Formation of Supported Lipid Bilayer Composition Arrays by Controlled Mixing and Surface Capture

Virtually all structures in living cells are defined by lipid bilayers. Many molecular components can diffuse along these 2-dimensional structures, sampling all or part of the membrane, and this diffusion is central to their function. Supported lipid bilayers capture this membrane fluidity and have been used to study a wide range of membrane-associated biomolecules.1-4 Recently, several techniques have been developed to microfabricate barriers on glass substrates to direct lipid bilayer selfassembly, facilitating the patterning of multiple, isolated membrane regions, or corrals, on a single substrate.5-12 The resultant corrals are typically of identical composition; however, a handful of strategies, including spatially directed photochemical transformations11 and membrane stamping,8,13 can be used to produce bilayer arrays with specific composition variations in different corrals. These composition arrays are desirable for investigating numerous biological systems, including how lipid and/or protein composition affects binding of membrane-associated receptors with their ligands or each other, cellular processes such as intracellular protein modification and trafficking, and cell activation. In this communication, we introduce a robust approach that, for a wide variety of situations, greatly simplifies the production of large-scale parallel bilayer arrays with defined variations in composition. The method, outlined in Figure 1, is based on exposing a prepatterned surface to a gradient of vesicles of various compositions created by flow and taking advantage of irreversible binding of vesicles to a substrate, subsequent fusion into a bilayer, and lipid mixing within each corralled region. For demonstration purposes, we fabricated a simple, millimeter-scale, converging channel out of poly(dimethylsiloxane) (PDMS, Sylgard 184; Dow Corning) elastomer (Figure 1A). One wall of the channel consisted of a glass coverslip containing microcontact-printed9,10,14-16 grid lines of fibronectin;17 however, any of a variety of materials can be used to create these barriers.5,8,10,12 The elastomer channel was filled with water, and then two solutions of vesicles were manually pumped through the channel as illustrated in Figure 1B. For visualization purposes, each solution contained vesicles supplemented with a small amount of a different fluorescently labeled lipid;18 conceptually, this technique is compatible with any biomolecule that can be incorporated into lipid bilayers by vesicle fusion. Downstream of the convergence, the two solutions partially mix by diffusion,19 exposing the micropatterned array to a controllable, spatially defined gradient of the two types of vesicles. The vesicles are irreversibly captured on the micropatterned coverslip, subsequently rupturing and fusing into a continuous supported lipid bilayer.3,4,21 The local composition of the bilayer reflects the distribution of vesicles exposed to the surface. Because the bilayer is a 2-dimensional fluid, any nonuniformity in membrane composition will be erased by lateral diffusion.10,11 The presence of barriers, however, limits this mixing to defined * Address correspondence to this author. Phone: (650) 723-4482. Fax: (650) 723-4817. E-mail: Sboxer@Stanford.edu. (1) Dori, Y. ; Bianco-Peled, H. ; Satija, S. K. ; Fields, G. B. ; McCarthy, J. B. ; Tirrell, M. J. Biomed. Mater. Res. 2000, 50, 75-81. (2) Grakoui, A. ; Bromley, S. K. ; Sumen, C. ; Davis, M. M. ; Shaw, A. S. ; Allen, P. M. ; Dustin, M. L. Science 1999, 285, 221-7. (3) McConnell, H. M. ; Watts, T. H. ; Weis, R. M. ; Brian, A. A. Biochim. Biophys. Acta 1986, 864, 95-106. (4) Sackmann, E. Science 1996, 271, 43-8. (5) Cremer, P. S. ; Yang, T. L. J. Am. Chem. Soc. 1999, 121, 8130-1. (6) Groves, J. T. ; Ulman, N. ; Boxer, S. G. Science 1997, 275, 651-3. (7) Cremer, P. S. ; Groves, J. T. ; Kung, L. A. ; Boxer, S. G. Langmuir 1999, 15, 3893-6. (8) Hovis, J. S. ; Boxer, S. G. Langmuir 2000, 16, 894-7. (9) Kam, L. ; Boxer, S. G. J. Biomed. Mater. Res. Accepted for publication. (10) Kung, L. A. ; Kam, L. ; Hovis, J. S. ; Boxer, S. G. Langmuir 2000, 16, 6773-6. (11) Kung, L. A. ; Groves, J. T. ; Ulman, N. ; Boxer, S. G. AdV. Mater. 2000, 12, 731-4. (12) van Oudenaarden, A. ; Boxer, S. G. Science 1999, 285, 1046-8. (13) Hovis, J. S. ; Boxer, S. G. In preparation. (14) Kumar, A. ; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 4. (15) Bernard, A. ; Delamarche, E. ; Schmid, H. ; Michel, B. ; Bosshard, H. R. ; Biebuyck, H. Langmuir 1998, 14, 2225-9. (16) James, C. D. ; Davis, R. ; Meyer, M. ; Turner, A. ; Turner, S. ; Withers, G. ; Kam, L. ; Banker, G. ; Craighead, H. ; Isaacson, M. ; Turner, J.; Shain, W. IEEE Trans. Biomed. Eng. 2000, 47, 17-21. (17) The use of proteins as barrier materials can confer additional biological activity to patterned lipid bilayer surfaces; we have used fibronectin barriers to promote adhesion of anchorage-dependent cells onto lipid bilayer substrates, directing the interaction of these cells with the membrane patches.9 Here, fibronectin was labeled with Cascade Blue (Molecular Probes) to facilitate visualization of the micropatterned barriers. (18) Small unilamellar vesicles (SUVs) were prepared from dilauroylphosphatidylcholine (DLPC.; Avanti) supplemented with either 1 mol % of TexasRed DHPE (TR-DHPE.; Molecular Probes), 2 mol % of NBD-DHPE (Avanti), or 0.5 mol % of DiD (Molecular Probes). Lipids were reconstituted in water (5 mg lipids/mL) then extruded10 through 50-nm-pore membranes (Avanti). These stock solutions were diluted 1:3 (v/v) in aqueous buffer (pH 7.0) prior to use. Images of fluorescently labeled lipids and proteins were collected using appropriate filter sets, and are presented in false color as indicated in the figure legends. Figure 1. (A) Schematic illustration of the converging flow configuration used to produce limited mixing of two types of vesicles in solution. Two layers of poly(dimethylsiloxane) elastomer were cut using a template, oxidized in an air plasma, and layered to produce three walls of the converging channel (the channel downstream of the convergence measured 1 × 5 × 10 mm3). A glass coverslip patterned with an array of corrals17 comprised the fourth wall of the channel. (B) Schematic diagram illustrating controlled mixing and surface capture onto a substrate subjected to the flow shown in panel A. The local composition of a supported lipid bilayer formed by vesicle fusion reflects the makeup and spatial distribution of vesicles (denoted here as red and green dots) that are flowed past a pre-patterned surface. This compositional profile is spatially averaged as the components within each corral diffuse and mix. 12901 J. Am. Chem. Soc. 2000, 122, 12901-12902