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Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface
Materials Chemistry and Physics xxx (2016) 1e8
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Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface Jan Pawłowski, Joanna Juhaniewicz, Sławomir Se˛ k* Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Zwirki i Wigury 101, 02-089, Warsaw, Poland
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STM and AFM methods were used to examine adsorption of model lipid on Au(111). Self-assembly of model lipid leads to formation of highly organized molecular film. The model is proposed which reproduces the STM contrast.
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Article history: Received 10 March 2016 Received in revised form 31 August 2016 Accepted 29 October 2016 Available online xxx
Although the adsorption of lipids on reconstructed Au(111) surface and formation of highly ordered stripe-like domains are well-known phenomena, the exact orientation of the molecules with respect to the substrate remains unclear. Therefore, in this study we have focused on the structure and arrangement of lipid molecules forming highly ordered stripe-like domains at gold electrode-electrolyte interface. N-palmitoyl-D-erythro-dihydroceramide-1-phosphate was selected as model compound since its ability to transform into hemimicellar structure is limited. This way it was possible to get very stable lipid film with characteristic stripe-like pattern. Application of complementary techniques such as atomic force microscopy and scanning tunneling microscopy enabled detailed characteristics of lipid adlayer adsorbed on Au(111) electrode. Based on careful analysis of the experimental results, we have proposed a model which describes the arrangement of the molecules within the film. In general, it assumes flat-lying orientation of the lipids but only one hydrocarbon chain of phosphorylated dihydroceramide is involved in direct interaction with gold. © 2016 Elsevier B.V. All rights reserved.
Keywords: Lipids Adsorption Gold electrode Atomic force microscopy Scanning tunneling microscopy
1. Introduction Interfacial self-assembly is one of the most commonly used
* Corresponding author. E-mail address: [email protected] (S. Se˛ k).
methods to obtain functionalized surfaces with purposely tailored properties [1]. The latter depend on chemical nature and the arrangement of the adsorbate molecules on the substrate. Molecular films immobilized at solid surfaces are particularly attractive and they play a crucial role in many domains of fundamental research as well as technological applications. These include surface modification for sensors [2,3], biofuel cells [4,5], corrosion
http://dx.doi.org/10.1016/j.matchemphys.2016.10.046 0254-0584/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046
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inhibition [6], heterogeneous catalysis [7], and investigation of mediated electron transfer [8,9]. Moreover, molecular layers adsorbed on a surface with well-defined crystallographic structure can act as a template for the construction of molecular electronics devices [10] and nanoscale level engineering [11]. Among many possible surface modifications, lipids self-assembled on metal substrates have emerged as an attractive platform to investigate membrane related processes [12e14] and design biosensing devices [15,16] or biomimetic interfaces [17,18]. Lipid monolayers and bilayers immobilized on gold surface already proved to be suitable systems for the studies of protein binding [19,20] interactions with antimicrobial peptides [21e25], as well as characterization of redox-active enzymes [26] and modelling of transmembrane ion transport [27,28]. Since the suitably tailored lipid films supported on metal substrates are increasingly used in a variety of fields, full control over their structure and properties becomes an important issue. Therefore, it is crucial to understand the fundamental mechanisms of lipid adsorption and film formation at solid-liquid interfaces. Recently, we have demonstrated that formation of lipid bilayer on Au(111) electrode by spreading of small unilamellar vesicles involves several steps [29]. These include deposition of the vesicles on gold surface followed by release of lipid molecules which adsorb with flat-lying orientation and form stripe-like domains. The latter serves as a template for development of hemimicellar film which facilitates further adsorption and rupture of SUVs and the bilayer is spread over hemimicellar film. Finally, a single planar bilayer is formed due to the fusion between coupled layers. In this paper, we have focused on the initial step of this mechanism that is adsorption of lipid molecules with long molecular axis parallel with respect to the Au(111) surface. So far, this phenomenon was observed for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) molecules either alone or in the presence of cholesterol and it was ascribed to preferential interactions between hydrophobic acyl chains and gold surface [30,31]. However, the exact orientation of the molecules with respect to the substrate plane and the extent of the molecule-substrate contact are still unclear. Moreover, the detailed examination of the stripe-like domains formed by DMPC is difficult due to their limited stability. As it was already mentioned, prolonged exposure of modified Au(111) electrode to lipid vesicles results in transition from stripe-like structure to hemimicellar film and then to planar bilayer. In order to suppress this transformation, we have selected phosphorylated dihydroceramide, which is Npalmitoyl-D-erythro-dihydroceramide-1-phosphate, as a model lipid. The size of the phosphate polar headgroup in this compound is smaller comparing with the diameter of the hydrophobic domain. Molecular shape affects supramolecular structural organization and therefore, the ability of the lipid to form hemimicellar structures will be limited. This assumption is reasonable since it was reported in the literature that closely related compounds such as phosphorylated ceramides, when added to lipid mixtures cause a reduction of the lamellar-to-inverted hexagonal phase transition temperature and have ability to induce negative membrane curvature [32]. Thus, by using N-palmitoyl-D-erythro-dihydroceramide-1-phosphate as a model lipid, we obtained stable stripe-like domains on reconstructed Au(111) surface. This allowed careful examination of their structure with scanning probe microscopy and based on it we have proposed detailed description of moleculesubstrate contact.
which was purchased from Avanti Polar Lipids Inc. In all experiments we have used distilled water passed through a Milli-Q water purification system and its final resistivity was 18.2 MU cm. Surface pressure - molecular area isotherms for N-palmitoyl-Derythro-dihydroceramide-1-phosphate monolayer at the air-water interface were recorded using a KSV LB trough 5000 (KSV Ltd., Finland) equipped with two movable Teflon barriers and a paper plate used as a surface pressure sensor. Trough and barriers were washed thoroughly before each experiment using the mixture of chloroform and methanol and finally rinsed with Milli-Q water. As a subphase 0.01 M phosphate buffer saline (PBS) aqueous solution was used. N-palmitoyl-D-erythro-dihydroceramide-1-phosphate was dissolved in a mixture of CHCl3:CH3OH:0.5N HCl (20:9:1). The resulting clear solution was spread on buffer surface and then it was left to evaporate the solvent. The compression of the monolayers was performed at the barriers speed of 10 mm/min at a constant temperature of 21 ± 1 C. In order to prepare the suspension of N-palmitoyl-D-erythrodihydroceramide-1-phosphate in 0.05 M KClO4 aqueous solution we have followed the same procedure as for preparation of small unilamellar vesicles [33]. However, the resulting product was a fine suspension of lipid aggregates. The protocol involved preparation of stock solution by dissolving 1.0 mg of N-palmitoyl-D-erythrodihydroceramide-1-phosphate in 5 ml of hexane/ethanol 2:1 mixture. An aliquot of the solution (0.2 ml) was placed in a test tube and the solvent was evaporated by vortexing under argon stream. Subsequently, the test tube with dried lipid cake was placed for approximately 3 h in vacuum desiccator to remove the solvent. Finally, 1.5 ml of 0.05 M KClO4 solution was added to the lipid cake and the sample was sonicated for at least 1 h. Portion of the resulting suspension was added to electrochemical cell of the microscope to give approximate lipid concentration of 4.0 106 M. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) images were obtained with 5500AFM (Keysight Technology) and Dimension Icon (Bruker) instruments respectively. The experiments were performed at 21 C. All images were recorded in 0.05 M KClO4 under electrochemical control with single crystal Au(111) (MaTeck) as a working electrode, miniaturized Ag/ AgCl (sat. KCl) as a reference and platinum wire as a counter electrode. The components of the electrochemical cell as well as metallic electrodes were cleaned in piranha solution (concentrated H2SO4/30% H2O2 3:1, v/v) for at least 2 h and then rinsed with copious amounts of Milli-Q ultrapure water. (CAUTION: piranha solution reacts violently with organic materials and should be handled with extreme care.) Single crystal Au(111) electrode was carefully flame annealed and then placed in electrochemical cell of the microscope. In order to facilitate the surface reconstruction, the electrode was held for approximately 30 min at the potential of 0.3 V directly before each experiment. For STM imaging, we have used electrochemically etched tungsten tips coated with polyethylene to minimize leakage currents. The AFM images were taken in PeakForce Tapping mode using qp-BioAC cantilevers (Nanosensors, CB2: nominal spring constant 0.06e0.18 N/m). Both STM and AFM imaging was performed on four independently prepared samples. N-palmitoyl-D-erythro-dihydroceramide-1-phosphate molecule was modelled using Spartan ’14 Version 1.1.4 (Wavefunction, Inc.). Molecular modeling was performed with the ab initio Hartree-Fock method using the 3-21G basis set available in the software package.
2. Experimental 3. Results and discussion The chemicals used in this work were purchased from SigmaAldrich and Avantor Performance Materials SA. The only exception was N-palmitoyl-D-erythro-dihydroceramide-1-phosphate,
The structure of N-palmitoyl-D-erythro-dihydroceramide-1phosphate molecule is illustrated in Scheme 1. It consists of
Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046
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Scheme 1. Structure of N-palmitoyl-D-erythro-dihydroceramide-1-phosphate.
sphinganine backbone and fatty acid residue in hydrophobic part and phosphate headgroup in a polar head region. Total length of the molecule is ~2.6 nm. Cross sectional area of the hydrophobic part of the lipid was estimated to be ~40 Å2, while the corresponding area of the phosphate group is ~26 Å2 assuming its van der Waals radius of 2.9 Å. This clearly shows the disproportion in size when comparing hydrophobic and hydrophilic parts of the lipid molecule. Based on simple geometric considerations, one can conclude that ability of phosphorylated dihydroceramide to form either micelles or small vesicles will be rather limited. This can be confirmed by estimation of packing parameter defined as p ¼ VC/(Ah lC), where VC denotes volume of the hydrophobic tails, Ah is the projection area of the headgroup and lC is the length of lipid chain. Resulting value determined for phosphorylated dihydroceramide is well above the unity, which means that the molecules will tend to form aggregates with negatively curved interfaces [34]. Disproportion in size between phosphate group and the hydrophobic part of the phosphorylated dihydroceramide can be verified using Langmuir technique where the single molecular film is spread at the air-water interface and the changes in surface pressure are monitored as a function of the area available for
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molecules. An exemplary isotherm recorded for N-palmitoyl-Derythro-dihydroceramide-1-phosphate film spread at the surface of 0.01 M PBS aqueous solution is presented in Fig. 1. It was found that the area available for single molecule within the organized monolayer of phosphorylated ceramide is 43.5 ± 0.5 Å2. This result suggests that the packing of acyl chains within the film is practically unaffected by the presence of phosphate group, since it roughly corresponds to the doubled area usually observed for organized monolayers of unionized saturated fatty acids and fatty amines of comparable length. For example, the area per molecule for monolayers of hexadecanoic acid and hexadecanamine is ~21 Å2 [35]. As can be seen in Fig. 1, the monolayer of phosphorylated dihydroceramide becomes unstable at surface pressure of ~46.0 mN/m where the plateau is observed representing the transition of the film into multilayer state. Interestingly, relatively dense packing of the molecules within the monolayer can also be confirmed by analysis of compression modulus (see inset in Fig. 1). Its maximum value of ~250 mN/m is indicative for existence of solid-like film. Thus the structural analysis as well as surface pressure measurements strongly suggest that N-palmitoyl-D-erythro-dihydroceramide-1-phosphate will have very limited tendency to form micellar aggregates with positive curvature. Therefore it could be expected that upon deposition on Au(111) transformation into hemimicellar structure will be suppressed enabling more detailed examination of lamellar phase created at the initial stages of lipid adsorption. Based on such assumption, we have examined the adsorption behavior of N-palmitoyl-D-erythro-dihydroceramide-1phosphate on Au(111) electrode. Fig. 2A shows an image recorded using electrochemical atomic force microscopy (EC-AFM) for bare gold electrode at the potential of þ0.25 V vs. Ag/AgCl(sat. KCl) in the absence of lipid molecules. The surface is flat with a step edge visible at the left side of the scanned area. The average surface roughness (Sq) determined from 1.5 1.5 mm2 scans taken from several spots on bare Au(111) electrode was 0.084 nm, which confirms good quality of the substrate. Fig. 2B presents an EC-AFM image of the same electrode recorded after injection of phosphorylated dihydroceramide suspension
Fig. 1. Surface pressure vs. area per molecule isotherm recorded for N-palmitoyl-D-erythro-dihydroceramide-1-phosphate film spread at the surface of 0.01 M PBS. The isotherm was recorded at 21 ± 1 C. Inset shows the changes in compression modulus as a function of surface pressure.
Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046
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Fig. 2. EC-AFM topography images recorded on bare Au(111) electrode (A) and after injection of the suspension of N-palmitoyl-D-erythro-dihydroceramide-1-phosphate and formation of adsorbed layer (B). The images were recorded in an aqueous solution of 0.05 M KClO4 at the potential of þ0.25 V vs. Ag/AgCl(sat. KCl). The distribution of the thickness determined for the lipid film from cross sectional analysis is shown in panel (C). Image of the N-palmitoyl-D-erythro-dihydroceramide-1-phosphate adlayer recorded after potential step to þ0.35 V vs. Ag/AgCl(sat. KCl) is shown in panel (D).
into the electrochemical cell of the microscope. The total concentration of the lipid in electrochemical cell was 4.0 106 M. The potential of the electrode was kept constant and it was adjusted to þ0.25 V vs. Ag/AgCl(sat. KCl). The images were taken directly after introduction of the lipid suspension into the electrochemical cell. Under such conditions lipid molecules tend to form an adlayer on Au(111) surface which is clearly resolved in AFM image. Importantly, the lipid film remains stable for several hours and does not undergo transition neither to hemimicellar state nor to the bilayer. This confirms that N-palmitoyl-D-erythro-dihydroceramide-1-phosphate has limited ability to form micelles. Interestingly, the adsorption of the phosphorylated dihydroceramide on gold surface is not random. As can be seen in Fig. 2B, numerous domain boundaries and unoccupied spaces appear as dark straight lines aligned with three particular directions rotated by 120 . Such symmetry is characteristic for Au(111) structure. Therefore, we conclude that the molecules within the adlayer follow crystallographic structure of the electrode surface. In order to assess the orientation of the adsorbed molecules with respect to the substrate, we have carried out cross sectional analysis of AFM images and determined the thickness of the lipid film. The spread of the determined values is shown in Fig. 2C and it represents data obtained from fifty individual profiles taken within different regions on four independently prepared samples. The mean thickness was found to be 0.8 ± 0.1 nm suggesting that phosphorylated dihydroceramide forms a monolayer and its molecules adopt orientation with their long molecular axis parallel to the substrate plane. Such behavior seems to be common for lipids as well as n-alkanes
and their derivatives [30,31,36]. However, the thickness of the lipid film reported here is higher than expected if both hydrophobic chains were in contact with the electrode surface. The diameter of the alkyl chain is about 0.5 nm, thus the thickness of the film would be close to this value. The discrepancy strongly suggests that the adsorption of double-chain lipids on Au(111) surface involves the contact only with one alkyl chain, while the other remains at certain distance from the surface contributing to the increased thickness of the film. Interestingly, we have found that the ordered adlayer of phosphorylated dihydroceramide is stable within the certain potential range. More specifically, at the potentials more negative than 0.40 V the adlayer becomes desorbed and bare gold surface is exposed, while at the potentials more positive than þ0.30 V the adlayer transforms into irregular structure as demonstrated in Fig. 2D. Similar behavior was reported for other amphiphiles adsorbed on Au(111) surface and it was ascribed to the changes in charge density at the electrode surface [37,38]. Nevertheless, the transition from highly ordered to random orientation often coincides with potential at which gold reconstruction is lifted. This suggests that interatomic distances at reconstructed surface of Au(111) are preferable for optimal packing of alkyl chains. In order to obtain more detailed information about ordered adlayer, molecular packing and orientation of lipid molecules with respect to the substrate were examined by high resolution imaging with electrochemical scanning tunneling microscopy (ECSTM). Fig. 3A shows an image of bare Au(111) electrode recorded at 0.3 V vs. Ag/AgCl(sat. KCl) in the absence of the lipid. Well-
Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046
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Fig. 3. (A) EC-STM topography image recorded for reconstructed Au(111) surface at the potential of 0.30 V vs. Ag/AgCl(sat. KCl) in the absence of lipid molecules. (B) EC-STM topography image of the same Au(111) electrode taken at þ0.25 V vs. Ag/AgCl(sat. KCl) after injection of the suspension of phosphorylated dihydroceramide into the electrochemical cell of the microscope. It reveals stripe-like domains formed by adsorbed lipid. (C) High resolution topography image (10 10 nm2) of the stripe-like domain illustrating the arrangement of the molecules with respect to the crystallographic directions of the substrate. (D) High resolution STM current image (8.5 8.5 nm2) of lipid molecules adsorbed on Au(111) surface demonstrating the differences in contrast over individual molecule (indicated by black and white arrows).
defined crystallographic structure of the electrode surface was confirmed by the presence of reconstruction lines. The latter are arranged in pairs which form reconstruction rows. The distance between paired lines is 2.5 nm, while the separation between the rows is 6.3 nm [39]. Since these values are well-known, the reconstruction lines can be used for precise calibration of the measured distances. Moreover, assuming that the lines are running h i parallel to 112 direction, it is possible to determine crystallographic directions on Au(111) electrode surface. Fig. 3B presents an image of Au(111) electrode taken at þ0.25 V vs. Ag/AgCl(sat. KCl) after injection of the suspension of phosphorylated dihydroceramide into the electrochemical cell of the microscope. The lipid molecules are organized into stripe-like structures, which form domains rotated by 120 . This confirms that the orientation of the molecules within the adlayer is affected by crystallographic structure of the substrate. Careful examination of high resolution image shown in Fig. 3C reveals that stripes are aligned with ½121 direction and consist of two rows of single lipid molecules oriented perpendicular to the stripe direction. The width of the individual stripe is ~5.0 nm, which roughly corresponds to the doubled length of phosphorylated dihydroceramide molecule. This indicates that lipids adopt antiparallel orientation with head-to-head and tail-totail packing order. Further analysis of STM image shows that long axis of lipid molecules is aligned with ½101 crystallographic direction. Such orientation is known to be favorable for n-alkanes and their derivatives adsorbed on Au(111) surface [37,40,41]. Therefore, it is reasonable to conclude that hydrophobic part of the lipid molecule is directly involved in the interactions with the substrate. At this point, it is important to consider exact orientation of the hydrocarbon skeleton with respect to the Au(111) substrate. For
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simple n-alkanes, the analysis of the potential energy difference between a molecule in an adlayer and a free molecule revealed that carbon skeleton is predominantly oriented parallel to the plane of gold surface [42]. This was also confirmed by infrared reflection absorption spectroscopic study, which proved that n-alkanes adopt flat-on orientation on gold at room temperature [43]. Therefore, it is reasonable to assume that adsorption of lipids on Au(111) involve parallel orientation of the hydrocarbon chain with respect to the surface of the substrate. However, phosphorylated dihydroceramide possesses two alkyl chains being parts of fatty acid residue and sphinganine backbone. Moreover, the C-C-C planes of these hydrocarbon chains are rotated with respect to each other. This implies that the adsorption of both chains would result in unfavorable stress and strain within the molecule. For this reason, the molecule-substrate interaction through single chain seems to be more likely. This would also explain the contrast observed in STM current images. As demonstrated in Fig. 3D, current flow is not uniform throughout the individual molecule. It consists of bright linear feature indicated by black arrow with closely located and less intense spots pointed by white arrow. Similar spots were observed in numerous STM images of n-alkanes and their derivatives on Au(111) and the interpretation of such contrast assumed parallel orientation of carbon skeleton plane with respect to the substrate [44]. Therefore, we have concluded that the presence of the spots in Fig. 3D results from flat-on orientation of one of the hydrocarbon chains in lipid molecule. Consequently, the linear feature was ascribed to the second hydrocarbon chain of lipid with C-C-C plane tilted with respect to the substrate. Thus the STM current contrast reflects varying tunneling conditions over differently oriented carbon backbones. Interestingly, the topography does not show such details as demonstrated in Fig. 3A. In this case the contrast is also quite different from that observed for single n-alkane molecules and the characteristic bumps are not clearly resolved within the hydrophobic region. This may indicate that the contributions from individual hydrocarbon chains are either averaged or dominated by single chain with tilted orientation. It is noteworthy that the hydrophobic part of the molecule is represented by single bright rod and the spacing between them measured along ½121 direction is 0.5 nm, which is also characteristic for n-alkanes and their derivatives adsorbed on Au(111) [37,42]. This may suggest that only one alkyl chain is in direct contact with gold surface, while the other is located slightly above the surface plane. Such configuration is represented by two molecules superimposed on STM image in Fig. 4A with fatty acid residue lying flat on gold and the sphinganine backbone tilted and located slightly above the surface. In such case the latter would have major contribution to the bright contrast produced in topography image within the hydrophobic region. Interestingly, the STM image in Fig. 4A also shows the location of the polar heads in a trough separating two rows of flat lying molecules. Phosphate groups from opposing rows are arranged in a zipperlike pattern. It is also noteworthy that the individual polar heads are not coaxial with respect to the long molecular axis of the hydrophobic part of lipids represented by bright rods and they are visibly shifted by approximately 0.25 nm. Interestingly, polar heads produce slightly darker contrast in topography image comparing with hydrocarbon part of the molecules. This indicates that they are located closer to the surface than topmost part of the hydrophobic region. This would support earlier assumption that hydrocarbon chains are located in two different planes with respect to the electrode surface. Moreover, the molecules seem to be bridged along ½121 direction within the region between polar heads and the hydrophobic tails. This may indicate possible hydrogen bonding between amide groups and/or hydroxyl groups from adjacent lipids.
Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046
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Fig. 4. (A) High resolution STM image recorded on Au(111) electrode modified with N-palmitoyl-D-erythro-dihydroceramide-1-phosphate adlayer. Individual molecules are clearly resolved and the distance between them along ½121 direction is 0.5 nm. White double arrow and black arrow mark hydrophobic part of the lipid molecule and polar head respectively. Two cartoon molecules are overlaid on STM image to demonstrate their orientation with respect to the substrate. (B) A model representing the arrangement of lipid molecules on Au(111) surface, which explains observed STM contrast. It can be superimposed on image in panel (A) by vertical translation (C) Side view along ½101 direction, which demonstrates that the C-C-C plane of the fatty acid chain is parallel to the electrode surface, while the skeleton of sphinganine backbone is tilted and located above the surface plane.
Based on the careful analysis of the AFM and STM data, we have proposed the model which explains the arrangement of the molecules within the adlayer and their detailed orientation with respect to the substrate plane. Fig. 4B presents the top-view of the model which reproduces STM contrast. The molecules are arranged in rows running along ½121 direction and adopt antiparallel orientation. Such pattern is quite typical for long chain amphiphilic molecules adsorbed on gold or highly oriented pyrolytic graphite since it allows optimization of the interactions within the adlayer [37,38]. Importantly, we have assumed that only one hydrocarbon chain is involved in direct interaction with the substrate, which is fatty acid residue. It adopts flat-lying orientation and its C-C-C plane is parallel to the Au(111) surface. Interaction of this particular hydrocarbon chain with gold enables closer approach of the phosphate groups to the electrode surface as it was observed on STM images. The orientation of the second alkyl chain from sphinganine backbone is more vertical. This is related to the fact that the C-C-C planes of the hydrocarbon chains within the lipid molecule are rotated with respect to each other. As a consequence the sphinganine backbone is located slightly above the surface and partially overlaps fatty acid chain from adjacent lipid. This is clearly visible in Fig. 4C where the side-view along the ½101 crystallographic direction is shown. Such arrangement allows optimal packing of the alkyl chains with a periodicity of 0.5 nm, which is the same as the distance determined from STM images. It also shows that the major contribution to the topographical contrast comes from sphinganine backbone. The latter explains misalignment between phosphate group and the long axis of bright rods reproducing hydrophobic region of lipid. Moreover, the estimated thickness of the film with this particular orientation of the molecules is around 0.72 nm, which is in a very good agreement with the results obtained from AFM measurements.
4. Conclusions Self-assembly of lipids at gold electrode-electrolyte interface leads to formation of molecular film which at certain potential range shows highly ordered stripe-like pattern. We have employed EC-AFM and EC-STM methods to examine its structure in detail and N-palmitoyl-D-erythro-dihydroceramide-1-phosphate was chosen as model lipid. We have found that upon adsorption on Au(111) surface the molecules are packed side-by-side and organize into lamellar structures running along ½121 direction. In order to optimize the interactions within the film, they adopt antiparallel orientation in neighboring rows. Importantly, long molecular axes of lipids are aligned with ½101 crystallographic direction of gold. Such arrangement is quite often observed for the monolayers of nalkanes and their derivatives on Au(111) surface [36e38,42]. Therefore, it is reasonable to conclude that van der Waals interactions between the alkyl chains and the gold substrate are the main driving forces for the formation of an ordered lipid adlayer. In order to describe exact orientation of the molecules with respect to the surface, we have provided a model, which assumes that only one hydrocarbon chain being part of fatty acid residue is involved in direct interaction with gold substrate. It is adsorbed with C-C-C plane parallel to Au (111) surface, while the sphinganine backbone is located slightly above the surface and its carbon skeleton plane is more vertical. The model nicely reproduces the STM contrast observed for stripe-like domains of two-chain lipids adsorbed on Au(111). Importantly, it can be applied to previously reported films containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine as well [29e31] and provides complementary description of recently reported mechanism of lipid adsorption and bilayer formation on gold surface. Nevertheless, our findings described in this paper may also be important for some applications related to templated deposition of nanostructures or nucleation in nanoconfinement
Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046
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[45e47]. We have demonstrated that within the certain range of potentials, phosphorylated dihydroceramide forms stable and ordered monolayer on gold which does not transform neither into hemimicellar film or the bilayer. In such case the surface pattern of the highly ordered monolayer can potentially control morphology, crystallographic orientation and spatial distribution of deposited nanostructures. The advantage of such approach is that the pattern transfer from the template to nanocrystal arrays can be completed with exceptionally high resolution which is impossible to achieve using lithography. Moreover, stripe-like pattern formed by phosphorylated dihydroceramide adsorbed on gold surface exposes cross section of the lipid bilayer lamellae. This offers unique platform for probing molecular interactions between lipids and biologically relevant species or drugs. Such approach was utilized by Mao and coworkers who used highly oriented pyrolytic graphite modified with phosphatidylethanolamine adlayer to probe the interactions between the lipid and aspirin [48]. Interestingly, the latter formed rod-like aggregates in register with underlying pattern, which simply shows that the template formed by lipid molecules can also be used as a template for growth of organic nanostructures. Another advantage of the system presented in this work is related to the fact that it offers biocompatible platform with possibility to work under electrochemical control of the substrate. Thus templated self-assembly can be performed in the presence of the external electric field. Acknowledgment This work was financially supported by Polish National Science Centre, Project No. 2012/05/B/ST4/01243. The study was carried out at the Biological and Chemical Research Centre, University of Warsaw, established within the project co-financed by European Union from the European Regional Development Fund under the Operational Program Innovative Economy, 2007e2013. References
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Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface Jan Pawłowski, Joanna Juhaniewicz, Sławomir Se˛ k* Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Zwirki i Wigury 101, 02-089, Warsaw, Poland
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
STM and AFM methods were used to examine adsorption of model lipid on Au(111). Self-assembly of model lipid leads to formation of highly organized molecular film. The model is proposed which reproduces the STM contrast.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 March 2016 Received in revised form 31 August 2016 Accepted 29 October 2016 Available online xxx
Although the adsorption of lipids on reconstructed Au(111) surface and formation of highly ordered stripe-like domains are well-known phenomena, the exact orientation of the molecules with respect to the substrate remains unclear. Therefore, in this study we have focused on the structure and arrangement of lipid molecules forming highly ordered stripe-like domains at gold electrode-electrolyte interface. N-palmitoyl-D-erythro-dihydroceramide-1-phosphate was selected as model compound since its ability to transform into hemimicellar structure is limited. This way it was possible to get very stable lipid film with characteristic stripe-like pattern. Application of complementary techniques such as atomic force microscopy and scanning tunneling microscopy enabled detailed characteristics of lipid adlayer adsorbed on Au(111) electrode. Based on careful analysis of the experimental results, we have proposed a model which describes the arrangement of the molecules within the film. In general, it assumes flat-lying orientation of the lipids but only one hydrocarbon chain of phosphorylated dihydroceramide is involved in direct interaction with gold. © 2016 Elsevier B.V. All rights reserved.
Keywords: Lipids Adsorption Gold electrode Atomic force microscopy Scanning tunneling microscopy
1. Introduction Interfacial self-assembly is one of the most commonly used
* Corresponding author. E-mail address: [email protected] (S. Se˛ k).
methods to obtain functionalized surfaces with purposely tailored properties [1]. The latter depend on chemical nature and the arrangement of the adsorbate molecules on the substrate. Molecular films immobilized at solid surfaces are particularly attractive and they play a crucial role in many domains of fundamental research as well as technological applications. These include surface modification for sensors [2,3], biofuel cells [4,5], corrosion
http://dx.doi.org/10.1016/j.matchemphys.2016.10.046 0254-0584/© 2016 Elsevier B.V. All rights reserved.
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inhibition [6], heterogeneous catalysis [7], and investigation of mediated electron transfer [8,9]. Moreover, molecular layers adsorbed on a surface with well-defined crystallographic structure can act as a template for the construction of molecular electronics devices [10] and nanoscale level engineering [11]. Among many possible surface modifications, lipids self-assembled on metal substrates have emerged as an attractive platform to investigate membrane related processes [12e14] and design biosensing devices [15,16] or biomimetic interfaces [17,18]. Lipid monolayers and bilayers immobilized on gold surface already proved to be suitable systems for the studies of protein binding [19,20] interactions with antimicrobial peptides [21e25], as well as characterization of redox-active enzymes [26] and modelling of transmembrane ion transport [27,28]. Since the suitably tailored lipid films supported on metal substrates are increasingly used in a variety of fields, full control over their structure and properties becomes an important issue. Therefore, it is crucial to understand the fundamental mechanisms of lipid adsorption and film formation at solid-liquid interfaces. Recently, we have demonstrated that formation of lipid bilayer on Au(111) electrode by spreading of small unilamellar vesicles involves several steps [29]. These include deposition of the vesicles on gold surface followed by release of lipid molecules which adsorb with flat-lying orientation and form stripe-like domains. The latter serves as a template for development of hemimicellar film which facilitates further adsorption and rupture of SUVs and the bilayer is spread over hemimicellar film. Finally, a single planar bilayer is formed due to the fusion between coupled layers. In this paper, we have focused on the initial step of this mechanism that is adsorption of lipid molecules with long molecular axis parallel with respect to the Au(111) surface. So far, this phenomenon was observed for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) molecules either alone or in the presence of cholesterol and it was ascribed to preferential interactions between hydrophobic acyl chains and gold surface [30,31]. However, the exact orientation of the molecules with respect to the substrate plane and the extent of the molecule-substrate contact are still unclear. Moreover, the detailed examination of the stripe-like domains formed by DMPC is difficult due to their limited stability. As it was already mentioned, prolonged exposure of modified Au(111) electrode to lipid vesicles results in transition from stripe-like structure to hemimicellar film and then to planar bilayer. In order to suppress this transformation, we have selected phosphorylated dihydroceramide, which is Npalmitoyl-D-erythro-dihydroceramide-1-phosphate, as a model lipid. The size of the phosphate polar headgroup in this compound is smaller comparing with the diameter of the hydrophobic domain. Molecular shape affects supramolecular structural organization and therefore, the ability of the lipid to form hemimicellar structures will be limited. This assumption is reasonable since it was reported in the literature that closely related compounds such as phosphorylated ceramides, when added to lipid mixtures cause a reduction of the lamellar-to-inverted hexagonal phase transition temperature and have ability to induce negative membrane curvature [32]. Thus, by using N-palmitoyl-D-erythro-dihydroceramide-1-phosphate as a model lipid, we obtained stable stripe-like domains on reconstructed Au(111) surface. This allowed careful examination of their structure with scanning probe microscopy and based on it we have proposed detailed description of moleculesubstrate contact.
which was purchased from Avanti Polar Lipids Inc. In all experiments we have used distilled water passed through a Milli-Q water purification system and its final resistivity was 18.2 MU cm. Surface pressure - molecular area isotherms for N-palmitoyl-Derythro-dihydroceramide-1-phosphate monolayer at the air-water interface were recorded using a KSV LB trough 5000 (KSV Ltd., Finland) equipped with two movable Teflon barriers and a paper plate used as a surface pressure sensor. Trough and barriers were washed thoroughly before each experiment using the mixture of chloroform and methanol and finally rinsed with Milli-Q water. As a subphase 0.01 M phosphate buffer saline (PBS) aqueous solution was used. N-palmitoyl-D-erythro-dihydroceramide-1-phosphate was dissolved in a mixture of CHCl3:CH3OH:0.5N HCl (20:9:1). The resulting clear solution was spread on buffer surface and then it was left to evaporate the solvent. The compression of the monolayers was performed at the barriers speed of 10 mm/min at a constant temperature of 21 ± 1 C. In order to prepare the suspension of N-palmitoyl-D-erythrodihydroceramide-1-phosphate in 0.05 M KClO4 aqueous solution we have followed the same procedure as for preparation of small unilamellar vesicles [33]. However, the resulting product was a fine suspension of lipid aggregates. The protocol involved preparation of stock solution by dissolving 1.0 mg of N-palmitoyl-D-erythrodihydroceramide-1-phosphate in 5 ml of hexane/ethanol 2:1 mixture. An aliquot of the solution (0.2 ml) was placed in a test tube and the solvent was evaporated by vortexing under argon stream. Subsequently, the test tube with dried lipid cake was placed for approximately 3 h in vacuum desiccator to remove the solvent. Finally, 1.5 ml of 0.05 M KClO4 solution was added to the lipid cake and the sample was sonicated for at least 1 h. Portion of the resulting suspension was added to electrochemical cell of the microscope to give approximate lipid concentration of 4.0 106 M. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) images were obtained with 5500AFM (Keysight Technology) and Dimension Icon (Bruker) instruments respectively. The experiments were performed at 21 C. All images were recorded in 0.05 M KClO4 under electrochemical control with single crystal Au(111) (MaTeck) as a working electrode, miniaturized Ag/ AgCl (sat. KCl) as a reference and platinum wire as a counter electrode. The components of the electrochemical cell as well as metallic electrodes were cleaned in piranha solution (concentrated H2SO4/30% H2O2 3:1, v/v) for at least 2 h and then rinsed with copious amounts of Milli-Q ultrapure water. (CAUTION: piranha solution reacts violently with organic materials and should be handled with extreme care.) Single crystal Au(111) electrode was carefully flame annealed and then placed in electrochemical cell of the microscope. In order to facilitate the surface reconstruction, the electrode was held for approximately 30 min at the potential of 0.3 V directly before each experiment. For STM imaging, we have used electrochemically etched tungsten tips coated with polyethylene to minimize leakage currents. The AFM images were taken in PeakForce Tapping mode using qp-BioAC cantilevers (Nanosensors, CB2: nominal spring constant 0.06e0.18 N/m). Both STM and AFM imaging was performed on four independently prepared samples. N-palmitoyl-D-erythro-dihydroceramide-1-phosphate molecule was modelled using Spartan ’14 Version 1.1.4 (Wavefunction, Inc.). Molecular modeling was performed with the ab initio Hartree-Fock method using the 3-21G basis set available in the software package.
2. Experimental 3. Results and discussion The chemicals used in this work were purchased from SigmaAldrich and Avantor Performance Materials SA. The only exception was N-palmitoyl-D-erythro-dihydroceramide-1-phosphate,
The structure of N-palmitoyl-D-erythro-dihydroceramide-1phosphate molecule is illustrated in Scheme 1. It consists of
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Scheme 1. Structure of N-palmitoyl-D-erythro-dihydroceramide-1-phosphate.
sphinganine backbone and fatty acid residue in hydrophobic part and phosphate headgroup in a polar head region. Total length of the molecule is ~2.6 nm. Cross sectional area of the hydrophobic part of the lipid was estimated to be ~40 Å2, while the corresponding area of the phosphate group is ~26 Å2 assuming its van der Waals radius of 2.9 Å. This clearly shows the disproportion in size when comparing hydrophobic and hydrophilic parts of the lipid molecule. Based on simple geometric considerations, one can conclude that ability of phosphorylated dihydroceramide to form either micelles or small vesicles will be rather limited. This can be confirmed by estimation of packing parameter defined as p ¼ VC/(Ah lC), where VC denotes volume of the hydrophobic tails, Ah is the projection area of the headgroup and lC is the length of lipid chain. Resulting value determined for phosphorylated dihydroceramide is well above the unity, which means that the molecules will tend to form aggregates with negatively curved interfaces [34]. Disproportion in size between phosphate group and the hydrophobic part of the phosphorylated dihydroceramide can be verified using Langmuir technique where the single molecular film is spread at the air-water interface and the changes in surface pressure are monitored as a function of the area available for
3
molecules. An exemplary isotherm recorded for N-palmitoyl-Derythro-dihydroceramide-1-phosphate film spread at the surface of 0.01 M PBS aqueous solution is presented in Fig. 1. It was found that the area available for single molecule within the organized monolayer of phosphorylated ceramide is 43.5 ± 0.5 Å2. This result suggests that the packing of acyl chains within the film is practically unaffected by the presence of phosphate group, since it roughly corresponds to the doubled area usually observed for organized monolayers of unionized saturated fatty acids and fatty amines of comparable length. For example, the area per molecule for monolayers of hexadecanoic acid and hexadecanamine is ~21 Å2 [35]. As can be seen in Fig. 1, the monolayer of phosphorylated dihydroceramide becomes unstable at surface pressure of ~46.0 mN/m where the plateau is observed representing the transition of the film into multilayer state. Interestingly, relatively dense packing of the molecules within the monolayer can also be confirmed by analysis of compression modulus (see inset in Fig. 1). Its maximum value of ~250 mN/m is indicative for existence of solid-like film. Thus the structural analysis as well as surface pressure measurements strongly suggest that N-palmitoyl-D-erythro-dihydroceramide-1-phosphate will have very limited tendency to form micellar aggregates with positive curvature. Therefore it could be expected that upon deposition on Au(111) transformation into hemimicellar structure will be suppressed enabling more detailed examination of lamellar phase created at the initial stages of lipid adsorption. Based on such assumption, we have examined the adsorption behavior of N-palmitoyl-D-erythro-dihydroceramide-1phosphate on Au(111) electrode. Fig. 2A shows an image recorded using electrochemical atomic force microscopy (EC-AFM) for bare gold electrode at the potential of þ0.25 V vs. Ag/AgCl(sat. KCl) in the absence of lipid molecules. The surface is flat with a step edge visible at the left side of the scanned area. The average surface roughness (Sq) determined from 1.5 1.5 mm2 scans taken from several spots on bare Au(111) electrode was 0.084 nm, which confirms good quality of the substrate. Fig. 2B presents an EC-AFM image of the same electrode recorded after injection of phosphorylated dihydroceramide suspension
Fig. 1. Surface pressure vs. area per molecule isotherm recorded for N-palmitoyl-D-erythro-dihydroceramide-1-phosphate film spread at the surface of 0.01 M PBS. The isotherm was recorded at 21 ± 1 C. Inset shows the changes in compression modulus as a function of surface pressure.
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Fig. 2. EC-AFM topography images recorded on bare Au(111) electrode (A) and after injection of the suspension of N-palmitoyl-D-erythro-dihydroceramide-1-phosphate and formation of adsorbed layer (B). The images were recorded in an aqueous solution of 0.05 M KClO4 at the potential of þ0.25 V vs. Ag/AgCl(sat. KCl). The distribution of the thickness determined for the lipid film from cross sectional analysis is shown in panel (C). Image of the N-palmitoyl-D-erythro-dihydroceramide-1-phosphate adlayer recorded after potential step to þ0.35 V vs. Ag/AgCl(sat. KCl) is shown in panel (D).
into the electrochemical cell of the microscope. The total concentration of the lipid in electrochemical cell was 4.0 106 M. The potential of the electrode was kept constant and it was adjusted to þ0.25 V vs. Ag/AgCl(sat. KCl). The images were taken directly after introduction of the lipid suspension into the electrochemical cell. Under such conditions lipid molecules tend to form an adlayer on Au(111) surface which is clearly resolved in AFM image. Importantly, the lipid film remains stable for several hours and does not undergo transition neither to hemimicellar state nor to the bilayer. This confirms that N-palmitoyl-D-erythro-dihydroceramide-1-phosphate has limited ability to form micelles. Interestingly, the adsorption of the phosphorylated dihydroceramide on gold surface is not random. As can be seen in Fig. 2B, numerous domain boundaries and unoccupied spaces appear as dark straight lines aligned with three particular directions rotated by 120 . Such symmetry is characteristic for Au(111) structure. Therefore, we conclude that the molecules within the adlayer follow crystallographic structure of the electrode surface. In order to assess the orientation of the adsorbed molecules with respect to the substrate, we have carried out cross sectional analysis of AFM images and determined the thickness of the lipid film. The spread of the determined values is shown in Fig. 2C and it represents data obtained from fifty individual profiles taken within different regions on four independently prepared samples. The mean thickness was found to be 0.8 ± 0.1 nm suggesting that phosphorylated dihydroceramide forms a monolayer and its molecules adopt orientation with their long molecular axis parallel to the substrate plane. Such behavior seems to be common for lipids as well as n-alkanes
and their derivatives [30,31,36]. However, the thickness of the lipid film reported here is higher than expected if both hydrophobic chains were in contact with the electrode surface. The diameter of the alkyl chain is about 0.5 nm, thus the thickness of the film would be close to this value. The discrepancy strongly suggests that the adsorption of double-chain lipids on Au(111) surface involves the contact only with one alkyl chain, while the other remains at certain distance from the surface contributing to the increased thickness of the film. Interestingly, we have found that the ordered adlayer of phosphorylated dihydroceramide is stable within the certain potential range. More specifically, at the potentials more negative than 0.40 V the adlayer becomes desorbed and bare gold surface is exposed, while at the potentials more positive than þ0.30 V the adlayer transforms into irregular structure as demonstrated in Fig. 2D. Similar behavior was reported for other amphiphiles adsorbed on Au(111) surface and it was ascribed to the changes in charge density at the electrode surface [37,38]. Nevertheless, the transition from highly ordered to random orientation often coincides with potential at which gold reconstruction is lifted. This suggests that interatomic distances at reconstructed surface of Au(111) are preferable for optimal packing of alkyl chains. In order to obtain more detailed information about ordered adlayer, molecular packing and orientation of lipid molecules with respect to the substrate were examined by high resolution imaging with electrochemical scanning tunneling microscopy (ECSTM). Fig. 3A shows an image of bare Au(111) electrode recorded at 0.3 V vs. Ag/AgCl(sat. KCl) in the absence of the lipid. Well-
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Fig. 3. (A) EC-STM topography image recorded for reconstructed Au(111) surface at the potential of 0.30 V vs. Ag/AgCl(sat. KCl) in the absence of lipid molecules. (B) EC-STM topography image of the same Au(111) electrode taken at þ0.25 V vs. Ag/AgCl(sat. KCl) after injection of the suspension of phosphorylated dihydroceramide into the electrochemical cell of the microscope. It reveals stripe-like domains formed by adsorbed lipid. (C) High resolution topography image (10 10 nm2) of the stripe-like domain illustrating the arrangement of the molecules with respect to the crystallographic directions of the substrate. (D) High resolution STM current image (8.5 8.5 nm2) of lipid molecules adsorbed on Au(111) surface demonstrating the differences in contrast over individual molecule (indicated by black and white arrows).
defined crystallographic structure of the electrode surface was confirmed by the presence of reconstruction lines. The latter are arranged in pairs which form reconstruction rows. The distance between paired lines is 2.5 nm, while the separation between the rows is 6.3 nm [39]. Since these values are well-known, the reconstruction lines can be used for precise calibration of the measured distances. Moreover, assuming that the lines are running h i parallel to 112 direction, it is possible to determine crystallographic directions on Au(111) electrode surface. Fig. 3B presents an image of Au(111) electrode taken at þ0.25 V vs. Ag/AgCl(sat. KCl) after injection of the suspension of phosphorylated dihydroceramide into the electrochemical cell of the microscope. The lipid molecules are organized into stripe-like structures, which form domains rotated by 120 . This confirms that the orientation of the molecules within the adlayer is affected by crystallographic structure of the substrate. Careful examination of high resolution image shown in Fig. 3C reveals that stripes are aligned with ½121 direction and consist of two rows of single lipid molecules oriented perpendicular to the stripe direction. The width of the individual stripe is ~5.0 nm, which roughly corresponds to the doubled length of phosphorylated dihydroceramide molecule. This indicates that lipids adopt antiparallel orientation with head-to-head and tail-totail packing order. Further analysis of STM image shows that long axis of lipid molecules is aligned with ½101 crystallographic direction. Such orientation is known to be favorable for n-alkanes and their derivatives adsorbed on Au(111) surface [37,40,41]. Therefore, it is reasonable to conclude that hydrophobic part of the lipid molecule is directly involved in the interactions with the substrate. At this point, it is important to consider exact orientation of the hydrocarbon skeleton with respect to the Au(111) substrate. For
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simple n-alkanes, the analysis of the potential energy difference between a molecule in an adlayer and a free molecule revealed that carbon skeleton is predominantly oriented parallel to the plane of gold surface [42]. This was also confirmed by infrared reflection absorption spectroscopic study, which proved that n-alkanes adopt flat-on orientation on gold at room temperature [43]. Therefore, it is reasonable to assume that adsorption of lipids on Au(111) involve parallel orientation of the hydrocarbon chain with respect to the surface of the substrate. However, phosphorylated dihydroceramide possesses two alkyl chains being parts of fatty acid residue and sphinganine backbone. Moreover, the C-C-C planes of these hydrocarbon chains are rotated with respect to each other. This implies that the adsorption of both chains would result in unfavorable stress and strain within the molecule. For this reason, the molecule-substrate interaction through single chain seems to be more likely. This would also explain the contrast observed in STM current images. As demonstrated in Fig. 3D, current flow is not uniform throughout the individual molecule. It consists of bright linear feature indicated by black arrow with closely located and less intense spots pointed by white arrow. Similar spots were observed in numerous STM images of n-alkanes and their derivatives on Au(111) and the interpretation of such contrast assumed parallel orientation of carbon skeleton plane with respect to the substrate [44]. Therefore, we have concluded that the presence of the spots in Fig. 3D results from flat-on orientation of one of the hydrocarbon chains in lipid molecule. Consequently, the linear feature was ascribed to the second hydrocarbon chain of lipid with C-C-C plane tilted with respect to the substrate. Thus the STM current contrast reflects varying tunneling conditions over differently oriented carbon backbones. Interestingly, the topography does not show such details as demonstrated in Fig. 3A. In this case the contrast is also quite different from that observed for single n-alkane molecules and the characteristic bumps are not clearly resolved within the hydrophobic region. This may indicate that the contributions from individual hydrocarbon chains are either averaged or dominated by single chain with tilted orientation. It is noteworthy that the hydrophobic part of the molecule is represented by single bright rod and the spacing between them measured along ½121 direction is 0.5 nm, which is also characteristic for n-alkanes and their derivatives adsorbed on Au(111) [37,42]. This may suggest that only one alkyl chain is in direct contact with gold surface, while the other is located slightly above the surface plane. Such configuration is represented by two molecules superimposed on STM image in Fig. 4A with fatty acid residue lying flat on gold and the sphinganine backbone tilted and located slightly above the surface. In such case the latter would have major contribution to the bright contrast produced in topography image within the hydrophobic region. Interestingly, the STM image in Fig. 4A also shows the location of the polar heads in a trough separating two rows of flat lying molecules. Phosphate groups from opposing rows are arranged in a zipperlike pattern. It is also noteworthy that the individual polar heads are not coaxial with respect to the long molecular axis of the hydrophobic part of lipids represented by bright rods and they are visibly shifted by approximately 0.25 nm. Interestingly, polar heads produce slightly darker contrast in topography image comparing with hydrocarbon part of the molecules. This indicates that they are located closer to the surface than topmost part of the hydrophobic region. This would support earlier assumption that hydrocarbon chains are located in two different planes with respect to the electrode surface. Moreover, the molecules seem to be bridged along ½121 direction within the region between polar heads and the hydrophobic tails. This may indicate possible hydrogen bonding between amide groups and/or hydroxyl groups from adjacent lipids.
Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046
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Fig. 4. (A) High resolution STM image recorded on Au(111) electrode modified with N-palmitoyl-D-erythro-dihydroceramide-1-phosphate adlayer. Individual molecules are clearly resolved and the distance between them along ½121 direction is 0.5 nm. White double arrow and black arrow mark hydrophobic part of the lipid molecule and polar head respectively. Two cartoon molecules are overlaid on STM image to demonstrate their orientation with respect to the substrate. (B) A model representing the arrangement of lipid molecules on Au(111) surface, which explains observed STM contrast. It can be superimposed on image in panel (A) by vertical translation (C) Side view along ½101 direction, which demonstrates that the C-C-C plane of the fatty acid chain is parallel to the electrode surface, while the skeleton of sphinganine backbone is tilted and located above the surface plane.
Based on the careful analysis of the AFM and STM data, we have proposed the model which explains the arrangement of the molecules within the adlayer and their detailed orientation with respect to the substrate plane. Fig. 4B presents the top-view of the model which reproduces STM contrast. The molecules are arranged in rows running along ½121 direction and adopt antiparallel orientation. Such pattern is quite typical for long chain amphiphilic molecules adsorbed on gold or highly oriented pyrolytic graphite since it allows optimization of the interactions within the adlayer [37,38]. Importantly, we have assumed that only one hydrocarbon chain is involved in direct interaction with the substrate, which is fatty acid residue. It adopts flat-lying orientation and its C-C-C plane is parallel to the Au(111) surface. Interaction of this particular hydrocarbon chain with gold enables closer approach of the phosphate groups to the electrode surface as it was observed on STM images. The orientation of the second alkyl chain from sphinganine backbone is more vertical. This is related to the fact that the C-C-C planes of the hydrocarbon chains within the lipid molecule are rotated with respect to each other. As a consequence the sphinganine backbone is located slightly above the surface and partially overlaps fatty acid chain from adjacent lipid. This is clearly visible in Fig. 4C where the side-view along the ½101 crystallographic direction is shown. Such arrangement allows optimal packing of the alkyl chains with a periodicity of 0.5 nm, which is the same as the distance determined from STM images. It also shows that the major contribution to the topographical contrast comes from sphinganine backbone. The latter explains misalignment between phosphate group and the long axis of bright rods reproducing hydrophobic region of lipid. Moreover, the estimated thickness of the film with this particular orientation of the molecules is around 0.72 nm, which is in a very good agreement with the results obtained from AFM measurements.
4. Conclusions Self-assembly of lipids at gold electrode-electrolyte interface leads to formation of molecular film which at certain potential range shows highly ordered stripe-like pattern. We have employed EC-AFM and EC-STM methods to examine its structure in detail and N-palmitoyl-D-erythro-dihydroceramide-1-phosphate was chosen as model lipid. We have found that upon adsorption on Au(111) surface the molecules are packed side-by-side and organize into lamellar structures running along ½121 direction. In order to optimize the interactions within the film, they adopt antiparallel orientation in neighboring rows. Importantly, long molecular axes of lipids are aligned with ½101 crystallographic direction of gold. Such arrangement is quite often observed for the monolayers of nalkanes and their derivatives on Au(111) surface [36e38,42]. Therefore, it is reasonable to conclude that van der Waals interactions between the alkyl chains and the gold substrate are the main driving forces for the formation of an ordered lipid adlayer. In order to describe exact orientation of the molecules with respect to the surface, we have provided a model, which assumes that only one hydrocarbon chain being part of fatty acid residue is involved in direct interaction with gold substrate. It is adsorbed with C-C-C plane parallel to Au (111) surface, while the sphinganine backbone is located slightly above the surface and its carbon skeleton plane is more vertical. The model nicely reproduces the STM contrast observed for stripe-like domains of two-chain lipids adsorbed on Au(111). Importantly, it can be applied to previously reported films containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine as well [29e31] and provides complementary description of recently reported mechanism of lipid adsorption and bilayer formation on gold surface. Nevertheless, our findings described in this paper may also be important for some applications related to templated deposition of nanostructures or nucleation in nanoconfinement
Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046
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[45e47]. We have demonstrated that within the certain range of potentials, phosphorylated dihydroceramide forms stable and ordered monolayer on gold which does not transform neither into hemimicellar film or the bilayer. In such case the surface pattern of the highly ordered monolayer can potentially control morphology, crystallographic orientation and spatial distribution of deposited nanostructures. The advantage of such approach is that the pattern transfer from the template to nanocrystal arrays can be completed with exceptionally high resolution which is impossible to achieve using lithography. Moreover, stripe-like pattern formed by phosphorylated dihydroceramide adsorbed on gold surface exposes cross section of the lipid bilayer lamellae. This offers unique platform for probing molecular interactions between lipids and biologically relevant species or drugs. Such approach was utilized by Mao and coworkers who used highly oriented pyrolytic graphite modified with phosphatidylethanolamine adlayer to probe the interactions between the lipid and aspirin [48]. Interestingly, the latter formed rod-like aggregates in register with underlying pattern, which simply shows that the template formed by lipid molecules can also be used as a template for growth of organic nanostructures. Another advantage of the system presented in this work is related to the fact that it offers biocompatible platform with possibility to work under electrochemical control of the substrate. Thus templated self-assembly can be performed in the presence of the external electric field. Acknowledgment This work was financially supported by Polish National Science Centre, Project No. 2012/05/B/ST4/01243. The study was carried out at the Biological and Chemical Research Centre, University of Warsaw, established within the project co-financed by European Union from the European Regional Development Fund under the Operational Program Innovative Economy, 2007e2013. References
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Please cite this article in press as: J. Pawłowski, et al., Self-assembly of phosphorylated dihydroceramide at Au(111) electrode surface, Materials Chemistry and Physics (2016), http://dx.doi.org/10.1016/j.matchemphys.2016.10.046