Formation of Ordered Phospholipid Monolayer on a Hydrophilically

Formation of Ordered Phospholipid Monolayer on a Hydrophilically Modified Au(111) Substrate Hiroaki Shimizu,†,‡ Soichiro Matsunaga,†,‡ Taro Yamada,*,† Toshihide Kobayashi,† and Maki Kawai†,‡ †

RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Department of Advanced Materials Science, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561, Japan

‡

ABSTRACT: The molecular arrangement of phospholipid molecules was investigated on a hydrophilically modified gold surface within an aqueous solution by scanning tunneling microscopy. By suspending phospholipid (1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) nanoparticles in the aqueous electrolyte surrounding a hydrophilically modified gold (111) substrate with 3mercaptopropionic acid (SH−C2H4−COOH, 3-MPA), well-ordered adlattices of POPC were observed. Traces of particle fusion were visualized before formation of the adlattice. Addition of cholesterol to the suspension seems to facilitate accommodation of POPC on this surface. The observed unit cells of POPC adlattices had dimensions of 0.5 nm × 1.9−2.5 nm. By high-resolution imaging, each unit cell was discerned to be occupied by one upright POPC molecule. The POPC + cholesterol suspension also leads to formation of a flat integrated POPC layer, which may be a lipid bilayer covering the surface. KEYWORDS: scanning tunneling microscopy, phospholipid, gold single-crystal surface, hydrophilic modification, high-resolution imaging

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surfaces. This is a physical trade-off with the high-magnification ratio visualizing angstrom-scale features. It is still technically not very simple to observe a soft living cell surface by STM or AFM. As a modest solution, we have to give in to this limitation and spread the phospholipid over a solid metal or oxide surface as a model system for the microscopic operation. Lipkowski et al. first presented in situ STM images of 1,2dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) molecules adsorbed on a bare Au(111) film surface immersed in 50 mM KClO4 solution under electrochemical potential control.4 The monolayer was formed by a process of vesicle fusion. Features of flatly lying DMPC individual molecules were discerned, forming striped patterns composed of arrays of molecules. Features attributed to a lipid hemimicellar structure were also observed. A series of reports using in situ electrochemical STM followed, dealing with Langmuir− Blodgett DMPC bilayers,5 DMPC−cholesterol mixed systems,6 and protein mixed system.7 They attempted construction and observation of lipid bilayers as a model cell membrane laid parallel along a metal substrate.8 For application of AFM, there

hospholipid molecules are the main components of natural cell membranes and organelles. The size of the phospholipid is 2−3 nm in length on average, mostly composed of one hydrophilic headgroup and two hydrophobic hydrocarbon chains. This special amphiphilic nature of phospholipid molecules drives the formation of a phospholipid bilayer in aqueous media, which is the elementary part of cell membranes. Due to the effects of a hydrophilic/hydrophobic interaction with the water interface, the integration of a phospholipid bilayer is so solid that even water molecules cannot go through it. It is desirable to observe angstrom-scale features of an individual phospholipid molecule, interacting with other lipid molecules and water, placed in a circumstance compatible to a living cell. It is easily imagined that all kinds of microscopies are limited in applicability in terms of compatibility with the target system of a living cell surface. When the ratio of magnification to visualize molecules of such size is considered, scanning probe methods, such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM), are the first choice. Scanning probe methods are basically applicable in aqueous media, and certainly so far, plenty of successful observations for biomolecules have been made.1−3 Nevertheless, STM and AFM are hindered by an essential condition where they should be operated over flat, rigid solid © 2016 American Chemical Society

Received: May 24, 2016 Accepted: August 5, 2016 Published: August 5, 2016 7811

DOI: 10.1021/acsnano.6b03421 ACS Nano 2016, 10, 7811−7820

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monolayer, POPC vesicle fusion, formation of POPC monolayer, and the final surface structure after the whole process. We also used cholesterol to see whether a mixed monolayer could be formed. In this series of experiments, we started from a well-ordered (√3 × √3)R30°-type 3-MPA monolayer on Au(111) in a phosphate buffer solution. Upon introduction of POPC suspension, we observed circular features that can be interpreted as traces of fusion of particles with an average diameter of 10 nm.14 The completion of monolayer formation exhibited patches of well-ordered stripes, in which arrays of individual POPC molecules were discerned. After a long period (>a few hours), the surface was in some cases covered with a flat featureless layer, which may have been a mobile double layer of POPC.

have been plenty of works visualizing model cell membranes spread over mica surfaces.9,10 Imaging of individual lipid molecules has not been performed very often. Exceptionally, frequency-modulated AFM was applied on a solid layer of phospholipid and realized molecular-scale imaging.11,12 We also adhere to in situ STM in studying model cell membranes for high-resolution observation. We observed a fluidic layer of 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) spread over Au(111) modified with 1-octanethiol in 0.05 M NH4ClO4 solution. We anticipated hydrophobic conformity of a 1-octyl brush on Au(111) and hexanol groups in DHPC, mimicking the structure of a cell membrane bilayer.13 By application of negative electrochemical potential, a solid feature was composed of stripes of 4−5 nm in width, matching the length of DHPC molecules. We could also visualize phospholipid nanoparticles/domains and their disassembly by the presence of a peptide.14,15 The present research planned to use hydrophilic modification of the Au(111) surface to realize the hydrophilic conformity between the surface modifier and the headgroups in the phospholipid. We anticipated that the hydrophilic surface modifier and the lipid headgroups come closer by the aid of hydrophilic conformity or hydrophobic force between the tail groups and bulk aqueous medium. The latter force would also induce lipid molecules in the solution to pile onto the surface, and as a result, a lipid bilayer would be formed. We put this idea into practice by modifying Au(111) with an well-established modifier of 3-mercaptopropionic acid (3MPA)16−22 and immersing the surface into a nanoparticle suspension of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in a phosphate buffer solution. Figure 1 shows the formulas of 3-MPA, POPC, and cholesterol studied in this work. We monitored the process of the formation of 3-MPA

RESULTS AND DISCUSSION Hydrophilic 3-MPA/Au(111) Surface. The observation every time was started by setting the 3-MPA/Au(111) bead crystal into the STM liquid cell and filling it with the buffer solution. Throughout this study, the substrate electrode potential was always controlled at 0 V versus a Pt quasireference electrode (0.22 ± 0.05 V versus Ag/AgCl) during STM observation. Figure 2a shows a typical lattice image of 3MPA-precovered Au(111) in the phosphate buffer. In Figure 2a, a monatomic step and vacancy islands23 with a Au(111) monatomic step height (=0.236 nm) are observed, and the terraces are covered with adlattice patches. The lattice structures in all patches look equivalent under three-fold rotational symmetry. The major adlattice constant obtained by in-plane two-dimensional Fourier transformation is 0.86 ± 0.05 nm. This lattice constant matches the (3 × 3) structure reported by Sawaguchi et al.18 in 0.05 M HClO4 solution. In Figure 2a, some minor adlattice patches are also seen, recognized as (√3 × √3)R30°. These unit cells are indicated in Figure 2b. Sawaguchi et al. described the (3 × 3) rhombic structure by systematic molecular rotation and displacement of 3-MPA molecules distributed in the (√3 × √3)R30° adlattice at 1/3 coverage with respect to the Au atoms of the underlying Au(111). The (3 × 3) rhombic structure is characterized by a pattern of stripes along [11̅0] and equivalent directions, composed of trimeric units of 3-MPA.18,19 The (3 × 3) structure is a variation of (√3 × √3)R30° structure, involving periodic 0°/120°/240° rotations and slight repositioning of 3MPA molecules along the surface. The geometrically characteristic features, such as 0.5 nm periodicity along [112̅] and equivalent directions (so-called “√3 directions”), are shared between those two. The hydrophilicity of 3-MPA/Au(111) is supported by the observation of a contact angle measurement of a pure water droplet in air. The average contact angle for 3-MPA/Au(111) was 38 ± 5°. The average contact angle for 1-C8H17SH/ Au(111) was 69 ± 5°. Compared to our former experience with 1-C8H17SH/Au(111) as a hydrophobic monolayer toward phospholipids,13,14 the present 3-MPA/Au(111) was obviously hydrophilic. Another microscopic evidence is given by our own infrared spectra of 3-MPA on the Au-evaporated film/Si prism measured in air. Figure 2c shows the multiple internal reflection Fourier transform infrared spectrum of the C−O stretching region, obtained by dividing the single-path spectrum with 3MPA by that of the substrate only. Figure 2c contains vibration signals for CO stretching (1693 cm−1) and O−C−O

Figure 1. (a) Formula of 3-MPA, (b) POPC, (c) cholesterol, and (d) atomic model structure of Au(111). 7812


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of H2O. In the atmosphere, this 3-MPA surface can adsorb some amount of H2O molecules. These frequency assignments were made by referring to the literature.24,25 This spectrum guarantees the existence of hydrophilic groups on Au(111). Time Course of STM Features on 3-MPA/Au(111) in Contact with POPC Suspension. Onto this 3-MPA/ Au(111) specimen immersed in the phosphate buffer was poured a pure POPC suspension. Figure 3 shows a series of STM images sequentially recorded. Before injection of POPC, the wide-range frame in Figure 3a involves vacancy islands on 3-MPA/Au(111). It has been experienced that 3-MPA/ Au(111) is stably observed by in situ STM in this potential range.16,18−20 Right after injection of the POPC suspension, ring-like features appeared sparsely distributed over the flat terraces (Figure 3b). The average outer diameter of the circles is approximately 15 nm. The features looked frozen during the scan of the whole frame in 135 s. The ring-like features attract our attention because their diameters are close to those of phospholipid particles we previously found on hydrophobically modified Au(111) in the same 200 μM POPC suspension.14 On 1-octanethiol-modified Au(111), we actually captured the features of 8−10 nm particles in STM images, and the process of particle fusion was triggered by addition of antibiotics.14 The POPC particles, which we called “minimal lipid particles (MLP)”, were vividly visualized as embodied entities on the surface. In the present case on this hydrophilic surface, we did not find POPC MLPs in Figures 3 and 4. However, we recognize the ring-like features as transient traces of MLPs being merged into the POPC monolayer. If the particles of this diameter are flattened, the diameter will be approximately 15 nm. The ring features were often associated with vacancy islands on the surface. The ring features might be mobile and could have been solidly visualized when they were immobilized by the perimeters of vacancy islands. The ring features seem to represent the POPC objects in the later stage of particle fusion on the hydrophilic surface. The rings are composed of dark 2−3 nm wide perimeter bands and inside areas as bright as the outside areas. The average depth of the dark perimeter bands in those images, measured on the STM image cross sections, is 0.03−0.04 nm. This contrast might be due to the flatly lying phospholipid molecules, in which the hydrocarbon chains are more electrically resistive, and the headgroups are more conductive. The dark perimeters are anticipated to be the hydrocarbon groups of POPC molecules. The ring features probably show the divergent flow of POPC molecules along the surface as an intermediate process of particle fusion. As time passed (in 40 min or so), the ring features were replaced by flat striped patches (Figure 3d). Depending on the position of the scanning frame as well as on time, the timing of ring disappearance was not at once. Figure 3d involves just one ring. Figure 4a,b shows the POPC striped patches spreading on the surfaces, although flat areas without visible stripes are left over. The directions of stripes were fixed over all of the surface, with each having 120° of the crossing angle. In some patches, the stripe is perpendicular to one of the Au(111) step lines, which tends to be parallel to the atom row ([11̅0] and equivalent directions). This indicates that the striped adlattices are rotationally fixed on the underlying three-fold symmetric Au(111) lattice. Then the stripes are parallel to [112̅] or equivalent directions. By two-dimensional Fourier trans-

Figure 2. (a) In situ electrochemical STM image of the 3-MPAcovered Au(111) bead crystal surface in 0.05 M sodium phosphate buffer (pH 7, from Na2HPO4 and NaH2PO4); 50 nm2; STM bias voltage = 400 mV (tip positive), preset tunneling current = 2.20 nA; scanned by a Pt/Ir tip. The atom-row direction ([110̅ ]) and the “√3” direction ([112̅]) are indicated by arrows. (b) Magnified image of the white square in (a). The inset parallelograms indicate the (3 × 3) unit cell (bottom) and (√3 × √3)R30° unit cell (top). (c) Internal multiple reflection−absorption infrared spectrum of 3MPA on Au-evaporated film/Si prism recorded in air, magnified in the C−O stretching region.

asymmetric and symmetric stretching (1555 and 1393 cm−1, respectively). They show proof for carboxylic acid and carbonate having dipole components vertical to the surface. The peak at 1630 cm−1 matches the H−O−H bending motion 7813


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Figure 3. In situ electrochemical STM images of the 3-MPA-covered Au(111) bead crystal surface in the 0.05 M sodium phosphate buffer (pH 7, from Na2HPO4 and NaH2PO4) in the course of POPC layer formation; scanned by a Pt/Ir tip. (a) Before injection of POPC suspension; 200 nm2; STM bias voltage = 200 mV (tip positive), preset tunneling current = 1.00 nA; (b) 14 min after injection of POPC suspension. The concentration of POPC in the STM solution cell was adjusted at 200 μM; 200 nm2. The STM bias voltage was fixed at −400 mV (tip negative) hereafter. Preset tunneling current = 1.30 nA. (c) After 44 min, 100 nm2, 1.30 nA. (d) After 176 min, 200 nm2, 1.60 nA. The positions of rings are indicated by arrows in (b) and (d).

will be discussed later. The periodicity along [112̅] directions was always 0.5 nm for POPC in this work, similarly to the previously reported case for DMPC.4,26 We also performed time-course observation of 3-MPA/ Au(111) in the sodium phosphate buffer solution homogenized with 200 μM POPC + 50 μM cholesterol. The interaction between phospholipids and cholesterol is a cell physiological topic in biomembranes,27 and physicochemical studies have been frequently made.28−30 Cholesterol influences lipid domain separation, fluid dynamic properties, and mechanical properties in membranes. Specifically for POPC, ligation of cholesterol was mechanistically discussed.31 Cholesterol is deeply related to amphiphilicity of phospholipid molecules, changing the trend of hydrophilic/hydrophobic assembling. Figure 4e shows a typical STM image in this POPC + cholesterol mixture. In the POPC + cholesterol suspension, the adlayer seems to be more uniform than in pure POPC. Ringlike features were never observed in several runs under STM monitoring, and stripe features similar to Figure 4a,b covered the whole surface within 2 h after the POPC + cholesterol injection. The directions of stripes are again seen parallel to [112̅] and equivalent directions, and the average stripe spacing is calculated to be 2.5 ± 0.3 nm. The size of the unit cell in Figure 4e seems to be the same as that for Figure 4a−d.

formation over the striped area on the major terrace in this image, the average spacing of the stripe lines is calculated to be 2.5 ± 0.1 nm. However, looking into this adlayer more in detail, we observe that the stripe spacing was not always the same. Figure 4c shows a close-up image with contrast adjustment, capturing the bottom of a monatomic chasm on a flat terrace. A single patch of adlattice, identical to those on the terrace, is seen covering the bottom. The level of the bottom is 0.24 ± 0.03 nm lower than the level of the terrace, indicating that the striped structure is tracking the underlying vacancy island. This image shows that the bottom of a monatomic vacancy island is also covered with a striped patch. The stripe spacing of this bottom patch is apparently shorter than those on the terrace. The lattices on the terrace do not look uniform under this magnification, either. Figure 4d shows a magnification of another part in Figure 4b, which contains a fine adlattice structure. The average repetition unit cell size is 2.4 nm × 0.5 nm. The 0.5 nm segments are parallel to [112̅] or equivalent direction (“√3” directions). It should be noted that the vertical tip displacement tracking all features in this image is smaller than 0.5 nm. The tip height is not equal to the morphological height of admolecules. The STM z-axis of admolecules within aqueous solution must be interpreted under consideration of electronic properties, which 7814


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Figure 4. In situ electrochemical STM images of the 3-MPA-covered Au(111) bead crystal surface in the 0.05 M sodium phosphate buffer (pH 7, from Na2HPO4 and NaH2PO4) in the course of POPC pattern formation in POPC suspension. The concentrations of POPC in the STM solution cell was adjusted at 200 μM. Scanned with a W tip. The STM bias voltage was hereafter fixed at −400 mV (tip negative): (a) 218 min after injection of POPC suspension; 200 nm2, preset tunneling current = 3.50 nA. (b) After 232 min, 100 nm2, 3.20 nA. (c) Close-up image of a vacancy island in (b). (d) Close-up image for molecular alignment in (b). The inset parallelogram indicates the 2.4 nm × 0.5 nm unit cell. (e) After 111 min, 200 nm2, 3.81 nA.

Namely, this adlattice in the POPC + cholesterol mixture did not contain cholesterol. This absence of ring-like features and rapid complete occupation by the stripe features are certainly due to coexisting cholesterol. Cholesterol probably controls the dynamic fluidity of POPC particle skins upon touching and disassembling onto the 3-MPA monolayer. Also, cholesterol seems to mobilize the

adsorbed POPC and promote rapid rearrangement of molecules toward better two-dimensional crystallinity. At a higher concentration of POPC in solution without cholesterol, a propagated form of ring-like objects was observed. Figure 5a,b depicts the process of merging ring-like patterns observed in a POPC suspension with higher concentration. These frames contain ellipses, dumbbell-like shapes, and more elongated shapes. These rubber band patterns 7815


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Figure 5. In situ electrochemical STM images of the 3-MPAcovered Au(111) bead crystal surface in the 0.05 M sodium phosphate buffer (pH 7, from Na2HPO4 and NaH2PO4) in the course of POPC layer formation. The concentration of POPC in the STM solution cell was adjusted at 3400 μM; scanned by a Pt/Ir tip; STM bias voltage = −400 mV (tip negative), preset tunneling current = 1.30 nA, 250 nm2: (a) 41 min after injection of POPC suspension; (b) 44 min after injection of POPC suspension.

Figure 6. In situ electrochemical STM images of the 3-MPAcovered Au(111) bead crystal surface in the 0.05 M NH4ClO4 (pH 7), in the course of POPC pattern formation in POPC + cholesterol suspension. The concentrations of POPC and cholesterol in the STM solution cell were adjusted to 200 and 50 μM, respectively. Scanned with a W tip. STM bias voltage was always −400 mV (tip negative); (a) 34 min after injection of POPC + cholesterol suspension; 200 nm2, preset tunneling current = 2.50 nA. (b) After 43 min, 10 nm2, 2.50 nA. The inset parallelogram indicates the 1.9 nm × 0.5 nm unit cell.

were probably formed by merger of the ring patterns driven by the high concentration of lipid particles. The bright spots with diameters around 10 nm match the features for intact lipid particles observed on hydrophobically modified Au(111).14 As the concentration of POPC particles was high, the particles before disassembling into the adlayer seem to be left over occasionally. Fine Structure of POPC Adlattices on 3-MPA/Au(111). As seen in the former sections, the POPC striped structure varies according to addition of cholesterol into the POPC suspension. Narrow-pitched stripes are homogeneously formed on the 3-MPA-modified terraces. By changing the content of the STM operational solution, we can anticipate some changes in the structure of the adlayer and the process of adlayer formation. To explore the resolvable structures of the adlattice, we chose 0.05 M NH4ClO4 (pH 7) as a different electrolyte from phosphate for the POPC + cholesterol suspension. HClO4 always exhibits complete dissociation in water32 and is anticipated to behave more independently with POPC than phosphate anions. Figure 6a shows the stripe patterns covering an area of 100 nm2 on the 3-MPA/Au(111) terraces, 34 min after immersion

in 200 μM POPC + 50 μM cholesterol suspended in 0.05 M NH4ClO4. The surface is covered with three equivalent lattices rotated by 120 and 240°, again reflecting commensuration with the 3-MPA adlattice and henceforward the substrate Au(111) lattice. In this POPC + cholesterol suspension, the ring-like features were not observed, and stripe features appeared much earlier. By zooming into a 10 nm2 area in Figure 6a, we could discern features of molecular arrays in 34 s for the whole frame scan, as shown in Figure 6c. This image is composed of identical bright dot arrays alternatively arranged with dark bands. The average spacing between the two nearest bright dot arrays is 1.9 ± 0.2 nm, and the average spacing between two nearest bright dots is 0.5 nm. The direction of bright dot arrays is determined to be the in-plane [112̅] direction of Au(111), judging from the stripe directions and the [11̅0] atom-row step lines in Figure 6a. In the POPC adlattice images in Figure 4 and Figure 6, the longer segment of the unit cell is in the range of 1.9−2.5 nm, whereas the shorter segment is constantly 0.5 nm. The shorter segment is parallel to the stripes, that is, the [112̅] direction. 7816


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Figure 7. (a) Schematic structure of the 3-MPA/Au(111)−(√3 × √3)R30° adlayer. The atoms are represented by space-filling color-coded balls: white, H; gray, C; red, O; yellow, S; dark yellow, Au. (b) Dimensions of an isolated POPC molecule optimized by Dmol3.33−35 DFT molecular orbital calculation package (Accelrys Ver. 4.0) installed in the supercomputing system at RIKEN Advanced Center for Computing and Communication. The geometric structure was energetically optimized with a DFT molecular orbital calculation with the doublenumerical plus d-function basis set (“DND” option) for all electrons. The local density approximation Perdue−Wang functional was employed. The orbital wave function was visualized for each of the energy eigenvalues on the basis of the optimized molecular structure. The atoms are represented by space-filling color-coded balls: white, H; gray, C; blue, N; red, O; purple, P. (c) Schematic structure of POPC overlayer on 3-MPA/Au(111)−(√3 × √3)R30°. The spacing between two neighboring POPC molecules is 0.5 nm, in accordance with the periodicity of underlying 3-MPA/Au(111)−(√3 × √3)R30°. The atoms are represented by space-filling color-coded balls: white, H; gray, C; blue, N; red, O; dark red, O in 3-MPA; yellow, S; purple, P; dark yellow, Au. The black arrow indicates the direction of view of panel (d). (d) Averaged STM top-view image of POPC unit cells and a model structure. The averaged image was obtained by overlaying 17 parts of less disturbed areas out of Figure 6c and numerically summing for each pixel. The unit cell is adjusted to a 1.95 nm × 0.5 nm rectangle. The model structure was drawn over 3-MPA/Au(111)−(√3 × √3)R30° schematic drawing with the head and tail views of a POPC isolated molecule generated by DMol3, with a minor adjustment for the periodicity of 0.5 nm.

Actually 0.5 nm = 0.288 nm (Au(111) atomic lattice constant) × √3 along [112̅] is equal to the unit cell segments of 3-MPA (√3 × √3)R30°. This geometry is illustrated in Figure 7a. The POPC features are arrayed in commensuration to the underlying 3-MPA [112̅] rows in the same periodicity. This 1.9 nm × 0.5 nm unit cell should be composed only of POPC without cholesterol because this unit cell is of the same kind as those in Figure 4a−d, which were acquired without cholesterol in the solution. The 0.5 nm segments along the [112̅] row are common for the unit cells in Figure 4 and Figure 6. This sort of cholesterol exclusion from the phospholipid adlattice was also found for DMPC.6,26

We estimated the size of a POPC molecule by structural optimization calculation using a commercial molecular orbital package, Dmol3, based on the density functional theory (DFT).33−35 This calculation yielded an outer shape of POPC supposed to be in vacuum, as shown in Figure 7b. The longitudinal size of this optimized POPC is 2.8 nm, and the width (the distance between the end C atoms terminating the two hydrocarbon chains) is 1.1 nm. The thickness, that is, the shortest dimension of the outer shape, exceeds 0.5 nm. To guarantee the POPC adlattice periodicity of 0.5 nm, we suppose a certain flexibility of the two hydrocarbon chains. The thickness and the angle between the two hydrocarbon chains were adjusted. 7817


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ACS Nano The orientation of the adsorbed POPC molecule can be derived under an assumption that the hydrophilic 3-MPA admolecules attract the POPC hydrophilic end within aqueous solution. This naturally leads to the upright position of the POPC molecule, with the choline group touching one 3-MPA admolecule, as shown in Figure 7c, with the periodicity along the [112̅] direction enforced at 0.5 nm. Since the POPC length exceeds the longer segment of the unit cell, one flatly laid POPC molecule cannot be accommodated in the cell. It is also impossible to squeeze a cholesterol molecule into the POPC unit cell because of the molecular sizes. We have to discuss the accountability of this structure on the basis of the high-resolution STM image in Figure 6b. To refine the individual molecular image, 17 pairs of neighboring unit cells without severe turbulence was selected within Figure 6b and numerically averaged for each pixel. The unit cell distortion was corrected to be a rectangle, being adjustable as a super cell of the (√3 × √3)R30°−3-MPA adlattice. The result is shown in Figure 7d. The unit cell contains some bright spots and less bright lines within an area of 1.95 nm × 0.5 nm. The repeated feature is not mirror symmetric along the [112̅] direction, matching the chiral L-type POPC reagent. Figure 7d contains a schematic top view of Figure 7c, indicating a fair similarity to the STM image. This model top view is of the hydrocarbon chains extending in a slanted direction from the end, as indicated by the arrow in Figure 7c. Therefore, in the top view, each of the chains looks to be approximately 1 nm long, which is shorter than the extended view of chains in Figure 7b. The bright spots are considered to correspond to the hydrophilic groups within a POPC molecule, bearing a high electric conductivity, and the tip can stay away from the surface to attain the preset tunneling current. The hydrocarbon chains are less conductive, and the tip needs to go closer to the surface and thus exhibits darker features. It is difficult to determine the geometrical alignment of those intramolecular groups along the vertical direction toward the surface. The observed heights of features are within 0.5 nm, which is apparently smaller than the length of the POPC molecule (2.7 nm). This is a general trend in observations of biological molecules by STM in aqueous solution.13,36,37 The intramolecular subgroups look threedimensionally overlapped in the top-view STM image. Stacking of more POPC/cholesterol molecules was not systematically observed over the ordered POPC monolayer. Although the model structure in Figure 7c,d involves hydrocarbon chains sticking out from the surface, the hydrophobic nature does not prevail overall because of the low molecular areal density of POPC. On a real cell membrane single leaflet, one phospholipid occupies just 0.4 nm2 at the minimum.38 In the model structure in Figure 7d, one POPC molecule occupies 0.98 nm2, and there must be room for H2O molecules to incorporate, making the hydrophobicity weaker than the densely populated membrane. Therefore, the hydrophobic ends of isolated POPC or cholesterol molecules is not considered to be attracted strongly by the POPC adlayer and to form a stabilized structure by the hydrophobic interaction. The Flat Layer. The POPC + cholesterol suspension sometimes introduced a flat overlayer over 3-MPA/Au(111) after a long immersion time. Figure 8a shows a bare 3-MPA/ Au(111) surface before lipid suspension was added. The surface is full of holes with a depth of Au(111) monatomic height and an average diameter of 5 nm. They are the vacancy islands. Figure 8b was recorded for 5 h after POPC + cholesterol suspension was added. It is notable that the vacancy islands

Figure 8. In situ electrochemical STM images of the 3-MPAcovered Au(111) bead crystal surface in the 0.05 M sodium phosphate buffer (pH 7, from Na2HPO4 and NaH2PO4) in the course of POPC pattern formation in POPC + cholesterol suspension. Scanned by a Pt/Ir tip: (a) 3-MPA/Au(111) surface before adding POPC + cholesterol suspension; 150 nm2; STM bias voltage = 200 mV (tip positive), preset tunneling current = 1.00 nA; (b) 292 min after injection of POPC + cholesterol suspension; 150 nm2. The concentrations of POPC and cholesterol in the STM solution cell were adjusted to 200 and 50 μM, respectively. STM bias voltage = −400 mV, preset tunneling current = 3.30 nA.

became completely invisible. The step lines in this image are all with Au(111) monatomic height, so the step terrace structure of underlying Au(111) is tracked by the overlayer. The invisibility of vacancy islands through the outermost surface can be explained by bridging of a tight lipid layer over the holes. In our previous report,13 sequential imaging of a mobile phospholipid layer discerned the vacancy islands passed over by the fringes of lipid layer openings (“lipid windows”). The vacancy islands were not visible through the lipid layer over them. The observation of a flat layer in Figure 8b indicates an integrated layer of POPC covering the 3-MPA/Au(111) terraces full of vacancy islands. The POPC integrated layer may include cholesterol because we have no experience imaging such a flat layer without cholesterol in the solution. Considering the hydrophilic headgroup and hydrophobic hydrocarbon chains, we can imagine an integrated POPC unit membrane (phospholipid bilayer) covering the 3-MPA/Au(111) surface. Both sides of this bilayer are skinned with the hydrophilic headgroups, conforming to hydrophilic 3-MPA and the bulk aqueous 7818


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electrode. The Pt quasi-reference electrode in this phosphate buffer was usually at 0.22 ± 0.05 V versus Ag/AgCl. As an atomically flat Au(111) surface, we utilized small (111) facets formed on a single-crystal gold bead with a diameter of approximately 3 mm, prepared by melting one end of the gold wire in a hydrogen− oxygen flame.39 This gold surface was covered with a 3-MPA selfassembled monolayer by immersing it into a 1 mM C2H5OH solution of 3-MPA for 24 h, followed by rinsing in pure C2H5OH. This piece of Au was fixed on the in situ STM measurement cell. The desired solutions were poured into the cell for successive STM observation. Additionally, we used a FTIR spectrometer (Tensor, Bruker Inc.) to confirm the hydrophilic groups of 3-MPA delivered on Au(111). We applied the internal multiple reflection configuration using a 45° wedge-cut Si prism (2 mm thick, 40 mm long along the light path). Approximately 20-fold internal interface reflection is expected. The reflection planes of the Si prism were covered with Au evaporated film (20 nm thick), which is mainly composed of (111)-oriented grains. The 3-MPA monolayer was formed in the same manner as Au bead crystals. The phospholipid (POPC) nanoparticle aqueous suspension was prepared by the previously reported procedure.14 In short, POPC solid flakes obtained by drying out the POPC CHCl3 solution were mixed with the phosphate buffer and sonicated with an ultrasonic homogenizer. The POPC concentration was adjusted to 200 μM. By this procedure, we obtained a POPC suspension that mainly contained POPC particles with an average diameter of 8−10 nm.14 Similarly, a POPC + cholesterol 4:1 (total concentration 50 μM) suspension was also prepared.

solution with energetic favorability. In this case, the wellordered adlattice of POPC, such as in Figure 6, is not laid underneath because of its weaker hydrophobicity, as discussed in the previous section. A mobile underlayer is anticipated to be composed of densely populated POPC with their headgroups near the 3-MPA surface. Although this integrated layer can be a model case of the phospholipid unit membrane, it took a long time to reach this sort of flat integrated monolayer, and we have no frequent chance to observe this surface under a stable condition. Contamination and instability of the tunneling gap were the problems to be solved in this observation. It is probably possible to accelerate the bilayer formation by adjusting the contents of phospholipid suspension.

CONCLUSIONS The microscopic features created on the hydrophilic 3-MPAcovered Au(111), immersed in aqueous POPC suspensions, were observed by in situ electrochemical STM. In suspensions with POPC only, we observed traces of vesicle fusion converting the POPC particles in the suspension to a monolayer of POPC on 3-MPA/Au(111) as ring-like or rubber-band-like features. Later, the flat terrace areas were covered by striped layers, composed of two-dimensional arrays of POPC molecules. The unit cell size is 1.9−2.5 nm × 0.5 nm, variable according to the contents of the suspension as well as the local geometry of the surface. The periodicity of 0.5 nm along the [112̅] direction of the substrate Au(111) is enforced by the periodicity of 3-MPA (√3 × √3)R30°, indicating a strong interaction between the hydrophilic groups on the both sides. Addition of cholesterol accelerated this formation of striped adlayers. By high-resolution observation, each unit cell is interpreted to be occupied with one POPC molecule with the hydrophilic headgroup bonded to a 3-MPA adlayer, forming a unit cell of 1.9 nm × 0.5 nm. In the POPC + cholesterol suspension, a flat layer was occasionally observed covering the underlying Au(111) terraces with vacancy islands after a long waiting period. This flat layer is considered to be an integrated POPC bilayer along the hydrophilic 3-MPA/Au(111) surface.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Author Contributions

H.S., S.M., and T.Y. designed, directed, and conducted the experiments. All authors discussed the results. T.Y., T.K., and M.K. co-wrote the manuscript with input from all authors. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was financially supported in part by RIKEN President Discretionary Fund (2004−2006, 2010−2012), RIKEN Lipid Dynamics Project, RIKEN Integrated Lipidology Program, and Grants-in-Aid for Scientific Research on Promotion of Novel Interdisciplinary Fields Based on Nanotechnology and Materials from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work is also supported by Kakenhi (Grant Nos. 19360024 and 25293015). Advanced Center for Computing and Communication (ACCC) of RIKEN is thanked for the supercomputational facility.

METHODS Chiral L-type POPC (purity >99%) and cholesterol (>98%) were obtained as CHCl3 solutions from Avanti Polar Lipids Inc. CHCl3 (>99%), C2H5OH (spectroscopic grade), Na2HPO4·12H2O (>99%), NaH2PO4·2H2O (>98%), NaOH (ultrapure grade), 3-SHC2H4COOH (3-MPA, >97%), and 1-C8H17SH (>97%) were obtained from Kanto Chemicals, Japan. Gold wire (diameter 1 mm, purity >99.999%, Furuya Metals, Japan) was used for the substrate Au material. The electrodes and wirings for the STM electrochemical cell were made from Pt (wires of 1 mm and 0.25 mm in diameter, and a sheet of 0.1 mm in thickness, purity >99.98%, Nilaco, Japan). All in situ STM images were obtained using Nanoscope E (Veeco instruments Inc.), with a scanning base of the electrochemical model. We utilized W tips (diameter 0.25 mm, Nilaco, Japan) sharpened by electrochemical etching as well as commercial ready-made Pt/Ir alloy tips (“Nano tip”, Bruker Inc.) The tip was coated with nail polish to minimize the Faraday current in the electrolyte. The measurement was performed in a Teflon−perfluoroalkoxyethylene cell (volume of liquid content = 2 mL), assembled with a Pt plate (6 mm × 20 mm) as the counter electrode and a Pt wire as a quasi-reference electrode. A sodium phosphate buffer (0.05 M, pH 7.0 ± 0.1, prepared by neutralization of Na2HPO4 and NaH2PO4 in Milli-Q purified water) was prepared as a stock and used as the supporting electrolyte. The quasi-reference Pt electrode was calibrated with a Ag/AgCl reference

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Formation of Ordered Phospholipid Monolayer on a Hydrophilically

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