Biomimetic Engineering of a Generic Cell-on-Membrane Architecture

May 3, 2012 - ABSTRACT: We develop a biomimetic cell-on-membrane architecture in close-volume format which allows the interfacial biocompatibility and...
0 downloads 0 Views 3MB Size
Download PDF
Letter pubs.acs.org/Langmuir

Biomimetic Engineering of a Generic Cell-on-Membrane Architecture by Microfluidic Engraving for On-Chip Bioassays Sang-Wook Lee,† Ji-Yoon Noh,‡ Seung Chul Park,† Jin-Ho Chung,‡ Byoungho Lee,† and Sin-Doo Lee*,† †

School of Electrical Engineering #032, Seoul National University, Kwanak P.O. Box 34, Seoul 151-600, South Korea College of Pharmacy, Seoul National University, Seoul 151-742, Korea

‡

S Supporting Information *

ABSTRACT: We develop a biomimetic cell-on-membrane architecture in close-volume format which allows the interfacial biocompatibility and the reagent delivery capability for on-chip bioassays. The key concept lies in the microfluidic engraving of lipid membranes together with biological cells on a supported substrate with topographic patterns. The simultaneous engraving process of a different class of fluids is promoted by the front propagation of an air−water interface inside a flow-cell. This highly parallel, microfluidic cell-on-membrane approach opens a door to the natural biocompatibility in mimicking cellular stimuli-response behavior essential for diverse on-chip bioassays that can be precisely controlled in the spatial and temporal manner.

T

referring to handling of liquids or gases in micrometer-length dimensions, have demonstrated three-dimensional flow control for single-cell experiments,16 the bubble-driven microfluidic transport for bioengineering,17 and the immobilization of a large number of picoliter-scale aqueous droplets in oil.18 However, the cellular environments on membranes, the compartmentalization of multiphase liquids including cells and lipids in array, and the fluid-addressable control over the delivery of reagents, all in a single platform, have not been established so far. We report on a microfluidic cell-on-membrane platform which avoids open-volume formats with free liquid−air interfaces and offers unique opportunities to precisely control the spatiotemporal attributes of cellular microenvironments. The underlying concept is based on microfluidic engraving of liquids inside a flow-cell which is preassembled with a bottom hydrophilic substrate having microwell patterns and a top hydrophobic substrate. This is fundamentally different from a microengraving method for rapid selection of single cells8 and multiparametric analysis of cells,19 which utilized open-volume formats during patterning and then sealed with a rigid substrate. Naturally biocompatible lipid membranes supported on the hydrophilic substrate are patterned simultaneously with biological cells through the movement of an air−water interface inside the flow-cell. The reagent delivery to the cell-onmembrane arrays can be precisely controlled by the introduction of a flow-stopper on the hydrophobic substrate in space and time without using complex channels or valves.

he development of a generic principle of creating biomimetic microenvironments in bioassays provides scientific insights into a wide range of cellular activities including adhesion, migration, differentiation, and apoptosis. Particularly, the physicochemical nature of a mechanical support for cells profoundly influences membrane-mediated phenomena1,2 and adhesion3−5 of the cells in contact, and the precise control of cell−surface interactions holds tremendous promise for applications in high-performance cell-based assays,6,7 ultrasensitive molecular detection,8 and drug screening.9 Advances in microfabrication and nanotechnology applied to biology have led to the construction of various functional surfaces for nanobio interfaces2,10 and versatile microfluidic devices for miniaturized biological assays.11,12 The prerequisite for the understanding of the essential features of the cellular interactions with the surface in contact and the biochemical processes mediated by external reagents is the creation of welldefined biomimetic microenvironments in the cell arrays with the properties of (i) the aqueous state sustaining the biological viability, (ii) the interfacial biocompatibility, (iii) the on-chip controllability of the vapor pressure, and (iv) the reagent delivery capability. Although much progress has been made toward micropatterning liquid biological substances into regular arrays, the majority of previous works are based on inkjet printing13−15 which is known to be effective for producing liquid patterns from solutions on dry surfaces such as glass, quartz, and polymer. In the inkjet printing case, the use of a humid chamber13 or a hydrated gel substrate14 is necessarily required for eliminating potential disruption of cell−surface interactions but it lacks the parallelism of registration and/or the reagent delivery to the cells. Recent works on microfluidics, © 2012 American Chemical Society

Received: March 12, 2012 Revised: April 22, 2012 Published: May 3, 2012 7585

dx.doi.org/10.1021/la3010695 | Langmuir 2012, 28, 7585−7590

Langmuir

Letter

Figure 1. On-chip microfluidic engraving for patterning droplets: (a) Schematic diagrams illustrating the microfluidic engraving on microstructures having w1 > 2l and w2 < 2l, where w and l are the lateral dimension of microstructures and the equilibrium length of the water meniscus, respectively. (b) SEM micrograph of a microstructure with w2 = 40 μm, where labels 1 and 2 denote two topographic edges. (c) Optical micrograph of droplets generated on two different microstructures with w1 = 100 μm (left) and w2 = 40 μm (the right). (d) Schematic diagrams representing the cross sections (i-i′ and ii-ii′ lines from (c)) of the droplets on two different microstructures. (e) SEM micrograph of the microwells, where labels 1−4 denote four topographic edges. (f) Optical micrograph of droplets generated on two different microwells with w1 = 100 μm (the left) and w2 = 40 μm (the right). Droplet iii and droplet iv indicate an empty microwell and a water-filled microwell, respectively. (g) Ratio of the water-filled microwells as a function of w (d = 2 μm).

vapor-saturated air inside the flow-cell provides aqueous microenvironments required for on-chip cell-based bioassays. Let us first describe the microfludic behavior of water in a continuous version of a flow-cell where periodic microstructures are produced as isolated barriers. We fabricated an array of two different rectangular microstructures whose lateral dimensions are 100 and 40 μm, respectively. The height was fixed as 2 μm. This topography provides a useful basis for constructing an array of microwells with different values of the width (w1 and w2) as shown in Figure 1a. Figure 1b shows the images of the rectangular microstructures (40 μm in lateral dimension and 2 μm in height) observed with a scanning electron microscopy. Two water menisci were held at two edges (label 1,2) of each microstructure. Clearly, for w1 = 100 μm, the menisci from adjacent edges were disjointed from each other, surrounding an individual microstructure (left in Figure 1c) while, for w2 = 40 μm, the neighboring menisci were bridged over five microstructures (right in Figure 1c). Figure 1d shows schematic diagrams showing the cross sections represented by the water menisci around two different microstructures in Figure 1c. From the geometrical argument together with θ ≈ 9.7° on a quartz,20 the meniscus length was estimated as l ≈ 23 μm. This is consistent with the experimentally measured value of 25 μm.

We first present a basic principle of microfluidic engraving of a liquid (water) inside a flow-cell with microwells on one of two substrates which is hydrophilic. The flow-cell consists of a relatively hydrophobic top substrate (polystyrene) and a relatively hydrophilic bottom substrate (quartz) with microwells as shown in Figure 1a. Suppose that the flow-cell is initially filled with water through an inlet from a reservoir. When water is subsequently drained from the flow-cell through an outlet by suction as shown in Figure 1a, a certain amount of water is left inside each microwell due to the surface hydrophilicity (or surface energy). The water meniscus is dictated by the hydrophilic strength, the microwell dimensions (the depth d and the width w), and the flow rate. In equilibrium, the meniscus length (l) from an edge of the microwell to a position converging to a flat water−air interface is simply given by l ≈ d/tan (θ/2) in a first-order approximation. Here, θ is the contact angle of water (the middle in Figure 1a). From a simple geometrical argument, two water menisci will be separated from each other for w1 > 2l while they will be bridged for w2 < 2l (the right in Figure 1a). Note that for given θ depending on the interfacial nature of the liquid, in principle, the geometrical parameters of the microwell, w and d, will determine the liquid patterns engraved in the flow-cell. This type of a closed-volume format with 7586

dx.doi.org/10.1021/la3010695 | Langmuir 2012, 28, 7585−7590

Langmuir

Letter

Figure 2. Micropatterning of solid-supported lipid bilayers through microfluidic engraving: (a) Schematic diagrams illustrating the patterning process of the SLBs through microfluidic engraving. (b) Epifluorescence micrograph showing the SLB micropatterning taken at an air−buffer interface observed in the Texas Red channel. (c) Epifluorescence micrograph of the resultant SLB microarray with several different values of w = 10, 20, 30, and 5 μm (inset) observed in the Texas Red channel.

Figure 3. Micropatterning of RBCs on SLBs through multiphase microfluidic engraving: (a) Schematic diagrams illustrating the patterning process of the RBCs by site-selective disassembly of the SLBs through microfluidic engraving. (b) Optical micrograph of he patterned RBCs through the process shown in (a). (c) Optical micrograph showing the randomly distributed RBCs without SLBs. (d) Combined image of an optical micrograph for RBCs and a fluorescence micrograph for SLBs taken from the same location.

In the light of the above idea, we fabricated an array of isolated microwells with four surrounding edges (label 1−4 in Figure 1e) for the purpose of patterning water into individual microwells with self-registration. Figure 1f shows a micrograph of water droplets patterned through microfluidic engraving in two different types of microwells having w1 = 100 μm and w2 = 40 μm for fixed d = 2 μm. It is interesting to note that even in the regime of w1 > 2l for disjointed menisci (label iii in Figure 1f), many of the microwells were fully filled with water (label iv in Figure 1f). This means that in the microwell case with four edges, the overlap of four menisci results in a more extended criterion for w1 than a first-order approximation where only two menisci are taken into account. Note that the condition of w2 < 2l is still sufficient to precisely pattern water into a variety of microarrays in the aqueous state. Figure 1g shows the ratio of the water-filled microwells to total number of microwells in an array as a function of w for given d = 2 μm. The large error bars observed for w larger than 100 μm implies that for given value of d = 2 μm, w should be less than 40 μm to obtain full engraving of water in the microwells. The critical dimension of w can be enlarged with increasing d. For biological applications, it is extremely important to prevent water droplets from drying during biochemical reactions. The reversible vapor saturation− condensation process in a water-filled microwell was demonstrated using the evolution of light interference patterns after

microfluidic engraving (see Supporting Information, Figure S1). The air inside the flow-cell was fully vapor-saturated, and the water meniscus in the flow-cell was maintained over 1 week, indicating that the flow-cell indeed yields the on-chip microenvironment for bioassays. In order to ensure the validity of microfluidic engraving in more general cases of liquids with the long-range structural order and the fluidity such as lipids and liquid crystals (LCs), we examine how the complex liquids can be patterned into microwells in our flow-cell and the structural order is reflected in the pattern symmetry. The supported lipid bilayers (SLBs) are an important class of lipid self-assemblies that mimic cell membranes.21 Moreover, the SLB provides a primary platform for in vitro studies of cell membrane-associated activities that are regulated through delicate interactions with a variety of substrates in aqueous environment.22−24 A schematic diagram showing the site-selective formation of the SLB from a buffer solution of small unilamellar vesicles in a microwell through microfluidic engraving is depicted in Figure 2a. Figure 2b shows a fluorescence micrograph taken at the air−buffer interface (white dotted line) where the SLBs doped with dye-conjugated lipids are selectively patterned in the water-filled microwells (two squares enclosed by white solid lines). The twodimesional fluidity of the SLBs was tested by fluorescence 7587

dx.doi.org/10.1021/la3010695 | Langmuir 2012, 28, 7585−7590

Langmuir

Letter

Figure 4. Cell-based bioassay upon reagent delivery: (a) Schematic diagrams illustrating the time-variable, sequential exposure of 1% ethanol solution to RBCs in the same flow-cell. (b−d) Optical micrographs showing the RBC microarrays exposed to ethanol for the elapsed time te = 5 min (b), te = 15 min (c), and te = 25 min (d), whose original biconcave structure transformed into spiculated shapes in a time dependent manner (the right).

= 13 μm and w = 40 μm, larger than the size of RBC which is about 8 μm) after microfluidic engraving. It should be noted that, without the underlying SLB, the RBCs in contact with the substrate surface were not fully removed due to strong adhesion (Figure 3c). Figure 3d shows a combined image of an optical micrograph for the RBCs and a fluorescence micrograph for the SLBs taken from the same location. Clearly, the SLBs as well as the RBCs were well positioned inside the topographical microwells with high accuracy of registration. Figure 4 shows the sequential reagent delivery to the cell-onmembrane microarray for on-chip bioassays in a temporally and spatially controlled fashion. The key idea is how to manipulate the flow of reagents in a specific, prescribed region inside the microarray. In our case, a deep trench (∼1 mm) was introduced on the top substrate for use as a flow stopper which prevents undesirable intrusion of reagents along the delivery channel by pinning the contact line. On the basis of this “flow−stop−flow” strategy, the cellular shape transformation,27 one of the important biomarkers for the determination of the pathological state of RBCs, was investigated in response to 1% ethanol solution at three different regions at the interval of 10 min (Figure 4a). Ethanol exposure to RBCs, commonly involved with alcohol consumption, is known to cause oxidative damage to the cells which leads to the loss of structural and functional integrity28,29 in time. Clearly, the RBC microarrays at the exposure time te = 5, 15, and 25 min (Figure 4b−d) taken in a single flow-cell show the transformation of the biconcave structure into spiculated shapes (inset in Figure 4b−d) with 8.3 ± 1.8, 20.6 ± 0.9, and 60.5 ± 3.6%, respectively. Note that our

recovery after photobleaching (FRAP)25 study (see Supporting Information, Figure S2). Figure 2c shows the resultant microarrays of the SLBs having different values of w = 10, 20, 30, and 5 μm (inset). Clearly, in the regime of w < 2l we studied, the SLBs together with water were precisely patterned in all types of the prescribed microwells. For a nematic LC possessing only the orientation order, well-defined patterns were spontaneously produced by microfluidic engraving. The 4fold symmetry observed under crossed polarizers (see Supporting Information, Figure S3) indicates that the LC molecules were aligned toward the normal direction of the LC−air interface which was curved inward in each microwell. The radial symmetry of the LC patterns comes from the orientation order of the molecules inside a microwell. We now explore the multiphasic patterning capability in a generic cell-on-membrane architecture which is a prerequisite for on-chip bioassays. The SLB inside the microwells provides a naturally biocompatible surface for cellular viability,3,4 whereas the SLB outside the microwells acts as a sacrificial layer for the site-selective detachment of cells through the disassembly of the bilayer structure (Figure 3a). This concept of multiphasic patterning can be directly applicable for various soft biomaterials such as proteins on the SLBs (see Supporting Information, Figure S4) that modify biomimetic environments, differentiating our approach from a conventional lift-off technique26 which is known to be very effective for solid materials in nonaqueous environments. The optical micrograph in Figure 3b shows a microarray of human red blood cells (RBCs) that are selectively preserved within the microwells (d 7588

dx.doi.org/10.1021/la3010695 | Langmuir 2012, 28, 7585−7590

Langmuir

Letter

array of a self-aligned liquid crystal; protein binding on SLBs. This material is available free of charge via the Internet at http://pubs.acs.org.

approach is not limited to one type of a reagent but it can be extended for a wide class of reagents in a highly parallel manner. In conclusion, the multiphasic engraving strategy described here provides scalable biomimetic platforms that promise to develop highly parallel hybrid nano- and biodevices and to detect small scale cellular activities, for example, enzyme kinetics and gene expression. Furthermore, it has potential to pattern proteins, antibodies and drugs, and other types of soft matters such as colloids and oils, and offers a viable way of delivering many different dynamic stimuli to living cells for bioassays.

■

■

*E-mail: [email protected]. Telephone: +82-2-880-1823. Fax: +82-2-874-9769. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported in part by the National Research Foundation and the Ministry of Education, Science and Technology of Korea through the Creative Research Initiatives Program (Active Plasmonics Application Systems).

EXPERIMENTAL SECTION

Fabrication of a Flow-Cell. The top substrate of the flow-cell shown in Figure 1a was fabricated using a polystyrene substrate (1 mm thick) with a hole for an inlet, and it was connected to a reservoir. Two types of the bottom substrates being used were (100) quartz wafers. In one type, rectangular microstructures (2 μm high) were produced using the standard photolithography and the chemical etching process with buffered HF (7:1 (v/v) NH4F/HF). The etching rate was 100 nm/min. In the other, microwells (13 μm deep) were produced with the help of an aluminum (Al) layer (1 μm thick) prepared on the quartz wafer. The Al layer was first patterned to define Al-covered and uncovered regions through the photolithography process. The uncovered regions were etched selectively by plasma of C4F8 and O2 using the plasma etching equipment (AOE, STS, U.K.) at the radio frequency of 13.56 MHz. The etching rate was 540 nm/min. The substrate was then cleaned with a piranha solution (3:1 (v/v) H2SO4/ H2O2) at 120 °C for more than 10 min to remove the Al patterns. The top and bottom substrates were finally assembled together with a glue spacer (120 μm thick). Formation and Observation of the SLBs. The phospholipid used as a base for the formation of the SLBs was 1,2-dioleoyl-snglycero-3-phophocholine (DOPC, Avanti Polar Lipids, Birmingham, AL). For imaging the SLBs, a red fluorescent dye labeled lipid, 1,2dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Texas RedDHPE, Molecular Probes, Eugene, OR), was mixed with DOPC at 1 mol %. All lipids were dissolved in chloroform. The rapid solvent exchange method was employed for evaporation of chloroform and hydration with Tris buffer (100 mM NaCl and 10 mM Tris at pH 8.0) simultaneously.30 The concentration of the lipid mixture used in this work was 0.2 mg/mL. Small unilamellar vesicles (SUVs) were produced by extruding (Mini-Extruder, Avanti Polar Lipids) 20 times through a filter with pores of 50 nm. When the cleaned substrate was incubated in the SUVs solution, the SLBs were formed via vesicle adsorption, rupture, and fusion. The SUVs were removed with deionized water after the SLB formation was completed. The Texas Red channel was monitored using epifluorescence microscopy (Eclipse E600-POL, Nikon). Preparation of the RBCs. For preparation of the RBCs, human blood was obtained from healthy male donors (18−28 years old) using a vacutainer with acid citrate dextrose and a 21-gauge needle (Becton Dickinson) on the day of each experiment. Platelet-rich plasma and buffy coat were removed by aspiration after centrifugation at 200g for 15 min. The centrifuged RBCs were washed three times with phosphate buffered saline (1 mM KH2PO4, 154 mM NaCl, 3 mM Na2HPO4, pH 7.4) and once with Tris buffer (15 mM Tris-HCl, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, pH 7.4). The washed RBCs were resuspended in Tris buffer and deposited on the prepared SLB by gravitational sedimentation at the concentration of 108 cells/mL for 30 min.

■

AUTHOR INFORMATION

Corresponding Author

■

REFERENCES

(1) Yoon, T.-Y.; Jeong, C.; Lee, S.-W.; Kim, J. H.; Choi, M. C.; Kim, S.-J.; Kim, M. W.; Lee, S.-D. Topographic control of lipid-raft reconstitution in model membranes. Nat. Mater. 2006, 5, 281−285. (2) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thomson, M. Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater. 2009, 8, 543−557. (3) Stroumpoulis, D.; Zhang, H.; Ruvalcava, L.; Gliem, J.; Tirrell, M. Cell adhesion and growth to peptide-patterned supported lipid membranes. Langmuir 2007, 23, 3849−3856. (4) Lee, S.-W.; Jeong, C.; Lee, S.-D. Elasticity-based patterning of red blood cells on undulated lipid membranes supported on porous topographic substrates. J. Phys. Chem. B 2009, 113, 3610−3614. (5) Sandrin, L.; Coche-Guérente, L.; Bernstein, A.; Basit, H.; Labbé, P.; Dumy, P.; Boturyn, D. Cell adhesion through clustered ligand on fluid supported lipid bilayers. Org. Biomol. Chem. 2010, 8, 1531−1534. (6) Park, M. C.; Hur, J. Y.; Kwon, K. W.; Park, S.-H.; Suh, K. Y. Pumpless, selective docking of yeast cells inside a microfluidic channel induced by receding meniscus. Lab Chip 2006, 6, 988−994. (7) Tavana, H.; Jovic, A.; Mosadegh, B.; Lee, Q. Y.; Liu, X.; Luker, K. E.; Luker, G. D.; Weiss, S. J.; Takayama, S. Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells. Nat. Mater. 2009, 8, 736−741. (8) Love, J. C.; Ronan, J. L.; Grotenbreg, G. M.; van der Veen, A. G.; Ploegh, H. L. A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat. Biotechnol. 2006, 24, 703−707. (9) Wu, L. Y.; Carlo, D. D.; Lee, L. P. Microfluidic self-assembly of tumor spheroids for anticancer drug discovery. Biomed. Microdevices 2008, 10, 197−202. (10) Alves, N. M.; Pashkuleva, I.; Reis, R. L.; Mano, J. F. Controlling cell behavior through the design of polymer surfaces. Small 2010, 6, 2208−2220. (11) Delamarche, E.; Juncker, D.; Schmid, H. Microfluidics for processing surfaces and miniaturizing biological assays. Adv. Mater. 2005, 17, 2911−2933. (12) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Cells on chips. Nature 2006, 442, 403−411. (13) Okamoto, T.; Suzuki, T.; Yamamoto, N. Microarray fabrication with covalent attachment of DNA using Bubble Jet technology. Nat. Biotechnol. 2000, 18, 438−441. (14) Xu, T.; Jin, J.; Gregory, C.; Hickman, J. J.; Boland, T. Inkjet printing of viable mammalian cells. Biomaterials 2005, 26, 93−99. (15) Calvert, P. Printing cells. Science 2007, 318, 208−209. (16) Peng, X. Y.; Li, P. C. H. A three-dimensional flow control concept for single-cell experiments on a microchip. 1. Cell selection, cell retention, cell culture, cell balancing, and cell scanning. Anal. Chem. 2004, 76, 5273−5281.

ASSOCIATED CONTENT

S Supporting Information *

Temporal evolution of the vapor saturation inside the flow-cell; fluorescence recovery after photobleaching on SLBs; micro7589

dx.doi.org/10.1021/la3010695 | Langmuir 2012, 28, 7585−7590

Langmuir

Letter

(17) Marmottant, P.; Hilgenfeldt, S. A bubble-driven microfluidic transport element for bioengineering. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9523−9527. (18) Schmitz, C. H. J.; Rowat, A. C.; Koster, S.; Weitz, D. A. Dropspots: a picoliter array in a microfluidic device. Lab Chip 2009, 9, 44−49. (19) Story, C. M.; Papa, E.; Hu, C.-C. A.; Ronan, J. L.; Herlihy, K.; Ploeph, H. L.; Love, J. C. Profiling antibody responses by multiparametric analysis of primary B cells. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 17902−17907. (20) Wang, W.; Yang, P.; Cheng, Z.; Hou, Z.; Li, C.; Lin, J. Patterning of red, green, and blue luminescent films based on CaWO4:Eu3+, CaWO4:Tb3+, and CaWO4 phosphors via microcontact printing route. ACS Appl. Mater. Interfaces 2011, 3, 3921−3928. (21) Sackmann, E. Supported membranes: Scientific and practical applications. Science 1996, 271, 43−48. (22) Yusko, E. C.; Johnson, J. M.; Majd, S.; Prangkio, P.; Rollings, R. C.; Li, J.; Yang, J.; Mayer, M. Controlling protein translocation through nanopores with bio-inspired fluid walls. Nat. Nanotechnol. 2011, 6, 253−260. (23) Zhou, X.; Moran-Mirabal, J. M.; Craighead, H. G.; McEuen, P. L. Supported lipid bilayer/carbon nanotube hybrids. Nat. Nanotechnol. 2007, 2, 185−190. (24) Lee, S.-W.; Na, Y.-J.; Lee, S.-D. Nondisruptive micropatterning of fluid membranes through selective vesicular adsorption and rupture by nanotopography. Langmuir 2009, 25, 5421−5425. (25) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 1976, 16, 1055−1069. (26) Suzuki, N.; Tanaka, H.; Yamanaka, S.; Kanai, M.; Lee, B. K.; Lee, H. Y.; Kawai, T. Epitaxial nanodot arrays of transition-metal oxides fabricated by dry deposition combined with a nanoimprintlithography-based molybdenum lift-off technique. Small 2008, 4, 1661−1665. (27) Lim, G. H. W.; Wortis, M.; Mukhopadhyay, R. Stomatocytediscocyte-echinocyte sequence of the human red blood cell: Evidence for the bilayer-couple hypothesis from membrane mechanics. Proc. Natl. Acad. Sci. U.S.A. 2002, 24, 16766−16769. (28) Gabbianelli, R.; Cifani, C.; Massi, M.; Polidori, C.; Falcioni, G. Oxidative damage in rat erythrocyte membranes following ethanol intake: Effect of ethyl pyruvate. Chem.-Biol. Interact. 2007, 169, 122− 131. (29) McLawhon, R. W.; Marikovsky, Y.; Thomas, N. J.; Weinstein, R. S. Ethanol-induced alterations in human-erythrocyte shape and surface-properties - modulatory role of prostaglandin-E1. J. Membr. Biol. 1987, 99, 73−78. (30) Buboltz, J. T.; Feigenson, G. W. A novel strategy for the preparation of liposomes: rapid solvent exchange. Biophys. Acta 1999, 1417, 232−245.

7590

dx.doi.org/10.1021/la3010695 | Langmuir 2012, 28, 7585−7590