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Langmuir 2007, 23, 1354-1358
A Novel Method To Fabricate Patterned Bilayer Lipid Membranes Xiaojun Han,† Kevin Critchley,† Lixin Zhang,‡ Singh N. D. Pradeep,‡ Richard J. Bushby,‡ and Stephen D. Evans*,† School of Physics and Astronomy, and Self-Organising Molecular Systems (SOMS) Centre, UniVersity of Leeds, LS2 9JT, Leeds, United Kingdom ReceiVed September 8, 2006. In Final Form: October 19, 2006 We introduce a new method for forming tethered bilayer lipid membranes on surfaces patterned using a photocleavable self-assembled monolayer (SAM). A SAM terminated with a hydrophobic fluorocarbon residue was bound to a gold surface through a link containing a photocleavable ortho-nitrobenzyl moiety. Hydrophilic regions were produced by irradiation with soft UV (365 nm) through a photomask. The patterned surface was characterized by scanning electron microscopy and electrochemical impedance spectroscopy. Tethered bilayer lipid membranes with well-defined bilayer and monolayer regions were then formed by exposure to egg PC vesicles. The membranes had resistance and capacitance values of 0.52 MΩ‚cm2 and 0.83 µF‚cm-2, respectively.
Introduction Lipid bilayers are ubiquitous in living organisms and serve to separate the environment inside a cell from that outside. They control material transport, enable signal transduction, and are essential for energy transduction. Due to the complexity of biomembranes, there has been extensive interest in employing model biomembranes to mimic biological cell membranes, including liposomes,1 bilayer lipid membranes (BLMs),2 supported bilayer lipid membranes, and so on. Among them, membranes supported on solids have been studied extensively as model systems of biological membranes, as well as offering potential for technological applications,3-7 such as biosensors based on electrical and optical detection. Supported membranes can be broadly classified into three types according to the functional nature of the substrate. In the first type, nonmetallic substrates (e.g., SiO2,8 glass,9-13 poly(dimethylsiloxane),14 or mica15,16) present hydrophilic surfaces on to which vesicles have been shown to adsorb, fuse, and rupture to form supported bilayer lipid membranes (sBLMs). The second type is based on the modification of gold, or Si, surfaces using * Corresponding author. Tel: 0044 113 343 3852. Fax: 0044 113 343 3900. E-mail:
[email protected]. † School of Physics and Astronomy. ‡ SOMS Centre. (1) Semple, S. C.; Chonn, A.; Cullis, P. R. Biochemistry 1996, 35, 25212525. (2) Griffin, S. D. C.; Beales, L. P.; Clarke, D. S.; Worsfold, O.; Evans, S. D.; Jaeger, J.; Harris, M. P. G.; Rowlands, D. J. FEBS Lett. 2003, 535, 34-38. (3) Sackmann, E. Science 1996, 271, 43-48. (4) Boxer, S. G. Curr. Opin. Chem. Biol. 2000, 4, 704-709. (5) Tanaka, M.; Sackmann, E. Nature 2005, 437, 656-663. (6) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58-64. (7) Cornell, B. A.; BraachMaksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580-583. (8) Tero, R.; Urisu, T.; Okawara, H.; Nagayama, K. J. Vac. Sci. Technol. A 2005, 23, 751-754. (9) Cremer, P. S.; Groves, J. T.; Kung, L. A.; Boxer, S. G. Langmuir 1999, 15, 3893-3896. (10) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554-2559. (11) Groves, J. T.; Wulfing, C.; Boxer, S. G. Biophys. J. 1996, 71, 27162723. (12) Hovis, J. S.; Boxer, S. G. Langmuir 2001, 17, 3400-3405. (13) Kam, L.; Boxer, S. G. Langmuir 2003, 19, 1624-1631. (14) Lenz, P.; jo-Franklin, C. M.; Boxer, S. G. Langmuir 2004, 20, 1109211099. (15) Leonenko, Z.; Finot, E.; Cramb, D. Biochim. Biophys. Acta 2006, 1758, 487-492. (16) Wang, L.; Song, Y. H.; Han, X. J.; Zhang, B. L.; Wang, E. K. Chem. Phys. Lipids 2003, 123, 177-185.
single-component self-assembled monolayers (SAMs) to confer hydrophobicity to the surface. For example, Plant et al.17-19 modified gold surfaces with simple alkanethiol derivatives to create hydrophobic surfaces which when incubated with vesicles led to the adsorption of a lipid monolayer. These hybrid bilayer membranes (HBMs), in which only the outer leaflet of the lipid membrane is mimicked, could be used to investigate pore formation17 and peripheral membrane binding, but are not necessarily suitable for studying membranes with large transmembrane proteins. In the third approach, gold surfaces are coated with mixed SAMs comprised of thiol-linked lipids and shortchain hydrophilic spacers. It is envisaged that, at sufficiently high surface concentrations of thiol-linked lipids, vesicles will adsorb, rupture, and form a bilayer with the thiol-linked lipids inserted into the lower leaflet of the bilayer and thus tethering the bilayer to the solid support. These “tethered bilayer lipid membranes” (tBLMs) have been used extensively to investigate membrane proteins and processes such as redox activity20,21 and ion transport across the membrane,22,23 antibiotic binding,24 and other applications.25-33 In a variation of this method we have shown that, with use of microcontact printing (µCP), it is possible (17) Plant, A. L. Langmuir 1993, 9, 2764-2767. (18) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 11261133. (19) Plant, A. L.; Brighamburke, M.; Petrella, E. C.; Oshannessy, D. J. Anal. Biochem. 1995, 226, 342-348. (20) Jeuken, L. J. C.; Connell, S. D.; Henderson, P. J. F.; Gennis, R. B.; Evans, S. D.; Bushby, R. J. J. Am. Chem. Soc. 2006, 128, 1711-1716. (21) Naumann, R.; Schmidt, E. K.; Jonczyk, A.; Fendler, K.; Kadenbach, B.; Liebermann, T.; Offenhausser, A.; Knoll, W. Biosens. Bioelectron. 1999, 14, 651-662. (22) Naumann, R.; Baumgart, T.; Gra¨ber, P.; Jonczyk, A.; Offenha¨sser, A.; Knoll, W. Biosens. Bioelectron. 2002, 17, 25-34. (23) Naumann, R.; Walz, D.; Schiller, S. M.; Knoll, W. J. Electroanal. Chem. 2003, 550, 241-252. (24) Spencelayh, M. J.; Cheng, Y. L.; Bushby, R. J.; Bugg, T. D. H.; Li, J. J.; Henderson, P. L.F.; O’Reilly, J.; Evans, S. D. Angew. Chem., Int. Ed. 2006, 45, 2111-2116. (25) Jeuken, L. J. C.; Connell, S. D.; Nurnabi, M.; O’Reilly, J.; Henderson, P. J. F.; Evans, S. D.; Bushby, R. J. Langmuir 2005, 21, 1481-1488. (26) Naumann, R.; Schiller, S. M.; Giess, F.; Grohe, B.; Hartman, K. B.; Karcher, I.; Koper, I.; Lubben, J.; Vasilev, K.; Knoll, W. Langmuir 2003, 19, 5435-5443. (27) Sackmann, E. Mol. Biotechnol. 2000, 74, 135-136. (28) Schiller, S. M.; Naumann, R.; Lovejoy, K.; Kunz, H.; Knoll, W. Angew. Chem., Int. Ed. 2003, 42, 208-211. (29) Sevin-Landais, A.; Rigler, P.; Tzartos, S.; Hucho, F.; Hovius, R.; Vogel, H. Biophys. Chem. 2000, 85, 141-152. (30) Terrettaz, S.; Mayer, M.; Vogel, H. Langmuir 2003, 19, 5567-5569. (31) Terrettaz, S.; Vogel, H. MRS Bull. 2005, 30, 207-210.
10.1021/la062636v CCC: $37.00 © 2007 American Chemical Society Published on Web 12/05/2006
Fabrication of Patterned Bilayer Lipid Membranes
Langmuir, Vol. 23, No. 3, 2007 1355
to create patterned SAMs containing hydrophilic and hydrophobic patches and that a lipid monolayer is formed on the hydrophobic patches and a bilayer above the hydrophilic regions.34,35 Such patterning brings potential benefits for electrical measurements in bilayers (reducing noise due to defects in the lipid layer) and the creation of regions in which the fluid bilayer regions are separated from one another by the HBM regions.34,35 To date there have been a variety of approaches to patterned bilayer formation; Morigaki et al. have formed bilayers from diacetylene-containing lipids. Irradiation through a mask leads to polymerization of the lipids, in the unmasked regions, thereby creating corals containing the mobile nonpolymerized lipids. The lipids in the corals can be subsequently exchanged with other lipids.36,37 Boxer and co-workers have followed an alternative approach whereby the substrate itself is patterned prior to bilayer formation by using either patterned metal oxides or photoresist.38-40 Following the incubation of these surfaces with vesicles, sBLMs are formed in which individual bilayer patches are separated by the oxide or resist barriers. In this paper, we present an alternative approach for fabricating patterned bilayers in which the SAM, designed to support the lipid bilayer, is itself patterned by exposure to soft UV light. Initially, a hydrophobic SAM is formed using derivatives that contain a photocleavable moiety. Irradiation through a mask leads to the cleavage of the hydrophobic groups, in the exposed regions, and yields a series of hydrophilic patches. Upon incubation with vesicles, HBMs are formed on the hydrophobic regions of SAMs while sBLMs are formed on the hydrophilic patches. A principal advantage of this approach is the significant reduction in the number of steps required to create a patterned bilayer over the µCP, and alternatively, schemes outlined above. A secondary advantage comes from the choice of a semifluorinated organothiol derivative to create the hydrophobic surface. Previous work by Silin et al. has demonstrated that HBMs formed on semifluorinated SAMs show improved electrical characteristics over those formed on hydrocarbon SAMs due to an increased packing density in the adsorbed lipid layer.41
respectively) using an Edwards Auto 306 evaporator. Glass slides were cut into approximately 1.5 cm2 for electrochemical measurement. After evaporation the gold electrodes were immediately immersed into 0.5 mM DTFE DCM solution for 24 h. On removal from solution, the substrates were rinsed thoroughly with DCM and then dried with nitrogen. The electrode was assembled into a electrochemical cell with a gold electrode area of 0.07 cm2. Photopatterning of SAMs. Details of the method used to pattern the SAMs is described elsewhere.42,43 A 365 nm UV lamp (BlakRay model B 100 AP) with nominal power, at the sample, of 7 mW‚cm-2 was used to irradiate the samples through a chromium/ quartz mask for 1 h. During irradiation, the sample was in contact with the mask. After the UV exposure, samples were rinsed with DCM, followed by Milli-Q water, and finally dried under a stream of nitrogen. Vesicle Preparation. Small unilamellar vesicles (SUVs) were prepared by tip sonication. A chloroform solution containing 5 mg of Egg PC was dried under nitrogen in a small glass bottle overnight. The dried PC film was then hydrated in 1 mL of 0.1 M KCl by vortexing. The cloudy solution was tip-sonicated at 1000 Hz data.20,34,46 The quality of the tBLMs described here, as determined from their electrical characteristics, appear to be better than those formed on microcontact printed SAMs which had total resistance and capacitance values of ∼0.1 MΩ‚cm2 and 0.9-1.0 µF‚cm-2, respectively.34 This improvement arises for two reasons: first, because the µCP stamped monolayers had more defects, which would decrease the resistance and increase the capacitance of the SAM; second, because the fluorocarbon surface promotes the formation of a more densely packed lipid layer.41 The quality of these patterned bilayers could undoubtedly be improved further by (i) reducing the diameter of the hydrophilic patches,47 (ii) using template-stripped gold as the underlying substrate,48 and (iii) having greater control over the photocleavage reaction. With respect to point (i), we note that the fully irradiated SAMs (∼50% cleavage) did not promote bilayer formation but required the presence of the hydrophobic “nonirradiated” regions surrounding the hydrophilic patches. This observation is in agreement with previous AFM studies on vesicle interactions with mCP systems.47 With regard to point (ii), Knoll and co(46) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenhausser, A. Langmuir 1997, 13, 7085-7091. (47) Jenkins, A. T. A.; Bushby, R. J.; Evans, S. D.; Knoll, W.; Offenhausser, A.; Ogier, S. D. Langmuir 2002, 18, 3176-3180. (48) He, L. H.; Robertson, J. W. F.; Li, J.; Karcher, I.; Schiller, S. M.; Knoll, W.; Naumann, R. Langmuir 2005, 21, 11666-11672.
Han et al.
workers found a dramatic improvement in the membrane resistance and capacitance values on template-stripped gold surfaces with bilayers yielding values of 7.4 MΩ‚cm2 and 0.73 µF‚cm-2 for resistance and capacitance, respectively.48
Conclusions Self-assembled monolayers were formed using DTFE derivatives that contained photocleavable units on gold supports. These hydrophobic SAMs supported the formation of hybrid bilayers in which a lipid monolayer was adsorbed at the hydrophobic SAM/subphase interface. Irradiation of the SAMs with soft UV light (365 nm, 3600 s) cleaved the fluorocarbon chains of the DTFE molecules to yield a mixed surface containing carboxylic acid and fluorinated functional groups. The electrical properties of bilayers formed on these photopatterned SAMs show an improvement over those previously made in our group on patterned SAMs formed using microcontact printing. This improvement is in part due to the use of the fluorocarbon surface for the HBM regions, which in itself has fewer defects but also promotes higher packing density in the overlying lipid phase.41 This photopatterning approach represents a simple and versatile system for creating patterned bilayer lipid membranes, of arbitrary shape and size, and requires fewer steps than any of the alternative techniques currently available for producing patterned bilayers. Improvements in the photocleavage reaction would be desirable to improve the control over the density of functional groups in the “hydrophilic” patches. Acknowledgment. We thank EPSRC for financial support: GR/S87195/01 and EP/C006755/1. LA062636V
