Lipid Bilayer Deposition and Patterning via Air Bubble Collapse

Aug 7, 2007 - In bubble collapse deposition (BCD), an air bubble is first “inked” with .... to form complex biomimetic supported lipid bilayers vi...
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Langmuir 2007, 23, 9369-9377

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Lipid Bilayer Deposition and Patterning via Air Bubble Collapse Morgan D. Mager and Nicholas A. Melosh* Department of Materials Science and Engineering, Stanford UniVersity, Stanford, California 94305 ReceiVed May 11, 2007. In Final Form: June 30, 2007 We report a new method for forming patterned lipid bilayers on solid substrates. In bubble collapse deposition (BCD), an air bubble is first “inked” with a monolayer of phospholipid molecules and then touched to the surface of a thermally oxidized silicon wafer and the air is slowly withdrawn. As the bubble shrinks, the lipid monolayer pressure increases. Once the monolayer exceeds the collapse pressure, it folds back on itself, depositing a stable lipid bilayer on the surface. These bilayer disks have lateral diffusion coefficients consistent with high quality supported bilayers. By sequentially depositing bilayers in overlapping areas, fluid connections between bilayers of different compositions are formed. Performing vesicle rupture on the open substrate surrounding this bilayer patch results in a fluid but spatially isolated bilayer. Very little intermixing was observed between the vesicle rupture and bubbledeposited bilayers.

Introduction In their natural role as the cell membrane, lipid bilayers are a complex platform for establishing ionic gradients, transducing chemical signals, and sending intercellular messages.1 Lipid bilayers supported on solid surfaces have been shown to preserve many aspects of their biological activity,2-4 while allowing the use of characterization techniques not possible in bulk solution, includingatomicforcemicroscopy,5-7 surfaceplasmonresonance,8-10 fluorescence interference contrast microscopy,11,12 and electrochemical impedance spectroscopy.13 Since supported lipid bilayers are bound to the substrate surface, it is possible to rapidly exchange the surrounding solution while continuously monitoring the bilayer.14 It has also been demonstrated that supported bilayers can be divided into spatially isolated regions on the substrate surface.15 Because of these properties, these systems have received significant attention in studies of protein binding,16 cell signaling,3 and fundamental lipid mechanics. There are currently two dominant methods for depositing substrate-supported lipid bilayers: Langmuir-Blodgett (LB) * To whom correspondence should be addressed. E-mail: nmelosh@ stanford.edu. (1) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman and Company: New York, 1995. (2) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159-6163. (3) Mossman, K. D.; Campi, G.; Groves, J. T.; Dustin, M. L. Science 2005, 310, 1191-1184. (4) Tamm, L. K. Biochemistry 1988, 27, 1450-1457. (5) Hui, S. W.; Viswanathan, R.; Zasadzinski, J. A.; Israelachvili, J. N. Biophys. J. 1995, 68, 171-178. (6) Koenig, B. W.; Krueger, S.; Orts, W. J.; Majkrzak, C. F.; Berk, N. F.; Silverton, J. V.; Gawrisch, K. Langmuir 1996, 12, 1343-1350. (7) Schonherr, H.; Johnson, J. M.; Lenz, P.; Frank, C. W.; Boxer, S. G. Langmuir 2004, 20, 11600-11606. (8) Reimhult, E.; Zach, M.; Hook, F.; Kasemo, B. Langmuir 2006, 22, 33133319. (9) Keller, C. A.; Glasmastar, K.; Zhdanov, V. P.; Kasemo, B. Phys. ReV. Lett. 2000, 84, 5443-5446. (10) Zhang, L.; Longo, M. L.; Stroeve, P. Langmuir 2000, 16, 5093-5099. (11) Crane, J. M.; Volker, K.; Tamm, L. K. Langmuir 2005, 21, 1377-1388. (12) Radler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4935-4548. (13) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 11261133. (14) Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 1477314781. (15) Cremer, P. S.; Groves, J. T.; Kung, L. A.; Boxer, S. G. Langmuir 1999, 15, 3893-3896. (16) Arispe, N.; Pollard, H. B.; Rojas, E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10573-10577.

deposition and vesicle rupture (VR). In the vesicle rupture technique, a solution of small unilamellar lipid vesicles (SUVs) is produced either by extrusion through a polycarbonate membrane or by sonication of larger vesicles. A substrate (usually silica) is then incubated with this SUV solution. The vesicles first adhere to the surface and then rupture on the surface. After rupture, the resultant bilayer discs fuse into a continuous fluid bilayer.8,17 The VR technique is relatively simple and inexpensive. By patterning the underlying substrate, it is possible to create patterns in VR bilayers. One of the earliest examples of patterning was the use of either scratches on a surface or lithographically defined “corrals” to prevent diffusion between adjacent bilayer patches, while allowing free diffusion within each patch.15,18 These corrals allowed the formation of localized concentration gradients within each region. The concept of patterning was later extended to allow the creation of bilayer and monolayer regions on the same surface by periodically altering the underlying substrate from hydrophilic to hydrophobic.19,20 In spite of the extensive studies of VR, there are still some outstanding questions and limitations. Since VR relies on adhesion between the vesicle and the substrate to initiate rupture, it can only be performed on a limited range of materials. The vast majority of VR studies have been performed on glass, oxidized silicon, or other surfaces composed mainly of SiO2 groups. Another limitation is that the mechanism of bilayer formation by VR is still not completely understood. Although recent work has done much to reveal the nature21-24 and magnitude7,25-27 of the adhesive force necessary for VR, there is still ongoing debate (17) Johnson, J. M.; Ha, T.; Chu, S.; Boxer, S. G. Biophys. J. 2002, 83, 33713379. (18) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651-653. (19) Lenz, P.; Ajo-Franklin, C. M.; Boxer, S. G. Langmuir 2004, 20, 1109211099. (20) Howland, M.; Sapuri-Butti, A.; Dixit, S.; Dattlebaum, A.; Shreve, A.; Parikh, A. N. J. Am. Chem. Soc. 2005, 127, 6752-6765. (21) Richter, R.; Mukhopadhyay, A.; Brisson, A. Biophys. J. 2003, 85, 30353047. (22) Cha, T.; Guo, A.; Zhu, X. Y. Biophys. J. 2006, 90, 1270-1274. (23) Reimhult, E.; Hook, F.; Kasemo, B. Langmuir 2003, 19, 1681-1691. (24) Rossetti, F. F.; Bally, M.; Michel, R.; Textor, M.; Reviakine, I. Langmuir 2005, 21, 6443-6450. (25) Egawa, H.; Furusawa, K. Langmuir 1999, 15, 1660-1666. (26) Zhdanov, V. P.; Dimitrievski, K.; Kasemo, B. Langmuir 2006, 22, 34773480. (27) Siefert, U.; Lipowsky, R. Phys. ReV. A 1990, 42, 4768-4771.

10.1021/la701372b CCC: $37.00 © 2007 American Chemical Society Published on Web 08/07/2007

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Figure 1. Diagram of the bubble collapse deposition (BCD) process. (A) The air bubble is coated with a lipid monolayer and moved to the area of interest. (B) The coated bubble is brought into contact with the substrate. (C) Air is removed to shrink the bubble, increasing the lipid monolayer pressure on the bubble surface. Once the monolayer collapse pressure is exceeded, the monolayer collapses, folding back on itself to deposit a second lipid leaflet on top of the first, forming a bilayer. (D) Upon completion of the bilayer in the center of the patch, the bubble snaps free of the surface, leaving a fluid bilayer patch behind.

about the structural transitions between vesicle and supported bilayer.14 In Langmuir-Blodgett (LB) deposition, a monolayer of lipid molecules at the air/water interface is compressed into an ordered state using a Teflon bar. A substrate is kept below the water surface until the desired surface pressure is obtained, and then it is drawn up through the air/water interface, depositing a monolayer on its surface. The second monolayer is deposited by passing the substrate back into the water, either vertically (LB) or horizontally (Langmuir-Schafer; LS), forming a fluid bilayer. Because this process is externally driven by monolayer compression and substrate movement, it is possible to deposit bilayers on a wider range of substrates than with VR.28-30 However, the LB technique creates spatially homogeneous bilayers that are more difficult to pattern. It can also be challenging to optically and electrically monitor bilayer properties during deposition with the LS method. In the following, we demonstrate a new technique for creating patterns of supported lipid bilayers in a fashion analogous to the LB technique. The method involves generating an air bubble at the end of a needle submerged in water and contacting the bubble with a preformed lipid bilayer. The high surface energy of the air/water interface drives the lipid to rapidly adsorb onto the bubble surface, forming a monolayer. The lipid-coated bubble is then placed at the desired deposition site. As the air is slowly withdrawn, the surface area of the bubble decreases, increasing the pressure of the lipid monolayer. When the pressure exceeds a critical threshold, the monolayer folds back on itself into a bilayer supported on the substrate. This is shown schematically in Figure 1. During continued collapse of the bubble, a sharp interface between the bilayer and monolayer regions continuously advances toward the center of the area in contact with the surface. As this interface coalesces at the center of the disc, the bilayer becomes complete and the bubble snaps free of the surface, leaving a fluid bilayer disc approximately the same size as the original contact area. The size of the lipid patches are thus readily (28) Csucs, G.; Ramsden, J. J. Biochim. Biophys. Acta 1998, 1369, 61-70. (29) Elender, G.; Kuhner, M.; Sackmann, E. Biosens. Bioelectron. 1996, 11, 565-77. (30) Kuhner, M.; Tampe, R.; Sackmann, E. Biophys. J. 1994, 67, 217-226.

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controlled by creating different initial bubble sizes before inking. Further size control can be achieved by varying the mechanical force exerted onto the bubble by the needle. Pressing the bubble with more force deforms a larger portion of the bubble into contact with the surface, resulting in a larger deposited bilayer patch. Mechanistically, this process shares many characteristics of the Langmuir-Blodgett technique, including compression of the monolayer at the air/water interface and bringing two opposing monolayers together to create the bilayer. However, bubble collapse deposition (BCD) has several advantages brought about by its simple instrumentation and localized deposition. Realtime observation of the entire deposition process is easily performed on a conventional inverted microscope, eliminating the additional equipment for real-time LB studies or the transportation time necessary to mount a fully formed LB sample in the microscope. Furthermore, sequential deposition of a single lipid or even multiple lipid compositions at different locations can readily create spatially and chemically heterogeneous patterns on a single substrate. Different bilayer compositions are patterned by contacting separate preformed bilayers of the desired composition and repeating BCD on the original substrate. Bilayer patches formed by bubble collapse can be spatially isolated from each other, or they can be deposited in contact with each other such that their contents intermix due to lateral diffusion. The substrate surface surrounding the BCD bilayer is left clean and unaltered after deposition, and it can be backfilled with a VR bilayer to also create multicomponent bilayer surfaces. Intriguingly, the bilayers formed by the two different techniques do not intermix. This phenomenon is attributed to a narrow band of disordered lipids ringing the outside of the BCD bilayer that inhibit vesicle adsorption and rupture. Unlike either VR or LB bilayers, BCD bilayers can be deposited multiple times at the same location. In this clean and replace process, the first bilayer is removed by contacting it with a clean air bubble (with no lipid monolayer) approximately the same size as the original. This bubble adsorbs all the lipids onto the surface, leaving a clean substrate. A flowing stream of bubbles can also be used to remove a larger area as necessary. Subsequently, another monolayer-coated bubble is placed in the same location and collapsed, leaving a fluid bilayer indistinguishable from the first. We have repeated this process 15 times in the same location with no significant change in bilayer uniformity or diffusion coefficient. This process may be a convenient mechanism for the cleaning and reapplication of bilayers for sensor-based devices Experimental Section Materials. All lipids and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL). Unless otherwise noted, the lipid used was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Fluorescent labeling was performed with 1 mol % of either Texas Red-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine or Oregon Green-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine purchased from Molecular Probes (Eugene, OR). Unless otherwise noted, all experiments were performed on silicon wafer substrates (University Wafer) with a 90 nm thermal oxide grown at 1000 °C with pyrolytic steam. Experiments done on an inverted microscope were performed with no. 1.5 glass coverslips (Fisher Scientific). Deionized water of 18.2 MΩ cm resistivity was obtained from a Milli-Q water purification system (Millipore, Billerica, MA). Hydrogen peroxide (30%) and sulfuric acid were CMOS grade and were purchased from JT Baker Analytical. All chemicals were used without further purification. The buffer used was 100 mM NaCl and 10 mM Tris-HCl, adjusted to pH 7.4 with NaOH.

Air Bubble Deposition of Lipid Bilayers Formation of Source Bilayers. Prior to lipid application, the chips were cleaned for at least 20 min in a 2:1 mixture of sulfuric acid and hydrogen peroxide maintained at 100 °C. (Caution! This mixture reacts Violently with organic compounds and should be handled with extreme care.) The chips were then rinsed extensively under deionized water and dried under a stream of nitrogen. Similar results were obtained when the substrates were cleaned for either 1 min in an oxygen plasma system or 30 min in a UV ozone cleaner. Lipids with either 1% Texas Red or 1% Oregon Green were first dissolved in chloroform at a concentration of 10 mg/mL. A 10µL drop of solution was placed on a clean Teflon surface, dried under a stream of nitrogen, and then desiccated under vacuum for at least 1 hour. The resulting lipid cake was then scraped onto a Teflon applicator and smeared across a limited portion of the chip. The chip was then rinsed vigorously for ∼1 min under a stream of deionized water. This results in a single lipid bilayer on the surface. The identity of this structure as a single lipid bilayer was evidenced by the fact that, under identical illumination conditions, it is the same intensity as a bilayer produced by BCD. Fluorescence recovery after photobleaching (FRAP) indicates that this source bilayer is laterally fluid but has a large immobile fraction.31 This bilayer has many defects, but these defects are removed once the lipids rearrange into a well-organized monolayer on the surface of the air bubble. The variations in fluorescent intensity evidenced by this source bilayer were not present in the resultant BCD bilayer. Thus, the quality of the source bilayer does not directly affect the quality of the eventual deposited bilayer. Bubble Formation/Manipulation. Air bubbles were formed with a home-built volume control system based on a NE-1000 syringe pump (New Era Pump Systems, Farmingdale, NY) with a 25 µL glass syringe purchased from Hamilton (Reno, NV). The needles used to form and manipulate air bubbles were straight-edge 20 gauge (Jensen Global, Santa Barbara, CA) and were cleaned with methanol and a nitrogen stream prior to each use. After cleaning, the needle, syringe, and connecting tubing were all completely filled with water. The plunger on the syringe was then withdrawn 10 µL while the needle tip was in air. This ensured that the air volume within the needle was the same each time to within the measurement error of the syringe ((0.2 µL). Movement of the needle was controlled with a Sutter Instruments MPC-385 micromanipulator. Bubbles were typically expanded at a rate of 1 µL/min to a final volume of 0.5 µL. The uncoated bubble was touched to a previously formed bilayer, thus coating the bubble with a monolayer. The monolayer-coated bubble was then moved to a new section of the substrate, and the air was slowly withdrawn, increasing the surface pressure of the monolayer. The speed of this collapse was varied from 0.5 to 50 µL/min and, unless noted, was 1 µL/min. Once the volume of the bubble was decreased by approximately one-half, the monolayer began collapsing on itself to form a bilayer on the surface. Throughout this process, the entire substrate and air bubble were kept under water. Backfilling Bilayers. The bubble collapse deposition of bilayers results in a circular patch of supported bilayer isolated on the substrate surface. The majority of the surrounding substrate is left unaltered and is capable of supporting bilayer formation. In some experiments, these surrounding areas were then coated with lipid bilayers via VR as described in detail elsewhere.2,32 This subsequent deposition will be referred to as backfilling. Briefly, 5 mg of POPC dissolved in chloroform was dried on the bottom of a glass vial and desiccated for several hours. The resulting lipid cake was then rehydrated with buffer and vortexed for several minutes. This suspension was then extruded 19 times through a polycarbonate membrane with 100 nm pores (Whatman Florham, NJ). After bilayer patches were formed on the sample surface with BCD, a small volume (approximately 50µL) of vesicle solution was delivered to the surface of the chip in a home-built flow cell. This solution was allowed to sit for 20 (31) Images and a further description of this source bilayer are available in the Supporting Information. (32) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316.

Langmuir, Vol. 23, No. 18, 2007 9371 min and was then rinsed from the surface with an extensive flow of buffer solution. Characterization. Microscopy was performed on a Zeiss AxoiSkop 2 upright fluorescence microscope (Jena, Germany) equipped with a mercury lamp source. Appropriate filter cubes for Texas Red and Oregon Green were purchased from Zeiss. Images were acquired on a Zeiss AxioCam HRm charge-coupled device (CCD) camera and processed with AxioVision software. Further processing, including image merging and false coloring to indicate dye species, was performed with ImageJ software from the National Institutes of Health. Where indicated, certain measurements were performed on an AxioVert inverted microscope (Zeiss).

Results and Discussion Bubble collapse deposition is mechanistically similar to LB deposition, but it allows highly localized bilayer formation and simple in situ imaging. In both cases, two opposing lipid monolayers are sequentially deposited from an air/water interface to form the bilayer. Unlike the LB method, where the lipid monolayer is formed at the planar air/water interface of a water trough, the BCD method uses the surface of a submerged air bubble, controlling the surface pressure of the monolayer by expanding or contracting the bubble itself. The lipid monolayer is added to the air bubble simply by bringing the bubble into contact with a pre-existing lipid bilayer, and then it is redeposited onto a substrate by collapsing the bubble. The BCD process thus consists of three distinct steps: (i) coating an air bubble with a lipid monolayer, (ii) placing the lipid monolayer-coated bubble on a clean substrate, and (iii) collapsing the air bubble to form a supported bilayer. This process is illustrated in Figure 1. The initial necessary step is adding a lipid monolayer onto the surface of an air bubble. Air bubbles were formed using a syringe pump to expand or contract the bubble at a fixed rate and positioned using a micromanipulator. Typical bubbles were 500700 µm in height (0.3-0.8 µL in volume). After formation, the bubble was touched to the surface of a previously formed supported lipid bilayer. Upon contact, the bubble was observed to rapidly expand at the chip surface, distorting the shape of the bubble. The elongated bubble then snapped free of the surface and returned to its spherical shape, though larger in size than the original bubble. This entire process occurred in ∼100 ms. The bubble removed a circular patch from the supported bilayer surrounding the contact point, but it often redeposited small regions of a bilayer near the center of this cleared region.33 We hypothesize this process occurs as diagrammed in Figure 2. Upon initial contact, a small region of the lipid bilayer ruptures to expose the hydrophobic tails to the air interface. The bubble then expands out along the middle of the bilayer, essentially cleaving it into two monolayers. This rapid expansion is driven by the high energy of the air/water interface on the bubble surface (72.8 mJ/m2).34 After this initial expansion, the bubble snaps back to a spherical shape, pulling the supported monolayer with it. This leaves behind a bare region of the substrate that has been stripped of the bilayer. The stripped region is usually larger than the bubble diameter, representing the extent to which the bubble expanded on the surface. To ensure complete saturation of the air/water interface with lipid molecules, the bubble is slowly dragged along the bilayer for a distance of ∼1 mm. If this is not performed, the eventual deposition will result in an incomplete bilayer or a patch with large holes in it, presumably corresponding to the areas of the bubble not covered in a monolayer. After saturation, the lipidinked bubble is lifted from the surface and ready to deposit. (33) Images and a further description of this source bilayer are available in the Supporting Information. (34) Harkins, W. D.; Brown, F. E. J. Am. Chem. Soc. 1919, 41, 499-524.

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Figure 2. Proposed mechanism of monolayer adsorption on the bubble surface. (A) A clean air bubble (no lipids present on the surface) is brought into contact with a supported lipid bilayer. (B) The air bubble opens an initial contact point and then rapidly expands between the two leaflets of the bilayer, coating the bubble surface with a monolayer. During this process, the air bubble is drawn out along the substrate surface further than its initial diameter. (C) After rapid expansion, the bubble snaps back to its original spherical shape, bringing the adsorbed lipid molecules with it and leaving behind a bare portion of substrate that no longer contains a bilayer. (D) The bubble is lifted from the surface and retains its monolayer coating.

Figure 3. (A-C) Sequential images from an inverted microscope as a bilayer is formed. (A) Monolayer-coated bubble in contact with a glass coverslip substrate. The uniform circular gray area is the supported monolayer at the substrate/monolayer/air interface, while the irregular background halo is due to the monolayer still on the bubble surface out of the focal plane. (B) As the bubble is collapsed, a sharp transition from monolayer to bilayer is observed. The single arrow indicates the outer perimeter of the monolayer and the double arrow indicates the outer perimeter of the bilayer. The dashed line corresponds to the line profile in (D). (C) Fully formed bilayer patch after the air bubble is withdrawn. At this point, the air bubble has been removed from the frame, so there is no background halo as before. Scale bar ) 150 µm. (D) Fluorescence intensity line profile taken from (B), showing the intensity jump from 1300 in the monolayer region to 3000 where a second leaflet has been deposited to form a bilayer.

Deposition proceeds by placing the inked bubble into contact with a glass coverslip or oxidized Si wafer at the desired location. An inverted microscope image of the monolayer-coated bubble sitting on a glass coverslip substrate is shown in Figure 3A, schematically corresponding to Figure 1B. The central gray circular region in Figure 3A is the supported monolayer, sandwiched between the air bubble on one side and the glass surface on the other. The diameter of this contact area was typically

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500-800 µm, but it could be easily varied by controlling the initial bubble size. The surrounding halo is from out-of-focus lipids on the unsupported surface of the air bubble. No lipids are present on uncontacted portions of the substrate, as seen from the lack of fluorescent intensity at the corners of the image. After this initial contact, a bilayer was deposited onto the glass surface by slowly shrinking the air bubble. As the air was withdrawn at a rate of ∼1 µL/min, the surface pressure within the monolayer increased until a critical pressure was exceeded and the upper monolayer was deposited on top of the first. This collapse occurred when the initial bubble volume had shrunk by approximately one-half. The transition from monolayer to bilayer can be clearly observed from the increase in fluorescence intensity, which begins at the outer perimeter and proceeds inward during shrinkage, as shown in Figure 3B. A line profile taken from Figure 3B is shown in Figure 3D. This profile shows approximately the 1:2 fluorescence intensity ratio expected for the transition from monolayer to bilayer. The boundary between these two intensity regions is extremely sharp, as is consistent with an abrupt transition from one to two monolayers. The interface between the monolayer and bilayer always maintained a circular perimeter as seen in Figure 3B. Note that the size of the background halo has decreased significantly from Figure 3A, since the volume of the air bubble has been reduced. After the bilayer was fully deposited, the remainder of the bubble pulled free from the surface and a completed bilayer disc remained supported on the substrate. From the time air removal was initiated until the bubble snapped free was typically around 20-30 s. Figure 3C shows the supported bilayer disc after completion. At that point, the needle had been removed, so the intensity of the bilayer was uniform across the disc. The diameter of the deposited bilayer patch was smaller than the area removed from the original source bilayer, since some lipids still remained upon the bubble after pulling free from the surface. The bilayer fluidity was characterized by fluorescence recovery after photobleaching (FRAP) at 25 °C using Texas Red as the fluorescent label. A spot ∼45 µm in diameter was bleached by exposure to high-intensity white light from a mercury lamp for 40 s. Because supported lipid bilayers are fluid in plane, the bleached molecules gradually diffuse out of the bleached area and are replaced with intact dye molecules. This recovery of fluorescent intensity was monitored over a period of several minutes, and the results were fit to FRAP expressions derived by Soumpasis assuming bleaching by a uniform circular light source and purely diffusive recovery.35 Representative images are shown in Figure 4A and B, and typical recovery data are plotted in Figure 4C. The calculated value of the diffusion coefficient from this experiment was 2.2 µm2/s, which is in good agreement with literature values from bilayers formed with other techniques.4,32 The experiments were conducted at 25 °C to ensure the lipids are well above the phase transition temperature of POPC; thus, the diffusion coefficient is not expected to vary strongly with temperature. Lipid Monolayer Formation on the Bubble. Since the compression of the lipid monolayer on the air/water interface is similar to that of the LB process, it is interesting to examine whether the actual surface pressure on absorption and collapse are similar to those observed for POPC on the LB trough. Assuming the monolayer is in thermodynamic equilibrium with the bubble, the monolayer pressure can be estimated from the change in bubble size after contact with the lipid source. When a lipid-free air bubble is brought into contact with a lipid source and picks up a monolayer, the volume of the bubble abruptly (35) Soumpasis, D. M. Biophys. J. 1983, 41, 95-7.

Air Bubble Deposition of Lipid Bilayers

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The total work done can be calculated by integrating this total force with respect to changes in h:

∆UV )

∫hh ∆p(h)Acap(h)(0.6dh) f

0

where hf is the final height, ∆UV is the work done expanding the bubble, and the factor of 0.6 is a geometric correction for the change in bubble shape during expansion.37 The driving force for this expansion is the reduction in interfacial energy, ∆Us, due to coating the bubble interface with a lipid monolayer:

∆Us ) γfAf - γ0Ai ) γfπ(a2 + hf2) - γ0π(a2 + h02)

Figure 4. FRAP measurements showing the bilayer (A) immediately after bleaching with a Hg lamp and (B) after 10 min of recovery. (C) Plotted intensity versus time, which was analyzed to give a diffusion coefficient of 2.2 µm2/s. Scale bars ) 20 µm.

increases even though the amount of air in the bubble and syringe is fixed. Assuming isothermal conditions, the work done by expanding the bubble must be equal to the reduction of the bubble’s surface energy upon coating it with lipid. The lateral pressure and density of lipid molecules within the monolayer can then be compared with surface pressure and molecular density in LB deposition. Figure 5A and B shows an optical image of the air bubble before and after picking up the lipid monolayer. Arrows indicate the bubble height, h, from the end of the needle tip. Approximating the bubble as a spherical cap,36 the surface area, Acap, and volume, Vcap, of the bubble are given by

Acap(h) ) π(a2 + h2) Vcap(h) ) 1/6πh(3a2 + h2) V(h) ) Vcap(h) + Vreservoir where a is the radius of the needle, V(h) is the total volume of air in the bubble and the needle, and Vreservoir is the initial volume of air inside the needle, which was 10.0 ( 0.2 µL. The remainder of the needle and tubing were filled with water. Assuming the air in the bubble behaves as an ideal gas under isothermal conditions, the change in pressure, ∆p, for a given volume change can be derived from the ideal gas law:

∆p(h) )

[

]

V(h) - 1 p0 V(h0)

where h0 is the initial height of the bubble. The initial pressure, p0, is assumed to be the atmospheric pressure of 1.01 × 105 Pa for T ) 298 K. The force, F, acting normal to the surface of the bubble can be found by multiplying this pressure by the surface area of the bubble:

F ) ∆p(h)Acap(h) (36) Oguz, H. N.; Prosperetti, A. J. Fluid Mech. 1993, 257, 111-145.

where γ0 and γf are the respective surface energies of the air bubble before and after picking up the lipid monolayer and Ai and Af are the surface areas of the bubble before and after adsorbing the lipid monolayer. Before adsorbing the monolayer, the surface of the bubble is a simple air/water interface with the surface energy γ0 assumed to be 72.8 mN/m.34 The energy gained by lowering the surface tension of the interface should be equal in magnitude but opposite in sign to the work done by volumetrically expanding the bubble. The resulting system of equations can be subsequently solved for the surface pressure, Π, of the lipid in the bubble-supported monolayer:

∆Us ) -∆UV Π ) γ0 - γf

[

Π ) γ0 + - πγ0(a2 + h02) +

]/

0.1πh02p0(R - 1)2(3a2 + h02(1 + R + R2))2 3πa2h0 + πh03 + 6Vreservoir

[π(a + R2h02)] 2

where R is the relative expansion ratio of the bubble, hf/h0. A plot of the observed expansion ratios R versus initial bubble height for seven separate experiments is shown in Figure 5C (squares). Overlaid are the calculated isostatic traces for monolayer surface pressures, Π, equal to 10, 20, 30, and 40 mJ/m2. Experimentally, the surface pressure in the monolayer was found to have an average value of 12 ( 3 mN/m regardless of initial bubble size, as shown in Figure 5D. The dashed lines indicate one standard deviation from the mean value. Surface pressure invariance with bubble volume is expected if there are sufficient lipids available when the bubble contacts the preformed bilayer to form an equilibrium packing density. The surface pressure of the lipid monolayer on the bubble is comparable to the middle of the liquid phase of the pressurearea isotherm on an LB trough. Figure 5E shows the LB isotherm for a POPC lipid at 25 °C. At a large area per molecule, the monolayer is in the gas phase, characterized by disordered molecular packing and an extremely low compression modulus.38 The low compression modulus is exhibited as a slope of nearly zero for areas greater than 220 cm2. At higher compression, the monolayer enters the liquid regime. This phase is the dominant (37) The scaling factor of 0.6 is applied to dh to account for the fact that expansion is not uniform around the perimeter of the bubble. For an increase in bubble height dh, the tip of the bubble will move a distance dh, while the base of the bubble is attached to the needle tip and will not expand at all. For the geometries considered here, the average displacement is approximately 0.6 times the maximum displacement. (38) Petty, M. C. Langmuir Blodgett Films; Cambridge University Press: Cambridge, 1996.

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Figure 5. Brightfield images of the air bubble (A) before and (B) after being coated with a monolayer. White arrows indicate the height of the bubble. Scale bars ) 300 µm. (C) Calculated and experimental values of the bubble extension ratio versus initial bubble size. Traces are given for Π ) 10 mN/m (s), 20 mN/m (- ‚ -), 30 mN/m (- - -), and 40 mN/m (‚ ‚ ‚). Experimental data are marked by filled squares. (D) Calculated surface pressure as a function of initial bubble size. No dependence was observed. The dashed lines are one standard deviation from the mean value of 12 mN/m. (E) Pressure-area isotherm obtained from a Langmuir-Blodgett trough with the same lipid. The arrow indicates the onset of monolayer collapse. The dashed lines correspond to the same values as those in (D).

feature in Figure 5E, comprising the bulk of the isotherm, from 220 to 120 cm2. Above a lateral pressure of 43 mN/m, the monolayer reaches its collapse pressure and begins to fold over on itself. This transition is indicated by a sudden decrease in the slope and is marked with an arrow. The range of lipid monolayer pressures on the bubble surface clearly lies in the liquid packing regime. The surface pressure of the monolayer during BCD was estimated to be greater than 40 mN/m based upon the volumetric contraction necessary to induce bilayer formation. After contacting the monolayer-coated bubble to a clean surface, it was necessary to shrink the bubble volume by approximately one-half to initiate deposition of the second monolayer leaflet. This corresponds to a decrease in the bubble surface area of ∼35%. This relative compression can then be compared to the LB isotherm in Figure 5E, with 12 mN/m or A ) 170 cm2 as the starting point. Upon 35% compression, A ) 110 cm2, corresponding to the collapse regime of the LB monolayer. From this comparison, the monolayer surface pressure during BCD should be near the LB collapse pressure of 43-45 mN/m. This result strongly supports that the mechanism involved in BCD is very similar to that of monolayer collapse in an LB film. The high deposition pressure during BCD is actually advantageous, as studies have shown that depositing the second leaflet of a LB bilayer as close to the collapse pressure as possible reduces subsequent defects in the bilayer.39,40 Processing Parameters. The conditions under which BCD was successful were investigated by varying bubble collapse rate, solution composition, substrate material, and substrate preparation. Most lipid bilayers produced with the LB technique are made with a dipper pull speed of 1-5 mm/min.39-41 Pull (39) Benz, M.; Gutsmann, T.; Chen, N. H.; Tadmor, R.; Israelachvili, J. Biophys. J. 2004, 86, 870-879. (40) Osborn, T. D.; Yager, P. Biophys. J. 1995, 68, 1364-1373. (41) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105-113.

rates significantly higher than this often result in incomplete monolayer transfer or lack of any transfer at all. The same is true of BCD. For a typical 500 µm diameter bubble, a withdrawal rate of 1 µL/min corresponds to a perimeter velocity of ∼0.3 mm/min. Bilayers were successfully deposited at rates of 0.140 µL/min equivalent to 0.03-12 mm/min. At 50 µL/min (15 mm/min) and above, no deposition was observed. FRAP measurements performed on the successful bilayers showed no significant trend in either the diffusion coefficient or mobile fraction as a function of the withdrawal rate. This indicates that the process has a binary type success: at all conditions under which a bilayer is formed, the bilayers are of high quality. The success rate and diffusion coefficient were also observed to be insensitive to the solution conditions, with no difference in the deionized water or high salt (0.1 M) buffer solution. The BCD technique was found to produce similar results on glass and thermally oxidized silicon, and thus, it appears to be generally applicable to silica surfaces. The success rate of BCD was found to be sensitive to the cleanliness of the substrate, so samples were always used within a few hours of being cleaned. Interestingly, residual lipids from previous bilayers had no such adverse effect on successful deposition. Once a bilayer patch had been deposited, it could be removed by contact with a monolayer-free air bubble. This removal is mostly, but not absolutely, complete, as there remains a very faint fluorescent signal in the area once occupied by the bilayer. A new monolayercoated bubble can then be brought in and collapsed on the site, and again a mobile, defect-free bilayer is formed. To test the reproducibility of these bilayers, this procedure was repeated 15 times in the same location with fluid bilayers resulting each time. In the course of these 15 trials, no significant trend in the diffusion coefficient or bilayer quality was observed.42 The (42) Images of the bilayers sequentially formed in the same location are available in the Supporting Information.

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Figure 7. (A) Low magnification (scale bar ) 200 µm) false color image of a circular BCD bilayer dyed with 1% Texas Red. The surrounding substrate has been backfilled by vesicle rupture with a bilayer dyed with 1% Oregon Green. The sharp boundary between the two colors indicates a lack of mixing. (B) High magnification (scale bar ) 25 µm) image of a BCD bilayer dyed with 1% Oregon Green surrounded by a VR bilayer dyed with 1% Texas Red. Reversal of the dye species between the two techniques did not induce mixing.

Figure 6. (A) Sequential deposition of two partially overlapping bilayer patches. The lipids in the upper bilayer patch were dyed with Texas Red, while those in the lower patch were dyed with Oregon Green. A fluid interface was formed between the two regions, and the respective fluorescent probes mixed over time. Scale bar ) 20 µm. (B) Intensity of the red dye signal at points A (- - -) and B (s) over time showing continued interdiffusion.

diffusion coefficient of the last bilayer formed was within 10% of the value for the first bilayer. With traditional patterning techniques, it is difficult to deposit arrays of more than two different types of lipid bilayer on one continuous surface. One advantage of BCD is the ability to deposit bilayers of different compositions on the same substrate, which potentially enables parallel studies of different lipid compositions. When two BCD bilayer patches of different compositions were deposited out of contact with each other, they remained isolated. To further investigate the multiple deposition process, two bilayer patches were deposited overlapping each other. One patch was dyed with Texas Red, and the other was dyed with Oregon Green. Figure 6A is a merged false color image of the overlapping region, showing the two probes freely interdiffusing between the regions. This indicates that when a bubble is brought down and collapsed in contact with an existing bilayer, it forms a continuous fluid bridge between the two, rather than depositing a multilayer or creating a disordered boundary region. The evolution of this interface over 2 min is plotted in Figure 6B, showing that the probes continue to mix over time. Further examination of Figure 6A reveals a fractal type edge on both bilayer patches. Over time, the bilayer patches were found to expand by a few percent into the surrounding area of the chip. This is consistent with the previously made observation that, upon removal of some portion of a VR bilayer, the remaining bilayer will expand into the free space as long as the exposed surface is left clean and without sharp topographic relief.15 In contrast to the smooth curve initially present at the edge of the

Figure 8. FRAP measurement of the interface between a bubbledeposited bilayer (upper region) and a bilayer made by vesicle rupture (lower region). Both lipids were dyed with Texas Red and could therefore be imaged simultaneously. (A) Prior to bleaching, there is a noticeable discontinuity beyond the edge of the bubble bilayer patch. (B) Photobleaching darkens both bilayers. (C) Near the interface, lipid material within the vesicle rupture region does not recover, indicating a lack of lateral contact. Further from the interface, the vesicle rupture bilayer does recover. Scale bars ) 10 µm.

bubble, this new interface was jagged and irregular, as has been observed in other lipid expansion experiments.43 Backfilling with Vesicle Rupture. BCD is compatible with traditional bilayer deposition methods, and it is possible to utilize multiple techniques on the same sample. After completion of a bubble-deposited bilayer patch, the surrounding area is still clean silica and may be amenable to backfilling with a VR bilayer. Do these two bilayers fuse or remain distinct after backfilling? To test the interactions between these different deposition techniques, a BCD patch was formed, and VR deposition was subsequently performed on the surrounding substrate. To investigate the fate of the separate lipid populations, different dyes (1% Oregon Green or Texas Red) were used in the VR and BCD bilayers. Mixing of the two bilayers would result in a homogeneous distribution of the two dyes, while a sharp boundary would indicate a lack of fusion. Figure 7 shows both low and high magnification images of a circular BCD bilayer surrounded by a VR bilayer. The boundaries between the two lipid populations remained sharp, even at micrometer length scales, indicating a lack of mixing. Figure 7 was taken from two different experiments, and the type of dye used in each bilayer was switched between the two experiments. This change was found to have no effect on the bilayer properties for either the BCD or VR bilayer, indicating that the identity of the dye molecules for the two different processes does not affect the outcome. This lack of bulk mixing is in contrast to the earlier results showing fusion between two overlapping BCD bilayer patches. We hypothesize that this effect is due to the presence of a “moat” region which has been previously observed in two separate studies of VR on chemically patterned substrates.19,20 The moat described (43) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 2554-2559.

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Figure 9. Schematic of the process of moat formation. (A) The lipid-coated air bubble is shrunk, increasing pressure in the monolayer. (B) Just before the critical collapse pressure is reached, some lipid molecules are deposited in a disordered manner on the chip surface. The monolayer subsequently folds back on itself and forms a bilayer. (C) The bilayer patch is completed, and the disordered lipid molecules remain on the surface past the bilayer edge. (D) The chip is exposed to a vesicle solution. Lipid vesicles adsorb to clean areas of the surface. Few or no vesicles adsorb in the region coated with disordered lipid molecules. (E) Adsorbed vesicles rupture, forming a supported bilayer, while the “moat” region between the vesicle rupture bilayer and the bubble-deposited bilayer prevents mixing between the two.

in these studies is an area between adjacent lipid membranes in which there is apparently little or no lipid present. The existence of these regions was attributed to a surface chemistry that is neither hydrophilic enough to support bilayer formation nor hydrophobic enough to support monolayer formation. The presence of this region was explained as a boundary between the masked and unmasked portions of the substrate that consequently received an intermediate degree of chemical modification. However, in the present experiment, no patterned chemical modification was performed on the substrate prior to bilayer deposition. To investigate whether a moat region is responsible for the lack of mixing between the VR and BCD bilayers, the backfilling experiment was repeated using Texas Red as the dye for both lipid populations. This modification allowed simultaneous imaging of both the BCD and VR bilayers during FRAP measurements as shown in Figure 8. The interface prior to any bleaching is shown in Figure 8A, where the BCD bilayer is in the upper half of the image and the VR bilayer is in the lower half. A slightly lower intensity band is observed between the two bilayers (∼5-7 µm wide). Figure 8B shows the same interface immediately after photobleaching. Both bilayers were bleached, but, as seen in Figure 8C, the bilayers did not recover equally. The bilayer deposited by bubble collapse was fully fluid up to its edge. The bilayer formed by vesicle rupture exhibited a more complicated behavior. The lipid material in the band region between the two types of bilayers was bleached, but it never recovered at all, indicating that it consisted either of unruptured vesicles or isolated patches of lipid. Regardless of the exact structure, it is clear that the material in this region is not fluidly connected to the bilayers on either side of the interface and is not mobile along its length. Further from the interface, the VR bilayer showed some recovery but with a large number of defects and immobile areas. FRAP measurements performed far from the interface in the bulk of the VR bilayer showed complete recovery with a normal diffusion coefficient. These results indicate that the lack of mixing observed earlier was due to an immobile region near the edge of the vesicle rupture bilayer. The proposed mechanism of formation of this region is shown in Figure 9. During deposition with the bubble collapse technique, shrinking the bubble initially causes the monolayer to shrink and recede across the surface without depositing a bilayer as shown in Figure 9A. We have observed that when a monolayer-coated bubble is moved across a clean silica surface without any air withdrawal, it will leave a faint fluorescent signal behind. This signal is more than 1 order of magnitude dimmer than that of a complete bilayer, but it indicates that a small amount of lipid is left. Immediately before collapse into a bilayer, the monolayer likely begins to deposit a small

Figure 10. Overexposed fluorescent image of the interface between a BCD bilayer (bright) and a VR bilayer (dark). The BCD bilayer patch imaged here was dyed with Oregon Green, while the surrounding VR bilayer was dyed with Texas Red. Three distinct mixing sites (indicated by arrows) are observed to cross the moat region. The plumes are Oregon Green dye diffusing from the BCD bilayer to the VR bilayer. Scale bar ) 10 µm.

number of molecules in a disordered state on the substrate surface. These molecules may passivate the surface, making it less favorable for more lipids to adhere. When the surface is then exposed to a vesicle solution, the vesicles will adsorb less onto this boundary region.19,20 When the vesicles then rupture and fuse to form a bilayer, there will be a gap region between the VR bilayer and the BCD bilayer. This result is perhaps not surprising, as the VR technique depends critically on the adhesion potential between the bilayer and substrate,7,22 which may be disrupted by a disordered population of adsorbed lipids. Unlike VR, BCD is not sensitive to the prior presence of disordered lipid molecules. The high energy air/water interface of the bubble is capable of removing lipid molecules from a surface and ordering them to form a monolayer. It is therefore not surprising that, if the moat region is comprised of a disordered layer of lipids adsorbed on the substrate, the air bubble would be able to remove these lipids and incorporate them into a subsequently deposited bilayer. Thus, although vesicle rupture failed in the region immediately surrounding a bubble-deposited bilayer, it was possible to make a fluid bilayer in that region by depositing another bubble-deposited patch. In fact, this moat region was not an absolute boundary. Mixing did occur, but it occurred only at a few highly localized regions around the perimeter. When an interface similar to that in Figure 7B was imaged at a higher excitation intensity, individual points

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of mixing spaced at the interface were seen. Figure 10 shows this mixing at such an interface. The BCD bilayer (lower right) was dyed with 1% Oregon Green and thus appears bright, while the surrounding VR bilayer (upper left) was dyed with Texas Red and appears dark in this filter set. The plumes, indicated by dark arrows, are points of mixing where Oregon Green is diffusing from the BCD bilayer into the VR bilayer. Some areas along the perimeter had higher densities of these contact points, and, through those regions, the composition of the circular patches equilibrated with that of the surrounding bilayer over a period of tens of hours.

surrounding substrate. Areas surrounding the bilayer deposited with this technique can be backfilled by vesicle rupture, and the resulting interface is stable for tens of hours. This stability is due to the failure of vesicle rupture to create a fluid bilayer near the interface. If, instead, another bubble-deposited bilayer is placed overlapping the first, a fluid and defect-free interface is produced and the contents of the two patches will begin mixing immediately. This ability to allow or prevent mixing between adjacent areas extends the versatility of the corral concept for supported lipid bilayers.

Conclusions

Acknowledgment. The authors thank Jason Fabbri for assistance with Langmuir-Blodgett measurements and Michael Preiner for help with numerical fitting routines. Financial support was provided by Arrowhead Research and the National Defense Science and Engineering Graduate Fellowship. Processing was performed with the support of the Stanford Nanofabrication Facility.

We have demonstrated a new method of patterning lipid bilayers on solid surfaces. This new technique is mechanistically similar to Langmuir-Blodgett deposition, but it offers several important advantages. Multiple bilayer compositions can be deposited on the same substrate with spatial selectivity. Deposition of the outer leaflet occurs at or near the collapse pressure of the monolayer, which has been shown to increase bilayer stability and decrease defect density.39,40 Also, bilayers can be deposited and removed from the same site many times without the need for harsh chemical treatments in between. This ability has important implications for microfluidic-based automated biosensors. As a patterning method, this technique is a simple single-step process requiring no chemical or photolytic modification of the

Supporting Information Available: Images and description of the source bilayer; images of an air bubble rapidly adsorbing a lipid monolayer; graph of solutions to the derivation of lipid monolayer pressure; and image of a bilayer that has been deposited on the site of a previously removed bilayer. This material is available free of charge via the Internet at http://pubs.acs.org. LA701372B