Gel-to-Fluid Phase Transformations in Solid-Supported Phospholipid

Article pubs.acs.org/JPCB

Gel-to-Fluid Phase Transformations in Solid-Supported Phospholipid Bilayers Assembled by the Langmuir−Blodgett Technique: Effect of the Langmuir Monolayer Phase State and Molecular Density Mohini Ramkaran† and Antonella Badia* Department of Chemistry, FRQNT Centre for Self-Assembled Chemical Structures, Université de Montréal, C.P. 6128 succursale Centre-ville, Montréal, QC H3C 3J7, Canada S Supporting Information *

ABSTRACT: Planar-supported phospholipid bilayers are increasingly used as synthetic membranes for scientific and practical applications. The thermotropic phase properties of supported bilayers are important for recreating biologically relevant situations. Unlike free-standing lipid membranes that undergo one gel-tofluid or main phase transition, mica-supported single bilayers have been found to undergo two separate leaflet transitions. Although the distinctive nature of the main transition in mica-supported bilayers has been attributed to different effects, determining their relevance has been problematic because vesicle fusion, the technique most widely used to prepare solid-supported bilayer membranes, does not allow one to readily control the lipid surface coverage and molecular density. To circumvent the limitations of the vesicle fusion method and systematically investigate the effects on the individual leaflet transitions of the lipid phase state and molecular density before deposition on the substrate, mica-supported single bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were prepared using the Langmuir−Blodgett technique. The gel-to-fluid transitions of the bilayer leaflets were tracked by controlled-temperature atomic force microscopy to determine the relative fractions of the gel and fluid phases as a function of temperature. The fraction of solid versus temperature data was fit to the van’t Hoff equation to determine the leaflet melting temperatures and transition enthalpies. The phase state and molecular density of the Langmuir monolayer precursor at the transfer pressure of 35 mN m−1 was found to have a greater effect on the main transition temperature and width of the distal (upper) leaflet than that of the proximal (lower) one. The contributions of substrate-mediated condensation, asymmetric lipid densities, and surface area available for thermal expansion of the bilayer are addressed. This work demonstrates the potential of the Langmuir−Blodgett technique as a tool for identifying and manipulating the factors that govern the phase transition properties of surface-confined lipid bilayers.

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behavior, such as decoupled gel−fluid phase transitions13−17 and reduced lateral mobility of the proximal leaflet lipids,18,19 which complicate the reproduction of biologically relevant situations in supported lipid membranes, as well as an asymmetric transbilayer distribution of charged lipids in mixed fluid bilayers of charged and zwitterionic phospholipids.20−22 Phospholipid bilayers undergo a series of thermotropic phase transitions. The gel-to-fluid or main phase transition is the one that occurs at the highest temperature, involving melting of the lipid acyl chains and extensive rotational motion.23 The main transition is a particularly significant consideration for producing and using solid-supported bilayers because the thermodynamic state of the lipid membrane is an important regulator of its physical structure, fluidity, and mechanical integrity. Gel−fluid transitions of the bilayer leaflets at different

INTRODUCTION Single bilayer films of lipids directly supported on planar solids are a type of biomembrane model of interest for biotechnological applications (electro-optical biosensors, microfluidic platforms, membrane microarrays) and are increasingly used in biophysical investigations of cell-membrane-based processes and membrane-anchored molecules by surface-sensitive techniques.1−4 An ultrathin water layer separates the adsorbed bilayer from the hydrophilic surface (e.g., mica, silicon oxide, borosilicate glass). The bilayer is physically held at the surface by a combination of hydration, van der Waals, and electrostatic forces and it is indefinitely stable as long as the system remains in an aqueous environment. The most common methods of preparation are vesicle fusion5−8 and monolayer transfer from the air/water interface using the Langmuir−Blodgett9,10 or Langmuir−Blodgett/Langmuir−Schaefer11,12 techniques. For both scientific and practical applications, solid-supported bilayers should mimic the physicochemical properties of freestanding lipid membranes. Recent studies, however, report notable substrate-induced differences in the single bilayer © XXXX American Chemical Society

Received: April 26, 2014 Revised: July 2, 2014

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dx.doi.org/10.1021/jp504092b | J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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Table 1. Physical Properties of Fully-Hydrated Multilamellar Membranes of DMPC and DPPC phase

gel (T = 10 °C for DMPC, T = 20−24 °C for DPPC) fluid (T = 30 °C for DMPC, T = 50 °C for DPPC)

property

DMPC

DPPC

TMLV (°C)28 m ΔHm (kJ mol−1)28 area/lipid (nm2)87,88 bilayer thickness (nm)33,88

23.5 ± 0.4 24.7 ± 2.7 0.472 ± 0.005 4.42

41.4 ± 0.5 34.8 ± 3.2 0.479 ± 0.002 4.78

area/lipid (nm2)33,89 bilayer thickness (nm)33,89

0.606 ± 0.005 3.63

0.64 3.85

headgroups15,39 or the ordered water layer sandwiched between the mica and bilayer film,14,42 (iii) higher lipid density in the proximal leaflet relative to the distal one,13,39 and (iv) melting at quasi-constant surface area and variable surface tension.13 Studies have in fact shown that the supported bilayer preparation method (vesicle fusion versus Langmuir−Blodgett deposition), solution ionic strength, substrate−vesicle incubation temperature, and substrate type modulate effect (i), resulting in either two (decoupled) phase transitions or a single (coupled) transition.19,39,41 More specifically, a salted imaging solution and the fusion of vesicles at a temperature greater than the proximal layer Tm lead from a decoupled to a coupled phase transition for phospholipid bilayers assembled onto mica.39 The former should decrease the electrostatic interaction between the mica and the proximal leaflet lipids whereas the effect of the vesicle−substrate incubation temperature is tentatively attributed to a variation in the lipid densities of the leaflets favoring the interdigitation of the lipid chains.39 A greater surface roughness (silicon oxide and glass versus mica) reduces the proximal leaflet−substrate coupling and favors a single phase transition.19,41 Scomparin et al. also showed an effect of the bilayer preparation method on the temperature dependence of the lipid diffusion coefficient.19 For example, glass-supported bilayers of dialkylphosphatidylcholines exhibit a single diffusion component as a function of temperature when assembled by Langmuir−Blodgett/Langmuir−Schaefer deposition whereas those formed by the fusion of fluid phase vesicles show two distinct components.19 Except for the investigation of Scomparin et al., which compared the lipid diffusion coefficients (lateral lipid mobilities) as a function of temperature in single bilayers prepared by vesicle fusion and the Langmuir−Blodgett technique,19 the aforementioned studies used the vesicle fusion method to form single-component or two-component phospholipid bilayers. It is difficult to control the reproducibility of the lipid adsorption/coverage (especially for phospholipids with high TMLV m ) and molecular density of solid-supported bilayers formed by vesicle fusion.8 The Langmuir−Blodgett technique, on the other hand, allows for the controlled deposition of monolayer films of precise molecular density and phase state (liquid-expanded or condensed phase) at a fixed surface pressure.43 Unlike the vesicle fusion method, supported bilayers with varying leaflet compositions and molecular packing densities can be prepared. A number of variables, such as the monolayer deposition temperature, surface pressure, lipid density, and monolayer transfer ratio can be systematically explored to gain a better understanding of the factors affecting the supported bilayer transition behavior. This article reports the controlled-temperature AFM investigation of gel-to-fluid phase transitions of fully hydrated mica-supported single bilayers of 1,2-dimyristoyl-sn-glycero-3-

temperatures and/or temperatures higher than those of freestanding bilayers (reference system) can lead to incomplete supported membrane fluidity/lipid mobility, as well as the presence of domain boundaries that act as passive defect sites.23,24 Membrane fluidity affects the dynamics of the interactions of lipid-linked bilayer constituents with their (macro-)molecular targets,25 whereas defect sites lead to nonspecific binding26 and compromise membrane electrical properties in biosensors with electrical detection.27 Much of our current understanding of phase transitions in biological membranes comes from experimental studies on aqueous dispersions of phospholipids in the form of unilamellar or multilamellar vesicles (MLVs) and theoretical calculations on free-standing bilayers.23,24,28,29 The gel-to-fluid transition in MLVs is a highly cooperative endothermic process (transition widths of 99%) from Avanti Polar Lipids (Alabaster, AL) and used without further purification. The phospholipids were dissolved in spectrograde chloroform (A&C American Chemicals, Montreal, QC) to a concentration of 1 mM. High-purity water (18.2 MΩ cm) obtained by further purification of distilled water with a Milli-Q Gradient system (Millipore, Bedford, MA) was used as the subphase for the Langmuir−Blodgett trough. Its surface tension was measured to be 72.1 mN m−1 at 22.0 °C. Ruby muscovite mica (ASTM grade 2, B&M Mica Co., Inc., Flushing, NY) was cleaved immediately before its immersion into the aqueous subphase. Langmuir Monolayer Isotherms and Langmuir− Blodgett Deposition. A 100 μL volume of DMPC or DPPC solution was spread on the water surface (area of 768 cm2) of a computer-controlled KSV 3000 trough equipped with a roughened platinum plate attached to a surface pressure sensor (KSV Instruments Ltd., Helsinki, Finland). The subphase temperature was regulated to within ±0.5 °C with an Isotemp model 1006D circulation bath (Fisher Scientific Limited, St-Laurent, QC). After waiting 20 min for the chloroform to evaporate, the phospholipid monolayer was symmetrically compressed at a rate of 0.01 nm2 molecule−1 min−1 up to the film collapse to record surface pressure (π) versus molecular area (A) isotherms. Bilayer films were deposited onto mica by Y-type vertical transfer at 35 mN m−1, unless otherwise specified. A surface pressure of 30−35 mN m−1 is believed to be appropriate to model lipids in bilayer membranes.44,45 The dialkylphosphatidylcholine monolayer was first compressed to 35 mN m−1 and maintained at this pressure for 20 min. It was then transferred from the air/water interface onto mica by pulling the immersed mica sheet upward through the interface (upstroke) at a velocity of 5 mm min−1. After waiting 20 min for the barrier movement and surface pressure to stabilize, the monolayercovered mica was vertically lowered (downstroke) through the air/phospholipid monolayer/water interface and into the aqueous subphase. Asymmetric DPPC bilayers were prepared by transferring a first monolayer (upstroke) at 35 mN m−1 and 20 °C, assembling a new monolayer at the air/water interface, and transferring this second film (downstroke) at 55 mN m−1. Bilayers were transferred under trough water to the thermoregulated fluid cell used for AFM imaging. Temperature-Controlled AFM Imaging. The micasupported phospholipid bilayers were imaged under water in intermittent-contact (tapping) mode with an extended Dimension 3100 scanning probe microscope and Nanoscope IIIa controller (Bruker Nano, Santa Barbara, CA) using silicon nitride microlevers (model no. MLCT or MSNL, Bruker AFM Probes, Camarillo, CA) of nominal spring constant of ∼0.1 N m−1, resonance frequency of ∼8 kHz in liquid, and a tip radius of ∼2 nm (MSNL) or ∼20 nm (MLCT). Height and phase

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RESULTS Langmuir Monolayer Isotherms and Langmuir− Blodgett Transfer. Surface pressure−molecular area (π−A) isotherms were recorded at the air/water interface at different subphase temperatures (TA/W): TA/W = 10, 15, and 20 °C for DMPC (TMLV = 23.5 °C, Figure 1A)28 and TA/W = 20 and 33 m °C for DPPC (TMLV = 41.4 °C, Figure 1B).28 The π−A m isotherms provide the lipid areal density as well as the phase state (liquid-expanded or condensed) of the Langmuir monolayer precursor at the film transfer pressure. The related data are summarized in Table 2. Except for DMPC at TA/W = 20 °C, the isotherms present either a plateau or shoulder indicative of a first-order transition from a fluid-like or liquidexpanded phase, in which the lipid heads are translationally disordered and the alkyl chains are conformationally disordered, at higher molecular area to a solid-like or condensed phase of closely packed and conformationally ordered lipid molecules at lower area.48 The alkyl chains in the condensed phase are tilted by ∼30° from the surface normal toward neighboring lipid molecules.48−50 Dialkylphosphatidylcholines adopt similar chain tilt angles in the gel phase of MLVs.33 The temperature dependence of the isotherms shown in Figure 1 is the same as that reported previously by different laboratories.51−53 As the aqueous subphase temperature increases, the liquid-expanded-to-condensed phase transition pressure (πLE‑C, Table 2) increases so that the lipid monolayers are in a more C



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Because the gel-to-fluid transition temperatures of MLVs of DMPC and DPPC are significantly different, and the critical transition temperature of DPPC is too high (Tc = 41−43 °C)51,54 to allow the assembly and transfer of monolayer films in the liquid-expanded state at 35 mN m−1 on our trough, the melting behaviors of mica-supported single bilayers prepared at a constant reduced temperature of 8.4−8.5 °C (i.e., TMLV − m TA/W) are compared in this article: TA/W = 15 °C for DMPC versus TA/W = 33 °C for DPPC. At these temperatures, π of 35 mN m−1 is situated just above (i.e., 6−9 mN m−1 above) the liquid-expanded-to-condensed phase transition regions of these lipids (Figure 1). Microscopy imaging has shown the remnants of liquid-expanded phase in this region of the isotherm,55,56 which is referred to as the “condensed-dominant phase” region.55 The mean areal density of DMPC at 15 °C is similar to that at 10 °C (Table 2). The mean molecular area of 0.54 nm2 of the DPPC monolayer at 33 °C is less than the lipid area of 0.64 nm2 in fluid phase MLVs of DPPC, but greater than the monomolecular lipid area at 20 °C (Table 2). Transfer ratios for the deposition of the proximal (bottom) and distal (top) monolayers onto mica at 35 mN m−1 are given in Table 2 as a function of TA/W. The transfer ratio (TR) is the ratio of the difference in the total area of the lipid monolayer at the air/water interface before and after transfer (ΔA) and the surface area of the mica (A) (i.e., TR = ΔA/A). In general, transfer ratios between 0.95 and 1.05 reflect that the monolayer is properly transferred to the substrate,43 and it is generally assumed that no significant desorption or condensation of the molecules occurs during transfer. Transfer ratios close to 1.00 were recorded for the deposition of the proximal layer at temperatures where the lipids are in the condensed phase at 35 mN m−1, i.e., TA/W = 10 °C for DMPC and 20 °C for DPPC. Transfer ratios ranging from 1.15 to 1.26 were obtained for proximal layer transfers of the liquid-expanded DMPC (TA/W = 20 °C) and condensed phase dominant DMPC (TA/W = 15 °C) and DPPC (TA/W = 33 °C), indicating that the lipids undergo substrate-mediated condensation so that the proximal layers are ∼15% to ∼26% denser on the mica than at the air/water interface. The substrate-mediated condensation and densification of phospholipids is a documented phenomenon that readily occurs for monolayer transfer in the liquid-expanded/ condensed phase coexistence region or at surface pressures below the liquid-expanded-to-condensed phase transition pressure, that is, directly from the pure liquid-expanded phase.57−61 Graf and Riegler report transfer ratios between 1.1 and 1.7 for the transfer of liquid-expanded 1,2-dimyristoylsn-glycero-3-phosphoethanolamine (DMPE) onto silicon oxide at surface pressures below the phase transition pressure, and

Figure 1. Surface pressure−area (π−A) isotherms of the phospholipid monolayers recorded on a pure water subphase held at different temperatures. (A) DMPC. (B) DPPC.

expanded state (higher molecular area) at the film transfer pressure of 35 mN m−1 (Alipid, Table 2). At TA/W of 20 °C, the shorter-chain DMPC is near its critical monolayer temperature (Tc) of 21−22 °C.53,54 The isotherm presents a break suggestive of a phase transition at ∼44 mN m−1, just below the film collapse at ∼45 mN m−1 (Figure 1A). DMPC is in the liquid-expanded state at the air/water interface at 35 mN m−1, occupying a molecular area of 0.53 nm2 that is less than the lipid density of 0.61 nm2 reported for fully hydrated MLVs of fluid phase DMPC (Table 1). Decreasing the subphase temperature to 10 °C decreases πLE‑C to ∼18 mN m−1 (Figure 1A), so that at 35 mN m−1, the monolayer is in a condensed phase where the lipid area of 0.47 nm2 is the same as that in gel phase multilamellar DMPC (Table 1). At TA/W of 20 °C, the longer-chain DPPC undergoes a liquid-expanded-to-condensed phase transition at 3.5 mN m−1 and the monolayer collapses at π of ∼71 mN m−1 (Figure 1B). DPPC is in the condensed state at 35 mN m1,55 and its molecular area of 0.46 nm2 is comparable to the lipid area in vesicles of gel phase DPPC (Table 1).

Table 2. Characteristics of the Langmuir Monolayer Precursors and Layer Transfer Ratios lipid

TA/Wa (°C)

DMPC

10 15 20 20 33

DPPC

πLE‑Cb (mN m−1)

phase state π = 35 mN m−1

Alipid (±0.01 nm2)c π = 35 mN m−1

TRproximald

TRdistald

± ± ± ±

condensed condensed-dominant liquid-expanded condensed condensed-dominant

0.47 0.48 0.53 0.46 0.54

1.03 1.22 1.26 1.02 1.15

0.97 0.99 0.83 0.95 1.05

18 29.0 44.0 3.7 25.5

1 0.4 0.5 0.5

a TA/W = aqueous subphase temperature for Langmuir monolayer assembly and transfer. bπLE‑C = liquid-expanded-to-condensed monolayer phase transition pressure (mean value and standard deviation). cAlipid = lipid area at the air/water interface. πLE‑C and Alipid were determined from π−A isotherms. Each π−A isotherm was repeated at least once, except for TA/W = 33 °C. The uncertainty of ±0.01 nm2 given for Alipid is based on the maximum variation obtained at 35 mN m−1 between repeated isotherms. dTRproximal and TRdistal = transfer ratios at π = 35 mN m−1 for the proximal and distal leaflets of the bilayers whose AFM images are shown in Figures 2 and 3.

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Figure 2. Intermittent-contact AFM images (5.0 μm × 2.5 μm) in pure water captured at various temperatures while heating DMPC single bilayers transferred onto mica at 35 mN m−1. (A) TA/W = 10 °C. (B) TA/W = 15 °C. (C) TA/W = 20 °C.

transfer ratios close to 1.0 for the transfer of condensed phase lipid.57 Attractive substrate/monolayer interactions and/or dehydration during deposition are believed to be the sources of this condensation process.57,59−61 Condensation of the proximal layers will become important later on for the discussion of their chain melting transitions. Transfer ratios of 0.95−1.05 were recorded for the distal layer transfers, except for the liquid-expanded DMPC transferred at TA/W = 20 °C (Table 2). The transfer ratio of 0.83 recorded for the distal layer is attributable to the desorption of some proximal DMPC molecules from the mica to the air/water interface during transfer of the second (distal) monolayer,9 and this is corroborated by the AFM images, which show a greater proportion of bilayer hole defects (Figure 2C). Mica-Supported Single Bilayer Film Topography. AFM height (topography) images of the mica-supported bilayer films acquired at the start of the heating runs (see top images in Figure 2) exhibit three distinct height levels, which correspond to solid (gel) phase lipid in both the proximal and distal leaflets (gold-yellow color), an intermediate phase in which the distal leaflet is melted or fluid and the proximal leaflet is solid (brown), and circular hole defects (brown-black), whose depths of ∼4 to ∼6 nm (Table 3) are comparable to the thickness of a single lipid bilayer.13,14,17,47,62−68 Mono- and bilayer-deep hole defects, whose edges are thought to be stabilized by phospholipids forming hemimicelles, have been previously observed in supported bilayers formed by the Langmuir−Blodgett technique, and their presence has been linked to the transfer of the second (distal) layer,9,10 as already mentioned in the previous section. The area fraction, shape,

Table 3. Characteristics of the Mica-Supported Phospholipid Single Bilayers Prepared by the Langmuir−Blodgett Technique at a Surface Pressure of 35 mN m−1 and Different Temperatures hole defectsa lipid

TA/W (° C)

DMPC

10 15 20 20 33

DPPC

surface coverage (%) 2 7 21 2 3

± ± ± ± ±

1 2 2 1 1

distal leafleta depth (nm)

% fluid

% solid

± ± ± ± ±

19 40 90 0 7

81 60 10 100 93

4.7 4.9 4.4 5.8 5.1

0.2 0.3 0.2 0.3 0.2

a

Hole defects and lipid phase coverages were analyzed using AFM images at the start of the heating runs, i.e., at TAFM = 16 ± 1 °C for DMPC or TAFM = 25 ± 4 °C for DPPC. Hole defect coverages are the average and standard deviation of AFM images acquired on different bilayers. The hole defect depths and % fluid and % solid phases are for the bilayers shown in Figures 2 and 3.

and size of the defects have been found to depend on the film transfer pressure, number of defects in the proximal layer, type of lipids used, and on the phase state of the lipids transferred. The initial area coverage of the hole defects in films of a given lipid increases with increasing TA/W (Table 3). The hole defect area coverage is smallest (∼2%) in bilayers formed via the transfer of condensed phase DMPC and DPPC and largest in films formed from the transfer of liquid-expanded DMPC (∼21%). The densely packed, conformationally ordered alkyl chains present in the condensed monolayer phase results in the E



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Table 4. Mica-Supported Bilayer Melting Dataa,b ΔHvH (kJ mol−1)

Tm (° C) lipid

TA/W (° C)

DMPC

10 15 20 20 33

DPPC

distal 20.0 16.8 10 °C gap between the distal and proximal leaflet meltings is not due to kinetics and that a slower heating rate would not narrow the gap. The AFM image acquired at T = 16.7 °C of the supported DMPC bilayer formed at TA/W = 20 °C (Figure 2C) shows an almost completely melted distal leaflet, which is expected given that it was transferred in the liquid-expanded state. The remaining solid phase occupies only 10% of the total lipidcovered area. The height difference of 4.4 ± 0.2 nm measured between the top of the solid phase and bottom of the hole defects is less than the mean of 4.7 ± 0.2 nm calculated from values determined by AFM for gel phase DMPC bilayers,13,14,17,47,62,64,65 but greater than the thickness of fluid phase bilayers of 3.6 ± 0.2 nm,47,64,65,67,68 suggesting that the holes contain some fluid (melted) lipid material. No solid phase remains at 19 °C. The proximal leaflet melts above 27 °C, where one observes the coexistence of pre-existing hole defects, a completely melted lipid phase, and a branch-like intermediate phase. The measured height difference between the intermediate and completely melted lipid phases is 0.7 ± 0.2 nm. The area fraction occupied by the hole defects decreases as these are filled with additional melted lipid. The proximal leaflet melting regime ends with the fluid phase and remaining hole defects dominating the bilayer topography (TAFM = 36.8 °C). We verified the reversibility of the melting transitions by cooling a melted DMPC bilayer transferred at TA/W = 10 °C from 34 to 17 °C (AFM images shown in Figure S4,

loss of fewer lipid molecules from the solid-supported proximal layer during the deposition of the distal layer, and hence to fewer hole defects. Likewise, the initial area coverage of the thinner intermediate lipid phase, usually in the form of dendritic or branch-like depressions that appear to run along the direction of substrate withdrawal from/immersion into the air/water interface, increases with increasing TA/W for a given lipid (% fluid phase in distal leaflet, Table 3). In DMPC bilayers transferred at 20 °C (liquid-expanded state), this intermediate phase constitutes 90% of the lipid-covered (i.e., nonhole) area. It is important to note that defects (holes, depressions, and cracks) are also present in solid-supported phospholipid bilayers prepared by the vesicle fusion method.8,13−15,17,38 Controlled-Temperature AFM Imaging. AFM height images of the mica-supported bilayers acquired at different temperatures while heating the samples are presented in Figures 2 (DMPC) and 3 (DPPC). The gel-to-fluid phase transformations/chain melting transitions are characterized by the coexistence of domains of different heights or thicknesses and phase contrast (Figure S1, Supporting Information). The melted or liquid-like regions appear thinner with respect to the solid-like or condensed domains. The observed height difference is a manifestation of the acyl chains of phospholipids in the fluid phase having gauche defects (e.g., 4−7 gauche bonds per acyl chain for a DMPC membrane),69 resulting in a less stretched (shorter) chain configuration, and hence a thinner layer, with respect to the all-trans conformation that is prevalent in the gel or solid-like phase (Table 1).32 The contrast in phase reflects the difference in mechanical properties between the condensed and fluid phases of the bilayer.17,38 Because it is difficult to optimize the intermittentcontact mode conditions for both stable height imaging and phase contrast, the bilayer melting transitions were monitored by height differences. The mica-supported DMPC bilayer for which the constituent monolayers were transferred from the air/water interface in the liquid-expanded state exhibited a single transition, involving melting of the proximal leaflet, in the temperature range 17−37 °C. The distal and proximal leaflets of DMPC and DPPC formed from Langmuir monolayers in the condensed-dominant or condensed state were found to undergo distinct gel-to-fluid transitions. Height differences ranging from 0.5 to 1 nm (depending on the tapping strength used for stable imaging in liquid) were measured by AFM between the coexisting fluid and gel phases in both the proximal and distal leaflet melting transitions (Figures S2 and S3, Supporting Information). These height differences are consistent with previously reported values of 0.4−0.9 nm measured by AFM.13,15,17,38,68 The DMPC and DPPC bilayers prepared by the Langmuir−Blodgett method undergo thermally induced changes in topography that are similar to those reported for mica-supported single bilayers F



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Supporting Information). Between 31 and 26 °C, the proximal leaflet solidification proceeds through the formation of irregularly shaped condensed domains that are smaller in size than those observed during the proximal layer melting. The distal leaflet solidification is accompanied by a restructuring of the bilayer between 24 and 21 °C, which yields a film topography consisting of an interconnected network of solid phase surrounded by an intermediate phase and irregularly shaped hole defects. The hole defect area coverage is 4.5 ± 1.4% and the mean hole depth (with respect to the solid phase) is 4.4 ± 0.2 nm between 21 and 17 °C. DPPC Single Bilayers. Langmuir−Blodgett deposition of the longer-chain DPPC onto mica produced bilayers with the presence of few hole defects (2−3% surface coverage). At TA/W = 20 °C, the film consists of an all-gel lipid phase at room temperature (Figure S3, Supporting Information). The hole depth of 5.8 ± 0.3 nm is in agreement with previously published AFM data for solid-supported DPPC,63,66 indicating that the hole defects extend down to the bare mica. The distal leaflet melting in the all-gel DPPC begins with the presence of linear depressions (TAFM = 34.1 °C, Figure 3A) that expand in area with increasing temperature (TAFM = 38.9 and 40.5 °C, Figure 3A). The distal leaflet melting of the DPPC bilayer transferred at TA/W = 33 °C (Figure 3B) proceeds in a manner comparable to that for the DMPC bilayers transferred in the condensed (Figure 2A) or condensed-dominant (Figure 2B)

phase. The topographic changes that occur during the proximal leaflet melting of DPPC (Figure 3) are generally similar to those observed for DMPC (Figure 2). Interestingly, the lipid areas at the outer peripheries of pre-existing hole defects, which are filled with fluid material, melt last in the DPPC bilayer transferred at TA/W = 33 °C (Figure 3B, TAFM = 48.4 °C), forming doughnut-like structures (see same spot images in Figure S5, Supporting Information) instead of the irregularly shaped domains observed in Figure 2. This feature has been captured during the proximal leaflet meltings of both DMPC and DPPC bilayers but is not the usually observed behavior. Parikh et al. have reported a related finding in which the gel-tofluid transition temperature of lipids located at the outer edge of glass-supported bilayer patches is higher than those located within the bulk of the patch.70 Although we point out this unusual phenomenon, a discussion of its origin is outside of the scope of this paper. A DPPC bilayer with asymmetric leaflet densities was also prepared at TA/W = 20 °C by depositing the proximal leaflet at the usual π of 35 mN m−1 and molecular area of 0.46 nm2 and the distal leaflet at 55 mN m−1 and area of 0.42 nm2 molecule−1. A sequence of AFM images acquired upon heating the bilayer is shown in Figure S6 (Supporting Information). The distal and proximal leaflets undergo the same temperatureinduced topographic changes as those of the symmetric bilayer (Figure 3A). Bilayer Melting Curves. Melting curves constructed for the distal and proximal leaflet transitions by plotting the fraction of the lipid-covered surface area occupied by the solid phase (fsolid) as a function of temperature (T) are shown in Figure 4. It is important to note that because the distal leaflet of the DMPC bilayer transferred in the liquid-expanded phase (TA/W = 20 °C) was almost entirely melted at the beginning of the heating run, a full fsolid−T plot could only be constructed for the proximal leaflet melting regime. Figure 4 shows two wellseparated transitions. In all cases, the fractional coverage of the solid phase is 0 at the end of the distal leaflet transition and the proximal leaflet melts only when the distal leaflet has completed its transition. Consistent with previous reports,13,16,17,39 the leaflet transition widths are significantly larger than the width of the main transition of MLVs, which can range from Tproximal m phase transitions at temperatures that are similar to those of bilayers prepared at an incubation temperature around the vesicle Tm (25 °C): transitions at 22 and 32 °C in bilayers assembled at an incubation temperature of 36 °C versus transitions at 22 and 30 °C for bilayers formed at 23 °C.39 Vesicle fusion, rupture, and spreading on the mica surface results in the simultaneous deposition of both leaflets.7 Adsorbed bilayer−vesicle interactions occur during bilayer formation. By contrast, preformed monolayers are individually transferred from the air/water interface by the Langmuir− Blodgett technique. These two methods of supported bilayer formation produce different thermotropic phase behaviors for deposition from the fluid phase. Two distinct melting transitions are observed by AFM for the DMPC and DPPC bilayers prepared by the transfer onto mica of Langmuir monolayers in the condensed-dominant or condensed phase. The Tm’s of the distal leaflets transferred in the condensed phase (TA/W = 10 °C for DMPC and 20 °C for DPPC) are higher than the ones transferred in the condenseddominant phase (TA/W = 15 °C for DMPC and 33 °C for DPPC) and the initial fraction of fluid or melted lipid in the distal leaflet is lower (Tables 3 and 4). As TA/W decreases, Tdistal m distal approaches TMLV (41.4 ± 0.2 °C) of the DPPC bilayer m . Tm of 41.4 ± 0.5 formed at TA/W = 20 °C is equivalent to TMLV m °C.28 Tdistal (20.0 ± 0.3 °C) of the DMPC bilayer prepared at m of 23.5 ± 0.4 °C.28 TA/W = 10 °C is, however, less than TMLV m MLV TA/W = 20 °C is situated 21.4 °C below Tm of DPPC and the monolayer transfer pressure of 35 mN m−1 is situated 31 mN m−1 above πLE‑C, whereas TA/W = 10 °C is 13.5 °C below TMLV m of DMPC and the monolayer is transferred at 17 mN m−1 above πLE‑C. At TA/W = 10 °C, we are not far enough above the transition region to transfer a completely ordered (solid) DMPC monolayer at 35 mN m−1. This is corroborated by the observation that at a reduced imaging temperature of 7.3−7.5 °C, the DMPC bilayer assembled at TA/W = 10 °C presents a significantly higher fraction of distal leaflet fluid phase (19%, TAFM = 16.0 °C, Figure 2A) than the DPPC bilayer transferred at TA/W = 20 °C (3%, TAFM = 34.1 °C, Figure 3A). Distal leaflet may require that its Langmuir melting of DMPC at TMLV m monolayer precursor be assembled and transferred at TA/W < 10 °C to further lower πLE‑C. There is no temperature effect on the proximal leaflet Tm of bilayers prepared from the condensed-dominant versus condensed phase (Table 4), most probably due to substratemediated condensation of the former. The mica-mediated condensation of the condensed-dominant monolayers produces a solid phase similar to that of the lateral monolayer compression to the condensed phase at the air/water interface, probably because the fluid material in the Langmuir monolayers before transfer is significantly less than in the case of transfer from the liquid-expanded phase. Because TA/W has a larger effect on the Tm of the distal leaflet than that of the proximal one, the gap between the distal and proximal leaflet Tm’s increases with increasing TA/W (Table 4). Moreover, ΔTm seems to be chain length dependent, because at a constant reduced temperature of 8.4−8.5 °C, ΔTm of the shorter-chain DMPC bilayer (16 °C) is larger than ΔTm of the longer-chain DPPC (9 °C). These results are in contrast to those of Gewirth et al., who report that the proximal leaflet transition occurs at + 5 °C, irrespective of the dialkylphosphatidylcholine Tdistal m

chain length (14, 15, and 16 carbons), in mica-supported bilayers prepared by vesicle fusion.14 Effect of TA/W on the Transition Width. Increasing TA/W broadens the distal leaflet transition but generally does not influence the proximal leaflet one (i.e., across the TA/W range investigated). A comparison of the fsolid−T curves of DPPC bilayers prepared at 20 °C (condensed phase at 35 mN m−1) versus 33 °C (condensed-dominant phase) best demonstrates this effect (Figure 4B). The temperature-induced broadening of the distal leaflet transition can be rationalized in terms of a decrease in the finite size of the cooperatively melting lipid cluster or domain.68,71 The liquid-expanded/condensed phase transition of lipids at the air/water interface is the monolayer equivalent of the gel/fluid transition in lipid membranes.45,51 The shorter and more sloped transition plateau in the π−A isotherm of the DPPC monolayer at TA/W of 33 °C versus 20 °C indicates a smaller cluster size (Figure 1B).54,72 The transfer ratios (Table 2) suggest that the distal layer does not undergo a substrate-mediated condensation/densification so that its phase structure should remain unchanged. If one assumes that the number of cooperatively transforming lipids in the Langmuir monolayer precursor is reflective of the cooperatively melting cluster size in the distal leaflet, the unit size should be smaller for the distal layer transferred at TA/W = 33 °C. The size of the cooperatively melting lipid cluster can be estimated from the (Table 1).39,67,71 For the ratio of ΔHvH (Table 4) and ΔHMLV m distal leaflet of DPPC, the cooperative unit size decreases from 32 ± 8 at 20 °C to 8 ± 1 at 33 °C. In the case of the proximal leaflet, substrate-mediated condensation decreases the DPPC molecular area to that of monolayers transferred at 20 °C. Consequently, the fsolid−T curve of the proximal leaflet of the DPPC bilayer at TA/W = 33 °C is similar to the proximal leaflet curve at TA/W = 20 °C (Figure 4B). The van’t Hoff transition enthalpies determined for the proximal leaflets of DMPC and DPPC are 2−6 times larger than those of the distal leaflets (Table 4), indicating an increased cooperative unit size, which is also reflected by the decreased widths of the proximal leaflet transitions (Figure 4). These findings suggest that the gel-tofluid transition widths are determined by the finite size of the cooperatively melting lipid clusters and that phospholipid-mica interactions increase the cooperativity of the gel-to-fluid transition of the proximal leaflet with respect to the distal leaflet. Monolayer Transfer Ratio and Bilayer Leaflet Density. An asymmetric lipid density has been invoked to explain the presence of two separate transitions in supported bilayers.13,39 To the best of our knowledge, there is no experimental proof for the existence of a substrate-induced lipid density imbalance in planar supported bilayers, nor is any given in the papers on decoupled gel-to-fluid transitions cited above. Such information is not readily obtainable for solid-supported bilayers formed by vesicle fusion, which remains the most prevalent preparation method. The results of theoretical simulations using a DPPC bilayer and a hydrophilic smooth substrate suggest that the lipid density in the proximal leaflet is up to 5% higher than that of the distal leaflet.73,74 The transfer ratio measured by the Langmuir−Blodgett technique allows the quantification of changes in the molecular density of a monolayer upon its transfer at a constant surface pressure from the air/water interface, where the molecular area is known from the π−A isotherm, to a solid substrate. Substrate-induced density changes of a few percent, as suggested from the simulations,73,74 are, however, within the experimental error of the I



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We therefore conclude that asymmetric lipid densities alone cannot explain the separate gel-to-fluid transitions exhibited by the mica-supported bilayers. A difference in molecular order might instead be at the origin of the decoupled bilayer phase transitions. Spectroscopic investigations of asymmetric bilayers of hydrogenated and deuterated DMPC deposited on bare gold and chemically modified, nanostructured silver surfaces by Langmuir− Blodgett/Langmuir−Schaefer transfer show differences in either the tilt angle or conformation of the acyl chains in the proximal and distal leaflets due to substrate−lipid interactions.77,78 Likewise, strong electrostatic interactions between the weakly hydrated mica3,79 and DMPC or DPPC could result in a more ordered proximal leaflet and a higher Tm. Gel-to-Fluid Transition at Quasi-Constant Surface Area. The fundamental question of how lipids transit from the solid to fluid phase on a constant or quasi-constant area surface has received little consideration thus far.13 The gel-tofluid transition in free-standing bilayers occurs at constant surface tension, resulting in lipid molecular area increases of 28% and 34% for DMPC and DPPC, respectively (Table 1). In solid-supported bilayers, the physical space for two-dimensional expansion is limited (i.e., domain boundaries and hole defects). For this reason, Charrier and Thibaudau propose that the melting transition in supported bilayers occurs at quasiconstant area and varying surface tension after observing two broadened gel-to-fluid transitions (i.e., transition widths of 5.5− 6.5 °C) for mica-supported DMPC bilayers with a hole defect coverage of ∼2% prepared by vesicle fusion, with Tm values (23.5 °C): Tdistal = 31 °C and significantly higher than TMLV m m 13 = 42 °C. The idea is that the inability of the leaflets to Tproximal m expand on the surface during their melting transition yields an increasing lateral pressure with temperature and a fluid phase that is compressed relative to the fluid phase in free-standing bilayers, resulting in a shift in Tm (by analogy with the Clausius−Clapeyron relation) and broadening of the transition width. The authors use a constant area and matter melting model as the core model (eq 6 in ref 13 and Figure S7, Supporting Information), which includes the area thermal expansion coefficients and area compressibilities in the gel and fluid phases, ratio of the molecular areas of the gel and fluid phases at Tm, and the reciprocal of the variation of the transition temperature with the bilayer surface tension (dπ/ dTm). Typical literature values for free-standing bilayers are used by the authors (although the transferability of bilayer vesicle values to surface-confined bilayers is questionable), except for dπ/dTm, which is taken from a Langmuir monolayer study. Because the model generates a melting curve (Figure S7A, Supporting Information) that does not fit their fsolid−T data, the authors refine the model to account for the small variation (∼4%) in the lipid area due to hole filling/closing during melting (i.e., quasi-constant surface area transition) and differences in the area compression of the distal and proximal leaflets.13 However, an inappropriate choice of parameters in the multiparameter core model significantly affects the applicability and fit of the model, or a refined version of it, to the experimental data (Figure S7, Supporting Information, for calculated curves). First, the authors incorrectly choose a dπ/ dTm value of 2.16 mN m−1 K−1 derived from twice dπLE‑C/dT of Langmuir monolayers of negatively charged 1,2-dipalmitoylsn-glycero-3-phosphoglycerol (DPPG) instead of twice dπLE‑C/ dT of zwitterionic dialkylphosphatidylcholine monolayers (i.e., dπLE‑C/dT doubles when a bilayer is considered).13,80 dπLE‑C/

transfer ratio determination and cannot be established with certainty. The interleaflet flip-flop of lipid molecules in planar supported bilayers depends on the acyl chain length, surface pressure, and temperature.12,75 It is an extremely slow process in the gel phase. Flip-flop rate constants ranging from 3.95 × 10−5 to 6.28 × 10−4 s−1 have been measured by sum-frequency vibrational spectroscopy (SFVS) at temperatures between 27.7 and 36.6 °C for silica-supported DPPC assembled by Langmuir−Blodgett/Langmuir−Schaefer at surface pressures between 30 and 36 mN m−1. Interleaflet lipid exchange is too rapid in the fluid bilayer to be measurable by SFVS.75 Laterally nanostriped phospholipid bilayer films with vertically superimposed condensed and fluid phases have been successfully prepared from two-component monolayers by Langmuir− Blodgett transfer on mica (these nanostructured bilayers also present bilayer hole defect coverages of 20−25%).76 Asymmetric transfers of single- and two-component bilayers of condensed and fluid phospholipids suggest that the stripe structure would not exist if there was a reorganization of the layers.76 We therefore exclude the interlayer transfer/rearrangement of lipid molecules during the deposition of the distal layer onto the lipid-covered mica. For monolayers transferred in the liquid-expanded or condensed-dominant state, there is clear evidence for the substrate-mediated densification of the proximal layer (Table 2). We limit ourselves to the DMPC bilayer prepared at TA/W = 15 °C and DPPC at 33 °C (i.e., constant reduced temperature) for which the proximal layer TR values reflect a 15% to 22% higher density on the mica relative to the air/water interface and the distal layer TR values are very close to 1.00, indicating that lipid loss during the distal layer transfer is small, as reflected by the low surface coverage of hole defects (∼3−7%). The transfer ratios suggest a significant lipid density imbalance in these two bilayers and differences of 16 and 9 °C are observed between the distal and proximal leaflet Tm’s for DMPC and DPPC, respectively. For the monolayers transferred in the condensed state (i.e., TA/W = 10 °C for DMPC and 20 °C for DPPC), the proximal layer TR values do not, within experimental uncertainty, differ from 1.00 (Table 2). The TR values of the corresponding distal leaflets are also close to 1.00, again suggesting that there is no appreciable difference between the molecular densities at the air/water interface and mica.12 Although the layer transfer ratios do not indicate any significant imbalances in the lipid densities of the two leaflets of these bilayers, there are differences of ∼13 °C and ∼6 °C in the leaflet Tm values of DMPC and DPPC, respectively (Table 4). We purposely prepared an asymmetric DPPC bilayer at TA/W = 20 °C by depositing the distal leaflet at a higher surface pressure (lower molecular area) than for the proximal leaflet (Figures S6 (Supporting Information) and 4B inset). Assuming again that the lipid densities of the Langmuir monolayer precursors are preserved on the mica support12 and that there is no redistribution between leaflets, the distal leaflet density is 8.7% higher than that of the proximal one. The higher lipid density of the distal leaflet deposited at π = 55 mN m−1 raises its Tm by ∼1 °C compared to the case of the distal leaflet transferred at 35 mN m−1, which is to be expected. Interestingly, the higher distal leaflet density raises the proximal leaflet melting temperature and this finding merits further investigation. More importantly, the gap between the distal and proximal leaflet melting temperatures remains and it is comparable (∼7 °C) to that of the symmetrical bilayer prepared at π = 35 mN m−1 and TA/W = 20 °C (Table 4). J



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dT is headgroup dependent and chain length independent.45 dπLE‑C/dT of DPPG (1.18 mN m−1 K−1) is half that of DMPC and DPPC (2.30−2.35 mN m−1 K−1).51,81 In fact, the thermoelastic shift obtained for GUVs of DMPC gives dπ/ dTm of 5.6 mN m−1 K−1, which is comparable to twice the dπLE‑C/dT of dialkylphosphatidylcholine monolayers.82 Using dπ/dTm of 2.16 mN m−1 K−1 instead of 5.6 mN m−1 K−1 in the constant area model proposed by Charrier and Thibaudau results in a higher Tm value and much broader transition width (Figure S7A, Supporting Information). Moreover, the model does not take into account the large increases in the area thermal expansion coefficient and area compressibility modulus near the phase transition,23,31,51,82 only the values in the gel and fluid phases are considered. The shifts in leaflet Tm’s with respect to TMLV obtained here m for two different alkyl chain lengths and different defect surface coverages are not consistent with the expectations of a (quasi)constant surface area transition. First, this model cannot explain melting of the all-solid distal leaflet of the DPPC bilayer (TA/W = 20 °C) with only 2% hole defect area at T = TMLV m . In 14−16 fact, several studies report distal layer melting at T ≈ TMLV m . Second, investigations of microstructured solid-supported lipid bilayers show that when there is sufficient lipid-free area on the surface, the single bilayers undergo reversible lateral area expansions during the solid-to-fluid transition of similar magnitude (∼25−30%) to those of the main transition of free-standing vesicles,47 suggesting that the phospholipid molecules are not pinned down to the surface and thus prevented from expanding. One would therefore expect a smaller shift of Tproximal with respect to TMLV as the bilayer-deep m m hole coverage (i.e., lipid-free area available to leaflet expansion) increases due to an expected smaller increase in lateral pressure during chain melting. We focus on the proximal leaflet because (i) its Tm is less affected by the phase state in which the Langmuir monolayer precursor is transferred from the air/ water interface and (ii) the melting leaflet should be more compressed than the distal one,13 the defect areas available for expansion being fully or partially occupied by melted material from the distal leaflet. The hole area of 21% in DMPC transferred at 20 °C is sufficient to accommodate the expansion of lipids in both leaflets during their respective transitions. Nevertheless, Tproximal is shifted by +6 °C from TMLV and the m m of the DPPC proximal transition width is ∼6 °C. Tproximal m bilayer prepared at TA/W of 20 °C with only 2% bilayer-deep and the transition holes is also shifted by +6 °C from TMLV m width is less (∼4 °C) than that of the DMPC, even though the available surface area is much too small to accommodate a bilayer expansion of the order of 30%.47 The bilayer with lower defect coverage should exhibit a larger shift in the leaflet Tm with respect to TMLV and larger transition width due to a larger m lateral pressure buildup and greater lipid compression during the leaflet transitions. However, this is not what we observe. Third, DMPC and DPPC bilayers with the same hole coverage (2%) exhibit different shifts in Tproximal versus TMLV m m , +9 °C for DMPC (TA/W = 10 °C) and +6 °C for DPPC (TA/W = 20 °C), even though the lipids occupy similar areas (Table 1) and have comparable area compressibilities and thermal expansion coefficients in the gel and fluid phases.31,33 The shift in the proximal leaflet Tm seems to depend on the lipid chain length, rather than on the surface area available for expansion. The possibility that the excess lateral pressure that develops in supported bilayers during chain melting is sufficient to eject material from the bilayer/water interface, analogous to the

collapse of Langmuir monolayers at high lateral pressures, must be considered. Possible mechanisms for monolayer collapse at the air/water interface include film folding and multilayer formation.83 Double-chain phospholipids are extremely insoluble in water. The critical micelle concentrations at 25 °C of DMPC and DPPC are 6 and 0.5 nM, respectively.28,84 The majority of the collapsed lipid molecules are expected to remain close to the mica surface so that they can readsorb/respread into a bilayer upon cooling. The hole coverage (4.5%) of the DMPC bilayer cooled to below room temperature after proximal leaflet melting (Figure S4, Supporting Information) is twice the coverage before heating (2%), suggesting loss of lipid from the mica surface during the leaflet transitions. In an early investigation of silicon oxide-supported single bilayers of DPPC prepared by Langmuir−Blodgett transfer using epifluorescence microscopy, Tamm and McConnell observed the formation of long tubular vesicles, ∼1 μm in diameter and length >200 μm, when the bilayers were heated through their chain-melting phase transition.11 The lipid tubules were found to grow from the planar bilayer, with one end remaining attached to the planar bilayer and the other end moving freely in the aqueous medium. The long tubules were stable for several minutes, after which they evolved into shorter tubules, helical structures, and spherical vesicles. Most of these structures remained attached to the supported bilayer. The underlying planar bilayer remained continuous and showed high lateral mobility. The release of lipid from the supported bilayer into vesicles was found to be partially reversible upon cooling (however, the degree of reversibility is not specified). The authors attribute the squeeze out of lipids from the planar bilayer surface at temperatures ≥ Tm to expansion of lipid on a fixed-area surface.11 Lipid squeeze-out and the concomitant formation of solution phase fluid vesicles would not necessarily be observed under the conditions required for high-resolution liquid imaging of planar supported bilayers by AFM. The vesicles occupy between ∼1% and ∼9% of the 5 × 103 μm2 area observed by epifluorescence microscopy,11 their two-dimensional size of 120 ± 85 μm2 is much larger than the 12.5 μm2 area imaged by AFM, and these probably form outside of the tip-contacted/scanned area. DPPC, the primary surface tension-lowering constituent of pulmonary surfactant, is capable of reaching surface tensions near 0 mN m−1 (π ∼ 69−72 mN m−1) when fully compressed at room temperature or 37 °C.85,86 In our Langmuir monolayer experiments (Figure 1B), DPPC collapses at π of ∼71 mN m−1 at 20 °C, as expected, but at lower pressure (∼50 mN m−1) at 33 °C because of the low-barrier compression rate used (0.01 nm2 molecule−1 min−1).85,86 By contrast, DMPC collapses at π of ∼45−48 mN m−1 for TA/W from 10 to 20 °C (Figure 1A). This difference in monolayer collapse pressure (i.e., monolayer stability) may explain the chain length dependence of the Tm shift of the proximal leaflets of bilayers with the same hole coverage (2%). DPPC can sustain a higher lateral pressure buildup from the melting chains than DMPC. We therefore conclude that lipid expulsion from the supported bilayer, akin to monolayer film collapse, rather than a quasi-constant surface area model, best accounts for our results.

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CONCLUSIONS We have shown that the preparation of solid-supported single bilayers by the Langmuir−Blodgett technique and quantification by high-resolution AFM of the surface coverage of the gel and fluid phases as a function of temperature to be useful for K



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(2) Castellana, E. T.; Cremer, P. S. Solid Supported Lipid Bilayers: From Biophysical Studies to Sensor Design. Surf. Sci. Rep. 2006, 61, 429−444. (3) Kiessling, V.; Domanska, M. K.; Murray, D.; Wan, C.; Tamm, L. K. Supported Lipid Bilayers. Wiley Encyclopedia of Chemical Biology; John Wiley & Sons, Inc.: Hoboken, NJ, 2008; Vol. 4, pp 411−422. (4) Groves, J. T.; Dustin, M. L. Supported Planar Bilayers in Studies on Immune Cell Adhesion and Communication. J. Immunol. Methods 2003, 278, 19−32. (5) Kalb, E.; Frey, S.; Tamm, L. K. Formation of Supported Planar Bilayers by Fusion of Vesicles to Supported Phospholipid Monolayers. Biochim. Biophys. Acta 1992, 1103, 307−316. (6) Egawa, H.; Furusawa, K. Liposome Adhesion on Mica Surface Studies by Atomic Force Microscopy. Langmuir 1999, 15, 1660−1666. (7) Richter, R. P.; Bérat, R.; Brisson, A. R. Formation of SolidSupported Lipid Bilayers: An Integrated View. Langmuir 2006, 22, 3497−3505. (8) Attwood, S.; Choi, Y.; Leonenko, Z. Preparation of DOPC and DPPC Supported Planar Lipid Bilayers for Atomic Force Microscopy and Atomic Force Spectroscopy. Int. J. Mol. Sci. 2013, 14, 3514−3539. (9) Bassereau, P.; Pincet, F. Quantitative Analysis of Holes in Supported Bilayers Providing the Adsorption Energy of Surfactants on Solid Substrate. Langmuir 1997, 13, 7003−7007. (10) Benz, M.; Gutsmann, T.; Chen, N.; Tadmor, R.; Israelachvili, J. Correlation of AFM and SFA Measurements Concerning the Stability of Supported Lipid Bilayers. Biophys. J. 2004, 86, 870−879. (11) Tamm, L. K.; McConnell, H. M. Supported Phospholipid Bilayers. Biophys. J. 1985, 47, 105−113. (12) Anglin, T. C.; Conboy, J. C. Lateral Pressure Dependence of the Phospholipid Transmembrane Diffusion Rate in Planar-Supported Lipid Bilayers. Biophys. J. 2008, 95, 186−193. (13) Charrier, A.; Thibaudau, F. Main Phase Transitions in Supported Lipid Single-Bilayer. Biophys. J. 2005, 89, 1094−1101. (14) Feng, Z. V.; Spurlin, T. A.; Gewirth, A. A. Direct Visualization of Asymmetric Behavior in Supported Lipid Bilayers at the Gel-Fluid Phase Transition. Biophys. J. 2005, 88, 2154−2164. (15) Keller, D.; Larsen, N. B.; Møller, I. M.; Mouritsen, O. G. Decoupled Phase Transitions and Grain-Boundary Melting in Supported Phospholipid Bilayers. Phys. Rev. Lett. 2005, 94, 0257011. (16) Yang, J.; Appleyard, J. The Main Phase Transition of MicaSupported Phosphatidylcholine Membranes. J. Phys. Chem. B 2000, 104, 8097−8100. (17) Garcia-Manyes, S.; Oncins, G.; Sanz, F. Effect of Temperature on the Nanomechanics of Lipid Bilayers Studied by Force Spectroscopy. Biophys. J. 2005, 89, 4261−4274. (18) Hetzer, M.; Heinz, S.; Grage, S.; Bayerl, T. M. Asymmetric Molecular Friction in Supported Phospholipid Bilayers Revealed by NMR Measurements of Lipid Diffusion. Langmuir 1998, 14, 982−984. (19) Scomparin, C.; Lecuyer, S.; Ferreira, M.; Charitat, T.; Tinland, B. Diffusion in Supported Lipid Bilayers: Influence of Substrate and Preparation Technique on the Internal Dynamics. Eur. Phys. J. E 2009, 28, 211−220. (20) Stanglmaier, S.; Hertrich, S.; Fritz, K.; Moulin, J.-F.; HaeseSeiller, M.; Rädler, J. O.; Nickel, B. Asymmetric Distribution of Anionic Phospholipids in Supported Lipid Bilayers. Langmuir 2012, 28, 10818−10821. (21) Rossetti, F. F.; Textor, M.; Reviakine, I. Asymmetric Distribution of Phosphatidyl Serine in Supported Phospholipid Bilayers on Titanium Dioxide. Langmuir 2006, 22, 3467−3473. (22) Richter, R. P.; Maury, N.; Brisson, A. R. On the Effect of the Solid Support on the Interleaflet Distribution of Lipids in Supported Lipid Bilayers. Langmuir 2004, 21, 299−304. (23) Mouritsen, O. G. Life - As a Matter of Fat: The Emerging Science of Lipidomics; Springer-Verlag: Berlin, 2005. (24) Mouritsen, O. G.; Jørgensen, K. A New Look at LipidMembrane Structure in Relation to Drug Research. Pharm. Res. 1998, 15, 1507−1519. (25) Menon, S.; Rosenberg, K.; Graham, S. A.; Ward, E. M.; Taylor, M. E.; Drickamer, K.; Leckband, D. E. Binding-Site Geometry and

establishing the effects of the monolayer phase state and molecular density prior to deposition on the solid surface and substrate-mediated lipid condensation on the thermotropic phase behavior. Although these parameters have been proposed to influence the gel-to-fluid transition in supported bilayers, past attempts to study and decouple their influence have been limited because it is difficult, if not impossible, to control and quantify the molecular density of the individual leaflets in bilayers formed by vesicle fusion. We have found that DMPC and DPPC bilayers deposited onto mica from monolayers in the condensed-dominant or condensed state at the bilayer− monolayer correspondence pressure of 35 mN m−1 undergo two separate transitions in which the distal leaflets melt around the Tm of free-standing multilamellar vesicles, whereas the proximal leaflets melt at higher temperature. The phase state of the Langmuir monolayer precursor has a greater effect on the Tm and transition width/cooperativity of the distal leaflet than those of the proximal leaflet due to substrate-mediated condensation and mica−lipid interactions. The distal and proximal layer transfer ratios obtained for the deposition of monolayers of the same molecular density and monolayers of different densities suggest that asymmetric leaflet densities alone cannot account for the proximal leaflets melting at higher temperature than the distal leaflets. Monolayer transfers carried out at a constant reduced temperature indicate that the difference in the leaflet Tm’s depends on the chain length. The melting curves acquired for bilayers with hole defect coverages (area for bilayer expansion) ranging from ∼2% to ∼20% are not consistent with expectations of a (quasi-)constant surface area and variable surface tension model. Future work will focus on the effects of the monolayer transfer pressure on the bilayer leaflet transitions and the role of thermally induced lipid squeeze out.

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ASSOCIATED CONTENT

S Supporting Information *

Additional AFM images and height profiles. DMPC melting curves calculated using a constant area melting transition model and related discussion. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*A. Badia. E-mail: [email protected]. Present Address

† M.R.: Department of Chemistry, McGill University, 801 Sherbrooke St. W., Montreal, QC H3A 2K6, Canada.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants to A.B. from the Natural Sciences and Engineering Research Council of Canada and Canada Research Chairs program and was made possible through access to the scanning probe microscopy facility of the Laboratoire de caractérisation des matériaux of the Université de Montréal.

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REFERENCES

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