Electrochemical SERS study of a biomimetic membrane supported at a nanocavity patterned Ag electrode

Electrochimica Acta 110 (2013) 120–132

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Electrochemical SERS study of a biomimetic membrane supported at a nanocavity patterned Ag electrode Mansoor Vezvaie a,b , Christa L. Brosseau a,c , Jacek Lipkowski a,∗ a b c

Department of Chemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1 National Research Council Canada, Canadian Neutron Beam Centre, Chalk River Laboratories, Building 459, Chalk River, Ontario, Canada K0J 1J0 Department of Chemistry, Saint Mary’s University, Halifax, Nova Scotia, Canada B3H 3C3

a r t i c l e

i n f o

Article history: Received 12 February 2013 Received in revised form 22 March 2013 Accepted 26 March 2013 Available online 6 May 2013 Keywords: Electrochemical-SERS Nanocavity patterned Ag Lipid-membrane Hybrid-lipid bilayer Silver-electrode

a b s t r a c t Lipid bilayers in which two leaflets were made of dimyristoyl-phosphatidylcholine (DMPC) and hybrid bilayers with one leaflet composed of hydrogenated lipid and another leaflet of deuterium substituted molecules (d63 -DMPC) were deposited on highly ordered nanocavity patterned Ag electrodes using LB–LS and vesicle fusion techniques. In situ electrochemical surface enhanced Raman scattering (EC-SERS) was then used to study potential driven changes in these model biological membranes. The nanocavity structures provided a SERS active substrate with uniformly distributed surface enhancement. To ensure that the bilayer was separated from the metal surface by a hydrophilic space layer, the electrodes were chemically modified with a self-assembled monolayer (SAM) of ␤-thioglucose (TG) molecules. The monolayer of TG ensured that the solid substrate surface was hydrophilic. The electrochemical properties of the bilayer were monitored by recording differential capacitance curves. EC-SERS indicated that at the silver surface modified by the monolayer of TG the lower leaflet (in contact with the support) is more ordered than the top leaflet that is contact with solution. However, both leaflets remained in the liquid crystalline (LC) state for the entire range of investigated potentials. The results of this study show that the DMPC bilayer at the nanocavity patterned Ag surface may be used as a good biomimetic membrane model in future SERS studies of membrane proteins. The information concerning the effect of the electrode potential on membrane stability may be useful for the development of biosensors. © 2013 Published by Elsevier Ltd.

1. Introduction Monolayers and bilayers of phospholipid molecules are popular models for mimicking plasma biomembranes [1,2]. Knowledge concerning properties of such ultrathin films has been important for bioprocessing and biomaterials [3–5], drug delivery [6], development of biosensors [2–4] and fuel cells [7]. The biologically most relevant thin film geometry is a planar lipid bilayer composed of two monolayer thick leaflets [8,9]. Several models of planar bilayers have been reported in the literature including black lipid membranes (BLM) [10,11], solid supported membranes (sBLM) [12], self-assembled films [2,13] and tethered bilayers [14]. Solid supported and tethered bilayers are by far the most popular model membrane models, and are often deposited on both hydrophilic and hydrophobic surfaces using techniques such as vesicle fusion, [15,16] self-assembly [16,17] and a combination of Langmuir–Blodgett (LB) – Langmuir–Schaeffer (LS) techniques

∗ Corresponding author. Tel.: +1 519 8244120. E-mail address: [email protected] (J. Lipkowski). 0013-4686/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.electacta.2013.03.139

[18,19]. Although the solid support decreases the fluidity of sBLMs and creates an anisotropic environment, it allows for in situ applications of microscopic, electrochemical and spectroscopic techniques to study the structure of such bilayers with molecular level resolution [20,21]. Surface enhanced Raman spectroscopy (SERS) has become a popular tool to study orientation and conformation of molecules in thin films adsorbed at the electrolyte/metal interface [22–26]. The most crucial point in SERS experiments is the choice of the SERS active substrate to gain detectable and reproducible signals. Silver nanoparticles are by far the most common SERS signal enhancers but the enhancement factor is highly irreproducible and their potential application is restricted [27–29]. The signal irreproducibility of nanoparticles as well as electrochemically roughened SERS active surfaces are assigned to the randomly distributed “hot spots” present on such surfaces [30–37]. Recently, a variety of differently engineered arrays of nanostructured materials with uniformly distributed patterns of localized surface plasmons have been developed to address the irreproducibility and non-uniformity of SERS signals. One of the most popular SERS active nanostructured patterns is the nanocavity array developed by the Southampton

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group [38–41]. Such nanocavity arrays offer the possibility of maximizing the SERS enhancement for any specific system by tuning the diameter and depth of the voids [38–41]. Jose et al. [42] have demonstrated recently that the nanocavities may provide water micro-reservoirs for the biomimetic membrane. When the phospholipid bilayer is deposited on such a water-filled nanocavity patterned substrate the bilayer is floating on such water nanoreservoirs and therefore functions as a more accurate model of a real cell membrane [42]. There are several publications in which SERS has been used to study biomembranes deposited at SERS active supports [43–45]. These studies demonstrated that SERS has the potential to study order in metal supported bilayers [43] and can be employed to study hybrid bilayers in which one leaflet is composed of hydrogen and another with hydrogen substituted lipids [44]. Biological applications of SERS have been reviewed in a recent article [45]. In our previous work [46], we described EC-SERS studies of a monolayer of ␤-Thioglucose (TG) self-assembled on nanocavity patterned silver electrodes. In this current work bilayers of dimyristoyl-phosphatidylcholine (DMPC) and hybrid bilayers with one leaflet composed of hydrogenated lipid and another leaflet of deuterium substituted lipid molecules (d63 -DMPC) were assembled on nanocavity patterned electrodes modified with a SAM of TG. The objective of these studies was to determine the stability of such membranes as a function of potential applied to the nanopatterned electrode. Model biomimetic membranes deposited at a metal electrode surface allow transduction of chemical changes taking place in the membrane to electrical signal such as current or changes of the membrane capacitance and resistance. The transduction of chemical to electrical information allows for development of biosensors with potential applications for fast drug screening and selective detection of ions and molecules in general. In order to develop such sensors the effect of potential applied to the electrode on the membrane stability needs to be determined. The SERS spectra recorded for these bilayers will provide information about the effect of electrode potential on the conformation of DMPC molecules and the phase state of the bilayer (liquid crystalline (LC) or gel state). The information about inter chain interactions and lateral packing of the acyl chains in the bilayers will be determined from the ratio of the intensities of the asymmetric CH2 (␯(CH2 )as ) to symmetric CH2 (␯(CH2 )s ) stretching bands I2880 /I2850 (I (CH2 )as /I (CH2 )s ) [47–51] The ratio of intensities of skeletal C C vibration bands at 1088 cm−1 , corresponding to gauche chain conformations, and at 1125 cm−1 corresponding to the trans conformation, I1088 /I1125 (Igauche /Itrans ) will be used as a measure of the ratio of gauche to trans conformations in the acyl chains[47,48]. Finally, the ratio of band intensities corresponding to vibrations of the O C C N+ fragment in the choline head group, at 712 cm−1 (gauche) and 770 cm−1 (trans) conformation I712 /I770 (Igauche /Itrans ) will be used to follow conformational changes in the polar head group of the DMPC molecules. Upon deuteration, the methylene stretching vibration shifts from ∼2800 to 3100 cm−1 to ∼2000 to 2300 cm−1 . Hybrid bilayers with one leaflet composed of hydrogenated lipid and the second leaflet composed of deuterated molecules will be used to study the layer-by-layer structure of these bilayers. This work will provide a background for future investigations of cholera toxin A insertion into such model membranes. The mechanism by which the A subunit of cholera toxin is transported across the membrane is not known and SERS has the potential to provide useful information to unravel this mechanism. The bilayers investigated in this study were deposited on the internal walls of the nanocavities and were not floating on the top of a water reservoir as demonstrated in [42]. The properties of such floating bilayers will be investigated in the future.

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2. Experimentals 2.1. Materials Hydrogenated DMPC (h-DMPC) and deuterated DMPC (d63 DMPC) were purchased from Sigma and Avanti Polar Lipids respectively and both compounds were used without further purification. Lipids were dissolved in chloroform to give a ∼2 mg mL−1 stock solution. The 1-Thio-␤-d-glucose sodium salt (purity 98+%), (Sigma, Oakville, ON), was used without further purification. Silver cyanide (purity 99%), potassium cyanide (purity 97+%) and potassium pyrophosphate (96+%) were obtained from Alfa Aesar (Ward Hill, MA). Silver wire (purity 99.99%) was obtained from Aldrich (Oakville, ON); suspensions of 300 nm diameter polystyrene spheres (PS) in water (8 wt%) were obtained from Duke Scientific Corporation (81 Wyman St. Waltham, MA 02454); suspensions of 500 and 750 nm diameter polystyrene spheres in water (2.5 wt%) were obtained from Alfa Aesar (Ward Hill, MA). The phosphate buffer for spectroelectrochemical experiments was prepared as follows; 57 ml of 0.2 M sodium phosphate monobasic dehydrate (15.6 g L−1 , purity 99+%, Aldrich, Oakville, ON) and 243 ml of 0.2 M sodium phosphate dibasic anhydrous (28.4 g L−1 purity 99+%, Aldrich, Oakville, ON) stock solutions were mixed and diluted to 600 ml to obtain a 0.1 M sodium phosphate buffer with pH=7.4. Ultra high purity water from Millipore system with resistance of 18 M cm was used to rinse glassware and prepare all solutions. All glassware was cleaned in hot mixed acid (HNO3 :H2 SO4 , 1:3, v/v ratio) and was rinsed thoroughly with Ultra high purity water. 2.2. Substrate preparation To fabricate silver nanocavities, a support electrode was prepared by vapor depositing 10 nm of chromium, followed by 200 nm of gold onto a pre-cleaned 1 mm thick glass microscope slide. This gold electrode was then cleaned by rinsing with deionized water and methanol, followed by ozone cleaning for 30 min in a UVozone chamber. To improve substrate wettability and to increase substrate hydrophilic interaction with partially charged PS microspheres, thioglucose was self-assembled onto the gold electrode by immersing the freshly cleaned substrate in a 20 mM methanolic solution of thioglucose at room temperature for 2 h. To prepare polystyrene (PS) templates, 10 ml of 8% suspension of 750 nm PS microspheres was centrifuged and the top water layer was replaced by 5 ml methanol followed by ∼10 min sonication. A drop of the final methanolic PS suspension was then added at the center of the rotating substrate. The single layer PS template was prepared by spin coating at high speeds of ∼3600–4000 rpm whereas the double and triple layers of PS microspheres could be achieved at lower speeds of ∼1200–2000 rpm. The 1.0 cm2 substrate spin coated by PS microsphere appeared in rainbow color when looked at different directions. Silver was deposited from a cyanide bath containing 0.1 M K4 P2 O7 , 0.1 M KCN, and 0.02 M AgCN using an all glass threeelectrode cell controlled by a HEKA PG590 potentiostat under galvanostatic pulse electroplating conditions [52]. The galvanostatic pulse plating was conducted by starting with a strong initial pulse to produce silver seeds at a current density of 20 mA cm−2 for 100 ms followed by ∼50 pulses of 5 mA cm−2 for 20 ms with a rest time of 1 s at zero current. The metal thickness was tuned to obtain the maximum surface enhancement of the electric field of the photon, as described in literature and summarized in the next section [38]. A large area platinum foil was used as the counter electrode and the reference electrode was a homebuilt Ag/AgCl electrode in saturated solution of KCl (−40 mV potential versus the saturated calomel electrode (SCE)). After deposition,

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Fig. 1. Scanning electron microscopy (SEM) images of the template and Nanocavities. SEM images of the polystyrene (PS) microsphere templates spin coated on the gold cover glass slide substrate (top images). The seven bottom images show the nanovacity structured Ag films prepared by pulse electrodeposition of Ag through the template followed by the solvent removal of the template. The thickness of the silver films was controlled by monitoring the charge passed electrodeposition. The Ag films are labeled using the corresponding reduced thickness parameter.

polystyrene templates were removed by dissolution in toluene. The dissolution process lasted several hours and was followed by sonication using a sonicator (Aquasonic Model 50D) for a period of 30–120 s. A Hitachi S-570 SEM was used to image both the polystyrene templates and the micro-porous metal films. To ensure that the SERS spectra recorded for DMPC deposited onto the nanocavities were free from spectral interferences due to contamination from the nanocavity fabrication procedure, selected spectra recorded at the nanopatterned electrodes were compared with similar spectra recorded on electrochemically roughened (ECR) silver electrode. The ECR silver electrodes were prepared using a three pulse technique in 0.2 M KCl solution [35]; the potential was initially held at −1 V for 5 min then swept back to +0.35 V (0.5 V/s) for 70 s then back to −0.35 V for 20 s. The last two pulses were repeated twice and at the end the potential was set at −0.3 V for 30 s. At the end of this treatment the roughened silver surface appeared dark brown, as is consistent with literature [35].

2.3. Pore size tuning of nanocavity patterned electrode for EC-SERS The enhancement produced by confined plasmons in nanocavity structures depends on the shape and the size of the void. As the void structure is defined by the normalized thickness t = d/2R (d is the metal thickness and R is the spherical void radii) [53–58], the silver thickness in the present experiments was controlled between t = 0.7–0.8. The SEM images of the controlled layers of PS templates and the resulting silver nanocavity structures after electrodeposition are shown in Fig. 1. The theoretical predictions of the enhancement in nanocavity structures described by Bartlett and coworkers [53,57,58] are consistent with the experimental data presented here. Although the high energy laser (e.g. 532 nm) could produce a more intense Raman signal, to avoid the destructive effect on silver substrate a low energy diode laser with wavelength 785 nm was used. At this wavelength, a maximum enhancement of the SERS signal was observed for t = 0.8. [53,57]. Consistent with this prediction for a film of DMPC deposited on the nanocavity array, a maximum enhancement was observed at approximately t = 0.8 value of the reduced thickness.

2.4. Thin film preparation methods 2.4.1. Langmuir–Blodgett film DMPC monolayers and bilayers were prepared using a Langmuir–Blodgett trough equipped with a Wilhelmy surface balance (KSV 5000, Finland) and a movable barrier. The whole system was controlled by KSV 5000 software and the temperature of the subphase was ∼25 ± 1 ◦ C (unless otherwise indicated). A few drops of DMPC solution were spread at the air/water interface, the solvent was allowed to evaporate, and a compression isotherm was recorded [59]. The compression isotherm showed a collapse pressure for pure DMPC at ∼47 mN m−1 . The monolayer was then transferred from the air/water interface onto the nanocavity electrode at a constant surface pressure of 40 mN m−1 . The first monolayer was transferred using the LB method by vertically withdrawing the electrode through the interface at a constant speed of 25 mm min−1 . The recorded transfer ratio was 1.0 ± 0.1. In this monolayer, the headgroups of DMPC faced the metal surface and the hydrophobic hydrocarbon chains were directed towards the air. After this emersion step, the monolayer-covered substrate was allowed to dry for ∼4 h. The second layer was transferred using the Langmuir–Schaeffer method. This second monolayer was compressed to a surface pressure of 40 mN m−1 . The monolayercovered nanocavity electrode was then brought into contact with the compressed monolayer at the air/water interface, allowing for transfer of the second leaflet onto the monolayer-covered electrode. The acyl chains of phospholipids in the second leaflet were directed toward the metal. The electrode covered by the second leaflet was allowed to dry for 4 h and then mounted in the spectroelectrochemical cell for further analysis. 2.4.2. Vesicle preparation and spreading A 2 mg/ml solution of DMPC in chloroform was used as the stock solution. 0.5 ml of the stock solution was dried by vortex mixing in a test tube under argon flow. Solvent residue was removed by placing the tube in a vacuum desiccator for more than 2 days. 2 ml of 50 mM NaF electrolyte was then added to the dry lipid and the mixture was sonicated at ∼35 ◦ C for at least 1 h. Usually the mixture became translucent after 20–30 min. It has been shown that 97% of DMPC vesicles prepared in this manner have a diameter in the range of 16–30 nm [60]. The nanocavity patterned substrates were

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Fig. 2. SERS of the hydrogenated (curve 2) and perdeuterated (curve 1) DMPC and the main peaks assignment in two spectral regions 500–1200 cm−1 (panel (b)) and 1800–3200 cm−1 (panel (a)).

exposed to the vesicle mixture for approximately 20 min, rinsed with water, and dried by argon stream. 2.5. Raman spectroscopy Raman spectra were obtained using a Renishaw model 2000 Raman microscope which allows high resolution confocal measurements. Laser excitation at 785 nm was achieved via a 300 mW NIR 780TF series Diode laser (Spectra-Physics). The spectrometer was equipped with a holographic notch filter and a near excitation filter (NExT filter). Raman scattering was detected via a 180◦ geometry using a RenCam CCD Detector (578 pixel × 385 pixel, from Renishaw). Leica water immersion objectives with magnifications ×50 and ×60 were used for SERS studies in solutions with a 10–12 ␮m experimentally measured laser spot on the sample surface. Spectra were acquired in pH 7.4 phosphate buffer electrolyte with 1 accumulation and an exposure time of 10–90 s. To avoid any damage to the system, the sample was not exposed to the laser during the time the electrode potential was changing. A holographic grating (1200 grooves mm−1 ) and a slit width of 50 ␮m allowed for a spectral resolution of ∼1 cm−1 . However, when the Raman band of a silicon wafer at 520.7 cm−1 was used to calibrate the spectrometer, the accuracy of the calibration was in the range of 2–6 cm−1 and was lower than the maximum spectral resolution. The Raman spectrometer was interfaced with a PC, and the spectral data were analyzed using the Renishaw WiRE software v. 1.2 based on the GRAMS/32C suite of programs (Galactic Industries). To increase the signal to noise ratio, 8–12 spectra collected under the same conditions were averaged. The selected spectral regions of SERS spectra for h63 -DMPC and d63 -DMPC with and without the TG layer were deconvoluted and the deconvoluted bands were assigned to the corresponding vibrational modes using literature sources [61–69]. 2.6. Electrochemical measurements and the spectroelectrochemical cell The electrochemical measurements were carried out in an all-glass three-electrode cell using the hanging meniscus configuration. A polycrystalline silver electrode served as the working electrode (WE) and a gold wire served as the counter electrode (CE). The reference electrode was a homemade Ag/AgCl in saturated solution of KCl electrode, and was connected to the cell via a salt bridge. Unless otherwise stated, all potentials are reported versus this reference electrode. The cleanliness of the system was

checked by recording cyclic voltammetry and differential capacitance curves using experimental procedures described previously [70]. A computer-controlled system consisting of a HEKA PG 590 potentiostat/galvanostat and a lock-in amplifier (EG&G Instruments 7265 DSP) was used to perform electrochemical experiments. All data were acquired via a plug-in acquisition board (National Instruments NI-DAQ BNC-2090) using a custom-written software provided by Professor Dan Bizzotto from the University of British Columbia. The electrochemical data on the nanocavity electrode were compared to the measurements on a smooth Ag electrode. This electrode was polished with 0.5 ␮m diamond paste to a mirror finish followed by stepwise washing with the following solutions; concentrated sulfuric acid for 5 min followed by rinsing with water, 3 min in perchloric acid 70%, 1 min in polishing solution (50 ml of 4 M of CrO3 + 50 ml of 0.6 M HCl) followed by rinsing with water; 5 min in concentrated ammonia with sonication followed by rinsing and then finally 5 min in concentrated sulfuric acid. The washed electrode was then transferred to the electrochemical cell under the protection of a drop of water to avoid oxidation by the presence of oxygen in air [71]. A homemade three electrode spectroelectrochemical cell was used. This cell was capable of holding a large amount of electrolyte (∼25 ml) to reduce the risk of temperature increase by the laser beam.

3. Result and discussion 3.1. SERS of DMPC bilayers deposited on nanocavity patterned Ag – spectral analysis Fig. 2 plots the SERS spectra for h63 -DMPC and d63 -DMPC bilayers deposited at the nanocavity patterned Ag, at the open circuit potential (∼−0.01 V vs the Ag/AgCl electrode). The shape and position of bands in the Raman spectra of the phospholipid bilayer depend on its dynamic behavior. Several types of lipid motions contribute to the overall dynamic behavior of the bilayer. Intramolecular chain disorder, resulting in the ‘fluid’ and ‘liquid crystalline’ behavior of the lipid matrix, arises from the formation of gauche conformers along the acyl chains. For membrane bilayers composed of phosphatidylcholine, spectroscopic and X-ray diffraction studies [72,73] showed non-equivalent conformations of the two acyl chains. The sn2 chain extends in a perpendicular direction to the linear sn1 chain where a gauche bond at the carbon

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Fig. 3. (a) Dependence of the asymmetric to symmetric methylene stretch Raman bands intensity ratio I2890 /I2850 on temperature (data for multilammelar vesicles taken from reference [65–67]). (b) Dependence of the C C stretching band intensity ratio for gauche and trans acyl chains conformations (IGauche /ITrans or I1088 /I1125 ratio). Data taken from Raman spectra of multibilayers of DMPC published in reference [64–66]. The experimental data obtained in this work for vesicle fused and LB–LS bilayers at 20 ◦ C are overlaid on each graph as large circles.

2 position enables the remaining fragment of the chain to pack parallel to sn1 chain [66]. At temperatures lower than the transition temperature (24 ◦ C), phosphatidylcholine bilayers are in a gel phase where the acyl chains assume an all-trans conformation and are packed parallel to each other [73]. At temperatures above the phase transition temperature the chains have gauche conformers and the bilayer has liquid crystalline (LC) and liquid like (LL) structure [47–49,62–65]. In the Raman spectra of the h63 -DMPC bilayer, three prominent bands sensitive to changes in bilayer characteristics are identified in the 2800–3100 cm−1 spectral region. The all-trans gelphase hydrocarbon chain methylene symmetric and asymmetric stretching modes are assigned to the 2850 and 2880 cm−1 bands, respectively. These bands lie on a background originating from the Fermi resonance interactions of the methylene deformation mode with the 2850 cm−1 methylene symmetric stretching. The peak at 2935 cm−1 includes a Fermi resonance component of the symmetric stretching modes of the lipid chain terminal methyl groups. Bands in the 1000–1200 cm−1 region are attributed to skeletal C C stretching vibrations, which are sensitive to the conformation of the hydrocarbon chain [72–74]. The intense band at 1088 cm−1 results from the C C stretching in the gauche conformation while the bands at 1060 cm−1 and 1125 cm−1 are assigned to C C stretching modes in the trans conformation. Raman spectra in the 1000–600 cm−1 region were used to identify conformation of the O C C N+ backbone of the phosphocholine moiety [75]. The Raman band attributed to the “totally” symmetric stretching gauche conformation of the O C C N+ (C N bonds of the quaternary ammonium group) appear at ∼715 cm−1 where as in the trans conformation this band shifts to about 770 cm−1 [43].

The parameters derived from the Raman band intensity can be used to assess the inter- and intra-molecular packing properties of the lipid bilayers. For example, changes in the 2850 and 2880 cm−1 peak-intensity ratio (I2880 /I2850 ) or (Ias (CH2 )/Is (CH2 )) reflect changes in inter chain interactions (lateral packing) within lipid assemblies [43,47–51,76]. Another important ordered/disordered parameter is the peak intensity ratios of I1088 /I1125 (frequently reported as Igauche /Itrans ) of the skeletal vibrations, considered as a measure of the number of gauche to trans conformations [73]. Finally, the ratio of the intensity of choline group vibrations at 715 and 770 cm−1 (Igauches /Itrans ) can be used as a measure of the changes in the conformation of the O C C N+ fragment of the polar head of DMPC. Therefore, the structure of the bilayer supported at the nanopatterned silver electrode surface can be conveniently determined with the help of SERS. There are several papers in which these ratios were measured as a function of temperature to describe the thermotropic behavior of DMPC bilayers in vesicles or stacks of multibilayers [43,47–51,76]. In Fig. 3, thermotropic data taken from the literature is plotted as calibration curves to identify the physical state of the bilayers deposited by vesicle fusion and by the combination of LB–LS techniques at the nanopatterned Ag electrode. The thermotropic data display a sharp step at 24 ◦ C which is the main phase transition temperature for DMPC. The data show that (I2880 /I2850 ) has high values at temperatures below the phase transition (where the membrane is in the gel state) and drops down at temperatures above the phase transition temperature (where the membrane is in the liquid crystalline state). In contrast the I1088 /I1125 ratio is small in the gel state (trans conformations dominant) and increases in the liquidcrystalline state where gauche conformations become pronounced.

Table 1 Intensity ratio parameters obtained from C H, and C C str. vibrations of DMPC and d63 -DMPC. I2880 /I2850 (I(CH2 )as /I(CH2 )s )

I1088 /I1125 (I(C

DMPC Crystalline LB–LS bilayer in solution LB–LS bilayer on Tg in solution Vesicle fused in solution Vesicle fused on TG in solution

C )G /I( C C )T )

1.43 0.91 1.01 1.07 1.09

0.75 1.03 0.9 0.9 0.8

4.4 2 3.2 1.8 3.3

Hybrid bilayer on TG (LB–LS) In solution (H bottom/D top) In solution (D bottom/H top)

1.23 0.87

0.71 0.68

3.1 2.7

I712 /I770 (I(OCCN+ )G /I(OCCN+ )T )

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Fig. 4. The normalized spectra of the C H stretching region for a hydrogenated leaflet of DMPC in hybrid bilayers in which one leaflet was hydrogenated and the second leaflet perdeuterated deposited at a TG monolayer modified nanopatterned Ag, panel (a); curve 1-hydrogenated leaflet is the bottom leaflet in contact with the SAM of TG, curve 2-hydrogenated leaflet is the top leaflet in contact with the electrolyte. The spectra were normalized using the peak at ∼1000 cm−1 corresponding to TG monolayer. Panel (b) shows the exponential decay of the relative enhancement as a function of distance from the surface calculated according to the method described in [78].

80

a

uFCm C C/ /µFcm

-2

60

40

20

1 2 3

0 0.0

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

-1.2

-1.4

E vs Ag/AgCl / (V)

1

b

160

120 -2

2

-2

CC// uFcm µFcm / Fcm C

The large dots in Fig. 3 represent the ratios for the two bilayers supported at the nanocavity patterned electrode determined at 20 ◦ C. When compared to the thermotropic data, the values of the (I2880 /I2850 ) for the supported bilayers are consistent with the numbers observed for the liquid-crystalline state. However, the I1088 /I1125 ratio is quite low and indicates that the chains have low concentrations of gauche conformers. Therefore the SERS data indicate that the bilayers are quite disordered. However, the disorder is reflected primarily in chain packing with the chains retaining the predominant trans conformation. We note that the thermotropic data in Fig. 3 were acquired from normal Raman spectra in which the relative band intensity may be different than in SERS spectra for bilayers supported at a metal surface. In the present case, the interaction between the lipid and the metal is very weak and the main source of the differences may be the orientation of the polarizability tensors for a given vibration and the surface normal. For molecules in the proximity of a metal, the bands that have large element of the polarizability tensor in the direction normal to the surface are enhanced and bands with large elements of polarizability tensor in the direction parallel to the surface are attenuated [36]. However, the acyl chains of DMPC molecules have rotational freedom (they rotate along the chain axis). In addition, their acyl chains are distributed on the surface of a cone whose angle with respect to the surface normal is the tilt angle (uniaxial distribution). Due to the chains rotation in their uniaxial distribution, the orientation of the methylene groups and hence the effective orientations of the polarizability tensors of the symmetric and asymmetric stretches are the average of many positions. This scrambling effect eliminates the differences between the orientation of the polarizability sensors of the symmetric and asymmetric stretches of the methylene group and validates the use of the thermotropic data to evaluate the order in the supported bilayers using the SERS spectra. This approach has previously been used by Leverette and Dluhy [43]. In order to assess the role of the hydrophilic SAM of TG, the DMPC bilayers were also formed by the vesicle fusion and LB–LS techniques directly at the nanocavity patterned Ag without the hydrophilic monolayer. The ratios of the band intensities determined from these experiments are compiled in Table 1. The top row in Table 1 shows the ratios of the band intensities for crystalline DMPC (well-ordered state) as a reference. These ratios can be used to gain important insight into the physical properties of model membranes and to examine the differences between the physical states of the membranes assembled by different methods. As seen in Table 1, for all the membranes built from hydrogenated DMPC, the ratio of I2880 /I2850 is smaller than 1.4 indicating that all

80

3 40

0 0.0

-0.2

-0.4

-0.6

-0.8

-1.0

E vs Ag/AgCl / (V) Fig. 5. (a) differential capacitance (DC) curves measured for, bare polycrystalline Ag electrode in 0.1 M NaF electrolyte (curve 1); nanocavity patterned Ag electrode coated by DMPC bilayer deposited using the LB–LS method (curve 2); nanocavity patterned Ag electrode coated with DMPC bilayer deposited using the vesicle fusion method (curve 3). (b) Differential capacitance curves for the silver electrode modified by SAM of TG (curve 1) and with DMPC bilayer deposited at the TG modified Ag using the LB–LS method (curve 2) and using the vesicle fusion method (curve 3).

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1 2

-1.3 -1.2 -1.1 -1.0 -0.9 -0.8

-2

-2 CC/ CµFcm / / uFCm Fcm

Raman Counts / a.u.

90

-0.7

Electrolyte a NaF DMPCBilayer Bilayer DMPC made by Vesicle Fusion 1

60

30

2

-0.6 -0.5

0 0.0

-0.2

-0.4

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

E vs Ag/AgCl / (V) 1.4

-0.3 -0.2 -0.1 ocp

b

3200

3000

II2880 2850 2880/I/I2850

1.2

2800

-1 Raman RamanShift Shift / cm -1

1.0

0.8

0.6 0.0

-1.1 -1.0

-0. 2

-0. 4

-0. 6

-0. 8

-1. 0

-1. 2

-1. 4

E vs Ag/AgCl / V

c

1.2

-0.9 -0.8

/ I11255 II1088 1088 /I112

Raman Counts / a.u.

-1.3

-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 ocp 1200

1.0

0.8

110 0

100 0 -1

Raman Shif // cm cm-1 Raman Shift

0.0

-0 .2

-0 .4

-0 .6

-0 .8

-1 .0

-1 .2

-1 .4

E vs Ag/AgCl / V

E vs. Ag/AgCl / V

Fig. 6. For a DMPC bilayer deposited at the bare nanocavity patterned Ag electrode; at selected electrode potentials bands in the C H stretch (top left panel) and C C stretch (bottom left panel) fragments of the SERS spectra. Panel (a) plots the differential capacitance curves for the bare electrode and the electrode with deposited DMPC bilayer. Panel (b) plots the I2880 /I2850 peaks intensity ratio and Panel (c) plots the I1080 /I1125 peaks intensity ratio as a function of the applied potential. The DMPC bilayer was deposited by vesicle fusion; supporting electrolyte was 0.1 M NaF solution.

these membranes are predominantly in the liquid-crystalline state [43,47–51,76]. However, one exception observed was the I1088 /I1125 ratio, which was less than 1. The small value of this ratio indicates that the acyl chains remain predominantly in the trans conformation. The best ordered bilayer, which is characterized by the highest I2880 /I2850 and the lowest I1088 /I1125 ratios, was formed by fusion of unilamellar vesicles on the thioglucose modified electrode. This bilayer is somewhat better ordered than the bilayer deposited on the TG modified Ag using the LB–LS techniques. In general, the bilayers assembled at the TG modified silver surface are more ordered than bilayers assembled directly at the metal

without protection of the hydrophilic monolayer. Clearly, contact with the metal enhances disorder in the bilayer. In addition, the bilayers formed by vesicle fusion are somewhat more ordered than bilayers formed by the LB–LS method. For all bilayers the disorder is reflected in chain packing with the number of gauche conformations in the chains being relatively small. The last column in Table 1 reports the ratio (I715 /I770 ) which is the ratio of the gauche to trans conformation of the O C C N+ fragment of the head group. In the crystalline state this ratio is high (4.4) indicating that the gauche conformation is predominant. For the supported bilayers, the I715 /I770 ratios are much lower, indicating the conformation

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Fig. 7. For a DMPC bilayer deposited at the bare nanocavity patterned Ag electrode; SERS bands at selected electrode potentials for the head group vibrations region (left panel). Panel (a) plots the differential capacitance curves for the bare electrode and the electrode with deposited DMPC bilayer. Panel (b) plots the I712 /I770 peaks intensity ratio as a function of the applied potential. The DMPC bilayer was deposited by vesicle fusion; supporting electrolyte was 0.1 M NaF solution.

of the polar head group is a mixture of gauche and trans conformations. However, this ratio is higher for the bilayer deposited at the TG modified Ag than on a bare metal surface indicating that the monolayer of hydrophilic SAM favors a gauche conformation of the head group. Although bilayers deposited using the LB–LS technique are slightly less ordered, this method allows one to build hybrid bilayers in which one leaflet consists of hydrogen and the second leaflet of deuterium substituted molecules. The SERS spectra then provide unique information concerning the order in individual leaflets of the hybrid bilayers. Therefore, SERS spectra were recorded for hybrid bilayers with one leaflet made of d63 -DMPC and the second leaflet made of h63 -DMPC. The phase transition of d63 -DMPC bilayer is 18 ◦ C (six degrees lower than for h63 -DMPC) [77] At 20 ◦ C the d63 -DMPC bilayer is already in the LC state. To investigate the order in individual layers of the bilayer, d63 -DMPC leaflet was used as a spacer and spectra were recorded for the h63 -DMPC leaflet only. The ratio of the corresponding band intensities determined for the hybrid bilayers are compiled in Table 1. The results show that surprisingly the bottom leaflet that is contact with the TG modified Ag surface has a larger ratio of I2880 /I2850 and hence is more ordered than the top leaflet that is contact with the electrolyte. The I1088 /I1125 ratios are low indicating that the acyl chains in the individual leaflets are predominantly in the trans configuration. The I715 /I770 ratios are quite large indicating that the O C C N+ fragment of the head group in the individual leaflet assumes predominantly gauche conformation. Another advantage of using hybrid bilayers is to evaluate the distance dependence of the enhancement factor. In order to illustrate the influence of the distance on the enhancement factor, Fig. 4a plots the Raman spectra of the hydrogenated leaflets of the hybrid

bilayers. In the first configuration, the leaflet composed of h63 DMPC is located at the top layer of the hybrid bilayer (facing the solution) whereas in the second configuration this layer is at the bottom (facing the metal). For both configurations, the C H band intensity was normalized by taking a ratio to the intensity of the 1000 cm−1 band (corresponding to the monolayer of TG). When h63 -DMPC constitutes the bottom leaflet, the spectrum is about two times stronger than the spectrum for the h63 -DMPC in the top leaflet. Similar value was observed for the ratio of C D bands intensities in d63 -DMPC leaflets. This behavior correlates very well with the theory. Fig. 4b shows the decay of the relative surface enhancement with the increase in the distance from the surface taken from literature [78]. If the average difference in the distance between the two leaflets in the bilayer is estimated as a thickness of a single DMPC layer (∼1.5 nm), the graph in the inset to Fig. 4b predicts a decrease of the surface enhancement by a factor of two [47–51,64]. This is an important result because it illustrates that SERS spectra could be used to determine a position of a biomolecule in the supported bilayer. This property will be used in the next paper to study potential controlled translocation of a protein through this model membrane. 3.2. SERS studies of the potential driven changes to the structure of the bilayer This section describes the effects of the electrode potential on the structure of the bilayer supported at the nanopatterned Ag surface. These measurements were performed in preparation for future studies of the potential-controlled protein translocation through the bilayer. Three model systems were investigated: (i) DMPC bilayer formed by vesicle fusion on a Ag bare electrode

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11::NaElec trolyte F 2::DMPC Bilayer on Tg by Vesicle Fusio 2 DM PC bilayer on Tg 2 : TG/DMPC bilaye rnTG

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vs. Ag/AgCl EEEvs Ag/AgCl///V V vs Ag/AgCl E vs. Ag/AgCl /VV

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Fig. 8. For a DMPC bilayer deposited at the TG SAM modified nanocavity patterned Ag electrode; at selected electrode potentials bands in the C H stretch (top left panel) and C C stretch (bottom left panel) fragments of the SERS spectra. Panel (a) plots the differential capacitance curves for the bare electrode and the electrode modified with TG and with deposited DMPC bilayer. Panel (b) plots the I2880 /I2850 peaks intensity ratio and Panel (c) plots the I1080 /I1125 peaks intensity ratio as a function of the applied potential. The DMPC bilayer was deposited by vesicle fusion; supporting electrolyte was 0.1 M NaF solution.

surface; (ii) DMPC bilayer formed by vesicle fusion on Ag modified with a SAM of thioglucose; (iii) hybrid bilayers formed by the LB–LS method on Ag modified by SAM of thioglucose in which hydrogenated DMPC constitutes one leaflet and the second leaflet was made of d63 -DMPC. 3.2.1. Electrochemical measurements Fig. 5a and b show the capacitance curves recorded at a silver electrode covered by DMPC membranes deposited by the vesicle fusion and LB–LS methods. The dashed line in Fig. 5a plots the differential capacitance curve recorded at the film-free polycrystalline silver electrode. The curve displays a characteristic minimum at E = −920 mV versus Ag/AgCl/Cl− corresponding to the position of

the diffuse layer minimum. The potential of this minimum is equal to the potential of zero charge (pzc). The silver surface is positively charged at potentials more positive than pzc and negatively charge at more negative potentials. Curves 2 and 3 in Fig. 5a show the capacitance curves for the electrode covered by the bilayer made by vesicle fusion and the LB–LS method, respectively. The curve corresponding to the film formed by vesicle fusion is characterized by somewhat smaller values of the capacitance, indicating that this film has less defects than the bilayer formed by the LB–LS technique. For the film formed by vesicle fusion the minimum of the capacitance amounts to ∼3.2 ␮F cm−2 and is observed at the potential of zero charge. The capacity remains low within the region ± 200 mV around the pzc and then slightly increases indicating that

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EEvs vs.Ag/AgCl Ag/AgCl/ V/ V Fig. 9. For a DMPC bilayer deposited at the TG SAM modified nanocavity patterned Ag electrode; SERS bands at selected electrode potentials for the head group vibrations region (left panel). Panel (a) plots the differential capacitance curves for the bare electrode and the electrode with deposited DMPC bilayer. Panel (b) plots the I712 /I770 peaks intensity ratio as a function of the applied potential. The DMPC bilayer was deposited by vesicle fusion; supporting electrolyte was 0.1 M NaF solution.

charging of the metal surface affects the film structure. The capacity increases steeply at potentials more negative than −1.2 V, indicating desorption of the film from the electrode surface. Fig. 5b shows the differential capacitance curves for the Thioglucose-covered Ag electrode in the absence and presence of DMPC bilayers formed by either vesicle fusion or the LB–LS technique. The differential capacitance curves recorded in the presence of the two films are quite similar to the curve recorded for a Ag surface covered by pure TG monolayer. Clearly, the stability of the bilayer formed on top the monolayer of TG is determined by the stability of the underlining monolayer of TG. The bilayer starts to desorb earlier at E ∼−0.8 V and this coincides with the onset of desorption of the monolayer of TG. The capacitance curve for the bilayer displays a small peak at −0.4 V which is also present in the capacitance curve recorded for the Ag electrode covered by the SAM and hence it reflects the reorientation of TG molecules in the SAM. 3.2.2. EC-SERS study of the DMPC bilayer formed at the bare Ag electrode The left panels in Fig. 6 show the C H and C C vibrational regions of the SERS spectra of a DMPC bilayer formed by vesicle fusion at a bare Ag electrode. The spectra were background corrected and were offset to be shown in the same scale. The right panels plot the differential capacitance curve and peak intensity ratios of I2880 /I2850 and I1088 /I1125 versus applied potential. These ratios will be used to explain the observed changes in the capacitance in terms of changes in the packing characteristics of the DMPC bilayer. The I2880 /I2850 ratio shows a broad maximum in the potential range between −0.3 and −0.9 V. At potentials close to the open

circuit potential (OCP) (∼0 V versus Ag/AgCl) the I2880 /I2850 ratio is ∼0.9 and as Fig. 3 shows this value is characteristic of the LC state of the bilayer. However, when potential becomes more negative the I2880 /I2850 ratio increases to a value ∼1.1–1.2 in the potential range between −0.4 and −0.9 V. The change in I2880 /I2850 ratio from 0.8 to ∼1.2 is characteristic for the transition between the liquid crystalline and a mixed gel/liquid crystalline state. For potentials lower than −0.9 V the ratio decreases again to a value between 0.9 and 0.8. This behavior indicates that at these negative potentials the bilayer is totally in the liquid crystalline state. The ratio of the C C skeletal vibrations intensities I1088 /I1125 , shown in Fig. 6c, is ∼1.1 at potentials more positive than −0.4 V. This value indicates that the acyl chains have quite a significant amount of gauche conformations at these positive potentials. At more negative potentials the ratio decreases quickly to attain a value of ∼0.8 at the pzc of the Ag electrode (at ∼−0.9 V). This low value of the ratio indicates that the chains are predominantly in the trans conformation. The left panel in Fig. 7 plots the fragments of SERS spectra in the 1000–750 cm−1 region where the bands corresponding to the vibrations of the head group are located. Panel A plots again the differential capacitance curve for this electrode while panel B plots the I770 /I715 peaks intensity ratio that is a measure of the gauche/trans ratio of the O C C N+ fragment of the head group. Table 1 shows that in the solid state where the O C C N+ fragment is predominantly in the gauche conformation this ratio is equal to ∼4.4. In Fig. 7b this ratio is ∼2.7 for a broad range of potentials from 0 to −1.1 V and it drops down steeply at potentials more negative than −1.1 V where desorption of the bilayer from the metal surface takes place. This data shows that in the bilayer supported at the

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Fig. 10. For the hybrid h-DMPC/d-DMPC bilayer deposited at the TG SAM modified nanocavity patterned Ag electrode using the LB–LS method, Panel (a) plots the I2880 /I2850 peaks intensity ratio and Panel (b) plots the I1080 /I1125 as a function of the applied potential; curve 1 shows the hydrogenated leaflet as the bottom layer in contact with TG; curve 2 shows the hydrogenated leaflet as the top layer in contact with the electrolyte. Cartoons on the right illustrate the structure of the h-DMPC/d-DMPC bilayer; supporting electrolyte was 0.1 M NaF solution.

nanocavity patterned Ag, the head group region of the bilayer is a mixture of gauche and trans conformers. The amount of trans conformers increases when bilayer becomes detached from the metal surface. The SERS data indicate that the bilayer is best ordered at potentials corresponding to the minimum on the capacitance curve (between −0.7 and −0.9 V) in the vicinity of the pzc of the Ag electrode. The I2935 /I2880 ratio is high, indicating a good packing of the acyl chains and the I1088 /I1125 ratio is low indicating that chains have predominantly trans conformations. However, this bilayer is a mixture of the gel and liquid crystalline states in which the phosphocholine head groups are also a mixture of gauche and trans conformers. The SERS data also demonstrate that the broad maximum seen on the capacity curve at ∼−0.4 V is not related to the reorientation of the polar head groups of DMPC molecules but corresponds to the order/disorder transition in the acyl chains. In fact the position of the capacitance maximum correlates with the inflection point observed for the plot of the I2880 /I2850 ratio. The acyl chains are better packed and have less gauche conformations at potentials more negative to the maximum and are less ordered at potentials more positive to the maximum (close to the ocp). 3.2.3. EC-SERS of DMPC bilayers formed on the TG modified nano patterned Ag The differential capacitance curves presented in Fig. 5a and b demonstrated that the bilayer formed by fusion of unilamellar vesicles of DMPC on a TG modified nanocavity Ag electrode behaved differently than the bilayer formed directly on the bare metal. The EC-SERS spectra were therefore acquired to determine the differences between the structures of the two types of bilayer. The

SERS spectra for the C H and C C stretching regions are plotted in the left panels of Fig. 8 while Fig. 8b and c plot the ratios of I2935 /I2880 and I1088 /I1125 respectively. For the benefit of discussion of the SERS data, the differential capacitance curves for the bare electrode and the electrode covered by the SAM of TG and the bilayer are shown in Fig. 8a. The I2880 /I2850 ratio is approximately equal to 1.1 for potentials higher than −0.8 V. This value indicates that the bilayer is a mixture of gel and liquid crystalline states and that the packing of the chains does not change in the broad potentials range from 0 to −0.8 V. At potentials more positive than −0.4 V, the I2880 /I2850 ratio is higher in the bilayer deposited at the TG modified electrode than at the bare electrode (compare to Fig. 6b), indicating that the chain–chain interactions are stronger. The plot of the I1088 /I1125 ratio shown in Fig. 8c displays a maximum at ∼−0.5 V which is an indication of a significant increase in the number of gauche conformers in the acyl chains. This maximum correlates with the position of the small maximum on the differential capacitance curve. As mentioned earlier, such a maximum is also observed in the capacitance curve of the monolayer of TG without the bilayer (see Fig. 5b). Therefore, one can conclude that the melting of the chains in the bilayer observed at these potentials is induced by a reorientation of TG molecules. The left panel in Fig. 9 plots SERS spectra in the 800–500 cm−1 region. Panel 9a plots the differential capacitance curve and panel 9B plots the ratio of intensities of the 712 and 770 cm−1 bands, which is a measure of the conformation of the O C C N+ fragment of the phosphocholine head group. Recalling that for an all-gauche conformation in the solid state the ratio is equal to 4.4 (Table 1), the data in Fig. 9b show that this ratio is high in

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the whole range of potentials where the bilayer is adsorbed at the electrode surface, increasing slowly from a value of ∼3.5 at ocp to a value ∼4.4 at ∼−0.7 V. This result indicates that the head groups are assuming a predominantly gauche conformation. At potentials more negative than ∼ −0.7 V the ratio drops down steeply to the value of ∼1.9, indicating that in the desorbed state the head groups are a mixture of gauche and trans conformations. The LB–LS technique allowed for the investigation of hybrid bilayers in which one leaflet was composed of hydrogenated and the other from deuterated DMPC molecules. These experiments provided complementary information about the structure of individual leaflets of the bilayer. The values of I2880 /I2850 and I1088 /I1125 determined for the hydrogenated monolayers constituted the top and the bottom leaflets of the bilayer are plotted in Fig. 10a and b, respectively. The data indicate that both leaflets are in the LC state. However, the I2880 /I2850 ratios are higher for the bottom leaflet indicating that the chain–chain interactions are stronger in the leaflet that is in contact with the TG monolayer rather than in the leaflet that is in contact with the solution. This is an unexpected result since intuitively one would expect a better packing of the chains in the top leaflet that are in contact with the solution than in the bottom leaflet that is in contact with the solid support. In conclusion the SERS data indicate that the bilayer formed on the TG monolayer is somewhat more ordered than the bilayer formed on the bare Ag surface. Apparently, interactions between the hydrophilic head group of DMPC and hydrophilic TG molecule assist in ordering of the bilayer. In general, the SERS studies presented here demonstrated that the properties of the bilayer are affected by the TG monolayer present underneath the bilayer.

4. Summary and conclusions SERS and EC-SERS spectra of DMPC bilayers assembled at the nanocavity patterned Ag electrode were recorded to determine the physical structure of these bilayers and changes of this structure as a function of the applied potential. In order to investigate the anisotropic properties of the phospholipid molecules in the two leaflets independently, we have assembled hybrid bilayers in which one leaflet was composed of hydrogenated lipid while the second leaflet was made of deuterated lipid molecules. The SERS experiments demonstrated that at a temperature of ∼20 ◦ C, all investigated bilayers were in the liquid crystalline state or their state consisted of a mixture of the liquid crystalline and gel states. Differential capacitance measurements showed that lipid bilayers deposited on a monolayer of TG exhibit a different behavior than bilayers assembled on bare electrodes. The electrochemical and SERS data indicated that these differences are dictated by the properties of TG monolayer. Bilayers assembled at the thioglucose modified Ag were more ordered than bilayers assembled at the bare Ag surface. In the case of hybrid bilayers, the leaflet in contact with the thioglucose monolayer was more ordered than the leaflet in contact with the electrolyte solution. It was demonstrated in this work that a combination of electrochemical and SERS measurements can provide unique molecular level understanding of the potential dependent properties of phospholipid bilayers supported at solid electrodes. These results show that, consistent with theory, the surface enhancement is distance dependent and the intensity of bands in the SERS spectra of the top layer are about two times lower than in the bottom layer. This is a unique property of SERS and in the next paper we will use it to monitor potentialinduced translocation of a protein across this phospholipid bilayer.

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Acknowledgement This work was supported by a grant from Natural Sciences and Engineering Research Council of Canada (NSERC). JL acknowledges a Canada Research Chair Award.

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