Langmuir 2003, 19, 8041-8049
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Nanoscale Stripe Patterns in Phospholipid Bilayers Formed by the Langmuir-Blodgett Technique Patricia Moraille and Antonella Badia* Department of Chemistry, Universite´ de Montre´ al, C.P. 6128, succursale Centre-ville, Montre´ al QC H3C 3J7, Canada Received April 29, 2003. In Final Form: June 8, 2003 A new methodology has been developed to create an extensive pattern of parallel stripes, ∼150-250 nm wide, in phospholipid bilayers supported on mica. These striped bilayers are prepared by the LangmuirBlodgett (LB) film technique. A striped monolayer consisting of two phospholipids in different states (condensed and liquid-expanded) is used to direct the deposition of the solid- and liquidlike phases of a second mixed monolayer during LB transfer. We also demonstrate that bilayer stripes can be generated by the condensation of phospholipids over the solidlike stripe domains of the underlying monolayer for a one-component film deposited just below the liquid-expanded-to-liquid-condensed phase transition pressure. Nonionic detergent extraction of the liquidlike phase from these LB films resulted in bilayerthick phospholipid stripes separated by a mica surface. A periodic array of grooves was produced by the selective adsorption of protein onto the mica regions of the detergent-treated bilayer. The LB film deposition of binary mixtures of solid-phase- and fluid-phase-forming phospholipids constitutes a novel strategy to create linear surface patterns that can be used to direct the deposition of molecules.
Introduction Recent examples have shown the potential of the Langmuir-Blodgett (LB) technique1,2 as an efficient and parallel process for preparing surface templates and masks that are chemically or physically differentiated on the micron to submicron scale.3-13 Laterally structured LB films14 are usually generated by the deposition onto solid substrates of regular 2D patterns formed at the air/water (A/W) interface by the phase separation or demixing of amphiphilic organic molecules. The dynamic wetting instabilities created under certain conditions during the vertical transfer process can also be used to produce regularly structured LB films from a homogeneous precursor monolayer8-11 or a phase-separated Langmuir monolayer that does not exhibit periodic lateral structures.12,13 In these cases, the controlled manipulation of the LB transfer parameters (transfer speed, temperature, surface pressure) should open new opportunities for generating extensively patterned surfaces, using different substrates and adsorbates, that are not based on selfassembly or lithography processes. * Corresponding author. E-mail:
[email protected]. (1) Blodgett, K. B. J. Am. Chem. Soc. 1934, 56, 495. (2) Blodgett, K. B. J. Am. Chem. Soc. 1935, 57, 1007-1022. (3) Duschl, C.; Liley, M.; Vogel, H. Angew. Chem., Int. Ed. Engl. 1994, 33, 1274-1276. (4) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229-1237. (5) Goren, M.; Lennox, R. B. Nano Lett. 2001, 1, 735-738. (6) Meli, M.-V.; Badia, A.; Gru¨tter, P.; Lennox, R. B. Nano Lett. 2002, 2, 131-135. (7) Fang, J.; Knobler, C. M. Langmuir 1996, 12, 1368-1374. (8) Mahnke, J.; Vollhardt, D.; Sto¨ckelhuber, K. W.; Meine, K.; Schulze, H. J. Langmuir 1999, 15, 8220-8224. (9) Gleiche, M.; Chi, L. F.; Fuchs, H. Nature 2000, 403, 173-175. (10) Gleiche, M.; Chi, L.; Gedig, E.; Fuchs, H. ChemPhysChem 2001, 3, 187-191. (11) Lu, N.; Gleiche, M.; Zheng, J.; Lenhert, S.; Xu, B.; Chi, L.; Fuchs, H. Adv. Mater. 2002, 14, 1812-1815. (12) Moraille, P.; Badia, A. Langmuir 2002, 18, 4414-4419. (13) Moraille, P.; Badia, A. Angew. Chem., Int. Ed. 2002, 41, 43034306. (14) Motschmann, H.; Mo¨hwald, H. In Handbook of Applied Surface and Colloid Chemistry; Holmberg, K., Ed.; John Wiley & Sons: Chichester, U.K., 2001; pp 629-648.
Gleiche and co-workers9,11 have demonstrated that regularly spaced parallel stripes (∼800 nm wide and ∼2 nm thick) and channels (∼200 nm wide) can be generated by the rapid transfer of a L-R-dipalmitoylphosphatidylcholine (L-R-DPPC) monolayer at surface pressures near (below) the liquid-expanded (LE)-to-liquid-condensed (LC) phase transition pressure. The stripes and channels (deposition-free gaps) result from periodic oscillations of the contact angle and meniscus height of the water subphase on the substrate. These wetting instabilities are caused by substrate-mediated condensation at the three-phase contact line (i.e., substrate/water subphase/ air), a process whereby the phospholipid monolayer is deposited onto the solid surface in a more condensed state (LC phase) compared to its state on the water surface (LE phase) due to attractive substrate/monolayer interactions.15-18 The reduced surface energy of the surfacecondensed phospholipid leads to an increase in the contact angle18 and, consequently, a decrease in the meniscus height of the water subphase on the solid substrate. Due to the continuous upward motion of the substrate, the meniscus height will however tend to exceed its equilibrium value, resulting in an accelerated adsorption on the substrate (noncondensing mode). Depending on the deposition speed used, alternating stripes of LC- and LE-rich phases15 or LC stripes and deposition-free gaps9 result. We recently showed that parallel stripes (∼100-200 nm in width) can be generated by the LB transfer of a mixed phospholipid monolayer from the A/W interface onto a mica substrate.12 In this case, the stripe pattern is composed of two saturated dialkylphosphatidylcholines, L-R-DPPC and L-R-dilauroylphosphatidylcholine (L-RDLPC), in different phases (Figure 1A). As in the stripes and channels formed from the single-component DPPC monolayer, the stripes generated from the DPPC/DLPC mixture are aligned perpendicular to the direction of (15) Spratte, K.; Chi, L. F.; Riegler, H. Europhys. Lett. 1994, 25, 211-217. (16) Spratte, K.; Riegler, H. Langmuir 1994, 10, 3161-3173. (17) Riegler, H.; Spratte, K. Thin Solid Films 1992, 210/211, 9-12. (18) Graf, K.; Riegler, H. Colloids Surf., A 1998, 131, 215-224.
10.1021/la034727n CCC: $25.00 © 2003 American Chemical Society Published on Web 08/08/2003
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Figure 1. Schematic representation of the phospholipid films supported on mica prepared for this study: (A) mixed DPPC/ DLPC monolayer in air; (B) symmetric DPPC/DLPC bilayer in water.
substrate withdrawal from the A/W interface (i.e., parallel to the three-phase contact line). By contrast, the DPPC/ DLPC stripe pattern is formed through a cyclical condensed-phase nucleation and depletion process that is coupled to dynamic wetting instabilities.12 Since stripe formation in the binary phospholipid system is not the result of a substrate-induced phase transition,9-11,15 the resulting pattern should be broadly accessible in terms of surface pressures, deposition speeds, and lipids. Although these DPPC/DLPC stripes have been used to generate well-defined protein and Au nanoparticle/protein patterns,13 the limited stability of solid-supported phospholipid monolayers in water and organic solvents restricts their application as surface templates. Furthermore, the orientation of the phospholipids in the LB monolayer (i.e., phosphocholine headgroups adsorbed on the substrate surface and lipid alkyl chains extended from the surface) excludes the possibility of using interactions with headgroup-functionalized phospholipids to fabricate novel biomolecular architectures. These limitations can, in principle, be circumvented by using a bilayer geometry (Figure 1B) which is stable in an aqueous environment. Herein, we report the conditions for preparing regular stripe patterns in phospholipid bilayers. The mechanism of stripe formation in these bilayer LB films differs from that involved in the formation of periodic ripples in twocomponent phospholipid bilayers19,20 and has the potential of being widely applicable. Experimental Section Materials. L-R-DPPC and L-R-DLPC were obtained as powders from Avanti Polar Lipids (Alabaster, AL) and used without further purification (chemical purity > 99%). Human-γ-globulins (from Cohn fraction II, III, 99%) and Triton X-100 (Ultrapure) were purchased from Sigma Chemical Co. (St. Louis, MO). Ruby muscovite mica (ASTM Grade 2) was from B&M Mica Co., Inc. (Flushing, NY). The high-purity water (18.2 MΩ cm) used for all experiments was prepared by passing water purified by reverse osmosis through a Milli-Q Gradient System (Millipore, Bedford, MA). Its surface tension measured at 22 °C was 72.1 mN m-1. Film Deposition. DPPC, DLPC, and DPPC/DLPC solutions were prepared using spectrograde chloroform to give a total phospholipid concentration of 1 mM. DPPC, DLPC, and mixed DPPC/DLPC monolayers were formed by spreading 100 µL of the appropriate solution on the water surface (area ) 768 cm2) of a KSV 3000 Langmuir-Blodgett trough equipped with a Wilhelmy plate sensing device (KSV Instruments, Helsinki, Finland). The subphase temperature was maintained at 20 ( 0.5 °C using an Isotemp 1006D circulation bath (Fisher Scientific). (19) Czajkowsky, D. M.; Huang, C.; Shao, Z. Biochemistry 1995, 34, 12501-12505. (20) Leidy, C.; Kaasgaard, T.; Crowe, J. H.; Mouritsen, O. G.; Jørgensen, K. Biophys. J. 2002, 83, 2625-2633.
Moraille and Badia Following solvent evaporation (15 min), the Langmuir monolayers were symmetrically compressed at a rate of 1 Å2 molecule-1 min-1 up to a deposition pressure of 25 mN m-1, unless otherwise mentioned. Monolayer films were deposited onto mica at a constant surface pressure by pulling the substrate upward through the A/W interface (upstroke) at 5 mm/min. Bilayer films were prepared by vertically lowering a phospholipid-monolayercovered mica substrate (freshly prepared) through a second monolayer film floating at the A/W interface and into the aqueous subphase (downstroke) at a surface pressure of 25 mN m-1. Bilayers were transferred under water to the liquid cell used for atomic force microscopy (AFM) imaging. Subsequent exposure of the bilayer films to air was avoided. Detergent Extraction. After AFM imaging, the bilayer temperature was lowered to 4 °C (measured with a thermocouple) by leaving the AFM liquid cell in the freezer for ∼10 min. The bilayer was then removed from the freezer, and ∼1 mL of an aqueous 1% v/v Triton X-100 solution (cooled to 4 °C before use) was immediately injected into the liquid cell which already contained ∼1 mL of water. The bilayer was left to incubate in Triton X-100 for 1 min at room temperature before rinsing with 4-5 times the cell volume (∼2 mL) with Milli-Q water.21 The sample was imaged at room temperature by AFM. Protein Adsorption. A 0.1 mg mL-1 human-γ-globulin (HGG) solution was prepared in Milli-Q water and centrifuged for 10 min at 14 000 rpm and 4 °C to remove protein aggregates. The bilayer (in the AFM liquid cell) was exposed to the HGG solution for 10-15 min at room temperature and rinsed with Milli-Q water before AFM imaging. AFM Imaging. The mica-supported DPPC/DLPC films were imaged at room temperature using tapping mode. Monolayers were imaged in air (Digital Instruments MultiMode microscope, Santa Barbara, CA) with etched silicon cantilevers of resonance frequency ∼300 kHz and spring constant ∼42 N m-1 (Olympus). Bilayers were imaged in Milli-Q water (Digital Instruments Dimension 3100 microscope) using sharpened silicon nitride microlevers (Veeco/TM Microscopes, Sunnyvale, CA) with a nominal spring constant of ∼0.1 N m-1 and a resonance frequency of ∼8 kHz in liquid. For each type of bilayer, images were obtained from two or three independently prepared samples, and several macroscopically separated areas were imaged on each sample. Representative images are presented.
Results and Discussion Striped Monolayers. All nanostructured bilayers were prepared using a mixed DPPC/DLPC monolayer as the lower layer. DPPC/DLPC films were transferred onto freshly cleaved mica at surface pressures of g15 mN m-1, where DPPC is in the condensed phase (either liquid- or solid-condensed)22,23 and DLPC is in the liquid-expanded or fluid state.12 A mean transfer ratio of 1.10 ( 0.06 was obtained for the deposition of mixed DPPC/DLPC monolayers onto mica, indicative of a good-quality transfer. AFM images of these monolayer films are presented in Figure 2. Parallel stripes (continuous and broken) protrude from the surrounding matrix by 0.6-0.8 nm. The thicker stripes exhibit a more positive phase shift in tapping mode AFM (see Supporting Information).12,13 Since the medium oscillation damping conditions (20-30%) used here for imaging in air provide contrast that is predominantly based on differences in elasticity,24,25 the larger phase lag indicates that the stripes are stiffer than the background matrix. The area fraction covered by the stripes, the measured step-height differences, and the mechanical (21) Rinia, H. A.; Snel, M. M. E.; van der Eerden, J. P. J. M.; de Kruijff, B. FEBS Lett. 2001, 501, 92-96. (22) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105-113. (23) Hwang, J.; Tamm, L. K.; Bo¨hm, C.; Ramalingam, T. S.; Betzig, E.; Edidin, M. Science 1995, 270, 610-614. (24) Magonov, S. N.; Elings, V.; Whangbo, M. H. Surf. Sci. 1997, 375, L385-L391. (25) Bar, G.; Thomann, Y.; Whangbo, M.-H. Langmuir 1998, 14, 1219-1226.
Striped Phospholipid Bilayers
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Figure 2. AFM images (in air) and cross-section analyses of monolayers on mica: (A) 0.15:0.85 (mol/mol) DPPC/DLPC transferred at 32 mN m-1; (B) 0.25:0.75 DPPC/DLPC transferred at 25 mN m-1; (C) 0.50:0.50 DPPC/DLPC transferred at 15 mN m-1. The step heights and stripe widths are (A) 0.74 ( 0.10 nm and 111 ( 32 nm, (B) 0.56 ( 0.05 nm and 153 ( 25 nm, and (C) 0.84 ( 0.08 nm and 197 ( 41 nm.
responses of the domains observed by AFM are consistent with phase separation into condensed DPPC-rich stripes and a liquidlike (fluid) DLPC-rich background.12,13 The stripes extend over an area of at least 100 µm × 100 µm, the largest area that can be imaged by AFM. Such an extensive array of parallel lines or stripes is difficult to produce using self-ordering and phase-separation processes (serpentine domains usually result)26-28 or by nanolithography techniques. The stripes form through the alignment and fusion of circular DPPC domains at the contact line between the water subphase and mica substrate.12 Parallel stripes can be generated at surface pressures and/or lipid compositions where condensed domains that are hundreds of nanometers in diameter exist at the A/W interface.29 The mean stripe widths (100-200 nm) and center-to-center spacings (0.8-2 µm) vary with the DPPC mole fraction and surface pressure (Figure 2), although a feature of all the patterns is that wider continuous lines are periodically interspersed among a more closely spaced series of narrower broken stripes.12 For compositions and pressures where the Langmuir monolayer consists of micron-size DPPC domains, an extensive stripe topography was not observed following LB deposition, presumably because these larger domains are not easily aligned and/or distorted by shear forces during the vertical transfer process.12 Interestingly, while stripe patterns can be easily generated by LB deposition of DPPC/DLPC monolayers onto freshly cleaved mica, we have not been able to form stripes on other substrates such as silicon/silicon oxide, gold, and 3-aminopropylethoxysilane-modified mica. We suspect that this is because the negative surface charge density on the mica (48 Å2)30 accommodates the density of the positively charged trimethylamine headgroups of condensed DPPC (i.e., at 20 °C, the molecular area of DPPC (26) Seul, M.; Chen, V. S. Phys. Rev. Lett. 1993, 70, 1658-1661. (27) Mansky, P.; DeRouchey, J.; Russell, T. P.; Mays, J.; Pitsikalis, M.; Morkved, T.; Jaeger, H. Macromolecules 1998, 31, 4399-4401. (28) Ahrens, H.; Baekmark, T. R.; Merkel, R.; Schmitt, J.; Graf, K.; Raiteri, R.; Helm, C. A. ChemPhysChem 2000, 2, 101-106. (29) Sanchez, J.; Badia, A. Thin Solid Films 2003, 440, 223-239. (30) Nishimura, S.; Tateyama, H.; Tsunematsu, K. J. Colloid Interface Sci. 1993, 159, 198-204.
varies from 51 Å2 at 15 mN/m to 47 Å2 at 32 mN/m). The charge density match would favor nucleation of the DPPC at the three-phase contact line. Striped Bilayers. The differences in molecular packing and interfacial energy between the more ordered DPPC stripes (∼23 mJ m-2)31 and the disordered DLPC matrix (∼31 mJ m-2)31 should allow one to control the deposition of a second DPPC/DLPC monolayer. To demonstrate this, a 0.25:0.75 (mol/mol) DPPC/DLPC mixture was used, although other stripe-forming compositions (and surface pressures) may work equally well.32 Figure 3A shows a typical AFM image of a bilayer prepared by the double LB deposition (upstroke/downstroke) of 0.25:0.75 DPPC/ DLPC at 25 mN m-1. Parallel stripes aligned perpendicular to the film transfer direction are observed; these are 260 ( 80 nm wide and 1.1 ( 0.2 nm higher than the surrounding matrix phase (Figure 3B). Bilayer-deep hole defects (3.9 ( 0.2 nm) are present in the surrounding phase and along the edges of the stripe domains.33 Such bilayerdeep holes are often observed in phospholipid bilayers prepared by the LB technique and arise via the desorption of phospholipid molecules from the first monolayer to the A/W interface during transfer of the second monolayer.34 The incoming phospholipids cover only the hydrophobic surface of the first monolayer, resulting in bare mica and lipid-bilayer-covered regions in contact with water.34 The number, shape, and size of the hole defects depend on the film transfer pressure, the number of defects in the lower layer, the type of lipids used, and the phase state of the lipids transferred (Supporting Information).34,35 The de(31) Berger, C. E. H.; van der Werf, K. O.; Kooyman, R. P. H.; de Grooth, B. G.; Greve, J. Langmuir 1995, 11, 4188-4192. (32) Striped bilayers were also prepared by the double LB deposition of 0.50:50 DPPC/DLPC at 15 mN m-1. Due to the lower surface pressure required to generate stripe patterns for this composition, significantly more and larger hole defects were present in the bilayer, and further work with 0.50:50 DPPC/DLPC was not pursued. No other compositions have been explored thus far for the double LB deposition. (33) The lipid bilayer thicknesses measured by X-ray diffraction for the lamellar phases of DPPC and DLPC at 25 °C are 3.16 and 4.71 nm, respectively (ref 46). (34) Bassereau, P.; Pincet, F. Langmuir 1997, 13, 7003-7007. (35) Rinia, H. A.; Demel, R. A.; van der Eerden, J. P. J. M.; de Kruijff, B. Biophys. J. 1999, 77, 1683-1693.
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Figure 3. AFM images (in H2O) of a bilayer prepared by the double deposition (upstroke/downstroke) of 0.25:0.75 DPPC/DLPC at 25 mN m-1: (A) lower and (B) higher magnification. Transfer ratio for the second deposition ) 0.48. The cross-section analysis in part B shows ∼1 nm thick stripes protruding from the surrounding phase and ∼4 nm deep holes in the background matrix. Shown in (C) is the bilayer after the adsorption of HGG.
sorption of lipid from the mica typically results in transfer ratios of
