Langmuir 2001, 17, 7951-7954
Patterned Supported Bilayers on Self-Assembled Monolayers: Confinement of Adjacent Mobile Bilayers M. P. Srinivasan,† T. V. Ratto, P. Stroeve, and M. L. Longo* Center on Polymer Interfaces and Macromolecular Assemblies, Department of Chemical Engineering and Materials Science, University of California, Davis, California 95616 Received May 25, 2001. In Final Form: September 18, 2001
Introduction Adsorption of lipid bilayers on surfaces has attracted much interest because of the possibility of constructing biomembrane-based sensors1 and for investigating fundamental biomembrane properties.2 To retain the fluid nature of the lipid which is important for cellular functions,3 various materials have been used to support the lipid bilayer and thereby minimize the effect of the solid substrate. Flexible polymer layers and polymer cushions,4-6 self-assembled monolayers (SAMs), and SAMsupported polycationic layers have served as supports for lipid bilayers.7-12 In addition to retention of fluidity, it is advantageous to confine the bilayer to prespecified areas on the surface in order to study multiple analyte-specific interactions. Partitioning of the fluid bilayer, so as to retain spatial information and control spatial geometry, has been accomplished by employing barriers to lipid diffusion such as metal lines and proteins that are deposited as patterns on the solid surface.13,14 Solid patterned lines and selective immobilization of lipids have also been employed as barriers to diffusion.15 Recently, a blotting and stamping method that exploits the self-limiting lateral expansion of the lipid bilayer and avoids the use of a second material on the solid surface has been developed.16 These methods * To whom correspondence should be addressed: Marjorie Longo, Department of Chemical Engineering and Materials Science, University of California, Davis, CA 965616. E-mail: mllongo@ ucdavis.edu. † On leave from The National University of Singapore, Singapore. (1) Lahiri, J.; Kalal, P.; Frutos, A. G.; Jonas, S. J.; Schaeffler, R. Langmuir 2000, 16, 7805-7810. (2) Sackmann, E. Science 1996, 271, 43-48. (3) Sen, A.; Ghosh, P. K.; Mukherjea, M. Mol. Cell. Biochem. 1998, 187, 183-190. (4) Knoll, W.; Frank, C. W.; Heibel, C.; Naumann, R.; Offenhauser, A.; Ruhe, J.; Schmidt, E. K.; Shen, W. W.; Sinner, A. Rev. Mol. Biotech. 2000, 74, 137-158. (5) Schmitt, J.; Danner, B.; Bayerl, T. M. Langmuir 2001, 17, 244246. (6) Wagner, M. L.; Tamm, L. K. Biophys. J. 2000, 79, 1400-1414. (7) Zhang, L. Q.; Booth, C. A., Stroeve, P. J. Colloid Interface Sci. 2000, 228, 82-89. (8) Zhang, L. Q.; Longo, M. L.; Stroeve, P. Langmuir 2000, 16, 50935099. (9) Schouten, S.; Stroeve, P.; Longo, M. L. Langmuir 1999, 15, 81338139. (10) Duschl, C.; Liley, M.; Coradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229-1237. (11) Plant, A. L. Langmuir 1993, 9, 2764-2767. (12) Cassier, T.; Sinner, A.; Offenhausser, A.; Mohwald, H. Colloids Surf., B 1999, 15, 215-225. (13) Kung, L. A.; Groves, J. T.; Ulman, N.; Boxer, S. G. Adv. Mater. 2000, 12, 731-734. (14) Kung, L. A.; Kam, L.; Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 6773-6776. (15) Groves, J. T.; Ulman, N.; Cremer, P. S.; Boxer, S. G. Langmuir 1998, 14, 3347-3350. (16) Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 894-897.
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enable production of bilayer arrays with variations in composition.17 Jenkins et al. have used a patterned array of SAMs as supports for phospholipid bilayers.18-20 Using surface plasmon microscopy, they investigated the adsorption of egg-phosphatidylcholine on a patterned array of mercaptoethanol patches surrounded by octadecanethiol SAM. They show that the rate of adsorption of the lipid on mercaptoethanol is higher than that on octadecanethiol. In this work, we have employed patterned SAMs as supports for two dissimilar lipid bilayers with the objective of investigating the feasibility of containing a bilayer within specified regions (but adjacent to a dissimilar lipid) by virtue of differences in the nature of the supporting thiol and of the bilayer. The pattern was created in the form of squares separated by grid lines. We show by fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) that mobility for the lipids is restricted within the patterned region due to the confinement of the lipids to their respective thiol SAM supports. The boundaries to mobility are sustained when a second, different lipid bilayer is deposited on a second thiol (different from the stamped thiol) that is in turn chemisorbed on the remaining bare gold surface of the stamped substrate. Experimental Section The thiols 2-mercaptoethylamine, or cysteamine (CA, Sigma, St. Louis, MO), and mercaptoundecanoic acid (MUA, Sigma) were used as the materials for self-assembly on the gold surface. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-stearoyl2-oleoyl-phosphatidylserine (SOPS) were used as the lipids (both from Avanti Polar Lipids, Alabaster, AL). 1-Palmitoyl-2-[6-[(7nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoserine (NBD-PS), 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphocholine (NBDPC) (both from Avanti), (Texas Red)-1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine triethylammonium salt (Texas Red-DHPE), and 4-(4-(didecyl-amino)styryl)-N-methylpyridinium iodide (4-Di-10-Asp) (both from Molecular Probes, Eugene OR) were used as the fluorescent probes. Tris-(hydroxymethyl) aminomethane (tris, 99.9%, Sigma) at pH ) 8.5 was used as the buffer. Microcontact printing of thiols was carried out as described in the literature.21 Poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning, Midland, MI) was patterned by pouring liquid PDMS on to transmission electron microscope (TEM) grids (SPI, West Chester, PA). After curing, the PDMS was peeled off the grids so that the pattern of the TEM grid was transferred to the PDMS. TEM grids of 100 and 200 mesh sizes (208 µm and 97 µm openings, respectively) were used to make the patterns. CA and MUA were made as 5 mM solutions in ethanol, and the PDMS stamps were inked with one of the ethanol solutions. The excess ethanol solution was removed by gently blowing a stream of nitrogen over the stamp. The inked stamps were pressed gently on the surface of gold-coated (50 nm thick) microscope glass slides that were previously coated with 3 nm of titanium to improve adhesion of gold. For experiments where the second lipid was to be deposited, the slide was subsequently soaked in (17) Kam, L.; Boxer, S. G. J. Am. Chem. Soc. 2000, 122, 1290112902. (18) Jenkins, A. T.; Bushby, R. J.; Boden, N.; Evans, S. D.; Knowles, P. F.; Liu, Q.; Miles, R. E.; Ogier, S. D. Langmuir 1998, 14, 4675-4678. (19) Jenkins, A. T.; Boden, N.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D.; Schonherr, H.; Vancso, G. J. J. Am. Chem. Soc. 1999, 121, 5274-5280. (20) Jenkins, A. T.; Neumann, T.; Offenhausser, A. Langmuir 2001, 17, 265-267. (21) Kumar, A.; Whitesides, G. M. Science 1994, 263, 60-62.
10.1021/la010776t CCC: $20.00 © 2001 American Chemical Society Published on Web 11/09/2001
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Figure 1. FRAP images of SOPS (2% NBD-PS) deposited on CA-stamped pattern: (a) fluorescence image before bleaching; (b) image after bleaching for 10 min; (c) image after recovery period of 60 min. 5 mM solution of the second thiol solution and then rinsed with ethanol and water. Typically, CA was stamped on the gold surface to form the discrete squares and MUA was chemisorbed on to the continuous grid lines. Lipid vesicle solutions were prepared by transferring 100 µL of the lipid (in chloroform) and a fluorescent probe (NBD-PS, NBD-PC, Texas Red-DHPE, or 4-Di-10-Asp) to the extent of 2% (mol) to a vial, and evaporating the chloroform in a gentle stream of N2. Two milliliters of buffer solution was then added to the vial (lipid concentration of 0.5 mg/mL), and the contents of the vial were warmed to 50 °C, vortexed, and sonicated at 20% output in continuous mode for 3 min intervals with a tip sonifier (Branson Sonifier Model 250, Danbury, CT) until the solution was clear. The vesicle solutions were deposited on the SAM-patterned gold-coated glass slides and allowed to stand for 6 h. The vesicle solutions were then washed with Tris and the lipid bilayer was contained in a Tris environment by sandwiching the buffer solution (in contact with the bilayer) between the gold slide and a cover slip using a PDMS spacer. The sandwiched assembly was used for the fluorescence bleaching experiments. A Nikon Diaphot 300 fluorescence microscope was used to examine the sandwiched assembly. For FRAP experiments, the NBD-labeled lipid was excited by exposure to blue light filtered from a 75 W xenon lamp for a period of 10 min. The shutter was closed at the end of the bleaching step and the observed fluorescence was recorded digitally at periodic intervals during the recovery stage. Bleaching was performed with a 40× lens and imaging was done with a 10× lens. Image-processing software (Scion Image, Scion Corp., Frederick, MD) was used to analyze the images and obtain information about recovery rates. For general imaging of NBD and 4-Di-10-Asp, the dyes were excited by blue light, and for imaging of Texas-red, green light was used.
Results and Discussion Figure 1 shows the sequence of FRAP images obtained for the case of SOPS containing NBD-PS deposited on CA. The CA was stamped on the gold slide as discrete squares and SOPS was subsequently deposited on the gold slide. Since the CA monolayer has a positive terminal charge, the anionic lipid bilayer fused preferentially on the CA-stamped surfaces, thus defining the pattern. Figure 1a shows the observed pattern prior to bleaching. One of the squares is completely dark at the end of the
Notes
Figure 2. FRAP images of SOPS (2% NBD-PS) deposited on CA-stamped pattern during recovery: (a) image at the end of bleaching; (b) image after 30 min of recovery; (c) image after 80 min of recovery.
bleaching period (Figure 1b) while the peripheral squares are partially bleached. Figure 1c shows the pattern 60 min after the end of the bleaching period. The figure shows complete mixing in the case of the peripheral squares as evidenced by the even brightness in those squares and no recovery in the case of the completely bleached square. This provides evidence that the lipids within each square are isolated from its neighboring squares and demonstrates the possibility of confining the lipid to a predetermined area in the absence of extrinsic barriers. The boundaries of the supporting SAM serve as self-limiting barriers to the diffusion of the lipid, and therefore expansion of the bilayer does not occur as observed on glass supports;22,23 this ensures the integrity of the pattern. Figure 2 shows the sequence of FRAP images obtained during the recovery stage for a bleached spot near the edge of a stamped square. The edge of the CA-stamped square is recovered intact. The extent of recovery was quantified by monitoring the fluorescence intensity of the exposed region normalized with the intensity of the unexposed region. The exposed region regained 80% of its fluorescence intensity at the end of 80 min. We then confined a zwitterionic lipid, POPC (doped with the cationic probe 4-Di-10-Asp), to the continuous grid lines by stamping CA onto the gold surface followed by chemisorption of MUA into the grid lines. Deposition of POPC onto the patterned surface resulted in confinement of the POPC to the continuous grid lines (Figure 3). Presumably, the positive charge of the probe played a role in preventing POPC from depositing on the CA-covered gold surface. Deposition of a second lipid onto a surface patterned with both CA and MUA was accomplished by deposition of SOPS followed by POPC on the pattern. When the two lipids were deposited with different fluorescent probes (Texas Red for SOPS and NBD for POPC) and the slide was imaged, it was possible to observe the green POPC regions (grid-line region in Figure 4a) and the red SOPS regions (squares in Figure 4b). The figure shows that in general, the boundaries between CA/SOPS regions and (22) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 25542559. (23) Hovis, J. S.; Boxer, S. G. Langmuir 2001, 17, 3400-3405.
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Figure 3. Fluorescent image showing POPC (channel region) deposited on MUA. The square regions were stamped with the cationic thiol, CA, which presumably repels the POPC bilayer which is doped with a cationic probe (4-Di-10-Asp). Note that a square is absent in the bottom right corner due to a defect in the PDMS stamp. Figure 5. Recovery data plotted as comparative intensity versus time for POPC on MUA-deposited regions and SOPS on CA-stamped regions. The relative intensity is calculated as IPOPC/ISOPS.
Figure 6. Recovery of (bleached) POPC on MUA-deposited Au substrate and SOPS on CA-deposited Au substrate. Figure 4. Fluorescent images showing the POPC (channel regions, a) and SOPS (squares, b) areas on the gold slide. NBDPC and Texas Red were used as fluorescent probes for POPC and SOPS, respectively. Panel a was obtained using blue light and panel b was obtained using green light. Spots of red fluorescence in the grid lines (b) can be explained by vapor phase deposition of CA during the stamping procedure as explained in the text.
MUA/POPC regions are intact when both lipid bilayers are present. These boundaries do not degrade for at least 24 h (longer times were not studied). In this case, we believe that mixing of the two lipids is mainly prevented by the repulsion between SOPS and MUA that retains SOPS on the CA monolayer. As has been discussed by Xia and Whitesides,24 we see evidence of transport of the thiol from the stamp to the surface in the noncontact regions through the vapor phase. This results in some sporadic degradation of the pattern where CA has deposited either diffusely or in spots in the grid lines. Subsequently, when SOPS and POPC are deposited, we observe sporadic regions of diffuse red fluorescence or spots (see Figure 4b) associated with SOPS within the continuous grid lines. These spots of red fluorescence are maintained for at least 24 h, again indicating that mixing does not occur between (24) Xia, Y.; Whitesides, G. M. Annu Rev. Mater. Sci. 1998, 28, 153184.
SOPS (confined by CA) and POPC. This observed sporadic pattern degradation could probably be almost completely avoided by replacing hand stamping, as performed here, by a more mechanical method in which the contact time and pressure could be more carefully controlled. The mobility of SOPS on CA residing on the squares relative to that of POPC on MUA on the grid lines was investigated. NBD-PS and NBD-PC were used as the fluorescent probes for SOPS and POPC, respectively. This was accomplished by focusing the incident radiation at a square grid line boundary that bleached part of a square and also the adjacent grid line. The fluorescence intensity for each region was monitored during the recovery period. Figure 5 shows a plot of relative fluorescence intensity obtained as the ratio of normalized intensities (IPOPC/ISOPS) as a function of time. SOPS recovers faster than does POPC and accounts for the decrease in relative intensity. The relative intensity increases at larger times as POPC now starts to show significant recovery. The difference in mobility between POPC and SOPS was independently verified by conducting FRAP experiments on the bilayers supported on the respective SAMs on different gold slides in the absence of stamping. The faster recovery observed in the case of SOPS (Figure 6) is consistent with the inference drawn from Figure 5. Diffusion coefficients obtained from the recovery curves
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by the method of Axelrod et al.25 were found to be 2.2 × 10-8 and 7.6 × 10-9 cm2/s, for SOPS and POPC, respectively. These values are consistent with measured diffusivities of bilayers on glass.23,26 Among the factors that may contribute to the difference in diffusivity are the inherent difference between the fluorescent markers and their charges, the thickness of the SAMs, or the water layer between the SAM and the bilayer. Thus, the difference in mobility between the dissimilar lipids in adjacent, confined regions, together with the retention of individual mobility properties of the lipids whether they are by themselves or adjacent to each other, has been used to show that, by varying the nature of the support, adjacent areas can be covered with dissimilar lipids while retaining the integrity of the boundary. This presents clear evidence that the demarcation provided by the dissimilar SAMs coupled with the inherent charge on the lipids is sufficient to contain the lipid bilayers to their respective regions. (25) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055-1069. (26) Yang, T.; Simanek, E. E.; Cremer, P. Anal. Chem. 2000, 72, 2587-2589.
Notes
Conclusions We have shown that lipid bilayers can be confined to prespecified regions by employing patterned SAMs as supports. The edge of the thiol support serves as a selflimiting boundary for the bilayer and enables deposition of dissimilar lipid bilayers on different thiol supports adjacent to each other. Some pattern degradation that we observe here is due to chemisorption of thiols from the vapor phase that could probably be virtually avoided by improved stamping technique. The observed difference in the mobilities of the dissimilar lipids deposited on adjacent thiol supports is consistent with measured diffusion coefficients and is indicative of the integrity of the boundary between the two regions. The self-limiting feature of the extent of bilayer occupancy on the thiol support suggests that it is feasible to reduce feature size without loss of discrimination. Acknowledgment. This work was supported by the MRSEC program of the National Science Foundation under Award DMR-9808677. M.L.L. acknowledges support by the Whitaker Foundation (RG-98-0276) and the NSF through the Career Program (BES-9733764). LA010776T
