Structure and Function of the Membrane Anchoring Self-Assembled

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Structure and Function of the Membrane Anchoring Self-Assembled Monolayers Bozena Rakovska,† Tadas Ragaliauskas,† Mindaugas Mickevicius,† Marija Jankunec,† Gediminas Niaura,† David J. Vanderah,‡,§ and Gintaras Valincius*,† †

Institute of Biochemistry, Vilnius University, Mokslininku 12, Vilnius 08662, Lithuania Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States § Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland 20850, United States ‡

S Supporting Information *

ABSTRACT: Structure of the self-assembled monolayers (SAMs) used to anchor phospholipid bilayers to surfaces affects the functional properties of the tethered bilayer membranes (tBLMs). SAMs of the same surface composition differing in the lateral distribution of the anchor molecule give rise to tBLMs of profoundly different defectiveness with residual conductance spanning 3 orders of magnitude. SAMs composed of anchors containing saturated alkyl chains, upon exposure to water (72 h), reconstruct to tightly packed clusters as deduced from reflection absorption infrared spectroscopy data and directly visualized by atomic force microscopy. The rearrangement into clusters results in an inability to establish highly insulating tBLMs on the same anchor layer. Unexpectedly, we also found that nanometer scale smooth gold film surfaces, populated predominantly with (111) facets, exhibit poor performance from the standpoint of the defectiveness of the anchored phospholipid bilayers, while corrugated (110) dominant surfaces produced SAMs with superior tethering quality. Although the detailed mechanism of cluster formation remains to be clarified, it appears that smooth surfaces favor lateral translocation of the molecular anchors, resulting in changes in functional properties of the SAMs. This work unequivocally establishes that conditions that favor cluster formation of the anchoring molecules in tBLM formation must be identified and avoided for the functional use of tBLMs in biomedical and diagnostic applications.

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INTRODUCTION The relationship between structure and function in nature is well-established. Structures of proteins, peptides, and nucleic acids are critically important for their biological function. Small structural changes in supramolecular complexes frequently lead to full inactivation of enzymes as well as are a trigger for life processes in organisms such as cell division, motility, or induce pathogenesis. The connection of structure to function may apply also to artificial self-assembled constructs such as tethered bilayer lipid membranes widely used for protein−membrane interaction studies (tBLMs).1−5 While the structural arrangement of phospholipids and sterols in tethered membranes affects their function and ability to interact with proteins,6 the role of structural arrangement of the tether remains obscure. Recent studies of polymer cushioned bilayers,7 as well as the coarse grain modeling,8 suggest that the surface state of tethers may play an important role in the properties of the attached bilayer. Our study initiates efforts to establish the relationships between the structure-function of the tBLMs and their enabling component, an initially formed anchoring self-assembled monolayer (SAM). Structurally tBLMs are a bilayer milieu anchored to a supporting surface via special lipid-like anchor molecules, which insert into the proximal leaflet of the bilayer via hydrophobic interactions. The molecular anchors keep the © 2014 American Chemical Society

membrane, completed with untethered phospholipids, at a certain distance from the solid substrate1,5 and support an exchangeable water (ionic) reservoir between the solid support and the bilayer.2,3,9 In contrast to freely suspended bilayers, tBLMs exhibit exceptional stability and excellent electric insulating properties and can be engineered into microchips,10 thus allowing usage of such constructs for biosensor engineering.11,12 The properties of tBLMs are dependent on their formation, a two-step process, initiating with the deposition of an anchoring self-assembled monolayer (SAM), a mixed SAM composed of a lipidic anchor and a small diluting molecule, followed by creation of the bilayer, completed with untethered lipids in both the inner and the outer leaflets. In the first step, typically thiol or disulfide chemistry is utilized to graft the anchoring synthetic lipid-like13 or cholesteryl molecules14 to a metal surface, in most cases, gold. The second step involves either of two well-established procedures: (a) the solvent exchange procedure1,5 or (b) the fusion of small unilamellar vesicles to the anchoring SAM.13,14 Various anchors1,4,13−16 are suggested for tethering the lipid bilayer; however, systematic studies have Received: September 17, 2014 Revised: November 9, 2014 Published: December 19, 2014 846

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Figure 1. EIS Bode spectra of DOPC tBLMs formed in the course of eight consecutive cycles of formation and removal on the same 30% WC14/ 70% βME anchor SAMs. Numbers indicate the sequence number of the tBLM-assembling experiment. (A) Impedance magnitude (module) versus frequency; (B) impedance phase (argument) versus frequency plots.

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not been undertaken to establish the relationship between the properties of the anchoring SAMs and the quality of the tBLMs. The structure of the SAM, the nature, composition, and distribution of the lipidic anchor molecule on the substrate surface, exerts dramatic effects on the properties of the tBLM. For example, it was recently demonstrated that usage of unsaturated molecular anchors exhibits significant advantages over saturated anchors, and produces more electrically insulating tBLMs.16 Electric insulation, that is, a tBLM with few or no defects, is an essential property, especially when the sensing element is a reconstituted pore-forming protein because it determines the detection limit12,17 and applicability for single molecule detection. The presence and nature of defects in tBLMs is exquisitely revealed with electrochemical impedance spectroscopy (EIS) or the atomic force microscopy (AFM)13,15 showing structural differences in tBLMs determined to be essentially 100% complete by surface plasmon resonance (SPR), quartz crystal microbalance (QCM), or neutron reflectometry (NR).4,18 Recently, Jeuken et al. demonstrated that the lateral phase separation may affect defectiveness of tBLMs.19 It was concluded that the presence of almost pure cholesteryl terminated domains is beneficial for tBLMs. However, whether or not such a conclusion may be extended to other, widely utilized linear hydrocarbon chain anchors remains unclear. In addition, the effect of exposure of the anchor SAMs to the water environment, in which these layers are designed to function, remains elusive. In this study, using earlier described4 molecular anchors, we aimed at establishing how lateral organization of molecular anchors affects the properties of the tBLMs. We demonstrate that favorable organization of the tethering SAMs may be achieved or lost depending on whether the conditions for lateral clustering of the molecular anchors exist. Factors such as exposure to water may trigger reorganization of the lateral arrangement of the molecular anchors, while the surface crystallography may also be favorable for cluster formation. This may influence many aspects of practical applications, such as regeneration, or multiple reuse of tBLMs. Knowing factors affecting critical properties, such as an electrical conductance and integrity, enables the development of controlled technologies, which may be important for sensor and other applications of tBLMs.

EXPERIMENTAL SECTION

Gold Film Deposition. For AFM, Au films were prepared on V-4 quality mica (SPI Supplies, U.S.). For reflection absorption infrared spectroscopy, as well as for electrochemical impedance spectroscopy (EIS), 25 × 75 mm glass slides from ThermoFischer Scientific (UK) were used. For SPR spectroscopy, BK7 glass slides (25 mm diameter, 1 mm thickness) were purchased from Autolab (Methorm, The Netherlands). Gold layers were deposited by the magnetron sputtering using PVD75 (Kurt J. Lesker Co., U.S.) system. Cr adhesion layer was 2 ± 0.5 nm, and variable thickness layer of gold was deposited under real-time quartz microbalance control. Sputtering parameters for 2 in. diameter metal targets were as follows: Cr, power 200 W, sputtering current 0.50 A at 4.5 mTorr argon pressure; Au, power 120 W, sputtering current 0.26 A at 4.2 mTorr argon pressure. Prior to coating, the deposition chamber was evacuated to 104 Hz) are an artifact arising from the capacitive coupling between the reference electrode lead and the ground. This distortion may be corrected (see Supporting Information of ref 18), but in this work we present unprocessed experimental data, because this has no effect on data interpretation. The mid frequency range contains an almost linear increasing trend as the frequency decreases. The linear increase of Z terminates or makes a pause at the inflection point that coincides with the negative of the phase minimum point on the frequency scale. Such behavior is typical for the EIS spectra of tBLMs containing defects.21 EI spectral changes seen in Figure 1, in particular, a downward movement of the inflection point on log Z versus log F curve, and phase extremum shift toward higher frequencies, are considered as evidence of tethered membranes losing their insulating properties, which may occur, for example, during the interaction of membranes with pore-forming peptides.1 Because gradual changes of the EIS spectra occur with repetitive exposure of anchor SAMs to water environment, we presume contact with water may be responsible for the transformations seen in Figure 1. Notably, even though changes in EIS response are quite considerable, the SPR experiment does not detect any difference in the amount of tethered material (data not shown) in an analogous series of experiments. In addressing the question of whether changes in anchor SAM properties may be responsible for degradation of the tBLM insulating properties, we tested how exposure to water affects the physical properties of the SAMs.

Figure 2. EIS complex capacitance plots of (A) self-assembled anchor monolayers (30% WC14/70βME) before and after incubation in water for 72 h, (B) tethered DOPC bilayers anchored to the same monolayers, and (C) Bode impedance phase plot of the same curves as in (B). Frequency range from 0.1 Hz to 20 kHz. Gold film thickness, 100 nm. Arrows indicate the frequency decrease direction.

transformations that occur during 72 h of incubation of anchors SAMs in water. Both the initial t = 0 and t = 72 h spectra exhibit almost ideal capacitive features. This may be inferred from the near perfect semicircular shape of the complex capacitance spectra seen in Figure 2A. The spectra in Figure 2A are modeled fairly well by a simple Rsol−CPE equivalent circuit, where Rsol is the solution resistance, and CPE is the constant phase element, with impedance defined as ZCPE = (CCPE)−1 × (iω)−α (α is the exponent of the CPE, i denotes the complex unit i = √−1, ω is the cyclic frequency, ω = 2πf, and f is a frequency measured in Hz). CCPE and α values calculated from 848

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Langmuir Table 1. Effect of Incubation in Water on Parameters of 30% WC14/70% βME SAMs and DOPC tBLMsa incubation time, h

CSAM

αSAM

contact angle, deg

Ymin,c μS/cm2

f min, Hz

SPR responseb to tBLM formation, mdeg

0 72

7.0 ± 0.35 11.8 ± 0.6

0.997 ± 0.001 0.984 ± 0.001

90.5 ± 0.2 85.4 ± 0.2

43.6 ± 2.2 (1.76 ± 0.09) × 105

1.4 ± 0.49 674 ± 38

420 ± 5 420 ± 5

a

tBLMS assembled via RSE methodology except for the SPR vesicle fusion experiment. Mean values with standard errors indicated. btBLMs assembled via vesicle fusion. cMagnitude of the complex admittance at f min, Ymin = Zmin−1.

Figure 3. SPR data. (A) Representative SPR traces (variation of the minimum of the SPR curve) of DOPC tBLM formation via vesicles fusion: red, on “dry” SAM; black, on SAM incubated for 72 h in water. Step at t = 1800 s reflects a refraction coefficient change upon flushing the cell with vesicle-free buffer. (B) Solid lines are SPR spectra of SAMs, and dotted lines are SPR spectra of vesicle fused tBLMs: red lines, on “dry” SAM; black lines, after incubation in water. SAMs were 30% WC14/70% βME.

on “dry” and “water incubated” SAMs produced identical shifts of the SPR absorption minimum (within limits of an error) of approximately 420 ± 5 mdeg (Table 1) consistent with the formation of tBLM in both cases. As demonstrated in a recent theoretical analysis,21 the frequency, f min, at which the negative of the impedance phase minimum is observed in the Bode plot is an indicator of the defect density in tBLMs. Figure 2 C and data in Table 1 show that tBLMs assembled on “dry” SAMs exhibit approximately 500 times lower f min values than tBLMs on water incubated SAMs. The same is true for the admittance values of the tBLMs at f min. The electrode admittance magnitude, calculated as Y = Z−1 at f min, of SAMs exposed to water for 72 h is 4100 times larger as compared to “dry” SAMs (Table 1), indicating that, while in both cases SPR detects the presence of intact membranes, the electrical properties of the two bilayers differ drastically. RAIRS. Figure 4 compares the RAIRS spectra before and after incubation of a WC14/βME monolayer in water for 72 h. C−H stretching vibrations of the methylene and the methyl groups contribute to the spectral features in the high frequency range. The bands at 2856 and 2927 cm−1 (Figure 4A) are assigned as the symmetric stretching mode (νs(CH2)) and asymmetric stretching mode (νas(CH2)) of the methylene groups, respectively.22,23 The weak feature near 2878 cm−1 belongs to a symmetric stretching mode (νs(CH3)) of the terminal methyl groups. The highest frequency component (2964−2967 cm−1) is associated with in-plane (ip) and out-ofplane (oop) asymmetric stretching vibrations of the methyl groups (νas(CH3)ip and νas(CH3)oop)).22 The difference spectrum in the C−H stretching frequency region reveals water-induced transformations in the hydrocarbon chain structure. The intensity of the νas(CH2) near 2930 cm−1 decreases (negative band in the difference spectrum), while its frequency slightly shifts to lower wavenumbers.

the Rsol−CPE model are tabulated in Table 1. The parameter α values are close to 1, confirming the near ideal capacitive nature of the interphase; consequently, the CPE coefficient CCPE may be regarded as a capacitance CSAM. Immediately after contact with water, the capacitance of the WC14/βME monolayer was 7.0 ± 0.35 μF/cm2, in good agreement with earlier data.16 Exposure of the SAMs to water triggers a gradual increase of the capacitance of the WC14/ βME SAMs with a value limit close to 11.8 ± 0.6 μF/cm2 (Table 1) and a slight decrease of α, which, for capacitive systems, reflects an increase in the heterogeneity of the interface (vide infra). Incubation of the anchor SAMs in water results in a contact angle decrease by approximately 5% (Table 1), indicating, along with the capacitance increase, a lowering of the hydrophobicity of the surfaces. Noteworthy, in contrast to EIS, the SPR spectra of the anchor SAMs do not change upon incubation (Figure 3B). The tBLMs exhibit quite different EIS spectral signatures depending on whether they have been assembled on unexposed (“dry”) or water-exposed SAMs, as seen from plots in Figure 2B. On “dry” SAMs, assembled tBLMs exhibit more than a 7fold smaller capacitance, as evidenced by the small semicircular part in the high frequency range of the EIS spectra (Figure 2B, blue curve). In contrast, the EIS spectra of tBLMs assembled on water-exposed SAMs exhibited large semicircular shapes (Figure 2B, red curve), comparable to the spectra of the anchor SAM only (Figure 2A, red curve). Changes in the complex capacitance EI spectra, upon assembly of tBLMs, for the water-exposed SAMs are, in effect, small to conclusively indicate the establishment of the tBLM. Evidence of a tBLM requires independent experimental techniques. Such a technique, sensitive to the amount of added lipid material, is SPR spectroscopy. Figure 3 shows the temporal traces of tBLM formation via vesicle fusion on “dry” and waterexposed SAMs. Surprisingly, as seen in Figure 3, vesicle fusion 849

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βME SAMs SAMs before and after exposure to water are displayed in Figure 5. The topographic image of the anchor SAMs indicates a flat surface with 3−5 nm high variation over the distance of 30−50 nm. There are almost no differences in the AFM topography images before and after incubation in water at 500 × 500 nm size resolution (Figure 5A and C). However, stark changes in the phase contrast AFM images can be seen for the water incubated samples (Figure 5B and D). “Dry” samples indicate, except for the grain boundaries, a relatively homogeneous AFM phase image (Figure 5B). After incubation in water (72 h), well-defined dark areas corresponding to a phase delay of up to −4o are visible (Figure 5D). At 500 × 500 nm resolution, we were not able to unambiguously relate delayed phase regions with the elements of the topography; however, at the higher 300 × 300 nm resolution, concurrence of the elevated features in the topography view (Figure 5E) and the phase delay regions in phase contrast image (Figure 5F) is found. Anchoring Properties of SAMs Are Affected by the Thickness of the Gold Substrate. In the course of the study, we found that differences in the gold substrate thickness gave rise to changes in the properties of the anchor SAMs, that parallel water induced effects, and consequently the function of the tBLMs. This unexpected parallelism prompted us to study more thoroughly the film thickness effect on the properties of the molecular anchors. Figure 6 shows the variation of the electrochemical parameters of the WC14/βME SAMs with the gold film thickness. While SAMs formed on both thin and thick gold layers exhibit water exposure effects, it is obvious that SAMs on thin films exhibit significantly higher CPESAM (which may be regarded as a capacitance) values and lower CPE exponents. The trend “the thinner the film the higher anchor SAM capacitance” is clearly seen in Figure 6 (red curves). The relative capacitance increase that occurs within 72 h of exposure to water is noticeably bigger for thick films (69% for 200 nm films) than for the thin films (42% for 10 nm films). The same is true for the variation of the CPE exponent (Figure 6, blue curves). To test if the observed capacitance increase is followed by the deterioration of the integrity and electrical insulation of the tBLMs, we investigated the EI parameters of tBLMs assembled on different thickness gold films. Figure 7 summarizes the results, which indicate that the capacitance increase that occurs due to a decrease of a gold film thickness is followed by a significant variation in the tBLM conductivity. In particular, gold film increase from 10 to 200 nm results in a membrane conductivity, evaluated as the tBLM admittance at f min, decrease by 3 orders of magnitude. The parameter f min follows the same trend. In general, one may claim that the gold thickness increase qualitatively results in the same effect as an exposure of the anchor SAMs to water. Different Thickness Films Exhibit Different Crystallographic Texture As Probed by Cyclic Voltammetry. To probe if the films of different thickness may have different structural features that may be responsible for the different functional properties of the tBLMs, we referred to cyclic voltammetry measurements. It is well-known that the pattern of electrochemical oxidation of the gold is sensitive to orientation of surface crystallographic facets.27,28 Figure 8 displays the cyclic voltammetry curves obtained on different thickness gold films. A different pattern of oxidation was observed for 10 and 200 nm gold films. Specifically, 200 nm gold films exhibit a more pronounced oxidation wave pattern at 1.15−1.20 V. In

Figure 4. RAIRS spectra of Au/(30% WC14/70% βME) selfassembled monolayers after (a) and before (b) incubation in water for 72 h in (A) high frequency and (B) fingerprint spectral regions. The difference spectra (c) are also shown. Gold film thickness, 100 nm.

Similarly, the frequency of νs(CH2) near 2856 cm−1 decreases (derivative-like feature in the difference spectrum 2850/2859 cm−1), while the intensity of the νas(CH3) at 2878 cm−1 increases. Water-induced changes are visible also in the fingerprint spectral region (Figure 4B), in which the most intense band is associated with the asymmetric stretching vibration of C−O−C group (νas(C−O−C)) in the ethylene oxide (EO) segment.16,24−26 After exposure to water, there is a considerable enhancement of the νas(C−O−C) mode at 1123 cm−1 (by a factor of 2.1) and a slight frequency shift to higher wavenumbers. The weak feature at 1463 cm−1 is associated with the scissoring bending mode of the methylene groups. Noteworthy, the negative control experiment, in which samples were kept for 72 h in a 100 Pa vacuum chamber of the spectrometer, indicated no spectral changes (see the Supporting Information). Atomic Force Microscopy. While RAIRS reveals the molecular level changes induced in the SAMs by water, it remains unclear if these changes lead to a collective, nanoscale level reorganization of the molecules on the surface. To address this question and directly detect possible lateral reorganization, we used AFM. The topography and phase images of WC14/ 850

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Figure 5. AFM 2D topography and phase images of 30% WC14/70% βME SAM surfaces on 200 nm gold substrates: (A, B) the topography and phase contrast of dry SAM sample; (C, D) the topography and phase contrast of the sample exposed to water for 72 h; (E, F) same as in (C) and (D) at higher resolution. Parts (G) and (H) show topography and phase contrast of two representative crystallites. AFM images were acquired in air. Scan parameters for (A)−(D), area size 500 nm × 500 nm, resolution at 1024 pixels per line, scan rate 1 Hz; for (E) and (F), area size is 300 nm × 300 nm, resolution at 4096 pixels per line, rate 2 Hz.

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the case of the 10 nm films this region is depressed, but at the higher potentials the curve transitions into a taller, more welldefined current peak around 1.4 V bias potential. Integration of the gold surface oxide reduction peak allows one to estimate the roughness factor, β, of the films, which indicates the ratio between the real surface area occupied by the gold atoms accessible to oxidation in electrolyte solution to the geometric surface area.29 The thin (10 nm) gold films exhibited a consistently lower β with the mean value of β = 1.31 ± 0.03 as compared to β = 1.39 ± 0.01 for the 200 nm films.

DISCUSSION Electrical Properties Attest for Drastic Changes in Anchor SAMs. In the tBLM regeneration experiments (Figure 1), every next cycle of tBLM formation results in a shift of the impedance negative of phase minima to a higher frequency range. This shift signals an increasingly larger density of defects that follows every attempt to regenerate the tBLM. Theoretical analysis21 allows one to estimate the density of defects in tBLM from the position of an impedance phase extremum on the frequency scale according to the formulas: 851

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Langmuir lg Ndef ≈ 0.93 lg fmin − lg k − 0.2 lg r0 − C

(1)

in which Ndef is the defect density in cm−2, f min is the frequency in Hz, at which the negative of impedance phase exhibits minimum, r0 is the defect size in cm, k = dsub/(ρsubCH), where dsub is the thickness of the submembrane layer, ρsub is the specific resistance of the submembrane layer, and CH is the Helmholtz capacitance of the interphase between solid electrode and the submembrane. Assuming k = 1.6 × 10−7 cm2/s, CH = 10−5 F/cm2, solution resistance between the working electrode and the reference electrode tip, Rsol = 30 Ω cm2, and r0 = 1 nm, the constant C = 1.24. The initial negative of phase versus frequency curve (Figure 1B) exhibits a minimum at f m < 0.50 Hz. According to eq 1, the corresponding defect density is Ndef < 3.8 × 106 cm−2, which is equivalent to less than four 1 nm size defects per 100 μm2. This calculation illustrates that defect density estimates depend on defect size. However, the defect size relatively weakly affects the defect density (see eq 1), and the theoretical analysis shows that the larger is the defect size the smaller is the defect density. Thus, for the assumption of 1 nm defect size, each time the tBLM is reassembled, the logarithm of the negative of impedance phase minimum shifts by approximately Δlg f min ≈ 0.2 (Figure 1B), so the defect density increases by a factor of approximately 1.6. After eight exchanges, the position of the f m ≈ 70 Hz (Figure 1B) and the defect density approaches 3.8 × 108 cm−2. Consequently, attempts to regenerate tBLMs utilizing the same anchor SAM result in membranes with increasingly higher defect densities. This means that the SAM loses its ability to properly tether the bilayer. Similarly, an exposure of SAMs, prior to the tBLM assembly, to water results in a gradual SAM capacitance increase (see the Supporting Information), which is followed by an approximately 500-fold shift of the f min (Table 1), and drastic changes in the Cole−Cole spectra of tBLMs (Figure 2B). All of these features attest for a significant increase of the defectiveness of tBLMs assembled on water-exposed SAMs. It is natural to assume that the tBLM property changes upon exposure to water are due to loss of material from the surface. However, the SPR attests to the fact that the same amount of organic material is deposited onto the surface in all rounds of regeneration (data not presented). This seeming inconsistency can explained by the fact that even at the largest defect densities, detectable by the EIS, the relative surface area coverage by the defects is small, only about 10−5, which is beyond the sensitivity limit of the SPR method. So, even though the SPR indicates the formation of an intact phospholipid bilayer, its integrity and the electrical insulation properties are strongly impaired in the course of each regeneration cycle. From these data, our working hypothesis was that the exposure to water affected the anchor SAMs in some way and resulted in the loss of its function to immobilize defect-free bilayers. To test this hypothesis, we carried out a series of experiments, during which mixed WC14/βME SAMs were incubated in water, while their properties were tested before and after. Figure 2A and data in Table 1 show that exposure to water (72 h) increases the capacitance of the anchor SAMs by a factor of 1.7. Within the same time interval, no changes in SPR thickness can be deduced from the almost identical SPR spectra of “dry” SAMs (Figure 3B). The SPR data indicate two important points: (i) no detectable amount of organic material is lost from the surface, and (ii) no detectable organic

Figure 6. Variation of the CPE (red curves, squares) and CPE exponent α (blue curves, triangles) of an anchor SAM’s Au/(30% WC14/70% βME) with thickness of the gold substrates. Open symbols, before exposure to water; closed symbols, after exposure to water for 72 h.

Figure 7. Effect of a gold substrate thickness on the admittance (Ymin = Zmin−1) and parameter f min of the DOPC tBLMs on 30% WC14/70% βME SAMs. Red line is the f m feature on the Bode plots of the EIS, and blue line is the tBLM admittance magnitude (Ymin) at f m.

Figure 8. Cyclic voltammetry curves of different thickness gold films: red, 10 nm; blue, 200 nm. Potential scan rate, 100 mV/s.

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Figure 9. Proposed model of a water-induced structural reorganization and cluster formation in anchor SAMs.

cluster formation would only result in a SAM capacitance increase and leave other qualitative features of EI spectra unchanged, which is clearly seen in Figure 2A. Spectral Changes Are Consistent with Structural Rearrangement. The structural rearrangement hypothesis (Figure 9) is also strongly supported by RAIRS, which provides detailed molecular level information on the structure of the adsorbed organic monolayers. In particular, the relative intensities and the frequencies of C−H stretching bands are sensitive to the orientation and packing of the alkyl chains.22,23 The difference spectrum of Figure 4A clearly shows waterinduced perturbations in parameters of the C−H stretching bands of the mixed WC14/βME SAMs. The positive feature near 2878 cm−1 indicates an increase in intensity of the νs(CH3) band. Because the transition dipole moment (TDM) of this mode lies in the direction of C−CH3 bond,22 the intensification of the methyl symmetric stretching band is evidence of reorientation of the methyl group so that the angle between the C−CH3 bond and the surface normal decreases, that is, adopting a more vertical orientation. The negative feature near 2930 cm−1, due to νas(CH2) vibration, is also consistent with a more vertical orientation. The TDM of this mode lies in the direction perpendicular to the CCC main axis and backbone plane.22,31 The positive feature near 2967 cm−1 is due to an enhancement of the intensity of asymmetric stretching vibration of methyl groups. This band is composed from two vibrational modes, νas(CH3)ip and νas(CH3)oop, which possess slightly different wavenumbers. For well-ordered alkanethiols in an essentially all-trans conformation, the ip and oop bands were observed at 2964 and 2954 cm−1, respectively.22 Importantly, the direction of the TDM for νas(CH3)ip mode is in-plane with the CCC backbone, while the TDM of νas(CH3)oop mode is aligned perpendicularly to the CCC backbone.22 In an earlier study, we showed that, for sparsely populated anchor monolayers prepared at low tether compound concentration (for example, 30% WC14), the alkyl chains are aligned near parallel with respect to the metal surface and the νas(CH3)oop mode ∼2961 cm−1 dominates.16 At higher concentrations, the tether molecules tend to adopt a more vertical orientation due to steric factors, so the orientation of alkyl chains changes to a more perpendicular orientation with respect to the surface and νas(CH3)ip mode at ∼2965 cm−1 becomes dominant.16 Similar changes clearly are visible in the water-induced difference spectra presented in Figure 4A. In addition, the downshift in νs(CH2) band frequency (derivativelike feature in the difference spectrum near 2850/2859 cm−1) indicates formation of clusters with more densely packed local structure upon interaction of monolayer with water.22,23 In the fingerprint region, the intensification of νas(C−O−C) mode is clearly visible (Figure 4B). An abrupt increase of the intensity of this mode is observed with increasing concentration of the WC14, forcing a more vertical alignment and clustering of polymethylene chains.16 Taken together, our RAIRS provides evidence for water-induced structural changes in

contaminants adsorbed to the surface during this prolonged period of exposure to water. Nevertheless, EIS indicates significant changes in the properties of the SAMs occur by the 70% increase in the capacitance. Despite this, the exponent of the constant phase element decreases only slightly after the incubation in water. Because it remains above 0.98 (Table 1, also see Figure 6) for “dry” and water exposed SAMs, one can assume near ideal capacitive behavior in both cases. The capacitance of a SAM modeled as a planar capacitor is CSAM = ε0 ×

εSAM dSAM

(2)

in which ε0 is the dielectric constant of a vacuum, and εSAM and dSAM are the dielectric constant and the thickness of SAMs, respectively. The increase of capacitance from 7 to 11.8 μF/cm2 upon exposure to water 72 h may occur, according to eq 2, as the result of a decrease of dSAM, an increase of εSAM, or both. A decrease in the thickness of the dielectric layer due to organic material loss from the surface is largely ruled out from the SPR data (Figure 3B). On the other hand, an increase of εSAM would result if water penetrated the anchor SAM. This highly possible process must be accompanied by a structural rearrangement of the polymethylene chains of anchor molecules, which may require considerable time to complete. Over the 72 h time period, the SAM capacitance approaches a value of 11.8 μF/ cm2, which equals almost exactly the value of SAMs formed only using the backfiller, βME.4 This prompts us to hypothesize that the capacitance increase and functional changes of the anchor SAMs are due to a rearrangement of the surface-bound molecules and not loss of material from the surface. The scheme in Figure 9 illustrates a cluster formation structural rearrangement hypothesis for the interaction of the anchor SAMs with water. The initial adsorption state of the anchors exhibits a fairly uniform distribution of disordered WC14 molecules with an average orientation far from the surface normal to maximize two favorable interactions contact with the βME/Au and intermolecular interactions along the polymethylene chains. Exposure to the water triggers interactions between the water and the βME and the ethylene oxide segments, decreasing the contact between the hydrophobic parts of anchors and underlying βME. However, the increased exposure of tetradecyl chains to water results in a force driving the anchor molecules to form clusters, maximizing the hydrophobic effect along the chains. Such a scenario is consistent with the features of the electrochemical impedance data (Figure 2). As the anchor molecules form local clusters, the capacitance should approach that for a pure βME SAM. The transition of the anchor molecules from a predominantly horizontal to predominantly vertical position will allow ions access to βME domains. These uncovered hydrophilic islands of 70% or more surface fraction provide no electric insulation, as was demonstrated by the finite element analysis of the EI spectra of previous tBLMs.30 Such 853

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Although high-purity water was used, the elevated clusters, islands of organic material, may be due to adsorption of residual contaminants during the prolonged exposure of the gold samples to water. However, if the contamination is responsible for the changes detected by AFM, one would expect (i) an SPR angle shift, consistent with an accumulation of organic material, (ii) an increase of the νas(CH2) band near 2927 cm−1, and (iii) a decrease in the electric capacitance. None of these effects were detected (Figures 1A, 3B, and 4). In fact, the opposite trends were found: (i) a decrease of the intensity of the νas(CH2) band (RAIRS) near 2927 cm−1 (Figure 4), and (ii) an increase of 1.7 in the capacitance of the SAMs (EIS, Figure 1A and Table 1). Excluding any adsorption of impurities, we conclude that the appearance of the round-shaped features in Figure 5C−F is consistent with the formation of surface clusters of WC14 molecules, supporting the hypothesis of structural changes that occur from the interaction of anchor SAMs and water. Surface Crystallography and Heterogeneity. The effect of gold film thickness on the properties of the anchor SAMs and the tBLMs is quite unexpected. From the cyclic voltammetry data, the roughness factor is 5−7% higher for the 200 nm films than for the 10 nm films. Roughness increases with thickness in magnetron sputtered films are well established,40,41 and corresponding higher capacitance values are expected for thicker films. However, the specific capacitance (normalized to the geometric surface area) of the anchor SAMs demonstrates the opposite trend. SAMs deposited on 200 nm gold films exhibit 30% lower specific capacitance (Figure 6) values as compared to the 10 nm films. A similar effect is also observed for the water incubated SAMs. In this case, the 200 nm films exhibited 20% smaller specific capacitance. Another interesting capacitive property of SAMs is the considerable variation of the constant phase element exponent. The deviation of α from 1.000 is relatively small (less than 0.005) in case of the 200 nm, gold (Figure 6, △), whereas for the 10 nm films, α approaches 0.983, noticeably lower. For ideally polarized electrodes, in the absence of the Faraday (electron transfer) processes, the deviation of α from 1 is due to surface heterogeneity. Noteworthy, according to Kerner and Pajkossy, heterogeneity on the atomic scale, not surface roughness, is the primary reason for constant phase element behavior of ideally polarized interfaces.42 The electrochemical properties of our SAMs are consistent with such an interpretation. The SAMs on the smoother 10 nm gold exhibited consistently lower α values (β = 1.31 ± 0.03) than SAMs on the rougher 200 nm (β = 1.39 ± 0.01). We conclude that the anchor SAMs on thin films are more heterogeneous than those on thick films. What factors may contribute to such an unusual effect? Our cyclic voltammetry data hint at two possible reasons. The thin films are smoother (see above roughness factor data and discussion) and exhibit a significantly higher fraction of atomically flat (111) facets on the surface. The latter is derived from the comparison of our cyclic voltammetry and data obtained on the monocrystalline gold films by others.27,28 Films with dominant (111) facets exhibit a single sharp oxidation peak, in our case observed at 1.4 V (Figure 8, red line). Gold surfaces more corrugated at the atomic scale such as the (110) planes exhibit earlier oxidation waves similar to that observed in our experiments at 1.15−1.20 V (Figure 8, blue line). We argue that atomically flat (111) faceted surfaces favor atomic-sized cluster formation resulting in a significant increase in the capacitance and an increase in local heterogeneity. In other

mixed WC14/βME SAMs, consistent with a change to a more vertical orientation and the formation of clusters of WC14 anchor molecules. The negative control experiment (see the Supporting Information) conclusively shows that exposure to water is the main factor responsible for the SAM properties changes in our experiments. Cluster Visualization. AFM as well as STM techniques were extensively used for studying formation of clusters in mixed SAMs. It was shown that even weakly interacting molecules exhibit a tendency to form nanometer size clusters on gold.32,33 On Au(111) surfaces, phase separation occurs spontaneously during overnight deposition of binary SAMs composed of different length alkanethiols.34 Even marginally different alkanethiols phase separate on thermally evaporated Au(111) after 4 days in the SAM forming solution.32 In contrast, the formation of phase separated domains in oligo(ethylene oxide) SAMs on Au (111) terraces required much longer time, and it occurred storing the samples under argon for as long as 4 weeks after incubation.35 In this study, the SAMs were assembled using considerably different size thiols. Neither the topography nor phase image of “as-deposited” SAMs on 200 nm thick Au films hint of the existence of phase separated regions (Figure 5A and B). The crystallites of the SAM covered Au are on average 40−60 nm. Height variations over an individual crystallite typically do not exceed 3−5 nm, indicating rather flat terraces. The ellipsometric thicknesses of pure βME and pure WC14 SAMs are 0.48 ± 0.05 and 3.37 ± 0.5 nm, respectively.4 The height difference of approximately 3 nm between phase separated domains, if they form spontaneously in the process of incubation, would be easily detected. Because they are not seen (Figure 5A), we contend that phase separated domains exceeding the AFM tip radius (10 nm) do not spontaneously form in the SAMs on 200 nm gold films. Phase contrast images map regions on the solid surface exhibiting different hardness, that is, the chemical nature of the sample.36 It is generally accepted37 that brighter domains in phase images correspond to hard materials when the interaction between tip and sample is dominated by a repulsive force. Phase imaging can also distinguish ordered and disordered regions within crystal structures by virtue of their differing mechanical responses.38 Ordered regions have a higher density and are harder, producing less energy loss per tapping mode cycle, giving rise to a lower phase shift (brighter), while disordered regions with lower density result in greater energy loss, greater phase shift, and appear darker.39 However, quantitative interpretation is always complicated because of the absence of information on the physical properties of the nanosized clusters. AFM images of “dry” anchor SAMs do not show any phase contrast (Figure 5B) except for grain boundaries. Incubation of these SAMs in water results in changes. The water-induced effects are barely visible in the topography views (Figure 5C). However, the phase view in Figure 5D reveals drastic changes on the surface of the SAMs, amplified in the higher resolution images of both the topography and the phase (Figure 5E and F). Round-shaped, well-contrasted features that resemble isolated islands of material protruding on average ∼2 nm above the crystallite surface are clearly seen. These elevated features corresponded to phase-lagged areas of the same size and shape (Figure 5G and H). These features appear only after exposure of the SAMs to water. 854

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Langmuir words, a homogeneous surface favors the formation of heterogeneous, clustered SAM structures. Notably, incubation of SAMs in water, another factor favoring cluster formation (vide supra), pushes the capacitance (CPE coefficient) upward, and the exponent of the constant phase element downward (Figure 6, □ and △). After incubation, the difference of the capacitive parameters (CPESAM and α) between thin and thick films decreases. This is especially well seen in the variation of α, reflecting surface heterogeneity. We conclude that after incubation in water SAMs on both thin and thick films become populated with clusters, as seen directly by the AFM (Figure 5). Ability to Anchor Bilayers. The formation of clusters, as depicted in Figure 9, whether they form spontaneously during the process of SAM deposition on thin Au or they form in the process of incubation of the SAMs in water, drastically affects the conductive properties of the tBLMs. The thickness of the substrate gold film, which affects capacitive properties only modestly (Figure 6), results in a variation of the defect density in tBLMs by almost 3 orders of magnitude. As a result, the residual conductivity of the tBLM estimated as the admittance Ymin at f min varies by the same extent (Figure 7). Evidently, clustering of the molecular anchors adversely affects the insulating properties of the tBLMs. This finding is in stark contrast to earlier studies, in which the cholesteryl terminated molecular anchors were utilized,19 suggesting linear chain anchors may exhibit a quite different tethering mechanism as compared to the compounds developed by the Leeds University group.43 The molecular level reasons as to why tethering anchor molecules lose their ability to anchor intact phospholipid bilayer remain elusive. One may argue that cluster formation creates vast areas on the surface containing few or no anchor molecules. Such areas may exhibit increased bilayer undulations, fluctuations of lipid density, and/or direct contact to the solid surface creating transient pores contributing to increased tBLM residual conductivity. Recently, utilizing a coarse-grained modeling technique, Liu and Faller demonstrated that clustering of the hydrophobic parts of the tether results in the spontaneous formation of transient pores and increased defectiveness of the tBLM systems.8 Notably, in this work,8 the driving force for cluster formation was the interaction between the hydrophobic parts of the immobilized molecular anchors inside the phospholipid milieu, while the length of the tether (hydrophilic part) facilitated cluster formation allowing the hydrophobic parts to freely move and get close to each other. This indicates that, in addition to interactions between the SAM and water and the structure features of the solid substrate, elucidated by the current study, interactions between the hydrophobic segments inside the membrane may contribute to cluster formation and poorer insulating properties of the tBLMs.

One of the most intriguing facts of this study is the sensitivity of the anchor SAM properties to the gold film thickness. SAMs on nanometer scale smooth gold film surfaces, populated predominantly with (111) facets, exhibited poor performance from the standpoint of the defectiveness of the anchored phospholipid bilayers, while corrugated (110) dominant surfaces produced SAMs with superior tethering quality. Our data strongly show that deterioration of the SAM function is primarily related to the formation of molecular anchor clusters and that atomically flat, homogeneous solid substrates possibly favor clustering, and are less suitable for the formation of the well-insulating tBLMs. While the detailed mechanism of the cluster formation is still not fully clear, it is beyond doubt that this process must be due to lateral movement of thiolate molecules. Such movement is possible if (a) the thiolate can move from one gold surface atom to another, or (b) gold thiolate complexes can move across the surface toward cluster sites. Recent scanning tunneling microscopy studies indicate the (111) gold surface undergoes significant reconstruction that includes formation of islands of gold adatoms.44 The formation of alkanethiolate doubly coordinated gold adatoms suggests the possibility of surface mobility of thiolate−gold complexes.45 Mechanisms involving such gold adatom may be responsible for the formation of lateral clusters of anchors observed in the current study. On the other hand, cluster formation by surface mobility of the thiolate only (with no gold adatom involved) would be dependent on the bond strength between the gold and the thiolate. Indeed, the electrochemical desorption experiments reveal stronger bonding of thiolates on Au(110) planes as compared to Au(111) planes.46 This parallels our data indicating increased cluster propensity for clustering and poor performance in tethering intact tBLMs on thin (10 nm) surfaces exhibiting higher content of Au (111) planes. We conclusively show that to accomplish highly insulating phospholipid bilayers, one needs to remove factors that favor cluster formation. The current work underscores the advantages obtained from reduced tether cluster formation concluded from earlier work, which showed that unsaturated anchor molecules, with one double bond in each alkyl chain, resulted in lower residual defect tBLMs relative to saturated anchors.16 The unsaturated anchors, which show greater disorder at high surface densities (RAIRS data), are less prone to form high-anchor-density clusters, consequently producing less defective tBLMs.16 The avoidance of cluster formation can be addressed further through alteration of the anchor molecule structure, for example, introducing branch sulfur atom containing groups ensuring multiple contact points with the gold lattice, thus reducing surface motility of the thiolate or anchor-gold complexes.

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CONCLUSIONS In this study, we demonstrate that different structural arrangements of the self-assembled molecular anchors on the solid support (Au) significantly affect their functional properties, in particular to tether intact phospholipid bilayers with minimal defects. A number of experimental techniques prove that a given composition (ratio) of anchor and backfiller molecules does not guarantee the formation of well-insulating intact bilayers. Same-composition anchor monolayers may tether phospholipid bilayers with defect densities differing by 3 orders of magnitude.

ASSOCIATED CONTENT

S Supporting Information *

RAIRS data of samples exposed to 100 Pa vacuum for 72 h, and SAM capacitance variation upon exposure to water from 0 to 96 h. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 855

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Langmuir Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the European Social Fund Agency, Lithuania (contract No. VP1-3.1-SMM-10-V-02-024). G.V. acknowledges the University of Maryland for granting access to the research facilities at the Institute for Bioscience and Biotechnology Research in Rocville, MD. G.N. acknowledges the Department of Organic Chemistry of the Center for Physical Sciences and Technology in Vilnius (Lithuania) for use of the Vertex 80v FTIR spectrometer in this study.

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