements.
Formation of the solvent-free membranes was ensured
by the use of the electroformation method in combination
with the electrospray technique, while the electrical access
to the both side of the membrane was realized by correctly
locating the membrane formation due to the integration of
the fence structure. The results examined the electrical
properties of the device demonstrated the potential for the
electrical recordings of ion-channel proteins.
Awaiting integration of solution-exchange components,
we believe that the developed solvent-free system will be
applied to wide varieties of ion-channel study.
ACKNOWLEDGEMENTS
The authors acknowledge the technical support provided by Ms. Maiko Onuki, Ms. Utae Nose, and Ms. Yoshimi Komaki. This work was partly supported by JST
(Strategic International Cooperative Program), Japan.
REFERENCES
[1] C. Miller, Ion Channel Reconstitution, Plenum Press,
New York, 1986.
[2] T. Osaki, H. Suzuki, B. Le Pioufle and S. Takeuchi,
Multichannel Simultaneous Measurements of SingleMolecule Translocation in α-Hemolysin Nanopore
Array, Anal. Chem., vol. 81, pp. 9866-9870, (2009).
[3] K. Funakoshi, H. Suzuki and S. Takeuchi, Lipid
Bilayer Formation by Contacting Monolayers in a
Microfluidic Device for Membrane Protein Analysis,
Anal. Chem., vol. 78, pp. 8169-8174, (2006).
[4] S. Kresák, T. Hianik and R. L. C. Naumann, Giga-seal Solvent-free Bilayer Lipid Membranes: from
Single Nanopores to Nanopore Array, Soft Matter,
vol. 5, pp. 4021-4032, (2009).
[5] K. Kuribayashi and S. Takeuchi, Electroformation
of Solvent-Free Lipid Membranes over Microaperture
Array, Proc. MEMS 2008, pp. 296-299, (2008).
Fig. 5 Confocal images of xy-plane (left column) and
xz-plane (right column). (a) Explanatory illustration.
(b) A solvent-free bilayer lipid-membrane dome formed
by gentle hydration. (c) A dome formed by electroformation. 1 wt% of a fluorescent lipid is mixed. (Intensity
bars at the right.)
CONTACT
T Osaki, Bio Microsystems Project, Kanagawa Academy
of Science and Technology (KAST), 3-2-1 Sakado, Takatsu, Kawasaki-city 213-0012, Japan, Tel: +81-44-819-2037,
Fig. 6 Current-time trace across a solvent-free bilayer
Email: [email protected]
lipid membrane with nanopore protein (α-hemolysin).
Applied voltage: 100 mV. The aqueous solution of
both chamber is deionized water.
*
542