pubs.acs.org/Langmuir © 2009 American Chemical Society
Free-Standing Lipid Bilayers in Silicon Chips-Membrane Stabilization Based on Microfabricated Apertures with a Nanometer-Scale Smoothness Ayumi Hirano-Iwata,*,†,‡ Kouji Aoto,§ Azusa Oshima,† Tasuku Taira,§ Ryo-taro Yamaguchi,§ Yasuo Kimura,§ and Michio Niwano†,§ †
Graduate School of Biomedical Engineering, Tohoku University, 6-6 Aoba, Aramaki, Aoba-ku, Sendai, 980-8579, Japan, ‡PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama, 332-0012, Japan, and §Laboratory for Nanoelectronics and Spintronics, Research Institute of Electrical Communication, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan Received July 11, 2009. Revised Manuscript Received August 29, 2009 In the present study, we propose a method for preparing stable free-standing bilayer lipid membranes (BLMs). The BLMs were prepared in a microfabricated aperture with a smoothly tapered edge, which was prepared in a nanometerthick Si3N4 septum by the wet etching method. Owing to this structure, the stress on lipid bilayers at the contact with the septum was minimized, leading to remarkable membrane stability. The BLMs were not broken by applying a constant voltage of (1 V. The membrane lifetime was 15-45 h with and without an incorporated gramicidin channel. Gramicidin single-channel currents were recorded from the same BLM preparation when the aqueous solutions surrounding the BLM were repeatedly exchanged, demonstrating the tolerance of the present BLM to repetitive solution exchanges. Such stable membranes enable analysis of channel functions under various solution conditions from the same BLM, which will open up a variety of applications including a high throughput drug screening for ion channels.
Introduction Reconstitution of ion channel proteins in free-standing bilayer lipid membranes (BLMs) provides an excellent system for understanding the physiological and pharmaceutical functions of the ion channels under chemically controlled conditions.1 Since ion channels are main drug targets, the BLM reconstitution system has attracted attention for drug discovery and protein/ligand screenings. In addition, the high affinities of channels to their specific ligands enable us to design an exquisite biosensor based on ion channel proteins.2,3 However, mechanical instability of BLMs hinders the widespread application of BLM systems.3 Extensive studies have been made to improve the stability of free-standing BLMs by preparing BLMs in microfabricated devices4-9 or supporting BLMs with hydrogels.10-12 These efforts lead to prolonged membrane lifetimes of several tens of *Corresponding author. Telephone: þ81-22-795-4865; fax: þ81-22-2175503; e-mail address:
[email protected].
(1) Miller, C. Ion Channel Reconstitution; Plenum Press: New York, 1986. (2) Sugawara, M.; Hirano, A.; B€uhlmann, P.; Umezawa, Y. Bull. Chem. Soc. Jpn. 2002, 75, 187–201. (3) Hirano-Iwata, A.; Niwano, M.; Sugawara, M. Trends Anal. Chem. 2008, 27, 512–520. (4) Schmidt, C.; Mayer, M.; Vogel, H. Angew. Chem., Int. Ed. 2000, 39, 3137– 3140. (5) Pantoja, R.; Sigg, D.; Blunck, R.; Bezanilla, F.; Heath, J. R. Biophys. J. 2001, 81, 2389–2394. (6) Suzuki, H.; Tabata, K. V.; Noji, H.; Takeuchi, S. Langmuir 2006, 22, 1937– 1942. (7) Pioufle, B. L.; Suzuki, H.; Tabata, K. V.; Noji, H.; Takeuchi, S. Anal. Chem. 2008, 80, 328–332. (8) Maurer, J. A.; White, V. E.; Dougherty, D. A.; Nadeau, J. L. Biosens. Bioelectron. 2007, 22, 2577–2584. (9) White, R. J.; Ervin, E. N.; Yang, T.; Chen, X.; Daniel, S.; Cremer, P. S.; White, H. S. J. Am. Chem. Soc. 2007, 129, 11766–11775. (10) Costello, R. F.; Peterson, I. R.; Heptinstall, J.; Byrne, N. G.; Miller, L. S. Adv. Mater. Opt. Electron. 1998, 8, 47–52. (11) Ide, T.; Ichikawa, T. Biosens. Bioelectron. 2005, 21, 672–677. (12) Shim, J. W.; Gu, L. Q. Anal. Chem. 2007, 79, 2207–2213. (13) Han, X.; Studer, A.; Sehr, H.; Geissb€uhler, I.; Berardino, M. D.; Winkler, F. K.; Tiefenauer, L. X. Adv. Mater. 2007, 19, 4466–4470. (14) Liu, B.; Rieck, D.; Wie, B. J. V.; Cheng, G. J.; Moffett, D. F.; Kidwell, D. A. Biosens. Bioelectron. 2009, 24, 1843–1849.
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hours.13,14 However, patch-clamped membranes with a lifetime of several hours are still more widely used in physiology fields than the BLM reconstitution systems. This is partly because drugs acting on ion channels in the patch-clamped membranes can be easily applied and removed by solution exchanges, which is a requisite for screening drug actions. Therefore, one of the most desirable goals of membrane stability for the free-standing BLMs is tolerance to solution exchanges while allowing channel-current recordings, if possible at the single-channel level, rather than just the improvement of membrane durability. Traditional free-standing BLMs are formed across apertures in Teflon or other plastic septa, by painting the apertures with lipid dissolved in organic solvent (usually n-decane).1 The solvent spontaneously drains toward the septum, finally forming a BLM surrounded by a thick annulus. On the basis of variational calculus, White demonstrated that the shape of the annulus plays a crucial role in the physiological characteristics of the BLMs.15 He proposed that the annulus-septum contact angle should be small for a stable arrangement. To reduce this angle, Wie et al. prepared septa having micrometer-scaled apertures with tapered sidewalls using polyimide16 or photoresist SU-8.14 BLMs formed in these apertures showed improved membrane stability with a lifetime of 50 h. However, they prepared BLMs from n-decane lipid solutions. It is often criticized that with membranes prepared using such unvolatile solvents, some amount of the solvent remains in the central hydrophobic area of the BLMs,17 which is likely to denature ion channel proteins. It is desirable to minimize organic solvent in membranes, although reduction of organic solvent may cause less stable BLMs than solvent-containing BLMs.18 (15) White, S. H. Biophys. J. 1972, 12, 432–445. (16) Eray, M.; Dogan, N. S.; Liu, L.; Koch, A. R.; Moffett, D. F.; Silber, M.; Wie, B. J. V. Biosens. Bioelectron. 1994, 9, 343–351. (17) Benz, R.; Fr€ohlich, O.; L€auger, P.; Montal, M. Biochim. Biophys. Acta 1975, 394, 323–334. (18) Batishchev, O. V.; Indenbom, A. V. Bioelectrochemistry 2008, 74, 22–25.
Published on Web 10/02/2009
DOI: 10.1021/la902522j
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Another classic method for BLM formation is folding up lipid monolayers spread at the air/water interface (folding method), which was introduced by Takagi et al.19 and later perfected by Montal and Mueller.20 Although this method still needs solvents to precoat the aperture edges, the amount of the solvents in BLMs by this method is much less than the solvent-containing BLMs.17 Ion channels that are susceptible to damages, such as glutamate receptor channels, have been reconstituted with their channel activities in reduced-solvent BLMs by the folding method21-23 as well as solvent-free BLMs.24-26 These reports strongly demonstrate the functionality of the reduced-solvent BLMs as a matrix for ion channel proteins. In the present study, we propose a method for preparing reduced-solvent BLMs that are stable enough for repetitive solution exchanges and allow single-channel recordings. The BLMs were prepared across a microfabricated aperture whose edge was thinned with a nanometer-scale smoothness. The aperture was prepared in a thin Si3N4 septum on a Si substrate (Figure 1a). By using the isotropic etching of Si3N4, the edge of the aperture was smoothly tapered in a nanometer-scale. Owing to the tapered structure of the aperture edge, the stress on lipid bilayers at the contact with the Si3N4 septum was minimized (Figure 1b), leading to membrane stability with resistance to repetitive solution exchanges. Gramicidin single-channel currents in different solution conditions were recorded from the same BLM preparation. Although solvent-containing BLMs have been considered more stable and often coupled with microfabricated devices,5-7,9,11 we showed a different approach to provide mechanical stability for reduced-solvent and more physiologically relevant BLMs. Such stable BLMs will open up a variety of applications including high-throughput analysis of ion-channel proteins.
Figure 1. (a) Schematic of a silicon chip fabricated in the present study. Blue lines represent SiO2 layers deposited on the Si3N4 and Si layers. (b) Schematic of a BLM formed across an aperture in the silicon chip.
Experimental Section Materials. L-R-Phosphatidylcholine (PC, chloroform solution) and L-R-phosphatidylethanolamine (PE, chloroform solution) were purchased from Avanti Polar Lipids, Inc. Cholesterol (Chol) was obtained from Wako Pure Chemicals Co. and recrystallized three times from methanol. Gramicidin was obtained from Sigma Chemical Co. A stock solution of gramicidin was prepared at 1 mg/mL in methanol and diluted 1000-fold with Kþ buffer (vide infra) just before use. Fabrication. Apertures with a diameter of 20-30 μm (Figure 1a) were fabricated in a FZ Si (100) wafer (>9000 Ω cm, 200 μm in thickness), one side of which was coated with a 240 nm thick Si3N4 layer (Semitec). Figure 2 shows the procedure for the fabrication of the Si chip having the apertures. The wafer was first thermally oxidized, and then the Si3N4 side was coated with a SiO2 layer using the sputtering method. The former oxide layer was photolithographically patterned, followed by anisotropic etching in 25% tetramethylammonium hydroxide at 90 °C. Then a SiO2 layer was deposited on the bare Si3N4 surfaces formed by the anisotropic etching. After photolithographic patterning, circular holes were fabricated in the Si3N4 layer by isotropic (19) Takagi, M.; Azuma, K.; Kishimoto, U. Annu. Rep. Biol. Works Fac. Sci. Osaka Univ. 1965, 13, 107–110. (20) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 3561–3566. (21) Minami, H.; Sugawara, M.; Odashima, K.; Umezawa, Y.; Uto, M.; Michaelis, E. K.; Kuwana, T. Anal. Chem. 1991, 63, 2787–2795. (22) Aistrup, G. L.; Szentirmay, M.; Kumar, K. N.; Babcock, K. K.; Schowen, R. L.; Michaelis, E. K. FEBS Lett. 1996, 394, 141–148. (23) Sugawara, M.; Hirano, A.; Rehak, M.; Nakanishi, J.; Kawai, K.; Sato, H.; Umezawa, Y. Biosens. Bioelectron. 1997, 12, 425–439. (24) Hirano, A.; Wakabayashi, M.; Sugawara, M.; Uchino, S.; NakajimaIijima, S. Anal. Biochem. 2000, 283, 258–265. (25) Kloda, A.; Lua, L.; Hall, R.; Adams, D. J.; Martinac, B. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1540–1545. (26) Favero, G.; Campanella, L.; Cavallo, S.; D’Annibale, A.; Perrela, M.; Mattei, E.; Ferri, T. J. Am. Chem. Soc. 2005, 127, 8103–8111.
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Figure 2. Aperture fabrication procedure: (1) thermal oxidation and sputtering of SiO2; (2) patterning and anisotropic etching of Si; (3) sputtering of SiO2; (4) isotropic etching of Si3N4; (5) SiO2 removal. etching in 85% phosphoric acid at 150 °C. Finally, the SiO2 layer beneath the holes was removed by 5% hydrofluoric acid to form apertures. BLM Formation and Current Recordings. The Si chip fabricated as above was silanized by treating with 2% (v/v) 3-cyanopropyldimethylchlorosilane (CPDS, Gelest) in acetonitrile for 24 h, and then set in the middle of a Teflon chamber. The chip separated two 1.5 mL compartments in the chamber. The Si3N4 layer was precoated with a thin layer of n-hexadecane by dropping a 10-μL aliquot of 0.1% n-hexadecane in chloroform. A 1400 μL portion of 2.0 M KCl solution containing 10 mM HEPES/KOH (pH 7.4, abbreviated as KCl buffer), filtered just before use through a cellulose acetate filter (pore size 0.20 μm; Advantec Toyo), was added to each side of the chamber. The water level in both compartments was set below the aperture. Then a small amount (10 μL) of a lipid solution was spread on the aqueous solutions. The composition of the lipid solution was 2 mg/mL PC:PE:Chol = 7:1:2 (w/w) in chloroform/n-hexane (1:1, v/v). After solvent evaporation, BLMs were formed by gradually raising the water level until it surpassed the aperture. The successful preparation of a BLM was known by observing an Langmuir 2010, 26(3), 1949–1952
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Figure 3. SEM image of an aperture edge. The aperture was fabricated in a Si3N4 septum by the isotropic etching. The range of the edge angle is superimposed on the image. increase in the membrane resistance from ∼10 kΩ to over 1 GΩ at an applied potential of ( 100 mV. BLM formation without precoating n-hexadecane was also investigated. The incorporation of gramicidin into the BLMs was performed by adding a 10-30 μL aliquot of 1 μg/mL gramicidin solution to the KCl buffer in both compartments under constant stirring. After successful incorporation of gramicidin, the aqueous solutions surrounding the BLMs were changed to 2.0 M CsCl solution containing 10 mM HEPES/CsOH (pH 7.4, abbreviated as CsCl buffer) by the repetition of sucking up an ∼400 μL portion of KCl buffer with tubes and adding the same volume of CsCl buffer with a micropipet. This process was repeated 5-15 times for a thorough solution exchange. Current recordings were performed with an Axopatch 200B patch-clamp amplifier (Molecular Devices). The signal was filtered at 1.0 kHz low-pass filter, digitized at 10 kHz, and stored online using a digital data acquisition system (Digidata 1440 and pCLAMP software ver. 10.2, Molecular Devices). The data were analyzed with a pCLAMP ver. 10.2 using a 500 Hz low-pass filter.
Results and Discussion Shape of the Aperture Edge and BLM Formation. Figure 3 shows a scanning electron microscopy (SEM) image of the edge of an aperture fabricated in a Si3N4 septum. The aperture edge was smoothly thinned with an edge angle of about 9-22° by the use of isotropic etching. Since the shape of the annulus that connects BLMs and the edge of the Si3N4 septum is important for the membrane stability and the annulus-septum contact angle should be small for a stable arrangement of BLMs,15 the tapered shape of the aperture in a 240-nm thick Si3N4 septum is suitable for stabilizing BLMs. BLM formation in the microfabricated apertures was examined after silanization of the chip surface with CPDS. The CPDS treatment made the chip surface hydrophobic, which was expected to accelerate deposition of a lipid monolayer with the hydrophobic tails oriented to the chip surface.9 Reduced-solvent BLMs were prepared by folding up two lipid monolayers across the aperture. BLMs having a resistance of 3 to >100 GΩ were formed in the apertures with a success probability of 92% (49 membranes out of 53 trials) when the Si3N4 septum around the aperture was precoated with a thin layer of n-hexadecane. Solvent-free BLMs were also formed without the precoating; however, the success probability was much lower (31%, 4 membranes out of 13 trials). Therefore, BLMs formed after the precoating were investigated for the following study. Although n-hexadecane coatings were necessary for reproducible formation of BLMs, the amount (10 nL) of n-hexadecane was much less than conventional rediced-solvent BLMs by the folding method,21,23 which is favorable for avoiding possible denaturation of ion channels in BLMs. The tapered apertures with a nanometer-scale smoothness (Figure 3) works to reduce the stress on the bilayer structure, leading to reduction in annulus volume required for supporting the contact between BLMs and the Si3N4 septa (Figure 1b). Langmuir 2010, 26(3), 1949–1952
Figure 4. (a) An average time-course of the membrane resistance after formation of the membrane. Mean ( SEM (n = 3-5). Error bars for 10 and 15 h are within the marker (O). (b) A time-course of the resistance obtained from the BLM that exhibited the longest lifetime of 45 h. For panels a and b, membrane resistance higher than 200 GΩ was counted as 200 GΩ. (c) An example of gramicidin single-channel current recorded 43 h after the BLM formation. The current recording was made from the same BLM as in panel b. Applied potential was -100 mV.
The present BLMs exhibited high mechanical stability as well as high-sealing property. The membranes were not broken by electrical shocks, such as plugging off and reconnecting the Ag/ AgCl electrodes inserted in the aqueous solutions surrounding the BLMs. Membranes survived when a constant voltage of (1 V was applied (100%, n = 9). Even when the applied potential was repeatedly switched from þ1 V to -1 V and vice versa, still 90% of the membranes (9 out of 10) survived these treatments. Membrane lifetime was 15-48 h (n = 2) without incorporated channels. When gramicidin ion channels were incorporated into BLMs, these membranes also showed a similar lifetime of 15-45 h (n = 3). Figure 4a shows an average time-course of membrane resistance obtained from all the five BLMs. All the membranes exhibited resistance higher than 100 GΩ up to 15 h after the membrane formation. The longest lifetime was obtained from a BLM containing a gramicidin channel, whose time-course is given DOI: 10.1021/la902522j
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in Figure 4b. The BLM exhibited resistance higher than 100 GΩ up to 43 h, and then the resistance decreased below 1 GΩ after 45 h. Figure 4c shows an example of the gramicidin single-channel current from the same BLM 43 h after the formation. Current steps corresponding to transition between the closed and open states were observed with a single-channel conductance of 22 pS in KCl (2.0 M) buffer. This conductance level is in the conductance range (20-25 pS in 2.0 M KCl) reported by others,27,28 suggesting the functionality of the present BLM even over 40 h after formation of the membrane. Tolerance to Solution Exchanges and Gramicidin SingleChannel Recordings. We next examined the tolerance to solution exchanges of the present BLMs containing a gramicidin single-channel. Figure 5 shows examples of single-channel currents from a BLM containing gramicidin when aqueous solutions were exchanged in series KClfCsClfKCl. In KCl (2.0 M) buffer, single-channel current with a conductance level of 20 pS was observed. Then the aqueous solutions surrounding the BLM were exchanged by sucking up KCl buffer with tubes and subsequently adding CsCl buffer with a micropipet. In order to rule out the possibility of membrane refolding, the water level was kept higher than the aperture in which the BLM was formed. After repeating this process 5-15 times for a thorough solution exchange, single-channel currents were still observed with a higher conductance level of 53 pS. The observed conductance levels (20 pS in 2.0 M KCl and 53 pS in 2.0 M CsCl) were very close to those reported for the gramicidin channel in 2 M Kþ (25 pS) and 2 M Csþ (55 pS) at 100 mV.27 Changing the aqueous solutions back to KCl buffer led to the observation of channel currents with the single-channel conductance of 22 pS. These results demonstrate that the present BLM containing a single gramicidin channel survived repetitive solution exchanges without any perturbation of single-channel conductance. When the solution exchange experiments were examined with different BLMs including a solvent-free membrane, all the BLMs containing gramicidin showed tolerance to solution exchanges (n=7), confirming the high stability of the present BLM system. The background current noise was 2-3 pA after being filtered at 500 Hz, which was relatively large for BLMs prepared in apertures of φ20-30 μm. This is probably due to large capacitance (mean ( SEM was 252 ( 19 pF) of the present system. Similar large capacitance of several hundred picofarads has also been reported for BLMs prepared on apertures in a Si chip.13 Since silicon has high dielectric constant of 11-12, the use of silicon resulted in a large capacitance of the total chip containing the BLMs. The open time of the gramicidin channel in the present BLMs was distributed in the range from 59 ms to 6.0 s with the mean open time of 0.35 ( 0.04 s (n = 191). The range of open time and mean open time was similar to those reported for gramicidin incorporated into BLMs prepared by the conventional method.29-31 Transient current changes were observed for the transitions between the closed and open states: 12 ( 2 ms for (27) Andersen, O. S. Biophys. J. 1983, 41, 119–133. (28) Busath, D. D.; Thulin, C. D.; Hendershot, R. W.; Phillips, L. R.; Maughan, P.; Cole, C. D.; Bingham, N. C.; Morrison, S.; Baird, L. C.; Hendershot, R. J.; Cotten, M.; Cross, T. A. Biophys. J. 1998, 75, 2830–2844. (29) Hirano, A.; Wakabayashi, M.; Matsuno, Y.; Sugawara, M. Biosens. Bioelectron. 2003, 18, 973–983. (30) Matsuno, Y.; Osono, C.; Hirano, A.; Sugawara, M. Anal. Sci. 2004, 20, 1217–1221. (31) Elliott, J. R.; Needham, D.; Dilger, J. P.; Haydon, D. A. Biochim. Biophys. Acta 1983, 735, 95–103.
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Figure 5. Single-channel currents recorded from a BLM containing a gramicidin channel at an applied potential of -100 mV. The aqueous solutions surrounding the BLM were exchanged from KCl to CsCl and then back to KCl by the repetition of sucking up the solutions and subsequently adding new solutions. The water level was kept higher than the aperture in which the BLM was formed.
openings and 13 ( 2 ms for closings (n = 191). The transients were slower than that (∼4 ms) obtained with conventional black films.27 The slower transient currents are again probably due to the large capacitance from silicon. Further improvement is necessary for recording channel currents with fast openTclose kinetics, for example, the application of a capacitance-reducing layer to the silicon chips, which can also work for reducing background current noise.
Conclusions In summary, we have succeeded in the preparation of reducedsolvent BLMs containing a single gramicidin channel that are stable enough for repetitive solution exchanges. Stable solventfree BLMs were also prepared, although the success probability of the membrane formation was lower than that of the reducedsolvent BLMs. BLMs based on unvolatile solvent, i.e., black films, have been considered more stable and are often coupled with microfabricated devices, however, membrane stability against solution exchange has not been demonstrated at the single-channel level. In contrast, we showed a different approach to stabilize membranes for reduced-solvent and more physiologically relevant BLMs. The key feature that stabilized BLMs is the nanometer-scale design of the microfabricated septum: an aperture with a smoothly tapered edge allows reduction of the stress on the lipid bilayer at the contact with the septum. The stable BLMs tolerable to solution exchanges enable analysis of channel functions under various solution conditions from the same BLM preparations. Since the fabrication process used in the present study is based on the wet process, our approach is suitable for fabrication of the apertures in an array format, which will realize a high throughput analysis of ion-channel proteins on Si chips. Acknowledgment. This work was supported by Grants-in-Aid from the Japan Society for the Promotion of Science (Grant Nos. 21350038 and 20246008) and JST PRESTO program. Financial support from the Takeda Science Foundation and The Asahi Glass Foundation are also acknowledged.
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