Solvent effects on composition and structure of thiolipid molecular anchors for tethering phospholipid bilayers

Applied Surface Science 509 (2020) 145268

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Full Length Article

Solvent effects on composition and structure of thiolipid molecular anchors for tethering phospholipid bilayers

T

Saulius Tumenasa,b,⁎, Tadas Ragaliauskasa, Tadas Penkauskasa, Audrone Valanciutea, Filipas Ambruleviciusa, Gintaras Valinciusa a b

Department of Bioelectrochemistry and Biospectroscopy, Life Sciences Center, Vilnius University, 10257 Vilnius, Lithuania Department of Optoelectronics, Center for Physical Sciences and Technology, 10257 Vilnius, Lithuania

ARTICLE INFO

ABSTRACT

Keywords: Solvent effect Multi-component self-assembled monolayers Electrochemical impedance spectroscopy Spectroscopic ellipsometry Polarization modulation infrared reflection absorption spectroscopy

This article reports the effects of solvent polarity on the formation and properties of multi-component selfassembled monolayers (SAMs) for surface immobilization of tethered bilayer membranes (tBLMs). Monolayers were formed on polycrystalline gold using a synthetic 1-thiahexa(ethylene oxide) thiolipid serving as molecular anchor mixed with the β-mercaptoethanol backfiller. The properties of SAMs and tBLMs prepared in different solvents were monitored by the electrochemical impedance spectroscopy, spectroscopic ellipsometry and polarization modulation infrared reflection absorption spectroscopy. We found that the solvent polarity allows controlling the surface concentration of molecular anchors on a surface in the range from 9% to 57% while keeping constant adsorbate concentration in solution. A solvent polarity also determines the structural arrangement of the molecular components. A low polarity environment favours sparser accommodation of molecular anchors and a more compact arrangement of backfillers, while the high polarity solvent favours clustering of the hydrophobic polymethylene parts, which adopt more vertical orientation tilted ~25° to a surface normal. Both composition and structure affect both the physical and functional properties of tBLMs. In general, the nature of the solvents used in the assembly of SAMs used in tBLM technologies should be taken into account if specific functional features of tBLMs are being sought.

1. Introduction Multi-component self-assembled monolayers (SAMs) derived from the adsorption of thiolipids and short-chain diluting molecules on gold provide a platform for designing novel artificial bio-membranes for biological and bioanalytical applications [1–7]. The structure of multicomponent SAMs, composition, and distribution of thiolipidic anchor molecules on the substrate surface affects the properties of the tethered bilayer lipid membranes (tBLM). It is important to know and control the surface concentration, orientation and conformation of tethering and diluting molecules on the gold surface in order to understand and control the lateral packing density of the lipids in the artificial biomembrane. One of the most known methods for preparing multi-component SAMs is an immersion of the metal substrate into an assembly solvent containing a mixture of thiols at room temperature. The mole fraction of adsorbate in the mix-SAM reflects the mole fraction of adsorbate in solution and can be changed by a choice of solvent if SAMs are formed from a mixture of polar and nonpolar molecules [8–10]. Multi⁎

component SAMs have been widely studied and revealed a dependence on different terminal groups, relative ratios of components, chain lengths, formation kinetics, and wetting properties of the surface [8,10–22]. A multi-component monolayer may adopt a more complex conformation rather than a homogeneously distributed thiolate. It could form micro- or macroscopically separated islands containing specific regions of order and disorder [11]. Recent studies have reported an influence of solvents on the self-assembly of thiols, showing that polar and/or hydrophilic solvents (e.g. ethanol) reduce the quality of SAMs with a polar or hydrophilic tail group (e.g. eOH, eNH2, and eCOOH) [23–25]. Zhang et al. showed that on copper the alkane chains of thiols are more disorganized in low polarity solvents (e.g. n-hexane) than in high polarity solvents [26]. The choice of solvent is an important parameter for determining the resulting quality of SAMs deposited from the solution. The thiolipidic molecule comprises a hydrophobic lipid tail and a hydrophilic spacer which ends with a thiol end-group that anchors the thiolipid to a gold surface. This architecture separates the hydrophobic portion of the tBLM from the metal surface by a hydrated hydrophilic

Corresponding author at: Department of Bioelectrochemistry and Biospectroscopy, Life Sciences Center, Vilnius University, 10257 Vilnius, Lithuania. E-mail address: [email protected] (S. Tumenas).

https://doi.org/10.1016/j.apsusc.2020.145268 Received 22 November 2019; Received in revised form 30 December 2019; Accepted 2 January 2020 Available online 07 January 2020 0169-4332/ © 2020 Elsevier B.V. All rights reserved.

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carried out. 2.3. Tethered bilayer lipid membranes by vesicle fusion

Fig. 1. Structures of molecules used for the mix-SAMs.

For tBLMs completed with multilamellar vesical fusion (MLV) 1,2dioleoyl-sn-glycero-3-phospho-choline (DOPC) and cholesterol mixture at a molar ratio of 6/4 was used [6]. For fusion, the vesicles were diluted to a final concentration of 1 mM. Mix-SAMs were exposed to the vesical solution for 30 min and then rinsed with a buffer.

spacer. McGillivray et al. introduced an attractive model for the biological membranes assembling tBLM by using the 1-thiahexa (ethylene oxide) lipid, 20-tetradecyloxy-3,6,9,12,15,18,22-heptaoxahexatricontane-1-thiol (WC14, shown as molecule 1 in Fig. 1) diluted by the backfiller [1]. The role of a diluting molecule is in the first place to control the surface concentration of the main thiolipid component and to increase the hydration of submembrane space in tBLM. The WC14 molecule has a thiol end-group, the hydrophilic oligo(ethylene oxide) (OEG) segment, which provides physical separation of the membrane from the solid surface, and the hydrophobic tail region formed by a pair of C14 n-alkane chains. In this article, we show that a favourable organisation of the tethering SAMs may be achieved or lost depending on the multi-component SAM preparation conditions. The effect of solvent polarity on mixSAMs of a synthetic 1-thiahexa(ethylene oxide) lipid, WC14, mixed with β-mercaptoethanol (βME, shown as molecule 2 in Fig. 1), was investigated. Information about the composition, density, and conformation of the investigated mix-SAMs was deduced from the spectroscopic ellipsometry (SE), polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and electrochemical impedance spectroscopy (EIS). The selected solvents for preparation of mix-SAMs were nonpolar n-hexane, polar aprotic 2-propanol, ethanol, ethanol/ methanol (molar ratio 1:1), methanol, and polar protic dimethyl sulfoxide (DMSO) (Table 1).

2.4. Spectroscopic ellipsometry (SE) SE data were recorded on an RC2 ellipsometer (J. A. Woollam Co., Inc., Lincoln, NE) in 210–1700 nm spectral range at angles of incidence 40–80° from the surface normal. Calculations were made using vendorsupplied software (CompleteEASE 5.19). SE measurements were performed in the air (ex-situ) at room temperature. 2.5. Electrochemical impedance spectroscopy (EIS) EIS measurements were performed using an electrochemical workstation Zennium (Zahner, Kronach, Germany) between 0.1 Hz and 100 kHz with 10 logarithmically distributed measurement points per decade [6]. A saturated silver–silver chloride [Ag/AgCl/NaCl (aq, sat)] microelectrode (M-401F, Microelectrodes, Bedford, NH) as a reference had a potential of +196 mV versus the standard hydrogen electrode. The auxiliary electrode was a 0.25 mm diameter platinum wire (99.99% purity, Aldrich) coiled around the barrel of the reference electrode. Measurements were carried out with 10 mV alternating current at 0 V bias versus the reference electrode in aerated solutions. The EIS measurement cell holder contained 12 separate vials (the working surface area is ~0.125 cm2 of each vial) that allowed carrying out 12 individual experiments on the same gold plated plate glass substrate.

2. Experimental section 2.1. Gold film deposition For polarization modulation reflection absorption infrared spectroscopy (PM-IRRAS), and for electrochemical impedance spectroscopy (EIS) and spectroscopic ellipsometry (SE), 25 × 75 mm glass slides from ThermoFischer Scientific (UK) were used. Gold layers were deposited by magnetron sputtering using PVD75 (Kurt J. Lesker Co., U.S.) system. Chromium adhesion layer was 2 ± 0.5 nm, and a variable thickness layer of gold was deposited under real-time quartz microbalance control.

2.6. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) The PM-IRRAS experiments were performed using a Vertex 70 V (Bruker) spectrometer, equipped with an external table-top optical mount PMA50, MCT-A detector, with a photoelastic modulator (PM-90 with II/ZS50 ZnSe 50 kHz optical head, Hinds Instruments, Hillsboro, OR) and a demodulator. The spectra were gained and processed using Opus 6.5 software. The PM-IRRAS measurements were performed in the air (ex-situ) at room temperature.

2.2. Self-assembled monolayers The details on the synthesis and properties of the anchor molecules used in this work were described previously [1]. Deposition of the anchor molecules was carried out from a solution containing mixtures of WC14 and βME (total thiol concentration = 0.1 mM). The selected solvents preparing SAMs were n-hexane, 2-propanol, ethanol, ethanol/ methanol (molar ratio 1:1), methanol, and dimethyl sulfoxide. Incubation was carried out for 12 h at room temperature. After incubation, samples were cleaned using the same solvent as for incubation and dried in a nitrogen stream and immediately used in all experiments

3. Results and discussion 3.1. Electrochemical impedance spectroscopy of SAMs Electrochemical impedance spectroscopy was used to evaluate the structural and functional properties of mixed SAMs. Fig. 2 shows the EIS spectra (Cole-Cole plot) of SAMs prepared from a constant molar ratio of 30% WC14 and 70% βME in solutions of different organic solvents (n-hexane, 2-propanol, ethanol, methanol and dimethyl

Table 1 Relative dielectric permittivity (ε) of solvents. Best-fit parameters to EIS spectra of SAMs, Rsol = 26 Ω cm2. Uncertainties are obtained from the covariance matrices. The fitting was performed using ZView software in the Data-Proportional mode. Solvent

ε

CPESAM (μF cm−2 s(α−1))

αSAM

Rdefect (kΩ cm2)

CPEdefect (μF cm−2 s(α−1))

αdefect

χ2, 10−4

n-hexane 2-propanol Ethanol Methanol DMSO

1.89 19.9 24.3 32.6 48.9

10.7 ± 0.3 7.4 ± 0.2 6.4 ± 0.1 2.2 ± 0.1 1.0 ± 0.1

0.984 0.995 0.990 0.990 0.978

>10,000 >10,000 >6000 0.07 ± 0.01 5.2 ± 3

– – – 3.3 ± 0.2 5.2 ± 0.2

– – – 0.983 0.912

4.68 2.79 0.97 4.50 6.04

2

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SAMs with the specific capacitance <1 µF·cm−2 comprise a 1.5 nm thick compact hydrophobic monolayer. Assuming the relative dielectric constant 2.2 for the tethradecylmethylene sheet the estimate of the specific capacitance is 1.2 µF·cm−2, which is slightly above the observed value. The reason of a modest discrepancy may be the inaccurate value of the relative dielectric constant as well as a presence of a hydrated (4% mol. water) 1.6 nm thick (neutron reflectometry estimate [1]) reservoir between the hydrophobic slab and the solid surface which capacitance was not taken into account. The EIS curves (methanol and DMSO solvents) can be fitted with relatively good fit qualities, χ2 ≈ 6·10−4 to a Model B. The Model B includes the electric equivalent elements CPESAM and Rdef. The latter two cannot be straightforwardly related to the physical properties of a monolayer or tBLM [27,28], though CPESAM with α > 0.9 is presumably reflecting the magnitude of the Helmholtz layer of the system, and Rdef – the defectiveness of the dielectric sheet. Using recently developed mathematical formalism for tethered bilayers [28] randomly populated with the ion-conductive defects [29] one can estimate both the capacitance of the hydrophobic sheet and the Helmholtz capacitance of the interface located between the solid surface and the submembrane water reservoir. The defect density and mean defect size can be estimated with the precision determined by certain assumptions about the value of specific resistance of submembrane reservoir [29]. We performed such analysis for a spectrum of the SAM deposited from DMSO. The fitting reveals quite a large defectiveness of the WC14 layers. The defect density was found to be 1.33 ± 0.13 µm−2. The medium defect size of defects is 2.55 ± 1.88 nm. Large defect density explains a specific shape of the DMSO curve in Fig. 2, specifically, the low frequency semicircular “tail” clearly distinguishing DMSO spectrums from all other in Fig. 2. The defect density in such SAM should decrease upon the formation of the phospholipid overlayer, which we observed in the current study (vide infra). As seen from Table 2, the estimated capacitance of the tetradecylmethylene sheet is 0.79 µF cm−2, which is lower compared to the estimate discussed above, 1.2 µF cm−2. Lower, than expected value hints at the formation of the non-covalently bound overlayers of WC14, which were observed earlier by neutron reflectometry [30]. Such overlayers are replaced by the overlayers of phospholipid when SAMs are exposed to the vesicle solution, forming a hybrid bilayer (vide infra). The Helmholtz capacitance of SAMs deposited from DMSO was found to be 6.27 ± 0.11 µF cm−2. This value is approximately 50% less than the capacitance of a 100% βME (11.9 µF cm−2) [1], suggesting the anchor molecules are occupying approximately 50% of the gold surface. In the current study, this estimate was verified by the spectroscopic ellipsometry (vide infra). Taken together, the data in Fig. 2, Tables 1 and 2 suggests that a polarity of a solvent plays an important role in defining the molecular ratio of SAMs components WC14 and βME on the surface. This tentative statement is further supported using spectroscopic ellipsometry (SE) technique.

Fig. 2. Cole-Cole plots of electrochemical impedance spectra (dots), normalized to electrode area, of SAMs, prepared from the solutions of 30% WC14 and 70% βME in various solvents. Experimental curves were fitted to model equivalent circuits shown in the inset.

sulfoxide). All adsorption solutions used had a total thiol concentration of 0.1 mM, and incubation time was 12 h. The plots of SAMs exhibit near-ideal semicircular shape in the entire range of frequencies, except for SAMs prepared from DMSO, which exhibit two semicircles, and methanol with noticeably deformed semicircular spectrum. Semicircular shapes of the plots in Fig. 2 imply that the films exhibit nearly ideal capacitive behaviour typical for insulating dielectric layers, and were observed earlier in numerous studies [1,6]. The semicircular plots are of different radii. This hints a different effective thickness and composition of the dielectric layers deposited from different solvents, which is confirmed in this study using optical techniques (vide infra). The semicircular curves in Fig. 2 can be fitted to a simple model denoted as Model A (inset in Fig. 2). The fitting data to a Model A is summarized in the first three lines of Table 1. The SAMs deposited from the low polarity hexane exhibit high specific capacitance >10 µF·cm−2. The capacitance data in our earlier studies [1] indicate that such high values are typical for the SAMs deposited from ethanol containing low < 10% fraction of molecular anchor WC14. As the polarity increases (2-propanol and ethanol in Table 1), the specific capacitance decreases. The value of the specific capacitance 6.4 µF·cm−2 in the case of ethanol deposited SAMs match well with the earlier work by others for 30% WC14 anchors, 7.18 µF·cm−2 [6]. The EIS response from SAMs deposited from the high polarity solvents (DMSO) cannot be fitted to a Model A. Instead, Model B, which was used in fitting EIS plots of tethered bilayers [1] yielded satisfactory fit of the EIS data in our study. The small, semicircular part of the spectra is associated with the element denoted as CPESAM, which reflects the capacitance of a single trans-extended tetradecylmethyl region of the WC14 anchor packed in a planar, almost compact single layer. The formation of a compact alkyl chain layer can be witnessed by >105° contact angle values (data not shown) and the comparison of a theoretical estimate of capacitance with the observed experimental capacitance. The neutron reflectometry data [1] indicate the WC14

3.2. Spectroscopic ellipsometry of SAMs Spectroscopic ellipsometry was applied as a convenient and precise means of determining the multi-component monolayer thickness and constituent parts of the films. Variable angle spectroscopic ellipsometry

Table 2 Physical parameters obtained from the analysis of the EIS spectra using the theoretical framework described in Refs. [28,29]. Cm – the capacitance of hydrophobic sheet, CH – the Holmholtz capacitance, rdefect – the defect radius, Ndefect – the mean defect concentration, and χ2 – the goodness of fit. Uncertainties are obtained from the dispersion of experimental results.

DMSO

Cm (μF cm−2)

CH (μF cm−2)

rdefect (nm)

Ndefect (μm−2)

χ2, 10−4

0.79 ± 0.10

6.27 ± 0.11

2.55 ± 1.88

1.33 ± 0.13

0.11

3

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determined parameters are presented in Fig. 3. As seen, with an increase of solvent polarity the volume fraction of WC14 increases from ~14% to ~80% and the βME thickness decreases from ~0.6 nm to ~0.2 nm. Decrease of βME thickness can be related to a conformation change from trans to gauche. The increase of volume fraction of WC14 indicate, that solvent polarity directly influences WC14 concentration on a gold surface. Knowing the volume fraction of WC14 allows one to quantitate the molar fraction of the WC14 in the hydrophobic part, Xtether, and a fraction of gold surface occupied by WC14 anchor, Xanchor. The sulfur atoms of unfunctionalized long-chain thiolates on Au (1 1 1) form a commensurate hexagonal overlayer with an idealized (defectfree film) packing density of 21.4 Å2/thiolate [32,40]. The alkyl chain cross-sectional area is 18.4 Å2 and comparing to crystalline OEG in its idealized helical form has a larger molecular cross-section of 21.3 Å2, which is the same as for sulfur atoms cross-sectional area. Taking into account WC14 and βME molecules structures (Fig. 1) and SE data, fractions of Xtether and Xanchor were determined and tabulated in Table 3. As seen from data in Table 3, that the molar fraction of WC14 hydrophobic part (an important parameter in applications of tBLMs) strongly depends on solvent polarity and is like solution molar fraction in a mixture of ethanol and methanol with 1:1 M ratio. The fraction of occupied gold by WC14 show the relatively large amount of βME filling the void areas between OEG chains. Assuming relation Xlipids + Xtether = 1 we can estimate the fraction of the transferable lipids, Xlipids, in the proximal leaflet of the tBLMs.

Fig. 3. SE thickness of βME (black dots) and volume fraction of WC14 (red dots) with respect to the relative dielectric permittivity of solvents. The vertical error bars indicate the standard deviation. Inset shows the optical model used.

(SE) data were analyzed taking into account EIS results and using the three-layer optical model ([WC14 + void]/βME/gold) presented as an inset in Fig. 3. Refractive indices of WC14 and βME were determined independently from SE measurements using the Levenberg-Marquardt algorithm for nonlinear regression on pure WC14 and βME samples prepared from DMSO and n-hexane solvents, respectively. The determined refractive index values of WC14 and βME were 1.512 and 1.487 (at 632.8 nm wavelength), respectively. Simultaneously, all SE data from all samples investigated were analyzed assuming constant WC14 and βME layer thickness of dWC14 + dβME = 3.36 nm and fitting the volume fraction of voids and thickness of βME. A constant sum of WC14 and βME layers thickness was assumed, based on the energetically more favourable helical conformation of OEG and a planar approximation of two alkyl chains of WC14 molecule on the gold surface what allowed to reduce cross-correlation of parameters in the optical model. The assumption is valid for SAMs prepared from low and high polarity solvents, even if PM-IRRAS data show the random orientation of WC14 in SAMs prepared from low polarity solvents (e.g. hexane and 2-propanol in Figs. 4 and 6). Advantage of SE investigating thin films on opaque substrates is the versatility of optical model used, it allows to separate constituent parts of a thin film with satisfactory accuracy if refractive indices are known. The void and WC14 were mixed using the EMA approach [31]. The

3.3. PM-IRRAS of SAMs In order to evaluate the contribution of WC14 main-chain conformation, orientation, and packing to the changes in multi-component monolayer structure as a function of solvent polarity, PM-IRRAS measurements were performed. Fig. 4 shows the PM-IRRAS spectra of the CeH stretching region for mix-SAMs on a gold surface formed from different solvents for 12 h immersion. The CeH stretching modes can be used to determine changes in the conformation and orientation of the mix-thiol monolayers. The CeH stretching region is dominated by the bands of the alkyl chains. As seen in Fig. 4, the symmetric methyl stretching vibration (νs (CH3)) at 2878 cm−1 intensity increases with an increase of solvent polarity because of an increase of WC14 on a gold surface. The bands at 2958 cm−1 and 2967 cm−1 corresponds to the asymmetric out-of-plane and in-plain stretching of the methyl groups. Due to a different orientation of these modes with respect to the surface normal, out-of-plane stretching is perpendicular to CeCH3 bond and CeCeC catenary plane, while in-plane perpendicular to CeCH3 bond and parallel to CeCeC catenary plane. The ratio of these vibrations gives the approximate orientation of WC14 hydrophobic part related to solvent polarity. For low polarity solvents, the out-of-plane peak is dominant showing that WC14 molecules overlie on βME and vice versa for high polarity solvents. However, an observable in-plane peak for low polarity solvents may indicate partial clustering of WC14 molecules. The bands at 2855 cm−1 and 2917–2927 cm−1 are ascribed to the symmetric and asymmetric CH2-stretching bands of the C14 methylene units, respectively. The position of bands for high polarity solvents (DMSO and methanol) are close to 2850 cm−1 and 2917 cm−1 showing that well-ordered thiolipid SAMs are formed [33]. For the mixSAMs formed in low polarity solvents (n-hexane, 2-propanol), the peaks of νs (CH2) and νas (CH2) appear at 2857 cm−1 and 2927 cm−1 and the vibration intensities of CH2 are relatively strong, showing that the alkyl chains of SAMs are disordered. However, the intensity decrease of symmetric and asymmetric CH2-stretching bands with an increase of solvent polarity is different because of WC14 conformation or orientation changes. Due to the complex nature of this spectral region the νs (CH2) and νas (CH2) modes were selected for structural interpretation. The orientation of alkyl chains at the metal surface can be determined if the direction of the transition dipole can be related to alkyl chain geometry. The transition dipole of symmetric and asymmetric

Fig. 4. PM-IRRAS spectra in the 3025–2775 cm−1 spectral region of mix-SAMs prepared from the solution with a constant ratio of 30% WC14 and 70% βME using various solvents. Deconvolution of IR spectra and peak positions are presented by thin black curves. Spectra are shifted for clarity. 4

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Table 3 The molar fraction of WC14 in mix-SAMs prepared from different solvents. Where meOH:etOH represent a mixture of ethanol and methanol with 1:1 M ratio (ε = 27.7).

Xtether (hydrophobic part) Xlipids (in proximal leaflet) Xanchor (fraction of occupied gold by WC14)%

n-Hexane

2-Propanol

Ethanol

meOH:etOH

Methanol

DMSO

0.16 ± 0.03 0.84 ± 0.03 9±3

0.17 ± 0.03 0.83 ± 0.03 10 ± 3

0.21 ± 0.03 0.79 ± 0.03 12 ± 3

0.44 ± 0.03 0.56 ± 0.03 26 ± 3

0.82 ± 0.03 0.18 ± 0.03 47 ± 3

0.99 ± 0.03 0.01 ± 0.03 57 ± 3

methylene vibrations are perpendicular to each other and orthogonal to the main alkyl chain. The tilt angles of the transition dipole moments of symmetric and asymmetric methylene vibrations modes are θs and θas. The tilt angle of the main chain axis θtilt is related by [34]

cos2

s

+ cos2

as

+ cos2

tilt

= 1.

The tilt angles of the transition dipole moments can be determined by calculating the spectrum of randomly oriented alkyl chains from the isotropic optical constants using the following relationship [35–37,41]:

cos2

=

Aexp d 3

Aiso d

where Aexp is the measured absorbance of the WC14 film adsorbed on the gold surface and Aiso is the absorbance calculated from the optical constants for reflection from gold using Fresnel equations. The intensity ratio of νs (CH2) and νas (CH2) can also be used to extract information on a twist of alkyl chain about its axis. The twist angle of the planar alltrans conformation of hydrocarbon chains is defined as the angle between the CeCeC plane and the plane formed by the chain axis and the surface normal and is defined as [38]

= tan

1

Fig. 5. Alkyl chain tilt and twist angles with respect to the relative dielectric permittivity of solvents. The vertical error bars indicate the standard deviation. The dashed black line shows the angle of random orientation, the dashed red line indicates the trivial twist angle, which corresponds to no preferential twisting of the alkyl chains.

cos as . cos s

optical response in PM-IRRAS spectra from OEG chain methylene vibrations will be more pronounced. Twisted methylene groups may arise from a helical structure of OEG chain where methylene groups of OEG are twisted about the main OEG chain axis. In the fingerprint region, the IR spectra mainly consist of oligo(ethylene oxide) CeC and CeO stretches and CH2 wagging, rocking, and twisting. Useful information concerning the orientation and conformation of the oligo(ethylene oxide) spacer can be determined from CeOeC stretching modes related to the helical or extended structure having the transition dipole moment either parallel, A2, or perpendicular, E1, to the OEG chain axis. The assignment of the IR bands for the molten and crystalline OEG was taken from Refs. [42–44]. Fig. 6 shows PM-IRRAS spectra in the 1400–900 cm−1 spectral region for mix-SAMs and βME prepared from n-hexane. Deconvolution of βME spectra show main peaks corresponding to all-trans conformation, CeC stretching at 1042 cm−1, CeO at 1062 cm−1, OH bending at 1253 cm−1, and CH2 waging at 1270 cm−1 [45,46]. For mix-SAMs peaks related to βME are masked by peaks related to WC14, however for low polarity solvents CeO peak apparent and indicate a conformation of βME. For low polarity solvents in mix-SAMs, βME adopts trans conformation and with an increase of solvent polarity gauche conformation starts dominating, as was observed in SE data analysis. The spectra for mix-SAMs show strong absorption bands at 1149 cm−1 and 1119 cm−1 which are ascribed to CeOeC stretching vibrations of WC14 indicating a crystalline helical structure of OEG moieties. However the CH2-rocking band at 945 cm−1, a -twisting bands at 1249 cm−1 and 1296 cm−1, and wagging modes at 1325 cm−1 and 1352 cm−1 are all characteristics for amorphous oligo(ethylene oxide) [47]. The IR spectra of the mix-SAMs show both helical and amorphous OEG moieties. Recent studies have reported that the OeCeCeO unit of the tetraethylene glycol spacer in DPTL SAM’s exist predominantly in the gauche conformation, the spacer is coiled but in the disordered state [41]. The conformation of OEG chains is also influenced by their packing

For calculations of Aiso, a thickness and voids fraction determined from SE measurements were used. The optical constants of gold were taken from ref. [39], the optical constants for βME and WC14 were determined in the IR range from transmittance spectra (spectra not shown). To minimize systematic errors the same functions for band fitting has always been used to deconvolute PM-IRRAS and spectra calculated from the optical constants. Our estimate of the error in the tilt or twist angles is ~5 deg. The integrated intensities of the deconvoluted bands were calculated and the tilt and twist angles of the alkyl chains for mix-SAMs prepared using various solvents were determined. The results are presented in Fig. 5. The alkyl chain tilt (θtilt) decreases with an increase of solvent polarity, for low-polarity solvents the θtilt value is near to the random angle (54.7°) and for high-polarity solvents θtilt were ~22° showing a planar approximation of alkyl chains [40]. Similar tilt angle values were obtained by Leitch et al. investigating conformation and orientation of a self-assembled monolayer of DPTL (2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-DL-α lipoic acid ester). Leitch et al. showed that DPTL tilt angle should be between 29 and 25° assuming the alltrans conformations for phytanyl chains [41]. The twist angle Ψ is calculated to be of ~45° for low polarity solvents, indicating no preferential twist, however with an increase of solvent polarity the twist angle decreases suggesting preferential twisting of alkyl chains. It is noteworthy that the difference in the conformation of the alkyl chain exists and depends on solvent polarity. With an increase of solvent polarity, WC14 molecule change orientation from random to ordered clusters with planar approximation. Decrease of twist angle indicates rather curved and twisted alkyl chains in the high polarity solvents, or this may be the contribution from OEG chains, which in crystalline OEG form have a helical structure. Taking into account that transition dipole moment of methylene vibrations of alkyl chains in the planar approximation is almost perpendicular (tilt angle ~22°) to surface normal, the 5

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Fig. 7. Cole-Cole plots of electrochemical impedance spectra (dots), normalized to electrode area, of DOPC-completed tBLM, prepared on mix-SAMs with a constant ratio of 30% WC14 and 70% βME. Experimental curves were fitted to a recently developed analytical algorithm [29].

defectiveness of tBLMs deposited on anchors from different solvents. Different defect densities should also inversely correlate with the densities of the anchor SAM, which as it follows from the data in Table 3 indicate an increase from 9% to 57% in the solvent sequence nhexane → DMSO. To assess the surface density and size (average value) of defects in tBLMs we solved the inverse problem using recently [29] developed an analytical algorithm for modelling EIS spectra of tBLMs heterogeneously populated with the nano-sized defects. The fitting to a model data is tabulated in Table 4. As expected, the vesicle fusion caused a significant, almost 1000fold decrease of defectiveness in the SAM formed from the DMSO, from 175·10−3 µm−2 to 0.26·10−3 µm−2. In general, the increase of the surface density of WC14 anchors results in a decrease of defectiveness in tBLMs from 4.25·10−3 µm−2 to 0.26·10−3 µm−2 (Table 4, column 4) in the sequence of SAM deposition solvents: n-hexane → DMSO. Such an effect can be expected not only because the surface density of the SAMs in this sequence increases but also, because of the decrease of exchangeable lipid in the proximal leaflet of tBLM. For example, the fraction of exchangeable lipid in SAMs deposited from DMSO is only 1%. In such case, the proximal to a surface anchor layer contains mostly dimiristoyl (C14) chains. Thus, the bilayers deposited on DMSO SAMs should be regarded as hybrid bilayers, in which a proximal to surface leaflet consists of saturated well-ordered polymethylene chains with the tilt angle of 22° as detected from PM-IRRAS (vide ultra). Atop of this, presumably, a solid-like monolayer, the layer of DOPC/CHOL (40%) mixture is deposited via the hydrophobic interaction. Such hybrid systems are known as extremely highly insulating and low defect density, but may not provide the necessary flexibility for functional reconstitution of membrane proteins [1]. While the density of defects varies significantly, the mean size of defects remains mostly constant with a varying radius of defects within the interval from 2.9 to 4.7 nm (assuming radial symmetry of the defects). Such similarity hints at the common origin of defects on the surface, which may be related to random defects in the surface lattice of a gold substrate. As seen from data in Table 4, the electric capacitance of bilayers indicates a modest downward trend in the solvent sequence from hexane to DMSO. As mentioned, this is the direction in which surface becomes more and more populated with saturated anchor molecules. Such a trend of Cm is expected because proximal to a surface saturated polymethylene sheet exhibits lower relative dielectric constant values than an unsaturated distal sheet of DOPC/CHOL. A simple estimate based on the structural data from neutron reflectometry [1] with dC14 = 1.5 nm and dDOPC = 1.6 nm, and εC14 = 2.2 and εDOPC = 2.8, yields a theoretical value of capacitance 0.69 µF cm−2. This estimate is

Fig. 6. PM-IRRAS spectra in the 1400–900 cm−1 spectral region of mix-SAMs prepared from the solution with a constant ratio of 30% WC14 and 70% βME using various solvents. Additionally, deconvoluted spectra of βME is shown. Peak positions are presented by dashed black curves and spectra are shifted for clarity.

density. When a mix-SAMs are prepared from high polarity solvents WC14 molecules are closer packed (molar fraction of WC14 prepared from DMSO in the hydrophobic part is of about 99%) and the two bulky alkyl chains (cross-sectional area of two alkyl chains is 45 Å2) provide sufficient space for OEG chains to coil. Consequently, the OEG spacer is coiled, while in the spacer layer the chains are packed irregularly and form a disordered structure. Gaps on a gold surface between OEG chains are filled with βME molecules (molar fraction of occupied gold by WC14 prepared from DMSO is of about 57%) with dominating gauche conformation. 3.4. Electrochemical impedance spectroscopy of tethered bilayers Mixed SAMs prepared from different solvents were used to form tethered bilayers by fusion of DOPC/CHOL(40%) vesicles. Exposure of SAM-coated Au/glass plates to vesicle solutions resulted in the EIS spectra shown in Fig. 7. An almost complete semicircular feature with the specific capacitance of 0.67–0.78 µF cm−2 in the high-frequency part of the EIS curve attests for the formation of complete tethered phospholipid bilayers in all cases. Some differences, however, can be seen in the spectra of different SAMs. SAMs deposited from the high polarity solvents (methanol and DMSO) resulted in EIS spectra, which are nearly perfect semicircles with very little “tails” in the low-frequency range of the spectra. While SAMs obtained from hexane, propanol and ethanol, which exhibited nearly ideal semicircular shape of EIS curves in Fig. 2, after vesicle fusion yield a complex-shaped spectrum with extended low-frequency parts (“tails”, [48,49]). Length of the low frequency “tails” is proportional to the defectiveness of tBLMs as was established by the finite element analysis modelling of the EIS spectra [48,49]. So, the spectra in Fig. 7 suggests a different level of 6

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Table 4 Best fit parameters of experimental EIS curves of vesicle fused tBLMs Cm – the capacitance of phospholipid sheet, CH – the Holmholtz capacitance, rdefect – the defect radius, Ndefect – the mean defect concentration, and χ2 – the goodness of fit. Uncertainties are obtained from the dispersion of experimental results. Solvent

Cm (μF cm−2)

CH (μF cm−2)

rdefect (nm)

Ndefect 10−3 (μm−2)

χ2, 10−4

n-Hexane 2-Propanol Ethanol Methanol DMSO

0.78 0.76 0.67 0.70 0.70

8.09 8.41 6.65 6.89 6.50

4.7 2.9 4.7 3.4 4.1

4.25 3.33 4.03 0.28 0.26

10.7 7.54 2.02 3.73 9.70

± ± ± ± ±

0.03 0.01 0.03 0.01 0.01

± ± ± ± ±

0.1 0.03 0.42 0.64 0.58

very close to the observed specific capacitances for tBLMs deposited on methanol and DMSO SAMs (Table 4). As the surface concentration of anchor decreases (down to 9% for n-hexane SAMs), the proximal leaflet is becoming populated with unsaturated DOPC/CHOL lipids. Replacing εC14 = 2.2 with εDOPC = 2.8, and dC14 = 1.5 nm with dDOPC = 1.6 nm leads to an estimated value of the capacitance 0.75 µF·cm−2, which is pretty close to observed values of capacitance in n-hexane (0.78) and propanol (0.76) deposited SAM systems. Such qualitative and quantitative correspondence of the experimental electric capacitance to theoretical estimates confirm the possibility to finely tune the structure and functional parameters of tBLMs by properly choosing deposition solutions of anchor SAMs. Finally, the Helmholtz capacitance obtained from fitting also indicates a slight downward trend, following an increase of anchor surface concentration in the range from n-hexane (9%) to DMSO (57%) (see Table 3). Such a trend is expected and qualitatively reflects an increase of organic material content of the interface as established by the SE (Fig. 3, and data in Table 3). Interestingly, quantitatively CH measured in tBLMs (Table 4) and direct measurements of CH on pure SAMs match quite poorly. For example, CH in tBLMs never reached the values observed for pristine SAMs (hexane solvent: 8.1 μF cm−2 in tBLMs and 10.7 in pristine SAMs). This suggests that the contact with water and the fusion of vesicles triggers massive structural reorganizations of anchor SAMs, including both anchor WC14 and βME molecules. These structural variations presumably explain specific CH values in tBLMs and pristine SAMs. Currently, there are quite limited experimental options for the real-time measurements of these changes by the IR techniques, in the process of vesicle fusion. Quite likely SEIRA is one of the perspective options. Knowing the molecular level details will provide important data for the directed design of a phospholipid bilayer based biosensors. Such an experiment is being designed in our group.

± ± ± ± ±

1.3 2.3 0.7 1.1 1.4

± ± ± ± ±

0.34 0.60 0.42 0.04 0.07

One of the major findings of the current work is a dependence of the tBLM defectiveness on the nature of solvents. Because of differences in surface concentrations of the molecular anchors solvent polarity allows controlling the defect concentration in tethered bilayers. High defect density is typical for tBLMs on SAMs formed from the non-polar solvents, such as hexane. On the other side of the spectrum, tBLMs formed on SAMs from polar solvents exhibit more than tenfold lower defectiveness. However, the specific capacitance of such tBLMs indicates a possibility of the formation of hybrid bilayers, which may not be suitable for functional reconstitution of proteins. Therefore, to produce a stable, low defect density, biologically relevant tBLMs one needs to fine-tune the polarity of the SAMs deposition solutions, which is possible given a wide range of organic liquids applicable for such purposes. Ellipsometry results show that concentration of adsorbates on a gold surface can be controlled in the wide range from 10% to 60% by adjusting the solvent polarity while keeping constant concentrations of adsorbing species in solution. The current study also revealed the conformational variations of βME molecules in mixed SAMs deposited form different solvents. The vibrational spectroscopy data show that βME adopts trans conformation which is consistent with more densely packed arrangement and increased thickness as detected by the spectroscopic ellipsometry. These variations may explain sometimes counterintuitive variations of the electrochemical capacitance data. In particular, the exposure of pristine SAMs to an aqueous solution of vesicles and triggered by this exposure fusion of vesicles should increase the Helmholtz capacitance because of expected increased hydration in a water environment. However, in experiments, the opposite is observed for SAMs deposited from low polarity hexane. Such unexpected variation of CH upon vesicle fusion is likely related to the structural rearrangement of backfiller molecules leading to a more compact and a thick layer consistent with observed CH decrease. The effect is more pronounced in SAMs deposited from hexane because the concentration of backfillers, in this case, is the highest as estimated by the SE. The PM-IRRAS data also show that an increase of the solvent polarity changes OEG conformation in molecular anchors from amorphous to mixed helical-amorphous moieties, while alkyl chains experience a change from random orientation to tilted ~25°. With an increased concentration of thiolipid molecules, the OEG chains show preferential twisting due to an optimization of van der Waals contact or due to a steric effect or both. In summary, we emphasize that the polarity of solvents used for the deposition of molecular anchors for tBLMs not only affect the concentration of the components on the surface but also their structural arrangement. However, despite quite obvious structural differences of molecular anchors tBLMs obtained by vesical fusion showed relatively modest variations of their electrical properties except for their defectiveness. The latter is one of the essential parameters for practical applicability of tBLMs as biosensors for membrane damaging agents. So, the directed development of tBLM based devices and their utility in applications may be more effective if the polarity of solvents from which the thiolipid anchors are formed is taken into account.

4. Conclusions Chemical nature of solvents has a profound effect on composition, structure and process of formation of multi-component thiol SAMs on a gold surface. The results of this study, in which n-hexane, 2-propanol, ethanol, methanol and DMSO were used as solvents, confirm that the solvent polarity is one of the effective instruments to control the ratio of WC14/βME on the gold surface. Different polarity is clearly affecting the chemisorption kinetics of thiols. High polarity solvents such as DMSO and methanol favours relatively polar component of SAM mixtures, i.e., βME, while the less polar component, WC14 tends to be expelled from solution, thus resulting in high concentrations of anchor molecules on the surface. In non-polar solvent the opposite is true. Such solvent favours less polar component WC14, while the polar βME is forced to the surface, thus yielding a low surface concentration of molecular SAMs. In those two cases, the same concentration (30%, mol) solutions of WC14 in DMSO and hexane results in surface concentration of WC14 57% and 9% correspondingly as established from the SE data. The values of the electrochemical capacitance of SAMs deposited form different solvents qualitatively match the trend. Specifically, high capacitances are typical for low surface concentrations SAMs from nonpolar solvents (hexane and propanol) and vice versa.

CRediT authorship contribution statement Saulius Tumenas: Conceptualization, Methodology, Investigation, 7

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Writing - original draft. Tadas Ragaliauskas: Investigation, Validation. Tadas Penkauskas: Methodology, Investigation. Audrone Valanciute: Methodology, Investigation. Filipas Andriulevicius: Formal analysis, Software. Gintaras Valincius: Conceptualization, Writing - review & editing, Supervision.

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