Nanomechanical Characterization of Phospholipid Bilayer Islands on

Jul 2, 2009 - School of Chemistry, Physics and Earth Sciences, Flinders UniVersity, Sturt Road, Bedford ... bilayer islands deposited on the flat subs...
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J. Phys. Chem. B 2009, 113, 10339–10347

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Nanomechanical Characterization of Phospholipid Bilayer Islands on Flat and Porous Substrates: A Force Spectroscopy Study Matthew R. Nussio,†,# Gerard Oncins,‡,§,|,# Ingrid Ridelis,† Endre Szili,† Joseph G. Shapter,† Fausto Sanz,*,§,|,⊥ and Nicolas H. Voelcker*,† School of Chemistry, Physics and Earth Sciences, Flinders UniVersity, Sturt Road, Bedford Park, Adelaide, SA 5001, Australia, Scientific-Technical SerVices, Nanometric Techniques Unit, UniVersity of Barcelona, Soler i Sabaris 1, 08028 Barcelona, Spain, Nanoprobes and Nanoswitches, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 13, 08028 Barcelona, Spain, Department of Physical Chemistry, Chemistry Faculty, UniVersity of Barcelona, Martı´ i Franque`s 1-11, 08028 Barcelona, Spain, and CIBER-BBN, Maria de Luna 11, 50018 Zaragoza, Spain ReceiVed: December 15, 2008; ReVised Manuscript ReceiVed: May 31, 2009

In this study, we compare for the first time the nanomechanical properties of lipid bilayer islands on flat and porous surfaces. 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero3-phosphatidylcholine (DPPC) bilayers were deposited on flat (silicon and mica) and porous silicon (pSi) substrate surfaces and examined using atomic force spectroscopy and force volume imaging. Force spectroscopy measurements revealed the effects of the underlying substrate and of the lipid phase on the nanomechanical properties of bilayers islands. For mica and silicon, significant differences in breakthrough force between the center and the edges of bilayer islands were observed for both phospolipids. These differences were more pronounced for DMPC than for DPPC, presumably due to melting effects at the edges of DMPC bilayers. In contrast, bilayer islands deposited on pSi yielded similar breakthrough forces in the central region and along the perimeter of the islands, and those values in turn were similar to those measured along the perimeter of bilayer islands deposited on the flat substrates. The study also demonstrates that pSi is suitable solid support for the formation of pore-spanning phospholipid bilayers with potential applications in transmembrane protein studies, drug delivery, and biosensing. Introduction Lipid bilayer membranes essentially “define” cells and organelles. Moreover, they participate in and regulate a host of cellular activities (e.g., biosynthesis, detoxification, metabolism, signaling, sorting, cell-cell interactions, motility, etc.). Previous studies have routinely used supported phospholipid bilayers (SPB) as a popular model system for the characterization of membrane structure,1-3 drug interactions,4-7 and the structure of membrane-associated proteins.8,9 These systems constitute an attractive model for studying membrane processes since they incorporate a nanometric lubricating layer of water between the bilayer and the substrate.10,11 However, many biological membranes feature an assortment of transmembrane proteins (ion pumps and ligand- or voltage-gated ion channels). These proteins contain extramembrane components which are likely to interact with the solid surface, affecting correct insertion of these proteins or their lateral mobility in the bilayer.12 Therefore, the ability to construct suitable biomimetic lipid interfaces to preserve protein activity is of significant interest. To minimize substrate interactions on SPBs, long hydrophilic spacer molecules have been utilized with the aim of extending * To whom correspondence should be addressed. (N.H.V.) E-mail: [email protected]. Tel.: +61-8-8201-5338. Fax: +61-8-82012905. (F.S.) E-mail: [email protected]. Tel.: +34-934-039-239. Fax: +34-934021-231. † Flinders University. ‡ Nanometric Techniques Unit, University of Barcelona. § Institute for Bioengineering of Catalonia. | Chemistry Faculty, University of Barcelona. ⊥ CIBER-BBN. # These authors contributed equally to this work.

the distance between the lower leaflet of the bilayer and the substrate. Most systems incorporate either short organic linkers13-19 or polymers20-22 to tether phospholipid bilayers to their supporting substrates. Otherwise known as tethered bilayer membranes (t-BLMs), these systems provide further stabilization of lipid bilayers as they are chemically linked to the solid support.16 Although structurally robust,19 the attachment to the solid support has inherent effects on the lateral mobility of constituent phospholipids. In fact, diffusion coefficients measured for t-BLMs23-25 are comparable to those measured for SPB on flat substrates.25-28 Measurements performed on freestanding lipid bilayers, however, demonstrate diffusion coefficients much greater than those observed for t-BLMs.26,27 Recent studies have also demonstrated porous solid supports to be beneficial for the assembly of phospholipid bilayers.29-31 The pores generate an interstitial free space below the phospholipid bilayer, providing a closer physiological mimic to the cell membrane scenario than SPB, which potentially preserves better transmembrane protein tertiary structure and activity. Experiments utilizing laser-structured micrometer and submicrosized pores have successfully spread phospholipids from organic solutions across the pores in order to provide a highly controlled environment for assessing the functional properties of ion channels.32,33 Pore-spanning lipid bilayers have also been routinely formed on porous alumina.30,34-39 Several methods have been adopted for the modification of porous alumina substrates with lipid bilayers. However, the most extensively used technique that has afforded long-term stability utilized a combination of a hybrid bilayer and a freestanding lipid bilayer, called nanoblack lipid membranes (nano-BLM).30,36-38 Bearing

10.1021/jp811035g CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

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gigaohm membrane resistances, these interfaces have yielded reliable systems for the analysis of single channel activities of peptides,36 such as gramicidin and alamethicin, and large proteins such as OmpF from E.coli.38 Nano-BLMs have also allowed the sensitive detection of light-induced proton currents from bacteriorhodopsin.37 The potential of pSi as a porous substrate for pore-spanning phospholipid bilayers has been recently demonstrated.29,31,40,41 A salient feature of pSi as a support material is its behavior as a one-dimensional photonic crystal. In particular, pSi with porespanning lipid bilayers has been shown to retain the optical reflectivity characteristics of pSi, suggesting that this composite structure can perform as a label-free photonic biosensor.29 In contrast, Kilian et al. have reported the formation of hybrid bilayers inside the pSi photonic crystal, prefaced by functionalization of pSi with a hydrophobic dodecane layer. This system allowed the group to study affinity capture of cholera toxin by membrane embedded GM1 glycolipid using shifts in the optical reflectivity.42 Also utilizing pore-spanning lipid bilayers, we have previously developed a pSi biosensor capable of detecting single integrin Rvβ3-expressing cells.31 This cell surface-bound adhesion receptor is important in cellular-cellular and cellularextracellular matrix interactions, and its expression in tumor cells is thought to be important in cell migration and therefore one of the targets for the inhibition of tumor growth.43 The described architectures all constitute attractive models for studying membrane physiological processes in a biomimetic environment which maintains membrane fluidity and is conducive to hosting a range of cell signaling transmembrane proteins and other biomolecules. The current study, however, aims to provide unprecedented information on the interaction forces and nanomechanical behavior of pore-spanning lipid bilayers on pSi, in direct comparison to typical SPBs formed on flat substrates, such as mica and silicon. pSi was selected as a substrate material due to its tunable porosity, versatile surface chemistry, its biodegradability, and finally its optical properties.44-47 With the aid of atomic force spectroscopy in force volume mode, the membrane interaction forces and nanomechanical behavior of SPBs on mica, silicon, and pore-spanning phospholipid bilayers on pSi were probed with nanometer and nanonewton resolution. The phospholipid bilayers investigated here comprised either DMPC or DPPC, differing only by the length of their alkyl chains (C14 vs C16). We are taking cues from recent studies, which have characterized both the effect of temperature and ionic strength on the nanomechanical properties of phospholipid bilayers.48-50 In regards to the current study, force-distance curves in the approach direction provided useful information regarding the maximum force that each membrane surface could withstand before breaking.51-53 Here, applying force in a direction perpendicular to the bilayer plane causes the bilayer to deform elastically until it is no longer able to withstand the force exerted by the AFM tip. At this point, a discontinuity in the approach force-distance curve is observed, which is indicative of the tip penetrating the membrane and leading to the onset of plastic deformation.54 The force at which this event occurs is known as the breakthrough force (jump), and can be used to probe cell membrane nanomechanics as well as interaction forces between neighboring lipid molecules in the membrane. The force value at which this jump takes place was used here to compare the membrane stability for each solid support. The current study also aims to demonstrate that pSi is a useful substrate for lipid bilayers from a nanomechanics point of view. We show that this porous material is conducive to the formation of pore-spanning phospholipid bilayers that better mimic

Nussio et al. physiological membranes than previous commonly used systems based on flat silicon or mica. Experimental Methods Materials. DMPC and DPPC were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. All aqueous solutions were prepared with Milli-Q grade reagent water with resistance ≈ 18.2 MΩ. Aqueous solutions were also filtered through a 0.2 µm membrane filter. Vesicle Preparation. Multilamellar vesicles (MLVs) were prepared by first dissolving aliquots of lipid (either DMPC or DPPC), in chloroform/methanol (3:1 v:v), followed by evaporation of the solvent under nitrogen. Lipid samples were suspended in 10 mM HEPES, 150 mM NaCl, 20 mM MgCl2 (pH 7.4). The final concentration of lipids was 0.5 mM. Samples were subjected to cycles of sonication and heating above the component transition temperature (TM) until a homogeneous mixture of small unilamellar vesicles (SUVs) (∼100 nm) was obtained. Samples were left to settle overnight and maintained at 4 °C. Substrate Preparation. P+2-type silicon wafers supplied by Virginia Semiconductors (3 inch, boron doped, 〈1-0-0〉, singlesided polished wafers with a resistivity 0.0005-0.001 Ωcm) were anodized in 1:1 HF/ethanol at a current density of 350 mAcm-2 using a 1.8 cm2 etching cell. The porous silicon films were then washed with ethanol, acetone, and finally dichloromethane before being dried under a stream of nitrogen gas. Ozone oxidation of pSi and plain silicon wafers was performed using a Fischer OZON, Ozon-Generator 500. All oxidations were run for 20 min at ozone flow rate of 3.25 g h-1. Commercial muscovite mica surfaces (Metafix, Montdidier, France) were cleaved prior to use, providing highly reproducible atomically flat surfaces for bilayer adsorption. Bilayer Formation. Formation of SPBs was achieved by depositing 80 µL of SUVs to either a freshly cleaved mica surface or silicon. For pore-spanning lipid bilayers, pSi was utilized. The prepared surfaces were then incubated above the TM of the component phospholipids for 35 min to promote bilayer formation. Prior to imaging, bilayer surfaces were washed several times with the working buffer (10 mM HEPES, 150 mM NaCl, 20 mM MgCl2 pH 7.4). Atomic Force Microscope. Images and force measurements were acquired using a Dimension 3100 microscope attached to a Nanoscope IV controller (Veeco Meteorology Group/ Digital Instruments DI, Santa Barbara, California) using V-shaped Si3N4 tips (OMCL TR400PSA, Olympus, Japan) with a nominal spring constant of 0.08 N m-1. Force curves were performed prior to acquiring topographic images (contact mode) in order to ensure that the minimum vertical force was applied and to avoid any damage to supported/pore-spanning lipid bilayers. The instrument was placed on a vibration isolation table and in an acoustic isolation box (TMC, Peabody, MA). Temperature was maintained between 19 and 21 °C. Vertical spring constant force calibration was performed with a force probe 1D MFP (Asylum Research, Santa Barbara, CA). Individual spring constants were calibrated using the equipartition theorem (thermal noise)55 after determination of the tip sensitivity (V nm-1), which was measured after several minutes of performing force plots to avoid hysteresis. The bilayer thickness was determined as follows: a phospholipid bilayer island was chosen and then a height histogram of the pixels over the bilayer island was compared to the histogram of the flat region surrounding the island (mica substrate). Bilayer thickness was determined as the height difference between the two histograms. This process was

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Figure 2. Schematic of a force volume experiment performed on a phospholipid bilayer island (A). Force curves performed along the perimeter are depicted in dark gray and force curves taken in the central region are shown in light gray squares. Between the two regions there is an intermediate region (white squares) where data was not analyzed. A typical force volume image (B) of a DMPC bilayer island on mica (5 × 5 µm2, 32 pixels × 32 pixels). Figure 1. AFM height image (3 × 3 µm2; Z-scale: 10 nm, 512 lines × 512 lines) of a (A) DMPC and (B) DPPC supported phospholipid bilayer on mica. Cross-sectional analysis gave a mean height of 4.36 ( 0.25 nm, and 4.69 ( 0.19 nm for (C) DMPC and (D) DPPC bilayers, respectively.

performed for islands coming from different regions of the sample and different samples in order to improve the quality of the presented data (at least 20 individual measurements were taken). Force spectroscopy measurements were acquired operating in force volume mode available in Nanoscope v.5.12 software (Digital Instruments, Santa Barbara, CA). Force volume permits acquisition of mechanical maps from a certain sample region. One force versus distance curve per position was performed. In this study, either 32 × 32 or 64 × 64 force versus distance curves per image were acquired. In addition to the force versus distance curves, the topographical information was also recorded for each location. A statistical data treatment was performed by fitting a Gaussian distribution to the histogram of the breakthrough forces obtained for each bilayer. Quoted force values correspond to the center value of a Gaussian fitting to the obtained histogram. These values are accompanied by the total number (n) of force measurements analyzed for each experiment. Force volume images retain the spatial information for each force curve and allow correlation with topographical data. In this context, “perimeter” force curves were considered to be the force curves performed on the perimeter of the island considering the perimeter to be 1 pixel thick. The data considered to be “center” or “central” corresponds to force curves performed in the central region of the bilayers. To prevent data intermixing, an intermediate region two pixels wide was defined between the “center” and the “perimeter” regions (Figure 2A). The number of force volume pixels along the perimeter of a bilayer island was lower than the number pixels in the central region, which led to a discrepancy in the number of force measurements. Result and Discussion Initial experiments investigated the surface topology of DMPC and DPPC SPBs on mica (Figure 1). Previous studies have demonstrated the surface coverage of SPBs to be dependent on the salt concentration and the pH of the aqueous environment.50 Here, experiments were performed with a high ionic strength (150 mM NaCl + 20 mM MgCl2) and pH 7.4 in order

to achieve a surface coverage of between 55% and 80%. Defects corresponding to the mica support allow the height of the SPBs to be measured, obtaining values of 4.36 ( 0.25 nm (n g 20), and 4.69 ( 0.19 nm for DMPC and DPPC SPBs, respectively. These values agree well with previous measurements performed on gel phase DMPC48,50,56,57 and DPPC48,58 bilayers on mica. Typical force spectroscopy measurements have previously been performed on either continuous SPBs or in locations close to the center of bilayer islands.48 In the current study however, force measurements taken from force volume images in the central region of the bilayer islands are distinguished from those taken along the perimeter (Figure 2). This was done to examine whether lateral interactions of the lipids, which are presumably weaker at the edges of a bilayer patch as compared to the center, have an influence on the breakthrough force. Only islands of 0.5 to 3 µm diameter were considered for force measurements. Measurements carried out in the central region of DMPC bilayer islands demonstrated a breakthrough force equivalent to 14.10 ( 1.36 nN (n ) 321) (Figure 3A). This was consistent with previous work performed by Garcia-Manyes et al. for compact gel DMPC bilayers under the same buffer medium.48,50 Along the perimeter of the SPB patches, the peak in the breakthrough force histogram (Figure 3B) was at 5.10 ( 3.16 nN (n ) 110). This corresponds to a 2.8 fold difference between center and perimeter. For DPPC, the difference in breakthrough force between the central region (28.63 ( 10.13 nN, n ) 206) and along the perimeter (20.53 ( 7.07 nN, n ) 156) was only 1.4 fold (Figure 3C,D). Breakthrough forces measured in the central region of DPPC bilayer islands are in accordance with previous work.48,49 Overall, a contrast in breakthrough force is observed between the perimeter and the central region of the bilayer islands of DMPC and DPPC (Table 1). This difference reflects a difference in packing density. Near the periphery, there is a greater degree of freedom for the lipids and this will lead to a looser packing yielding lower breakthrough forces and at the same time a greater variation in the chemical environment. This is also consistent with the greater dispersion in the breakthrough forces observed near the periphery in comparison to the center. To further investigate this trend, experiments were also performed with ozone-oxidized silicon substrates as they comprise comparable surface chemistry to mica.59,60 Indeed, DMPC and DPPC SPBs (Figure 4) show similar coverage (55-80%) as on mica. The measured heights of DMPC and DPPC were 4.77 ( 0.37 nm and 5.32 ( 0.26 nm, respectively. These values are slightly

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Figure 3. Histograms of the breakthrough force values for DMPC in the central region (A) (14.10 ( 1.36 nN) and along the perimeter (B) (5.10 ( 3.16 nN) of supported bilayer islands on mica. Histograms of the yield threshold value for DPPC in the central region (C) (28.63 ( 10.13 nN) and along the perimeter (D) (20.53 ( 7.07 nN) of supported bilayer islands on mica. All measurements were performed in 10 mM HEPES, 150 mM NaCl, 20 mM MgCl2 (pH 7.4).

TABLE 1: Calculated Bilayer Breakthrough Forces for Mica, Silicon, and pSi breakthrough force (nN) DMPC substrate mica silicon pSi

center

perimeter

DPPC center

perimeter

14.10 ( 1.36 5.10 ( 3.16 28.63 ( 10.13 20.53 ( 7.07 13.83 ( 2.49 5.47 ( 2.38 24.28 ( 6.69 18.20 ( 6.28 4.96 ( 1.84 4.59 ( 1.74 19.58 ( 6.69 17.64 ( 6.28

higher than on mica. Also consistent with mica, a 2.5-fold difference in breakthrough force was measured between the central region (13.83 ( 2.49 nN, n ) 192) and edges (5.47 ( 2.38 nN, n ) 90) of the DMPC bilayer islands (Figure 5A,B). Force measurements performed on DPPC bilayer islands (Figure 5C,D) showed only a 1.3 fold difference in breakthrough force between the central region (24.28 ( 6.69 nN (n ) 185)) and the perimeter (18.20 ( 6.28 nN (n ) 235)). In general, measurements of breakthrough forces in the central region of bilayer islands were comparable to those observed on mica (Table 1). An earlier study performed for DMPC SPBs in low ionic strength buffer medium also gave the same breakthrough force for mica and silicon substrates.50 The only structural difference between DMPC and DPPC is the length of their saturated alkyl chains. The two carbon length difference causes the two lipids to exhibit very different transition temperatures, 23 and 41 °C for DMPC and DPPC, respectively.48,61 On both mica and silicon, the breakthrough force for DPPC is almost twice as high as for DMPC. This is attributed to the increased compactness of the DPPC bilayer due to longer hydrocarbon chains giving rise to stronger van der Waals interactions. At high ionic strength buffer medium, the polar headgroup interaction is also attractive; the Na+ ions

Figure 4. AFM height image (2 × 2 µm2; Z-scale: 10 nm, 256 lines × 256 lines) of a (A) DMPC and (B) DPPC supported phospholipid bilayer on silicon. Cross-sectional analysis demonstrated a mean height of 4.77 ( 0.37 nm and 5.32 ( 0.26 nm for (C) DMPC and (D) DPPC bilayers, respectively.

are able to penetrate the phosphatidylcholine (PC) polar headgroups, creating a pseudoionic lattice with the lipid phosphate groups.62-64 Since the van der Waals interactions are stronger between DPPC hydrocarbons due to the shorter intermolecular distance, there is an increased electrostatic interaction between the polar PC head groups, thus also contributing to the higher breakthrough force observed for DPPC SPBs. Along the same vein, we attribute the smaller differences in breakthrough force between the edge and the center of DPPC

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Figure 5. Histograms of the breakthrough force values for DMPC in the central region (A) (13.83 ( 2.49 nN) and along the perimeter (B) (5.47 ( 2.38 nN) of supported bilayer islands on silicon. Histograms of the yield threshold value for DPPC in the central region (C) (24.28 ( 6.69 nN) and along the perimeter (D) (18.20 ( 6.28 nN) of supported bilayer islands on silicon. All measurements were performed in 10 mM HEPES, 150 mM NaCl, 20 mM MgCl2 (pH 7.4).

bilayer islands in comparison to DMPC to the more homogeneous intermolecular distances for DPPC phospholipids over the whole bilayer island than in the case of DMPC. These intermolecular interactions of DPPC lipids near the edge of the bilayer appear to be conserved to a greater extent than in the case of DMPC bilayers. The DMPC bilayer at the periphery of the island may be in a near-to-melting situation. On the other hand, DMPC bilayers in the central regions of the islands are stabilized by substrate interactions. It should be noted that the contrast in breakthrough force between edge and center is very similar for both mica and silicon substrates. Under the experimental conditions, mica is overall negatively charged, consisting of negatively charged oxide groups.65,66 Similarly, ozone-treated silicon features a negatively charged oxide layer of several nanometers thickness. Both substrates therefore provide appropriate chemical properties for phospholipid bilayer deposition.67 Both surfaces are superhydrophilic with sessile drop water contact angles below 10°. The AFM roughness for the substrates is slightly lower for mica (0.08 nm) than for silicon (0.13 nm). The lower bilayer height on mica in comparison to silicon can be attributed to the difference in surface chemistry between silicon and mica. Ozone treated silicon has a higher density of oxide groups in comparison to mica where the oxygens are shielded by potassium ions. The increased oxide density results in an increased packing density of PC SPBs, leading to increased bilayer height. To investigate the interplay between the bilayer membrane and the supported substrate architecture and the influence of the latter on the breakthrough force of the bilayer membrane, experiments were conducted using pSi. For the present study, pSi was fabricated by anodization followed by ozone oxidation to stabilize the substrate against hydrolysis in aqueous medium. The surface chemistry is therefore very similar to ozone-treated

flat silicon. Indeed, the ozone-oxidized pSi is also superhydrophilic showing sessile drop water contact angles below 10°. The dimensions of the porous architectures and the thickness of the porous layer can be varied by adjusting the anodic current density and the time of anodization. Under experimental conditions for the current study, cross-sectional analysis of the pores measured by AFM and SEM demonstrated pore diameters equivalent to 20 ( 5 nm (Figure 6A,D). Pores of such size are difficult to visualize using a standard AFM tip such as the one used here because of the mismatch in aspect ratio between porous silicon pores and the AFM tip as well as because of the similar dimensions of the radius of curvature of the AFM tip used and the pore diameter.31 Because of this tip convolution effect, the AFM roughness of the pSi surface was only 0.22 nm, approximately double the roughness of flat silicon. The porosity of pSi (as determined by gravimetry) is 65%. Bilayer formation inside of the pores was avoided by size exclusion; the diameter of the SUVs (∼100 nm) exceeded by far the pore size of the substrate.31 This facilitated the formation of bilayers suspended across the pores of the substrate as Figure 6 clearly shows for DMPC (Figure 6B,E) and DPPC (Figure C,F). In most cases, experiments utilizing DMPC resulted in continuous pore-spanning lipid bilayers. However, for the current study we wanted isolated islands and those could be achieved by shortening the incubation time. Islands height was typically 4.52 ( 0.27 and 4.61 ( 0.23 nm for DMPC and DPPC bilayers, respectively. These values were very similar to those measured on mica. The bilayers also demonstrated good lateral stability. This was confirmed by examining bilayer formation on macroporous silicon (pore diameter of around 50 nm), which showed less surface coverage of the pore-spanning lipid bilayer (data not shown). The breakthrough forces along the perimeter and in the central region of the DMPC bilayer islands on pSi were similar, at 4.96

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Figure 6. AFM height image of (A) pSi with pore widths of ∼20 nm (0.5 × 0.5 µm2; Z-scale, 5 nm; 512 lines × 512 lines) and with (B) DMPC bilayers (1.5 × 1.5 µm2; Z-scale, 10 nm; 256 lines × 256 lines) and (C) DPPC bilayers (3.0 × 3.0 µm2; Z-scale, 10 nm; 512 lines × 512 lines) suspended over the substrate surface. (D) SEM cross-section: the vertical distance is ∼2.5 µm (scale bar, 1 µm). Cross-sectional analysis of AFM images of pore-spanning lipid bilayers demonstrated a mean height of 4.52 ( 0.27 nm and 4.61 ( 0.23 nm for (E) DMPC and (F) DPPC, respectively.

Figure 7. Histograms of the breakthrough force values for DMPC in the central region (A) (4.96 ( 1.84 nN) and along the perimeter (B) (4.59 ( 1.74 nN) of pore-spanning bilayer islands on pSi. (C) Force-penetration curve performed in the central region of a DMPC island on pSi. Inset: corresponding approach force-distance curve. All measurements were performed in 10 mM HEPES, 150 mM NaCl, 20 mM MgCl2 (pH 7.4). (D) AFM height image (4 × 4 µm2; Z-scale, 10 nm; 128 lines × 128 lines) of DMPC bilayer on pSi imaged at close to breakthrough force.

( 1.84 nN (n ) 130) and 4.59 ( 1.74 nN (n ) 118), respectively (Figure 7). These forces are sightly lower than the breakthrough forces measured along the perimeter of DMPC bilayer islands on mica and silicon. The breakthrough force in the center of an island on pSi is around 65% lower than that measured on mica and silicon. The force-penetration curves for DMPC bilayer islands in Figure 7C show a total penetration of ∼8 nm before the breakthrough event. This penetration value is not fully due to the sample deformation but also to longrange hydration, steric and electrostatic forces. In fact, since

both DMPC and DPPC bilayers and the silicon nitride tip are negatively charged under the experimental conditions used here, a noticeable electrostatic repulsion arises as the tip approaches the sample.50,68 There is no reliable way to determine this electrostatic interaction accurately since it is experimentally difficult to calculate the tip charge density69 and the bilayer charge density once deposited on the substrate. After that, the breakthrough event consists of a ∼3 nm penetration. This value is lower than the total bilayer thickness since part of the deformation is included in the penetration before the break-

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J. Phys. Chem. B, Vol. 113, No. 30, 2009 10345 the bilayer,26,27,41 which in turn increases the area per lipid molecule. Consequently, lateral interactions between neighboring molecules are reduced and it is easier for the AFM tip to break the bilayer surface.48 Consistent with the work presented, research examining the elastic properties of porespanning lipid bilayers has observed a mechanical contrast as a function of distance to the pore rim,39 emphasizing the contribution of substrate interactions to the nanomechanical properties of lipid bilayers. Work probing the nanomechanical properties of native pore-spanning lipid bilayers on nanowells (200 nm diameter) has also afforded breakthrough forces equivalent to those measured for DMPC in the current study.70 Conclusions

Figure 8. Histograms of the breakthrough force values for DPPC in the central region (A) (19.58 ( 6.69 nN) and along the perimeter (B) (17.64 ( 6.28 nN) of pore-spanning lipid bilayer islands on pSi. All measurements were performed in 10 mM HEPES, 150 mM NaCl, 20 mM MgCl2 (pH 7.4).

through event. Therefore, the total penetration value observed in the force-penetration curve can be reconciled with the height measured of 4.61 ( 0.23 nm by cross-sectional analysis of the atomic force microscopy images (Figure 6E). If DMPC bilayer islands on pSi were imaged at close to breakthrough force, the bilayer is partly indented and the silicon pores become more apparent as a result (Figure 7D). However, height measurements on the bilayer under those conditions are not reflective of the true bilayer height and were therefore not performed. Force measurements performed on pore-spanning DPPC bilayers on pSi substrates gave breakthrough forces of 19.58 ( 6.98 nN (n ) 314) and 17.64 ( 4.47 nN (n ) 329) for the central region and along the perimeter of the bilayer islands, respectively (Figure 8). The values show that, just like in the case of DMPC bilayers on pSi, the nanomechanical properties of DPPC bilayers on pSi do not change substantially for the region being tested (i.e., the edge of the bilayer or its center). The breakthrough force values are also remarkably similar to those measured for DPPC bilayers along the perimeter of islands on mica and flat silicon. Breakthrough forces measured on all three substrates are summarized in Table 1. Our results show that the substrate pSi only weakly influences the nanomechanical properties of the lipid bilayer, judging by the lack of discrimination in nanomechanical stability between the center and the edge of bilayer islands (Table 1). We attribute the lower breakthrough force measured for DMPC and DPPC in the center of the bilayer island on pSi as compared to flat silicon to weak substrate interactions. Essentially, as the bilayer has less contact points with the substrate surface on a porous substrate than on a flat substrate, we expect an increase in surface mobility of

Single component DMPC and DPPC SPBs on mica and silicon substrates were prepared via the vesicle spreading technique. The breakthrough force data analysis proved that different nanomechanical stabilities can be observed in the center and along the perimeter of the bilayer islands. Furthermore, the preparation of pore-spanning lipid bilayers on porous silicon substrates was successfully demonstrated. In particular, the pore spanning bilayers were sufficiently stable to allow the nanoscale imaging of the bilayer architecture. Force spectroscopy measurements quantitatively revealed the effects of substrate on the nanomechanical properties of supported bilayers. For mica and silicon, we observed a significant decrease in the breakthrough force along the perimeter as compared to the center of a bilayer island. In contrast to the results for mica and silicon, measurements performed at the center of DMPC and DPPC bilayer islands deposited on pSi yielded breakthrough forces across the bilayer islands which were as low as the values measured along the perimeter of islands formed on the flat substrates. These results were related to a softening of the bilayer, which is indicative of and attributed to minimal substrate interactions. This in turn suggests a reduction of lateral interactions between neighboring phospholipid molecules due to the mechanical tension, with a consequent increase in surface mobility of the bilayer. In fact, substrates play two different stabilizing roles; first of all, the substrate gives a physical support to the bilayer, partly counteracting the pressure applied by the tip. Second, it can stabilize the bilayer by means of electrostatic interactions. Substrate stabilization was particularly effective for DMPC bilayers on flat substrate as judged by the more than 2.5-fold higher breakthrough force in the central region as compared to the perimeter. Another outcome of this study is that pSi solid supports have been shown to be suitable interfaces for the formation of porespanning lipid bilayers. The interstitial free space below the membrane bilayer facilitates a more fluid behavior, a fact that was clearly demonstrated by the substantial decrease in breakthrough force for pore-spanning lipid bilayers in comparison to those formed on flat supports. Providing a relevant model system for physiological cell membranes, these systems constitute valuable tools for the analysis of trans-membrane protein structure and activity. The biocompatibility of pSi, together with its ability to suspend lipid bilayers, also has important implications for potential applications in drug delivery and biosensing. Acknowledgment. Financial support from the Australian Research Council and from the Oz Nano2Life network is kindly acknowledged. References and Notes (1) Domenech, O.; Morros, A.; Cabanas, M. E.; Montero, M. T.; Hernandez-Borrell, J. Supported planar bilayers from hexagonal phases. Biochim. Biophys. Acta 2007, 1768, 100–106.

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