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J. Phys. Chem. C 2009, 113, 2187–2196

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Role of Oxide Surface Chemistry and Phospholipid Phase on Adsorption and Self-Assembly: Isotherms and Atomic Force Microscopy Jie Xu,† Mark J. Stevens,†,| Timothy A. Oleson,† Julie A. Last,‡ and Nita Sahai*,†,§ Department of Geology and Geophysics, 1215 West Dayton Street, Department of Surgical Sciences, 2015 Linden DriVe, Department of Chemistry, 1101 UniVersity AVenue, UniVersity of Wisconsin, Madison, Wisconsin 53706 ReceiVed: August 28, 2008; ReVised Manuscript ReceiVed: December 18, 2008

We have examined the effects of metal oxide surface chemistry and lipid phase on phospholipid adsorption affinity and self-assembly. Adsorption isotherms of ditridecanoylphosphocholine (DTPC) at 40 °C and pH 7.2 on quartz (R-SiO2), rutile (R-TiO2), and corundum (R-Al2O3) particle suspensions indicated oxide-dependent adsorption affinity that decreased as rutile > corundum = quartz at low concentrations and corundum > rutile = quartz at higher concentrations. Significantly, atomic force microscopy of DTPC and dipalmitoylphosphocholine (DPPC) at high concentration on planar oxide surfaces at 25 °C in liquid-crystal and gel phases, respectively, also showed oxide-specific adsorption. Multiple bilayers formed on corundum (100), indicating greater coverage compared to single bilayers, bilayer patches, or supported vesicle layers on the negatively charged surfaces of mica (001), rutile (100), and amorphous silica glass (fused quartz plate). Thus, the amount and self-assembly of adsorbed phospholipid was found to be oxide-dependent regardless of the lipid phase. Significantly, both experimental methods show multiple bilayer formation as a unique feature of the positively charged alumina surface. The observed oxide affinity sequences are interpreted as controlled by van der Waals and electrostatic forces between the oxide surface and the negatively charged (-R(PO4-)R′-) portion of the phosphocholine headgroup. Our results have implications for the interactions of amphiphilic molecules with mineral surfaces in diverse biogeochemical, biomedical, and industrial processes, including membrane-bound biomineralization, cell membrane stability during early evolution of life, organic matter burial in ocean sediments, the design of supported lipid bilayers and biomimetic cell membranes for medical implant devices, enhanced oil recovery, and ore extraction by froth flotation. 1. Introduction A fundamental question in the interactions of amphiphilic molecules with solid oxide surfaces is whether and how the specific and nonspecific surface chemistry of the oxide substrate affects the stability of the adsorbed, self-assembled amphiphilic structures. The interactions of amphiphilic molecules with inorganic solid substrates are of interest in many biogeochemical, environmental, industrial, and biomedical processes. For example, the earliest protocell membranes formed during the prebiotic stages of evolution of life must have involved micelles of simple single-chain amphiphiles such as fatty acids and their esters.1,2 We suggest that the stability of such micelles in contact with different silicate, oxide, carbonate, or sulfide minerals may have depended on the mineral surface chemistry as well as on the phase of the amphiphilic molecule but, to our knowledge, these remain unexplored questions. Intimate lipid-mineral interactions also occur in controlled biomineralization of amorphous silica by diatoms, calcite spicules by sponges, magnetite by bacteria, calcium oxalate (as kidney stones) in humans, etc., where precipitation occurs within a volume of space defined by phospholipid membranes.3,4 The ability of * Corresponding author. Tel: 608-262-4972. Fax: 608-262-0693. Email: [email protected]. † Department of Geology and Geophysics. ‡ Department of Surgical Sciences. § Department of Chemistry. | Current address: 645 Science Drive, Madison, Wisconsin 53711.

amphiphiles to adsorb at mineral surfaces also has environmental implications such as inhibiting the oxidation of pyrite (FeS2), which causes acid mine drainage;5 organic matter burial in ocean sediments, which is a sink for global CO2; and industrial applications such as enhanced ore mineral recovery by froth flotation,6 corrosion inhibition,7 lubrication,7,8 and the formation of micropatterned electronics.9 Phospholipid-oxide substrate interactions are also fundamental to creating biomimetic membranes known as supported lipid bilayers (SLBs)10,11 with a range of biomedical applications, including drug delivery,12,13 biomimetic catalysts, DNA hybridization kinetics,14 the creation of artificial cells,15 and biosensor technology.16-22 Furthermore, oxide- and silicate-based bioceramics such as titania (TiO2) and Bioglass are used as prosthetic implants in the human body, and even aluminum and titanium metal-based orthopedic implants develop a surface layer of the corresponding oxide when exposed to air or solution.23,24 It is important to understand how cell membranes, including those of the immune system, will react to these oxide implants.25 The stability of the SLB depends on a host of substrate-, phospholipid-, or solution-specific parameters, which control the thermodynamics and pathways of phospholipid self-assembly. Some of the parameters studied to date include substrate type,26-37 surface roughness,5,26 solution chemical composition (e.g., salts, EDTA),28,37-41 temperature,31,33,41-44 lipid tail properties,36 charge of the lipid headgroup,29,30,38,39 concentration of lipid,28,39 ratio of different lipids in a mixed system,29,35,41,43,45 vesicle size,31,32,35 ionic strength of solution,27,40 osmotic pressure,31 pH,27,46,47 and the buffer

10.1021/jp807680d CCC: $40.75  2009 American Chemical Society Published on Web 01/12/2009

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TABLE 1: Relevant Physical and Chemical Characteristics of the Oxide Particles Used in Isotherm Study

oxide

pHPZC67-69

Hamaker constant70 (10-20 J)

quartz (R-SiO2) rutile (R-TiO2) corundum (R-Al2O3)

2.9 5.8 9.4

0.63 26 4.2

mean particle diameter66a (nm)

polydispersity66,a

BET specific surface area (m2 · g-1)66

1369 ( 146 977 ( 26 2500b

0.256 0.231 N/A

6.04 3.26 1.17

a Z-average diameter and polydispersity are mean of 7 measurements (see Supporting Information). b Manufacturer-provided (Sigma-Aldrich) value.

TABLE 2: Point of Zero Charge and Surface Roughness of Solid Substrates Used in AFM Study substrate

crystal face

Amorphous SiO2 (fused quartz) Rutile (R-TiO2) Mica (KAl2(AlSi3O10)(OH)2) Corundum (R-Al2O3)

N/A (100) (001) (100)

PZC 3.5 67-69 5.9 67-69 6.6 67-69 5.9-9.4 67-69,72,73 a

roughness (nm) (1 × 1 µm2area)74,b 0.206 0.096 0.041 0.072

a The PZC of corundum (100) is not known accurately. A single determination of PZC of 5.972 using AFM has been made, but this value is very far from the value of PZC ) 9.4, also measured using AFM, for randomly oriented polycrystalline corundum PZC.73 The latter is consistent with the well-established value of ∼9.2 for corundum powders.67-69 b Calculated using Nanoscope Control software; average measurements in four different areas.

used.27 Accordingly, a wide range of analytical techniques has been employed such as atomic force microscopy (AFM),5,26-29,37-39,42,43,48-51 quartz crystal microbalance with dissipation monitoring (QCMD),14,30-34,38,39,43 ellipsometry,29,38 e-beam lithography,52 fluorescence imaging,35,36,40,53 differential interference contrast imaging,35 fluorescence pattern photobleaching recovery,19,35 neutron reflectivity,44,46,54 surface plasmon resonance,14 vibrational sum frequency spectroscopy,55 and infrared spectroscopy.56,57 Substrates studied include mica,26-28,30,38,41-43,47,50 amine-functionalized mica,27 silica,14,26-28,31-37,39,40,43,46,54 silica-coated QCMD sensors,30 quartz,36,44,54 glassslides,36 hydrogelbeads,57 TiO2,32,33,35,58 SrTiO3,35 oxidized Pt,29,31 gold,27,34,51alkane thiol-functionalized gold,34,60 Si3N4,31 polymer films,36 pyrite,5 and graphite.61-63 These studies have yielded a wealth of information but have focused primarily on the effects of phospholipid chemistry. Very limited quantitative studies of adsorption affinities on different oxides have been conducted. 64-66 Futhermore, it is not clear how the lipid phase may affect or modify the effects of oxide surface chemistry on membrane stability. We have previously established by quantitative isotherms that adsorption of 1,2dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) on oxide particles is dependent on oxide surface chemistry (chemical composition and surface charge).66 The main motivations for the present study were to determine quantitatively whether the oxide specificity holds for other phosphatidylcholine (PC) molecules and whether the oxide specificity can be confirmed on planar surfaces by examining the morphology of the self-assembled aggregates formed. We also aimed to establish whether such behavior depends on the lipid phase (liquid-crystal versus gel). Specifically, we examined 1,2-ditridecanoyl-sn-glycerol-3-phosphatidylcholine (DTPC) and DPPC that have 13 and 16 carbon tails, respectively, and both have the PC headgroup. These are common cell membraneforming zwitterionic lipids. At room temperature, DTPC and DPPC are in liquid-crystal and gel phase, respectively, with transition temperatures of 14 and 41 °C. The oxides we examined were silica, titania, and alumina. We used quartz (R-SiO2), rutile (R-TiO2), and corundum (RAl2O3) particle suspensions to measure DTPC adsorption isotherms at 40 °C and pH 7.2 to determine quantitatively the amount of adsorption on the different oxides (Table 1).66-70 We chose this temperature because it is biogeochemically relevant for early Earth scenarios and is also close to physiologic

temperature, and for comparison with previously measured DPPC adsorption isotherms at 55 °C. 66 The morphologies of the adsorbed self-assembled lipid aggregates and the extent of surface coverage were determined by atomic force microscopy of liquid-crystal phase DTPC and gel-phase DPPC at 25 °C on the following planar surfaces: amorphous silica glass (SiO2, fused quartz plate) and oriented single crystal faces of rutile (100), mica (001), and corundum (100) (Table 2).71-74 These oxides were chosen because of their range of characteristic surface properties in terms of chemical composition, point of zero charge, and Hamakar constant (Table 1). These characteristic properties affect electrostatic forces (surface charge) and van der Waals forces that, in turn, can influence phospholipid adsorption.75,76 Furthermore, these oxides are common components of minerals in the natural geochemical environment and are commonly used as substrates for SLBs and as bioceramic implants. Additionally, SLBs on the atomically smooth (001) cleavage plane of muscovite mica have been extensively characterized previously and provide a bench-mark for our study. 2. Materials and Methods 2.1. Materials. DTPC and DPPC were purchased from Avanti Polar Lipids (Alabastar, AL), and N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) was from Fisher Scientific (Hampton, NH). All other reagents (ACS grade) were purchased from Sigma-Aldrich (St Louis, MO). Prior to use in adsorption experiments, particles of corundum (R-Al2O3, 99.7% purity, Sigma-Aldrich), rutile (R-TiO2, 99.9% purity, SigmaAldrich), and Min-U-Sil5 natural quartz (R-SiO2, 98.3% purity, U.S. Silica, Berkeley Springs, WV) were dialyzed in 18 MΩ-m deionized (DI) water. Particles were characterized for specific surface area using N2 gas, multipoint, BET adsorption isotherms (Quantachrome Intruments, Boynton Beach, FL) and for particle size distributions using dynamic light scattering (Zetasizer, Malvern Instruments, U.K.) (Table 1). 66 The planar surfaces used for AFM study were plates of fused quartz (SiO2, 12.7 mm diameter × 1.58 mm; Quartz Plus, Inc., Brookline, NH), sapphire windows of (100) orientation (12.7 mm × 12.7 mm × 0.5 mm; MTI Corporation, Richmond, CA), and rutile windows of (100) orientation (10 mm × 10 mm × 0.5 mm; MTI Corporation). These surfaces were cleaned by the following procedure before each experiment:74 sonication in 2% Hellmanex II (Hellma Cells, Inc.) solution for 15 min,

Oxides, Phospholipids, Adsorption, Self-Assembly followed by a rinse in DI water; subsequently sonication in acetone for 5 min, followed by a rinse in DI water; and, finally, sonication in DI water for 5 min and blown dry in nitrogen flow. Muscovite mica sheets (Ted Pella, Inc., Redding, CA) were freshly cleaved before each use and did not require cleaning. 2.2. Preparation of PC Vesicles. All glassware used in the preparation was initially silanized with Sigmacote (SigmaAldrich) to minimize lipid loss by sorption to the glass surface. HEPES buffer solution was prepared according to the biochemical research standard77 with a final pH adjusted to 7.2 using 0.5 M NaOH. HEPES was chosen because it has less effect on the kinetics and adsorption of PC on SiO2 than does tris(hydroxymethyl)aminomethane) (TRIS) buffer.78 In addition, HEPES does not interfere with the quantitative inorganic phosphorus assay64-66 for PC used in the adsorption isotherm experiments. To prepare aqueous DTPC or DPPC solutions, the manufacturer-provided PC in chloroform solvent (concentration ∼20 mg PC/mL chloroform) was dispensed into a small glass vial, and the solvent was removed under a light nitrogen flow and then under vacuum in a desiccator. The dried PC film was hydrated with preheated (45 °C) buffer solution to obtain a stock lipid concentration of 1-1.5 mM and thermostatted at 45 °C for 2 h. Prior to use, solutions were bath-sonicated at 45 °C until maximum clarity was achieved (typically, 2-2.5 h) and then centrifuged (Beckman Coulter, Inc., Fullerton, CA) at 50000g and 25 °C for 1 h. The supernatant consisting of small unilamellar vesicles (SUV) was removed to a clean vial for use. The average size of the vortexed vesicles was ∼100 nm in diameter for DTPC and ∼120 nm for DPPC, as determined by dynamic light scattering (Zetasizer, Malvern Instruments, U.K.). The vortexed SUV solutions were used for the bulk adsorption isotherms. Solutions for the AFM study were started with the above supernatant (before vortexing) and were extruded (Avanti MiniExtruder, Avanti Polar Lipids, Inc.) eleven times through a 100nm-pore-sized membrane (NucleoPore, Corning, NY) at room temperature (∼25 °C) before injection.74 The average extruded vesicle size was 87.5 nm for DTPC and 120 nm for DPPC bydynamic light scattering (Table 1 of the Supporting Information). 2.3. Adsorption Isotherms. DTPC isotherms were obtained at 40 °C to ensure that the lipid was in liquid-crystal phase. Oxide suspensions were prepared by adding 750 µL of 0.05 M HEPES solution into 1.5-mL microcentrifuge tubes containing known masses (1.2-1.5 mg) of oxide, followed by vortexing and sonicating to disperse the particles. Vesicle solutions of DTPC of varying concentrations were prepared by dilution from the stock solution. Aliquots were reserved for measuring initial DTPC concentration, [DTPC]initial. Then 250 µL of DTPC solution was added to each tube containing oxide suspension, and identical blanks without oxide particles were prepared for each vesicle concentration. The samples were vortexed and thermostatted at 40 °C in a water bath for 12 h. The equilibrated solutions were centrifuged, and the supernatant was collected for measurement of final DTPC concentration, [DTPC]final. The difference between initial and final DTPC concentrations was taken as the adsorbed concentration.66 The DTPC concentrations were determined by an UV-Vis spectrophotometric method (UV-Mini 1240 UV-Vis spectrophotometer, Shimadzu Corp., Kyoto). Each experiment was conducted in triplicate, and triplicate spectrophotometric measurements were made on each. Thus, the error bars in the adsorption isotherm graphs below represent the standard deviation for nine separate measurements at each DTPC concentration.

J. Phys. Chem. C, Vol. 113, No. 6, 2009 2189 2.4. Atomic Force Microscopy. We used a Nanoscope IVMultiMode atomic force microscope (Digital Instruments Inc., Santa Barbara, CA) equipped with an “E” scanner (12 µm). In flow-through reactions with DTPC and with DPPC, 1 mL of PC solution was slowly injected through the fluid cell and allowed to interact with the oxide substrate for 30 min before imaging. The fluid-cell and o-rings used were washed extensively with DI water, 95% ethanol, and DI water again before each experiment. Note that the 30 min reaction time was chosen on the basis of preliminary results and showed no difference in adsorbed lipid morphologies after up to 8 h of reaction,74 consistent with previous QCM-D studies, in which the maximum adsorption was obtained for egg PC and dimyristoylphoshatidylcholine (DMPC; 14-carbon chain) on silica and occurs within ∼8 and 4 min,79,80 respectively, and egg PC on TiO2 with 100 nm vesicles in ∼20 min.32 Preliminary results showed that gel-phase DPPC vesicles adsorbed without rupturing at 25 °C;74 therefore, a second type of experiment (“batch experiment”) was conducted, for which the intent of the protocol was to intentionally induce physical rupture of the adsorbed DPPC vesicles by passing the vesicles through the air-water interface and blowing on it with nitrogen gas. Other methods such as adding salts,37 applying mechanical agitation,81 or varying lipid composition have been reported often in the literature to induce formation of bilayers.82 In the DPPC batch reaction, 1 mL of 1 mM DPPC buffer solution was extruded and immediately reacted with an oxide substrate using the Teflon-coated plastic cap (PTFE-lined, National Scientific Company) of a 10 mL glass storage vial as a reaction vessel. After 30 min of reaction time, the oxide was rinsed in DI water flowing at a moderate rate for 10 s. The DPPC solution left in the reaction vessel was replaced with DI water, and the rinsed substrate was then returned to the reaction vessel. The oxide was then transported to the AFM laboratory for surface imaging. Upon removing the oxide from the DI water bath, the oxide substrate was tilted, and the resulting drop of water was absorbed with a Kimwipe. Then a stream of nitrogen gas (supplied from evaporating liquid nitrogen) was blown over the oxide to evaporate any remaining fluid on its surface. Standard silicon nitride probes (Digital Instruments, Inc.) with a cantilever length of 100 µm, a nominal spring constant of 0.32 N m-1, and a nominal vibrational frequency of 56 Hz were installed in a TappingMode fluid cell (Digital Instruments, Inc.) to carry out imaging in situ.74 Several images were obtained at different areas on each reacted surface. “Blank” substrates in HEPES buffer solution without DTPC vesicles were also characterized in identical setups (Figure 1 of the Supporting Information). The surface roughness of each substrate was calculated using the Nanoscope Control software, using an area of 1 × 1 µm2 selected from the blank surfaces (Figure 1). The reported surface roughness is an average of four measurements (Table 2).74 3. Results 3.1. Adsorption Isotherms. Adsorption of DTPC on quartz, rutile, and corundum particles is shown in Figure 1. Below [DTPC] ∼0.65 mM, adsorption on rutile is slightly greater than on quartz and corundum, but at high concentrations, the trend is corundum > rutile ∼ quartz. In detail, adsorption on all the oxides reaches to ∼7-8 µM m-2 at low solution concentrations, then increases again and reaches a second plateau at high concentration (1 mM). On rutile and quartz, the second adsorption plateau occurs at 11-13 µmol m-2, as compared to a much higher value of ∼22-25 µmol m-2 on corundum. Using

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Xu et al. silica, 5-6 nm high bilayer patches or complete DPPC bilayers with small defects were observed (Figures 4 a). At least one complete bilayer with large connected overlying patches almost forming a second layer was seen on rutile (Figure 4b). Mica almost always displayed at least one complete bilayer, often overlain by large bilayer patches (Figure 4c). Multiple aggregates, each of 5-6 nm height up to a total ∼12-17 nm, were observed commonly on corundum (100) and were interpreted as multiple bilayers (Figures 4d).

Figure 1. Equilibrium adsorption of DTPC from bulk solution onto quartz, rutile, and corundum particles at 40 °C for pH 7.2 in 50 mM HEPES buffer with 20 mM [Na+]. Error bars represent the standard deviation of triplicate analyses of each data point conducted in triplicate.

a headgroup size of ∼0.5-0.6 nm2 for phosphocholine,83 we calculate that a single bilayer corresponds to ∼5-6 µM m-2. Thus, two bilayers adsorb on quartz and rutile at high solution concentration, as compared to multiple bilayers on corundum. 3.2. AFM Images and Height Information for DTPC. Planar views of 4 × 4 µm2 areas of the surfaces reacted with 1 mM solutions are shown in Figure 2 (see Figure 2 of the Supporting Information for the corresponding angled, 3-dimensional views). The vertical cross sections of the planar view surfaces yield height profiles, where the steps indicate the thickness of surface layers (Figure 2). The thickness of one DPPC (16-carbon chain) bilayer is ∼6 nm83,84 and DMPC (14carbon chain) is ∼4.6 nm.85 DTPC is shorter (13-carbon chain), so the bilayer thickness should be pHexpt) (Table 1). The greater uptake of DTPC on corundum suggests that the negatively charged -R-PO4--R′- moiety of the phosphocholine headgroup is attracted electrostatically to the corundum surface and is repelled from the quartz and rutile surfaces.66 Specific, inner-sphere surface complexation of the -RPO4--R′ group is ruled out because of the presence of a 0.4-1 nm water layer between the oxide surface and the lipid bilayer.66,75,86-90 The positively charged sR-N(CH3)3+ moiety of the headgroup is inferred not to be involved in electrostatic attraction or repulsion. This difference in the two moieties of the lipid headgroup is because of the greater negative charge density associated with the smaller -R-PO4--R′ group, as compared to a lesser positive charge density associated with the larger -R-N(CH3)3+ group.66 We have observed very similar behavior for DPPC adsorption on oxide particle suspensions and have proposed a model to explain the observed adsorption of two bilayers on the quartz and rutile surfaces and of multiple bilayers on corundum.66 In brief, the adsorption of the first DTPC or DPPC bilayer is driven by van der Waals attraction between the oxide and the lipid vesicles. Formation of the first bilayer results in expulsion of charge-balancing Na+ or Cl- ions from the vicinity of the oxide surfaces, thus extending the thickness of the diffuse layer of counterions. A second bilayer can adsorb within the thickened diffuse layer by van der Waals attraction between the first lipid layer and additional vesicles. Adsorption on the negatively charged surfaces is limited at this stage because van der Waals forces are too weak to operate beyond the second bilayer, which is estimated to be ∼15-18 Å away from the surface. 66 For corundum, however, additional adsorption can occur because of the electrostatic attraction between the positively charged surface and the phosphate ester of the lipid headgroup. It is significant that the difference in lipid affinity between negatively and positively charged oxide surfaces obtained in bulk particle suspensions is confirmed by the DTPC images and height profiles obtained by AFM on planar surfaces (Figure 2). Being in the liquid-crystal phase, DTPC vesicles can rupture, fuse, and self-assemble after adsorption into bilayer patches or complete bilayers, depending on the charge in the oxide surface.

Oxides, Phospholipids, Adsorption, Self-Assembly

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Figure 2. Planar view AFM images and height profiles of reacted substrates in the flow-through setup, at [DTPC] ) 1 mM and pH 7.2, for fused quartz plate (amorphous silica glass) (a), rutile (100) (b), mica (001) (c), and corundum (100) (d). Amorphous silica showed sparse bilayer patches compared to dense bilayer patches (∼90%) or a complete bilayer on rutile. Mica showed a complete bilayer, whereas multiple bilayers formed on corundum.

Thus, the AFM images and height profiles provide direct visual confirmation of oxide-dependent adsorption as well as multiple bilayer formation on corundum. We recognize that the adsorption isotherms on particles indicate one extra layer of lipid adsorption, as compared to the AFM images for each planar surface, and the difference may be due to the smaller specific surface area as compared to the particles. Nevertheless, it is significant that both types of

experiments yielded the same result in terms of indicating oxidedependent extent of adsorption and greater adsorption on surfaces with lower negative surface charge. 4.2. Effect of Lipid Phase. We were interested in determining whether the oxide-dependent behavior of phospholipid adsorption is affected by the gel phase. We found that the incomplete coverage by deformed DPPC vesicles and bilayer patches on negatively charged surfaces in the gel phase (flow-

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Figure 3. Planar view AFM images and height profiles of reacted substrates in the flow-through setup, at [DPPC] )1 mM and pH 7.2, for fused quartz plate (amorphous silica glass) (a), rutile (100) (b), mica (001) (c), and corundum (100) (d). Silica exhibited a supported vesicle layer and bilayer patches formed on rutile. A supported vesicle layer was observed on mica and bilayer patches on corundum. The lower half of the image in part b is slightly distorted because of disturbance by a passing-by train outside.

through experiment) relative to greater bilayer coverage on corundum was consistent with the results obtained in the liquidcrystal phase with DTPC. Thus, even though vesicle rupture was limited on silica and rutile, the amount of coverage or lipid affinity was also lower compared to alumina. The limited rupture of vesicles observed in flow-through DPPC experiments has been reported previously in the literature. The overall adsorption, fusion, and rupture of vesicles into bilayers depends on vesicle-substrate adhesion energy, vesiclebending energy, and water-substrate adhesion energy.91-93 Even if thermodynamically favorable, fusion is sometimes kinetically slow.94 A critical coverage of vesicles is sometimes needed

before vesicles will rupture due to favorable fusion of smaller vesicles into larger ones,26,30,32,95 but if the vesicle concentration is too high, steric hindrance may stabilize vesicles and inhibit their rupture.31,45 It has also been proposed that rupture of bilayers on an isotropic hydrophilic surface is not possible without the aid of an external force,93 which may be the reason that adding salts,26 applying mechanical agitation,82 or varying lipid composition is often required to induce formation of bilayers.83 In our system, rupture was likely limited in the flowthrough experiment because of the lack of mobility in the gel phase. Rupture and subsequent fusion into bilayers could only be promoted in the batch protocol by moving the adsorbed

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Figure 4. Planar view AFM images and height profiles of reacted substrates in the batch reaction setup, at [DPPC] ) 1 mM and pH 7.2, for fused quartz plate (amorphous silica glass) (a), rutile (100) (b), mica (001) (c), and corundum (100) (d). Bilayer patches were observed on silica and a bilayer with large irregular overlying bilayer patches on rutile. A complete bilayer with large overlying bilayer patches was observed on mica, and multiple bilayers on corundum.

vesicles through the air-water interface and blowing nitrogen gas. The salient point here is that regardless of SVL or SBL formation in the flow-through and batch-experiments, respectively, lipid adsorption was oxide-dependent, with greatest uptake on corundum, followed by mica, rutile, and quartz. Results of previous AFM studies on DPPC adsorption at pH ∼7.2-7.4 have also indicated different self-assembled morphologies on silica,38,51,79,28,31,38,40 rutile,33,35,59 and mica28,50,96,97

depending on the DPPC concentration, solution ionic strength, and the presence of divalent cations (Table 3). However, except for pyrite (FeS2) in an experimental setup similar to that in the present study,9 and on mica using a different experimental procedure,97 multilayers have not been reported previously in the literature for alumina. Indeed, vesicles were found not to adsorb at all onto an electron-beam-evaporated thin film of aluminum oxide.98 Thus, we have demonstrated that phospho-

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TABLE 3: Summary of Results from Studies of Phosphocholine Adsorption on Oxide and Mica Substratesa lipid, concn

substrate

analytical instrument

buffer soln

SiO2

DOPC, 0.1 mg mL-1

AFM QCM-D

SiO2

DOPC, 0.02-0.1 mM

ellipsometry

SiO2

egg PC

QCM-D

not stated

SiO2

egg PC

QCM-D

not stated

SiO2

QCM-D

SiO2

egg PC, POPC, 13 mg mL-1 DTPC

SiO2

DPPC

AFM

SiO2

DPPC

pH 8, 100 mM NaCl, 10 mM Tris pH 7.2, 10 mM HEPES pH 7.2, 10 mM HEPES pH 7.2, 10 mM HEPES pH 7.4, 50 mM HEPES, 150 mM NaCl, 4 mM EDTA pH 7.4, 150 mM NaCl, 10 mM HEPES

AFM

AFM -1

mica (001)

DPPC, 1 mg mL

AFM

mica (001)

DOPC, 0.02-0.1 mM

ellipsometry

mica (001)

DMPC, DPPC, 0.5 mM

AFM

mica (001) mica (001)

DMPC, 1 mg mL-1 DMPC, 0.5 mM

mica (001)

DOPC, 0.1 mg mL-1

mica (001)

DTPC

mica (001)

DPPC

mica (001)

DPPC

TiO2 (unspecified egg PC phase) TiO2 (unspecified egg PC phase) TiO2 (100) DTPC

pH 7.4, 150 mM NaCl, 2 mM NaNO3,b 10 mM HEPES ( 2 mM Ca or EDTA pH 7.4, 150 mM NaCl, 10 mM HEPES

pH 7.4, 150 MM NaCl, 20 mM MgCl2, 10 mM HEPES AFM pH 7.0, 10 mM NaCl AFM pH 7.4, 0-150 mM NaCl, 20 mM MgCl2, 10 mM HEPES QCM-D, AFM, pH 7.4, 150 mM ellipsometry NaCl, 3 mM NaNO3,b 10 mM HEPES ( 2 mM Ca or EDTA AFM pH 7.2, 10 mM HEPES AFM pH 7.2, 10 mM HEPES AFM pH 7.2, 10 mM HEPES QCM-D not stated QCM-D

not stated

AFM

pH 7.2, 10 mM HEPES pH 7.2, 10 HEPES pH 7.2, 10 HEPES pH 7.2, 10 HEPES pH 7.2, 10 HEPES pH 7.2, 10 HEPES

TiO2 (100)

DPPC

AFM

TiO2 (100)

DPPC

AFM

corundum (100)

DTPC

AFM

corundum (100)

DPPC

AFM

corundum (100)

DPPC

AFM

vesicle preparation method, size

reaction type

result

ref 2+

SUV

flow-through

SLB without Ca , SLB with Ca2+

SUV, 25 nm

flow-through

SUV and EUV, 25-200 nm SUV, 25 nm; EUV, 40 nm SUV and EUV, 30-200 nm EUV, 120 nm

flow-through

SLB and vesicles on 94 silica with and without Ca2+ SLB 32

flow-through

SLB

33

flow-through

SLB

31

flow-through

sparse bilayer patches SVL

present study

bilayer patches

present study

EUV, 100 nm EUV, 100 nm EUV, 100 nm

flow-through (gel phase) batch (gel phase) batch, 30 s reaction time

SUV, 25 nm

flow-through

SUV EUV, 100 nm SUV

batch (below transition temperature) batch batch

SUV

26

present study

SLB with some 41 visible monolayers SVL on mica 94 without Ca2+, SLB and vesicles on mica with Ca2+ SLB 85 SLB SLB

81 91

flow-through

SVL without Ca2+, SLB with Ca2+

38

EUV, 120 nm

flow-through

SLB

present study

EUV, 100 nm

flow-through SVL (gel phase) batch (gel phase) SLB with large, overlying patches flow-through SVL

present study

flow-through

SVL

33

flow-through

dense bilayer patches to almost complete SLB bilayer patches

present study

EUV, 100 nm SUV and EUV, 25-200 nm SUV, 25 nm; EUV, 40 nm EUV, 120 nm

mM

EUV, 100 nm

mM

EUV, 100 nm

mM

EUV, 120 nm

mM

EUV, 100 nm

mM

EUV, 100 nm

present study

32

flow-through (gel phase) batch (gel phase) bilayer with overlying patches flow-through multiple SLBs

present study

flow-through bilayer patches (gel phase) batch (gel phase) multiple SLBs

present study

present study present study

present study

a Abbreviations: sonicated unilamelar vesicles, SUV; extruded unilamelar vesicle, EUV; supported vesicle layer, SVL; supported lipid bilayer, SLB. b Ref 26 states the salt is NaN3; we have assumed here that it was a typographical error and the salt used was actually NaNO3.

lipid adsorption affinity and self-assembly are oxide-dependent and that the oxide dependence is not modified or altered by the gel or liquid-crystal phase of the lipid. 5. Conclusions The results of our systematic study establish that phospholipid adsorption on oxide surfaces depends on surface chemistry regardless of the lipid phase. Significantly, the AFM results on planar surface confirm the quantitative adsorption isotherm results on particle suspensions. We show for the first time that

multiple bilayers are formed on the positively charged alumina surface. Among others, one interesting question that arises from these results is whether the earliest amphiphilic micelles of protocells would have had preferential stability in contact with specific minerals. Efforts are currently underway in our laboratory to examine this idea. Acknowledgment. This research was funded by an NSF CAREER Award (EAR 0346689), the ACS Petroleum Research Fund (41777-AC2), a NASA Astrobiology Institute DDF grant,

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