2009 APL Poulos Lipid Bilayer EWOD

8/6/2019 2009 APL Poulos Lipid Bilayer EWOD

1/3

Electrowetting on dielectric-based microfluidics for integrated lipid bilayerformation and measurement

Jason L. Poulos,1 Wyatt C. Nelson,2 Tae-Joon Jeon,3 Chang-Jin CJ Kim,2,4 andJacob J. Schmidt1,4,a1Department of Bioengineering, University of California Los Angeles, Los Angeles, California 90095, USA

2Department of Mechanical and Aerospace Engineering, University of California Los Angeles,

Los Angeles, California 90095, USA3

Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea4California Nanosystems Institute (CNSI), University of California Los Angeles, Los Angeles,California, USA

Received 19 May 2009; accepted 3 June 2009; published online 8 July 2009

We present a microfluidic platform for the formation and electrical measurement of lipid bilayermembranes. Using electrowetting on dielectric EWOD, two or more aqueous droplets surroundedby a lipid-containing organic phase were manipulated into contact to form a lipid bilayer at theirinterface. Thin-film Ag/AgCl electrodes integrated into the device enabled electrical measurementof membrane formation and the incorporation of gramicidin channels of two bilayers in parallel. 2009 American Institute of Physics. DOI: 10.1063/1.3167283

Artificially reconstituted lipid bilayer membranes havebeen used to provide an easily manipulated and highly con-trollable environment for the study of ion channels at thesingle molecule level since their development over 40 yearsago.1 Measurements of ion channels in planar lipid bilayershave been pursued for a wide range of applications in bio-sensing, single molecule mass spectrometry, and DNAsequencing.26 Unfortunately, practical embodiments of ionchannel-based devices are limited by the shortcomings of thelipid bilayer scaffold containing the ion channel.

Initially, formation of reconstituted lipid bilayer mem-branes required the deposition of lipids or a lipid-containingsolution over an orifice in an insulating partition separating

two electrolyte reservoirs. The resultant bilayers have char-acteristically high resistance G and support the mea-surement of ion channels at the single molecule level. How-ever, their characteristic short lifetime and mechanicalinstability limit any technological applications. Bilayer for-mation over a mechanically stabilizing support such as asolid surface7 or a porous hydrogel8 have been shown tosignificantly extend bilayer lifetime. However, measurementof solid-supported bilayers is somewhat complicated by theirlimited volume on one side, preventing dc measurements.Although hydrogel-supported bilayers do not share thisshortcoming, they cannot withstand transport and must becreated on site by a skilled operator.912

Although a shippable lipid bilayer membrane platformhas been demonstrated,13 there is also interest in automatedon-demand bilayer formation because of potential for highlyintegrated compact devices with small sample volumes andcompletely electronic system control enabled by use of mi-croelectromechanical systems fabrication technologies. Anumber of microfluidic devices constructed have used thetraditional MuellerRudin bilayer formation method14,15 andrequired the creation and manipulation of a solvent boluswithin an aqueous-filled microfluidic channel to form a lipidbilayer, a process problematic to automate. Microfluidic

devices that lessen this burden have also been exploredrecently.16,17

Recently, a bilayer formation method based on mechani-cal union of self-assembled lipid monolayers has simplifiedthe process of bilayer formation within microfluidicdevices1719 leading to the possibility of extremely highthroughput.20,21 An application of this technique by Aghdaeiet al.

22 used dielectrophoresis DEP to drive bilayer forma-tion in a microfluidic device.

Electrowetting on dielectric EWOD is an alternativemicrofluidic driving mechanism by which samples are ma-nipulated solely via electrical signals.23 EWOD is exception-ally well suited for lab-on-chip applications because highly

concentrated electrolyte solutions can be manipulated with-out joule heating, which can limit the applicability of DEP.24

In EWOD-driven droplet motion, electric fields are appliedlocally across a hydrophobic-coated dielectric, increasing thewettability of selected regions of the droplet on the substrate,resulting in droplet motion from induced differential surfacetension. This method requires low power and fabrication issimple and scalable, making devices amenable to integrationwith myriad other on-chip transduction mechanisms.

Here we describe a device that combines the EWODdriving mechanism with on-chip thin-film electrodes for par-allel formation and measurement of artificial lipid bilayerarrays. Electrowetting is used to facilitate the contact of

separate aqueous droplets immersed within a lipid-containing alkane solution, resulting in functional lipid bi-layer membranes able to host ion channels. Furthermore, byintegrating fabrication of Ag/AgCl electrodes into the device,our EWOD chip allowed automatic and direct access todroplets for multiplexed electrical measurements. The con-tacting monolayer method on an EWOD chip with integratedAg/AgCl electrodes represents an attractive and scalableplatform that allows automated formation of lipid bilayersand simultaneous monitoring of ion channels in an array for-mat.

Devices were fabricated from a glass wafer coated with140 nm of indium tin oxide ITO Tech Gophers Corpora-

tion. The ITO layer was patterned and etched using standardaElectronic mail: [email protected].

APPLIED PHYSICS LETTERS 95, 013706 2009

0003-6951/2009/951 /013706/3/$25.00 2009 American Institute of Physics95, 013706-1

Downloaded 14 Jul 2009 to 128.97.83.241. Redistribution subject to AIP license or copyright; see http://apl.aip.org/apl/copyright.jsp
http://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1063/1.3167283


2/3

photolithography processes to create the underlying elec-trodes, enabling movement of droplets by EWOD.25 Afterpatterning the ITO, a 1-m-thick silicon nitride layer wasdeposited by plasma-enhanced chemical vapor deposition,insulating the ITO electrodes from the rest of the chip above.Next, 300 nm of silver was evaporated and patterned bylift-off to define the electrodes for measurement of mem-brane and ion channel activity. Finally, 200 nm of CytopAsahi Glass Co. was spun onto the wafer and patternedusing oxygen plasma etching, exposing regions of the silver

electrodes. Figure 1 shows a top view of the final device; seeEPAPS supplementary material in Ref. 26 for a schematic ofthe process flow.

Prior to use of the device, 0.5 l droplets of standardbleach were placed on the exposed silver electrodes for ap-proximately 30 s to create a Ag/AgCl electrode. The devicewas then rinsed with deionized water and blown dry. Next,2 5 l aqueous droplets 1M KCl, 10 mM Tris-HCl, 1 mMEDTA, pH 8.0 were placed on the exposed Ag/AgCl elec-trodes. A Cytop-coated ITO glass plate was then placed ontop of the droplets using double-sided tape as spacers suchthat the gap between the upper and lower hydrophobiccoated plates was 400 m.

The droplets were moved over the Ag/AgCl electrodesby activating the ITO EWOD electrodes with 30 to 60 V rmsat 1 kHz.27 A solution of 5% 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine Avanti Polar Lipids in n-decane MPBiomedicals, a standard formulation for conventionallyformed MuellerRudin lipid bilayers, was then pipetted intothe side of the device where it easily wetted the Cytopsurface and filled the enclosed volume. To prevent the aque-ous droplets from merging during the addition of the organicsolvent, the ITO EWOD electrodes were actuated, which im-mobilized the droplets. For experiments in which ion channelincorporation was measured, gramicidin A gA, Sigma wasdissolved into the organic phase to a final concentration of

100 ng/ml. The number of ion channels incorporated into thebilayer could be roughly controlled by adjusting this concen-tration.

After the initial placement of the aqueous droplets andinjection of the organic phase, a monolayer of lipid mol-ecules begins to self-assemble at the aqueous-organicinterface.18 This is a time-dependent process; therefore, be-fore final droplet contact was made the droplets were entirelysurrounded by the organic solvent lipid mixture for 5 min. Ifthe droplets were brought together without taking this self-assembly time, the droplets fused and no bilayer was formed.

To form a bilayer, the ITO EWOD electrodes were acti-vated to move the droplets toward each other at a speed1 mm/s Figs. 1c1e. During this movement, each

water droplet was deformed in the direction of movement,flattening the leading interface. When the electrodes weredeactivated, the droplet interface relaxed into a circularshape. The movement and subsequent relaxation were se-quentially employed to move the droplets together and intocontact, forming lipid bilayers. Specifically, the dropletswere positioned so that the relaxation of the droplet inter-faces caused the two monolayers to come into contact, whileeach droplet volume was in contact with a separate indi-vidual Ag/AgCl electrode. The device could be used in sev-eral different configurations to form multiple individualmembranes simultaneously Fig. 1b. Devices could becleaned for reuse by soaking and rinsing in acetone, metha-

nol, isopropanol, and deionized water and baking at 200 Covernight.The bilayer resistance and capacitance were measured,

as well as the conductance of any ion channel incorporatedinto the bilayer, using Axopatch 200B and DigiData 1322AAxon Instruments connected to the Ag/AgCl electrodes.Membrane formation was monitored by measuring the ca-pacitive current flowing in response to an applied 20 mVpeak-to-peak 8 Hz triangle wave. The constant backgroundcapacitance was measured while the droplets were separatedin the initial placement of the aqueous droplets on the deviceFig. 2a. Compared to traditional bilayer platforms, thebackground capacitance was large, a result of the device de-

sign and electrode geometry.When the droplets were moved into contact, the capaci-

tance increased and eventually stabilized at a larger value

FIG. 1. Color online Device structure and droplet movement usingEWOD. The device contains three pairs of Ag/AgCl electrodes black areaswith underlying ITO electrodes dark gray areas, with the rightmost ITOelectrodes outlined in dotted lines for droplet movement a. In b, aqueousdroplets surrounded by the lipid-containing organic phase are being posi-tioned prior to an experiment. A bilayer has formed on the left, while theright droplets are still being moved into position. Activation of the ITOelectrodes causes aqueous droplet movement cd. After droplet posi-tioning, the electrodes are deactivated and the interfaces of the aqueousdroplets relax, forming a bilayer e. For scale, the length of the square endsof each Ag/AgCl electrode is 300 m.

FIG. 2. Electrical measurements of membrane formation and ion channelincorporation. With the initially large droplet separation, the measured back-ground capacitance proportional to the amplitude of the measured wave-form is large a, but less than the total capacitance measured upon bilayerformation b. c Gramicidin incorporation was also measured in two bi-layers simultaneously 70 mV applied potential.

013706-2 Poulos et al. Appl. Phys. Lett. 95, 013706 2009



3/3

Fig. 2b. The bilayer contribution to this total capacitancewas determined by subtracting the background from the ca-pacitance measured when the droplets were in contact. Thecapacitance of the bilayer in Fig. 1b was determined in thisway to be 650 pF. The in-plane dimension of the bilayer inFig. 1b was visually estimated to be 450 m; with theaforementioned vertical gap of 400 m, a bilayer area of0.18 mm2 would result. This area, with the measured bi-

layer capacitance, yields a membrane specific capacitance ofapproximately 0.36 F /cm2, consistent with literaturevalues.28,29 After bilayer formation, the measured resistanceswere greater than 2 G.

We verified bilayer formation and functionality by mea-suring the incorporation of the pore forming peptide grami-cidin A. Gramicidin A forms a monolayer-spanning monova-lent cation-selective channel with a pore diameter of4 .30 Gramicidin can be incorporated into artificial bilay-ers by adding it to the aqueous or organic phase, as it issoluble in both. Here, we add it to the organic phase becausebilayer formation is immediately indicated by gA channelmeasurement. Measurements of incorporated gA were ob-

tained for several hours, limited by the bilayer lifetime. Al-though we did not extensively explore bilayer lifetimes inthis study, those we observed ranged from 2 to 12 h, compa-rable to those in previous microfluidic devices.14,31

We also explored the simultaneous formation and mea-surement of two bilayers in a single device Fig. 1b. Wemeasured the bilayers with a custom-built electronic packagethat automatically switched between each channel at speci-fied time intervals. This was accomplished using 2 SPDTMicromini switches RadioShack controlled by an ArduinoDiecimila microcontroller Arduino Inc.. The switches re-peatedly connected and disconnected each of the two bilay-ers to the Axopatch input and output. After bilayer formation,

the characteristic dimer formation and dissociation of gAwas seen in the measurement of each bilayer Fig. 2c. Theuse of additional amplifiers would allow for simultaneousmeasurement of a larger number of bilayers.14

The process of lipid bilayer formation through contact-ing monolayers is more controllable than conventional arti-ficial bilayer formation techniques because the removal ofthe solvent between the monolayers preceding bilayer forma-tion is directly achieved mechanically and, as a result, is wellsuited for use in microfluidic devices. When the aqueousphases are droplets very small sample volumes are obtain-able, enabling a high degree of miniaturization and parallel-ization with minimal reagent needs. EWOD is ideally suited

to manipulate these droplets requiring almost no fluidic han-dling apparatus. An EWOD device with integrated Ag/AgClelectrodes enables measurement of lipid bilayer formationand incorporation of ion channels in an extremely compactdevice without requirement of an operator. We have takenthe first steps toward such a system with the device presentedhere, capable of parallel creation and measurement of twolipid bilayers and incorporated ion channels. Scaling of this

device is straightforward and future work will be directedtoward application of this platform to ion channel-based bio-sensing.

Wyatt Nelson and Jason Poulos contributed equally tothis work. This work was supported by NSF IntegrativeGraduate Education and Research Traineeship IGERTthrough the UCLA Materials Creation Training Program

MCTP and NSF CAREER award Grant No. 0644442.1P. Mueller, D. O. Rudin, H. T. Tien, and W. C. Wescott, Nature 194, 9791962.

2H. Bayley, Curr. Opin. Chem. Biol. 10, 628 2006.3R. Capone, S. Blake, M. R. Restrepo, J. Yang, and M. Mayer, J. Am.Chem. Soc. 129, 9737 2007.

4L.-Q. Gu, O. Braha, S. Conlan, S. Cheley, and H. Bayley, Nature Lon-don 398, 686 1999.

5V. A. Karginov, E. M. Nestorovich, M. Moayeri, S. H. Leppla, and S. M.Bezrukov, Proc. Natl. Acad. Sci. U.S.A. 102, 15075 2005.

6J. W. Robertson, C. G. Rodrigues, V. M. Stanford, K. A. Rubinson, O. V.Krasilnikov, and J. J. Kasianowicz, Proc. Natl. Acad. Sci. U.S.A. 104,8207 2007.

7I. Koper, Mol. Biosyst. 3, 651 2007.8

R. F. Costello, I. R. Peterson, J. Heptinstall, and D. J. Walton, Biosens.Bioelectron. 14, 265 1999.9T. J. Jeon, N. Malmstadt, J. L. Poulos, and J. J. Schmidt, BioInterphases 3,Fa96 2008.

10T. J. Jeon, N. Malmstadt, and J. J. Schmidt, J. Am. Chem. Soc. 128, 422006.

11X. F. Kang, S. Cheley, A. C. Rice-Ficht, and H. Bayley, J. Am. Chem.Soc. 129, 4701 2007.

12J. W. Shim and L. Q. Gu, Anal. Chem. 79, 2207 2007.13T. J. Jeon, J. L. Poulos, and J. J. Schmidt, Lab Chip 8, 1742 2008.14B. Le Pioufle, H. Suzuki, K. V. Tabata, H. Noji, and S. Takeuchi, Anal.

Chem. 80, 328 2008.15M. Zagnoni, M. E. Sandison, and H. Morgan, Biosens. Bioelectron. 24,

1235 2009.16N. Malmstadt, M. A. Nash, R. F. Purnell, and J. J. Schmidt, Nano Lett. 6,

1961 2006.17K. Funakoshi, H. Suzuki, and S. Takeuchi, Anal. Chem. 78, 8169 2006.18L. M. Tsofina, E. A. Liberman, and A. V. Babakov, Nature 212, 681

1966.19M. A. Holden, D. Needham, and H. Bayley, J. Am. Chem. Soc. 129, 8650

2007.20A. J. Heron, J. R. Thompson, A. E. Mason, and M. I. Wallace, J. Am.

Chem. Soc. 129, 16042 2007.21J. L. Poulos, T. J. Jeon, R. Damoiseaux, E. J. Gillespie, K. A. Bradley, and

J. J. Schmidt, Biosens. Bioelectron. 24, 1806 2009.22S. Aghdaei, M. E. Sandison, M. Zagnoni, N. G. Green, and H. Morgan,

Lab Chip 8, 1617 2008.23J. Lee, H. Moon, J. Fowler, T. Schoellhammer, and C.-J. Kim, Sens.

Actuators, A 95, 259 2002.24T. B. Jones, K. L. Wang, and D. J. Yao, Langmuir 20, 2813 2004.25H.-W. Lu, F. Bottausci, J. D. Fowler, A. L. Bertozzi, C. Meinhart, and

C.-J. Kim, Lab Chip 8, 456 2008.26See EPAPS supplementary material at http://dx.doi.org/10.1063/

1.3167283 for a schematic of the process flow.27U.-C. Yi and C.-J. Kim, J. Micromech. Microeng. 16, 2053 2006.28T. Hanai, D. A. Haydon, and J. Taylor, J. Theor. Biol. 9, 422 1965.29K. Janko and R. Benz, Biochim. Biophys. Acta 470, 8 1977.30D. W. Urry, M. C. Goodall, J. D. Glickson, and D. F. Mayers, Proc. Natl.

Acad. Sci. U.S.A. 68, 1907 1971.31M. E. Sandison, M. Zagnoni, and H. Morgan, Langmuir 23, 8277 2007.

013706-3 Poulos et al. Appl. Phys. Lett. 95, 013706 2009

http://dx.doi.org/10.1038/194979a0http://dx.doi.org/10.1016/j.cbpa.2006.10.040http://dx.doi.org/10.1021/ja0711819http://dx.doi.org/10.1021/ja0711819http://dx.doi.org/10.1038/19491http://dx.doi.org/10.1038/19491http://dx.doi.org/10.1038/19491http://dx.doi.org/10.1038/19491http://dx.doi.org/10.1038/19491http://dx.doi.org/10.1073/pnas.0507488102http://dx.doi.org/10.1073/pnas.0611085104http://dx.doi.org/10.1039/b707168jhttp://dx.doi.org/10.1016/S0956-5663(99)00003-2http://dx.doi.org/10.1016/S0956-5663(99)00003-2http://dx.doi.org/10.1116/1.2948314http://dx.doi.org/10.1021/ja056901vhttp://dx.doi.org/10.1021/ja068654ghttp://dx.doi.org/10.1021/ja068654ghttp://dx.doi.org/10.1021/ac0614285http://dx.doi.org/10.1039/b807932chttp://dx.doi.org/10.1021/ac7016635http://dx.doi.org/10.1021/ac7016635http://dx.doi.org/10.1016/j.bios.2008.07.022http://dx.doi.org/10.1021/nl0611034http://dx.doi.org/10.1021/ac0613479http://dx.doi.org/10.1038/212681a0http://dx.doi.org/10.1021/ja072292ahttp://dx.doi.org/10.1021/ja075715hhttp://dx.doi.org/10.1021/ja075715hhttp://dx.doi.org/10.1016/j.bios.2008.08.041http://dx.doi.org/10.1039/b807374khttp://dx.doi.org/10.1016/S0924-4247(01)00734-8http://dx.doi.org/10.1016/S0924-4247(01)00734-8http://dx.doi.org/10.1021/la035982ahttp://dx.doi.org/10.1039/b717141bhttp://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1088/0960-1317/16/10/018http://dx.doi.org/10.1016/0022-5193(65)90041-Xhttp://dx.doi.org/10.1016/0005-2736(77)90057-8http://dx.doi.org/10.1073/pnas.68.8.1907http://dx.doi.org/10.1073/pnas.68.8.1907http://dx.doi.org/10.1021/la7007528http://dx.doi.org/10.1021/la7007528http://dx.doi.org/10.1073/pnas.68.8.1907http://dx.doi.org/10.1073/pnas.68.8.1907http://dx.doi.org/10.1016/0005-2736(77)90057-8http://dx.doi.org/10.1016/0022-5193(65)90041-Xhttp://dx.doi.org/10.1088/0960-1317/16/10/018http://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1063/1.3167283http://dx.doi.org/10.1039/b717141bhttp://dx.doi.org/10.1021/la035982ahttp://dx.doi.org/10.1016/S0924-4247(01)00734-8http://dx.doi.org/10.1016/S0924-4247(01)00734-8http://dx.doi.org/10.1039/b807374khttp://dx.doi.org/10.1016/j.bios.2008.08.041http://dx.doi.org/10.1021/ja075715hhttp://dx.doi.org/10.1021/ja075715hhttp://dx.doi.org/10.1021/ja072292ahttp://dx.doi.org/10.1038/212681a0http://dx.doi.org/10.1021/ac0613479http://dx.doi.org/10.1021/nl0611034http://dx.doi.org/10.1016/j.bios.2008.07.022http://dx.doi.org/10.1021/ac7016635http://dx.doi.org/10.1021/ac7016635http://dx.doi.org/10.1039/b807932chttp://dx.doi.org/10.1021/ac0614285http://dx.doi.org/10.1021/ja068654ghttp://dx.doi.org/10.1021/ja068654ghttp://dx.doi.org/10.1021/ja056901vhttp://dx.doi.org/10.1116/1.2948314http://dx.doi.org/10.1016/S0956-5663(99)00003-2http://dx.doi.org/10.1016/S0956-5663(99)00003-2http://dx.doi.org/10.1039/b707168jhttp://dx.doi.org/10.1073/pnas.0611085104http://dx.doi.org/10.1073/pnas.0507488102http://dx.doi.org/10.1038/19491http://dx.doi.org/10.1038/19491http://dx.doi.org/10.1021/ja0711819http://dx.doi.org/10.1021/ja0711819http://dx.doi.org/10.1016/j.cbpa.2006.10.040http://dx.doi.org/10.1038/194979a0

Documents

2009 APL Poulos Lipid Bilayer EWOD