Electrodes for membrane surface science. Bilayer lipid membranes

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Electrodes for membrane surface science. Bilayer lipid membranes tethered by commercial surfactants on electrochemical sensors. Ophélie Squillace, Charles Esnault, Jean-François Pilard, and Guillaume Brotons ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00267 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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ACS Sensors

Electrodes for membrane surface science. Bilayer lipid membranes tethered by commercial surfactants on electrochemical sensors. Ophélie Squillace, Charles Esnault, Jean-François Pilard and Guillaume Brotons*. IMMM, Institut des Molécules et Matériaux du Mans, Université du Maine - UFR Sciences et Techniques, Avenue Olivier Messiaen, 72085 Le Mans, France. Membrane Biosensors; Electrochemical Impedance Spectroscopy; Biomimetic Lipid Membrane; Sparsely Tethered Phospholipid Bilayer; Non-Ionic Diblock Copolymer and Oligomeric Surfactants, Brij and CiEj.

Commercial surfactants, inexpensive and abundant, were covalently grafted to flat and transparent electrodes and it appears to be a simple functionalization route to design bio-membrane sensors at large scale production. Sparsely tethered bilayer lipid membranes (stBLM) were stabilized from such molecular coatings composed of diluted anchor-harpoon surfactants that grab the membrane with an alkyl chain out of a PEGylated-hydrogel layer which acts as a soft hydration cushion. Avoiding the synthesis of complex organic molecules to scale up sensors realization was achieved here by grafting non-ionic diblock oligomers (Brij58: CxH2x+1(OCH2CH2)nOH with x=16 and n=23), as well as PEO short chains ((OCH2CH2)nOH with n=9 and n=23) from their hydroxyl (-OH) end-moiety to a monolayer of -Ar–SO2Cl groups, easy to form on electrodes (metals, semi-conducting materials, …) from aryl-diazonium salts reduction. A hybrid molecular coating on gold with scarcely Ar-SO2-Brij58 and PEO oligomers, was used to monitor immobilization and fusion kinetics of DOPC small uni-lamellar vesicles (SUV) by both quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) techniques. Using flat and transparent thin chromium film electrodes, we designed biosensors to couple surface sensitive techniques for membranes, including: X-ray reflectivity (XRR) and atomic force microscopy (AFM) with sub-nanometer resolution; optical microscopy such as fluorescence recovery after photo-bleaching measurements (FRAP) in addition to electrochemistry techniques (cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS)). The advantages of this biomembrane sensing platforms is discussed for research and applications.

Phospholipids assemble the backbone of biological membranes and their simplified biomimetic models, such as lipid vesicles, black lipid membranes and solid supported bilayers were established to mimic and study these fundamental architectures. Biomimetic membranes were designed to assess the mechanisms of regulation of ionic channels and other transmembrane proteins1-2. At the solid-liquid interface, supported membranes are good models to design sensors for the study of adhesion and cell interaction.3-5 Those latter represent a particularly advantageous platform, because they are stabilized and amenable to characterization by a wide range of surface-sensitive techniques6. They are currently by far the most used cell membrane models especially with the development of hydrophilic surfaces such as polymer cushions with diluted synthesized lipid7-9 or amphiphilic10 anchors which enable their partial immobilization at the same time as it favors their fluidity5. Such tethered membranes11, provide a platform to study membrane proteins’ functions, ion transport, pores, that will benefit the understanding of fundamental mechanisms. In these cases, an interstitial aqueous volume between the membrane and the substrate is a prerequisite12. Thus, stBLM became a promising model systems but suffered from the difficulty in synthesizing the “anchor-harpoon” molecules and in controlling their den-

sity, as well as the hydration of the cushion that prevents membranes from pinning to the solid13-14. The chemical structure of the spacer of commonly used anchor-lipids is mainly based on ethylene oxide oligomers bound to lipids which requires cumbersome chemistry14-15. As the outreach of fundamental studies have extended to applications, transmembrane proteins are involved in a wide range of fundamental physiological processes such as: smell16 that can be exploited for the development of supported olfactive sensors; antibiotics targeting the cell membrane itself are developed for novel antibacterial treatments that could reduce the development of drug resistance17; organ-on-chips have held the attention as promising human in vitro functions mimicking human normal and pathological physiology within sensor platforms that have high measurement accessibility.18 In this regard, new routes for biomimetic membranes on electrodes are needed for the development of biosensors on a large scale. Grafted on conductors and semi-conductors, inexpensive and available in large quantities, commercial surfactant molecules are particularly promising and well adapted to biosensors applications. We previously demonstrated that Brij or CiEj and PEO chains can be easily grafted from their mixtures in solution, and lead to different electrode surface states19. In the present paper we tested their tethering ability from the insertion of an alkyl chain in the

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hydrophobic bilayer’s core, while keeping a hydrated layer underneath by diluting those anchor-harpoon surfactants with shorter chain hydrophilic backfilling polymers. Lipid bilayer vesicles were formed, deposition and fusion on flat electrodes was studied without drying steps. The kinetics of formation has been monitored by QCM20 and SPR21. EIS was used for investigation of the membrane properties and surrounding ions dynamics since it can provide valuable insight and direct field of application and study. The electrodes supporting the tethered membrane were also designed from chromium in order to carry out structural investigation of the bilayer and of the hybrid coatings using scattering and simultaneously electrochemical techniques. Our aim to combine several experimental techniques on the same biosensor has imposed certain constraints, like a low electron contrast of the surface with membrane in order to carry out structural investigation with x-ray reflectivity, which is impossible on high electron density electrodes such as gold 19. Optical transparency was also imposed for FRAP experiments to measure the lateral lipids dynamics using fluorescent lipid probes inserted in the membrane. First fluorescence experiences are shown here to underpin the versatility of techniques that can be used to characterize the membrane. Besides, as models of biomimetic membranes are expanding, development in the field of (micro)fluidics play an important role as it can offers unprecedented control over culture conditions, provide stimuli such as chemical gradients22, spatial homogeneity23, time-dependent biochemical stimulations, and substrate mechanical properties 2425 . In the effort to deploy several techniques simultaneously on a same substrate and therefore membrane, experiments were carried out in a milli-fluidic cell that was designed for performing EIS and neutron reflectivity (NR) experiments.

EXPERIMENTAL SECTION Chemicals and Materials. Ethanol (EtOH, 99.8%), acetone (99.5 %), dichloromethane (99.9%), acetonitrile (99.8%), methanol (99.7%), hydrochloric acid (HCl, 37%), sodium nitrite (NaNO2), Brij58: (C16H33(OCH2CH2)20OH), PEO9-CH3 (H(OCH2)9CH3), PEO23-OH (H(OCH2)23OH), 1,2-Dioleoyl-cnglycero-3-phosphocholine (DOPC) and a chain-labelled 18:112:0 phosphatidylcholine (NDB-PC) were purchased from Sigma–Aldrich (St Quentin Fallavier, France) and used without further purification. Milli-Q water (Versol, Aguettant, Lyon, France) were used. Electrodes and sensors elaboration. Lipid membranes were tethered to three types of working electrodes: 1) commercial gold coated quartz for microbalance experiments (from BiologicsTM, optical quality sensors). The gold thin film covers a cylindrical patch of 0.25cm radius (SWE=0.196 cm² and RMS = 1.2 nm). The specific cleaning protocol developed previously19 was used prior to the QCM gold sensors experiments to reduce the dispersion of the EIS data; 2) SPR biochips coated with a 28nm thick gold layer on top of a 2nm thick layer of chromium; 3) chromium thin films of 8 to 30 nm thick, evaporated 19 on microscopy cover glasses (thickness ~170 m and optical index ~1.5 from Cole-PalmerTM) with a functionalized area SWE=2.25cm² defined from the electrochemical cell geometry, roughness RMS  1 nm and conductivity 0.5.103 -1cm-1. All electrodes were functionalized identically via a surface chemistry which is effective on gold, chromium and chromium oxides

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(Figure 1A)19. The electrode reactivity was obtained by the reduction of aryldiazonium salts that were formed in-situ from sulfanilic acid (5 mM) in the presence of one equivalent of NaNO2 in HCl (1 M) aqueous solution. Then -Ar-SO3H surface groups were changed to -Ar-SO2Cl by immersion in a PCl5/CH2Cl2 solution (5 mM). This -SO2Cl group is known to strongly react with weak nucleophilic nitrogen or oxygen groups, to form respectively sulphonamides or sulfonic ester linkers. Activated electrodes were dip in an acetonitrile solution of solubilised surfactants at 8 10 -5 M. Long dips of 17 to 25 hours were used with Brij58, or PEO or in mixtures of both (then keeping the total molar concentration fixed but varying molar ratios: Birj/PEO: 25 %, 50 % and 75 %). Some electrodes were also prepared using a first long dip in a 100% Brij58 solution, then rinsed in acetone and dried before a second dip in a 100 % PEO solution for a day. Membrane deposition via fusion of SUVs. A single BLM was formed using the spontaneous fusion of SUVs on the electrodes 26-27. DOPC lipid, dispersed in chloroform was first evaporated and pulled under vacuum for 4 hours, and rehydrated in NaCl 150mM overnight to get a concentration of 1mg/mL. The solution was then sonicated at 60°C for 20min and filtered through a membrane with pore size of 200nm (PALL LIFE SCIENCES GHP Acrodisc GF 13mm syringe filter). Lightscattering measurements (DLS) showed that stable vesicles with a typical size of  80 ±15 nm were obtained and could be used over weeks when stored at 4 °C. For FRAP experiments, the SUVs in suspension were prepared mixing DOPC with 3 % of NDB-PC, a chain-labeled 18:1-12:0 phosphatidylcholine. The SUVs solutions were gently injected in the flow cells and let in contact with the functionalized surface for at least 4 hours before thorough rinsing with the solvent used afterwards to eliminate SUVs in excess (NaCl 0.15M). Quartz Crystal Microbalance. The kinetics of vesicles adsorption were monitored using a standard QCM setup (Princeton applied research, model QCM922 supplied by Bio-Logic) that measures the SUVs immobilized mass per unit surface on the previously described QCM sensors. The data was set to pg/mm2 units following the data analysis described in the SI II). Surface Plasmon Resonance. The same functionalizations were obtained on the gold SPR sensors and kinetics of formation of a single tBLM were measured from DOPC vesicles fusion using a HORIBA scientific SPR-imager 28 in the Kretschman’s configuration. Small changes in the refractive index close to the water/gold interface were monitored through the angular shift of total internal reflection of light from the back of the SPR sensor. The data also translates to pg/mm2 units as described in the SI II). AFM, CA, XRR and FRAP. The functionalized sensors covered with a single stBLM were also studied after drying using AFM, Contact angle measurements, X-ray reflectivity and FRAP. These methods are described in the SI III) and IV). Electrochemical impedance spectroscopy (EIS). EIS experiments were carried out at room temperature with a three electrodes flow cell connected to a potentiostat/galvanostat model VMP2/Z system (Bio-LogicTM). Non-faradaic measurements were carried out in NaCl 0.15 M. A 25 mV amplitude sine wave was applied to the working electrode at 0 V bias versus the reference electrode (a saturated silver-silver chloride [Ag|AgCl|NaCl(aq,sat)] microelectrode from Bio-LogicTM,


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ACS Sensors model RE-3V) that was screwed in between the counter and the working electrode (chromium-coated substrate). For the auxiliary electrode, a 1cm² platinum electrode was set at 5 mm from the working electrode. The frequency range goes from 0.1 Hz to 200 kHz and was always scanned from high to low frequencies. The high resistance of cell (Rs) gives a restricted access to the high frequencies domain (f < 𝑓c2 = 1⁄(R ci R S Q⁄(R ci + R S ))1⁄𝛼 2π above the impedance that follows Rs). Data were fitted using a homemade Matlab program and theoretical models described below. Data are presented in the Nyquist plot (Im(C) vs Re(C), where C=C(ω) is the frequency-dependent capacitance of the electrode where ω is the angular frequency, ω=2πf, with f the frequency in Hertz). All the data could be well fitted using the Rs(Rci-CPEdc) equivalent circuit in non-faradaic mode, as described in the SI I).

RESULTS AND DISCUSSION The sensors surface chemistry used to build the mixed surfactant coatings is described by the scheme in Figure 1A and was well characterized previously 19. It is based on the covalent bonding of non-ionic Brij surfactants and short PEO chains on the functionalized sensor surface. Brij58 harpoon-anchor molecules, or PEO hydrogel forming molecules or in mixtures of both (varying here molar ratios: Birj58/PEO: 25 %, 50 % and 75 %) were grafted on thin film chromium ultra-flat electrodes on glass. These coatings prove to be able to stabilize a single lipid bilayer whose electrochemical properties and structure are the object of this paper. Tethered membranes on Brij functionalized gold QCM and SPR sensors. SPR experiments were conducted to measure the kinetics of the membrane formation using SPR gold films functionalized with the hybrid coating (Au-Ar-SO2-Brij58 completed with PEO). Figure 1B shows the mass intake after contact with DOPC SUVs of 75 nm radius in NaCl 0.15M solution (red line). The gain in mass reached a plateau at 4107.7  479 pg/mm². Considering the molar mass of DOPC molecules, MDOPC = 786.15 g/mol and a head-group area SDOPC = 0.725 nm² at 20°C 29-30, a single completed bilayer corresponds to m1Bi-DOPC = 3601.2 pg/mm². Thus, the SPR signal saturates at Nbi = 1.14  0.13 bilayers, in good agreement with a single stBLM considering that the head-group area, molecular density and SPR sensor active area used for the calculation come with such uncertainty. The presence of extra material, such as adsorbed non fused vesicles and/or multi-layered surface patches in very small amounts, cannot be excluded. We also measured the membrane forming kinetics on gold QCM sensors functionalized identically. The SUVs were let into contact with the surface for an hour and the solvent was gently flushed out with NaCl solution in order to remove possible closely floating vesicles (vertical line in the data curve). Figure 1B shows a plateau mass intake of 4069  138.4 pg/mm² (black line) that also corresponds to a single lipid bilayer (N bi = 1.13  0.05). Reimhult et al.31 commented the complementary aspects of both techniques. The SPR evanescent wave probes the optical index roughly over 100 nm and its variations translate in a gain in mass at the interface. The probed depth is comparable with the diameter of non-fused vesicles so that the SPR signal does not count properly the mass of large analytes and the SUVs inner solvent does not contribute to the effective SPR signal either. At the opposite, the SUV mass signal from QCM

measurements comes from variations of the quartz oscillating frequency and includes the lipids plus the inner and bound water molecules32-33. Thus, the QCM signal of 2-3% of intact vesicles at the interface could be misinterpret as the mass of a stBLM, which would give a negligible signal in SPR. The fact that both techniques gave the same mass plateau confirms that a stBLM formed and excludes the hypothesis that a layer of non-fused vesicle remained. The presence of membrane was easily confirmed by CA (Figure S-1) and AFM (Figure S-2) since the tBLM is destabilized once exposed to air leaving typical patterns of dry membrane on the surface with hydrophobic side of the lipid from the upper leaflet exposed to air. EIS detection of the membranes. The EI spectra measured in non-Faradaic mode in NaCl 0.15M are plotted Figure 1C and best fit parameters are reported first block Table 1. The coatings electrochemical behaviour was extensively studied previously19 and we recap some results in the SI for bare and functionalized sensors without membranes. The PEO9-CH3 coating is the more compact, homogeneous and thin, as expected. It forms a hydration layer, corresponding to the dielectric capacity gap of our model circuit, which is hard to go through for the ionic species of the double layer that accumulate at the interface under Efield. In comparison, the coverage of pure Brji58 is much sparser but offers a thicker layer with possible diffusion zones for the double layer ions, due to its hydrophobic alkyl chains and longest molecule. Even at high fractions of Brij58, interfacial capacities are 2 to 20 fold higher (Q~25µF cm-2 s(-1)) with a lower resistance Rci (of few hundreds of k cm²) than the values drawn from the harpoon-anchor molecules synthetized in the literature (PEO-lipid SAMs34-36 eg. WC14: PEO6-(-O-C14H29)2, FC16: PEO6-(-O-C14H29)2). In contrast, Brij58 molecules do not form a dense monolayer and have a higher relative permittivity so that sparse Brij58 plus PEO coatings form much more hydrophilic coatings than the lipid-PEO SAMs cited. The dynamics of ions near the functionalized electrode are greatly affected by the presence of DOPC SUVs, by their fusion, and the formation of a single stBLM. The EI spectra of the 6 types of surface: Cr-Ar-SO2Cl; Cr-Ar-SO-(Brij58x:PEOx-1) with x=0; 25%; 50%; 75%; 100%; after contact with SUVs were recorded over time and are plotted in Figure 1 C2-4. The best fit parameters of the data are shown Table 1. A complete defect-free lipid membrane would act as a pure capacitor, but charge transport can take place across the bilayer through defects in the membrane, modeled in the form of a membrane resistance that decreases with the presence pores. Two main behaviours stand out regarding the lipid bilayers formed on the six different surfaces. One embraces pure PEO and Brij5825:PEO75 (x=25%) functionalized surfaces (respectively right triangles yellow and left triangle rose curves) which are the most hydrophilic systems, the other includes Brij58x:PEOx-1 (x= 50%, 75%) and pure Brij58 surfaces (respectively diamond violet, down triangle red, up triangle green curves). The EI spectra from the first category (pure PEO type) do not significantly change over time. The spectra after rinsing are similar to those before contact with SUVs. They depict a Rs(RciCPE) behaviour that is very close to the one of a RS CPEdl circuit specially because of the main capacitive regime (high Rci values, >721.9 k cm² for pure PEO) with fewer leakage currents than the more diluted systems. One can notes that for all


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of these surfaces, an increase in resistance and decrease in capacitance after SUVs injection were recorded and are in agree-

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ment with what other authors have observed during vesicles fusion due to the additional thickness of the lipid bilayer newly formed37.

Figure 1. A) Scheme of the functionalization leading to the covalent binding of PEO n and Brij58 B) QCM and SPR adsorption kinetics of DOPC SUVs on a hybrid coating of Ar-SO2-Brij58 and PEO graft to a functionalized gold electrode. The vertical line in the QCM curve corresponds to rinsing stage after which the stBLM only remains. The sketch illustrates the surface functionalization and shows a single membrane tethered on the coated electrode. C) Complex plane plots of total capacitance of chromium and functionalized electrodes: AuArSO2Cl, Au-ArSO-Brij58(x): PEO9-CH3(1-x), with x the ratio of Brij58 and 1-x the ratio of PEO9-CH3. 1) in 150 mM NaCl before injection of SUVs, 2) straight after the injection of DOPC SUVs (t=0), 3) after 4h of contact with the SUVs, 4) after rinsing.

The Cr-Ar-SO-(Brij5850:PEO50) functionalized surface follows the same behaviour at first, but after 4h, it rather follows the trend of the surfaces with higher fractions of Brij58. Over time these latter become similar to the Rs(RciCPE) behaviour with Rci that drops of about one order of magnitude with the formation of stBLM (from 890.2 k cm² to 88.1 k cm²), and exhibit low-frequency signal tails that increase in length and continue to lift off the x-axis after rinsing. To a lesser extent, the same evolution is observed for the Cr-Ar-SO2Cl functionalization which does not prevent membrane from adsorbing to the surface in the opposite of pure PEO surfaces. However, this surface exhibit only mere changes before (t=0) and after rinsing which is not the case for the surfaces of the second category (the mean standard deviation of the fit parameters is of 91.4; 99.7; 108.7; 129.0 respectively for Cr-Ar-SO2Cl; pure Brij; Brij5875:PEO75 and Brij5850:PEO50). Cr-Ar-SO-Brij5825:PEO75 (x=25%) is the other surfaces that exhibits the least changes (with a mean standard deviation of 95.9) and with pure PEO these surfaces do not display long low frequency tails after rinsing. The effect of rinsing has been reported by authors and it also resulted in a reduction of the membrane impedance for all tether density/membrane compositions (up to a factor of 10). It

was attributed to the decreased ability of divalent cations to cross the hydrophobic membrane core, resulting in a change of the electrical properties of the tethered membrane, rather than to structural changes of the membrane. Similarly, the slight increase of capacitance after rinsing show that a bilayer is well formed on the sensor (in agreement with the QCM and SPR data) but it has eliminated the vesicles floating closely to the interface and above the bilayer (a system distributed in height would result in smaller values of the capacitance) letting easier access for ions to the pathways created by the defects in the membrane and generating the observed decrease of R ci. The behaviour for dense Brij58 surfaces resembles to the one described by the model of Valencius et al. that takes into account defects’ density and size in the stBLMs38. According to this model, an increase of the density of pores and/or their size results in a decrease of Rci, an increase of the imaginary part of the low-frequency capacitance and a lifting off the axis of the capacitance’s mid-frequency extremum. Besides, areas with membrane are thicker than areas with defaults and possess a low dielectric constant. They would therefore exhibit a lower capacity (C=/d) than the defect areas that are thin with a high dielec-


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ACS Sensors tric constant (close to water). The presence of defects gives additional current parallel to the electrode interface in the submembrane reservoir. These currents are at the origin of the EI spectra’s shape modeled by Valicius et al38. The spectra exhibit a typical “two semi-circle” shape, characteristic of the presence Surfaces

Fit and calculated parameters (RS(RCiCPEdc)) Q (µF s-1cm-2) C (µF cm-2)

CrCoating

CrCoatingDOPC, t=0

CrCoatingDOPC, t=4h

CrCoatingDOPC, rinsed

 Rci (k cm²) Rc ( cm²) fc1 et fc2 (Hz) Adjustment rmse Q (µF s-1cm-2) C (µF cm-2)  Rci (k cm²) Rc ( cm²) fc1 et fc2 (Hz) Adjustment rmse Q (µF s-1cm-2) C (µF cm-2)  Rci (k cm²) Rc ( cm²) fc1 et fc2 (Hz) Adjustment rmse Q (µF s-1cm-2) C (µF cm-2)  Rci (k cm²) Rc ( cm²) fc1 et fc2 (Hz) Adjustment rmse

Cr-Ar-SO2Cl                            

Cr-Ar-SO2Brij58 (100%)                            

of defects in tBLMs. The transition from one to another occurs in the ∼50-500 Hz range, (depending on the parameters of the system). The extremum formed

Cr-Ar-SO2Brij58 (75%)                            

Cr-Ar-SO2Brij58 (50%)                            

Cr-Ar-SO2Brij58 (25%)                            

Cr-Ar-SO2PEO9 (no Brij58)                            

Table 1. Best-fit parameters obtained for the EIS data measured in the non-faradaic mode in 0.15M NaCl on chromium electrodes, pre-functionalized (-ArSO2Cl) and functionalized (-ArSO2-Brij58(x) : -ArSO2-PEO9-CH3(1-x) with x = 1; 0.75; 0.5; 0.25 ; 0). Surfactant concentration used for functionalization: C = 0.09 mmol/L. Fitted parameters are: Q, pseudo-capacity; Rct and Rs charge transfer resistance and cell resistance (~7 cm², invariable); s, mass transfer coefficient. Calculated associated characteristic frequencies of the circuit are also given: f c1 at low frequencies and fD at high frequencies, and the electrode surface coverage for pre-functionalizations. by the boundary between two semicircles moves “north and west” in the complex plane (ReCtot -ImCtot) as the conductance of the individual membrane defect increases. In the case of the functionalization with Brij, the values of the capacity of the chromium stBLM are so high (20.4 µF cm-2 in average against 70%) WC14 functionalized gold, the capacity barely vary34. In the case of our surfactant tethered system the same behavior is observed before and

after DOPC stBLM formation (with a mean standard deviation of the capacity of 2.4 whatever the density of Brijs) but with higher capacity values. In light of literature, these EIS data monitored on stBLMs show that Brij58 functionalized surfaces do not constitute dense anchor-harpoon layers even for the highest ratios as it was previously described by the EIS data of the sensor functionalization without membrane. Curled up alkyl chains would explain the high values, the distribution and the low evolution of the interfacial capacity when stBLMs were formed with a certain amount of pores. This falls in agreement with the previous EIS analysis carried on that revealed a low density of anchor-harpoons molecules19. In the overall, the im-


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pedance properties of the tBLMs formed on the different functionalized surfaces show that the membranes do not form absolutely sealed bilayers and present a certain amount of pores. Surfaces from the first category (pure PEO type) are more likely to lead to membrane weak adsorption than immobilization, a result that is consistent with what has been observed by AFM and contact angle measurements as no dried membrane patterns were noticed (stable contact angles), whilst systems of the second category with more anchors, though still sparse, have well tethered membranes from SUVs fusion. The purpose of developing substrates that could result in the formation of fluid membranes with good sealing properties has led to the synthesis and investigations of new tethering molecules. As an example, for Anderson et al. no observable correlation between tether length and bilayer resistance was observed in the tBLMs formed on synthesized anchorlipids (DPhyPC, …) and Heinrich et. al showed that differences between FC16based and WC14-based stBLMs in their electrical parameters are rather moderate and that in structural terms, bilayers formed on FC16 are extremely well defined and virtually defect-free down to a tether fraction of 25%35. The diluted systems having a reduce number of carbonyl groups in the submembrane space were also noticed to probably allow for an easier flux of the ions out of the spacer region. Systems whose harpoons are made of one alkyl chain, such as Brij surfactants, result in membranes with lower sealing properties than using tailor made lipid tethers with a double alkyl chain. Results reported for fully tethered and diluted systems have resistance and capacitance properties in the range of M and below 1 Fcm-2 with NaCl solution37. Note that lower resistances and higher capacitances were reported while changing the NaCl solution for KCl. Bilayers comprised of DPhyPC formed sealing membranes at tethering densities as low as 60% on the base of thiolipid tethers diluted with mercaptoethanol molecules. The lower resistance of the amphiphilic system is most likely due to the increased fluidity of sparsely tethered membranes, which favors certain types of defect formation. However, it must also be taken into account that the stBLMs were formed on surfaces whose surface energy is much lower than that of the thiolipid tethers diluted with ME which displayed high water contact angles (close to 90)39. In order to study ion transport processes trough channels, a pure membrane resistance of 1-10 M cm² is desirable40 since at lower resistance, defect sites allows for background ion leakage across the bilayer. Here, molecular coatings with PEO and Brijs anchor-harpoon surfactants give hydrophilic electrode suitable to support tethered lipid bilayers providing a fluid biomimetic model for membranes with a good equilibration of ions and solvent between sup and sub compartments. Single membrane electro-sensors designed to study its structure and dynamics. In this section we discuss the advantages of building such cheap and easy to form hybrid coatings, on multi-purpose electrochemical sensors designed for being also suitable to membrane surface sensitive techniques. Thin chromium film electrodes were prepared so that X-ray reflectivity, AFM, and FRAP experiments could be carried out on the same sample, as well as electrochemistry. X-ray and Neutron Reflectivity. Thanks to: i) the relatively low electron density of chromium (eCr 1.99 e-/Å3) and its oxides; ii) to its conductivity allowing one to deposit low thicknesses (8 to 32 nm films); iii) to their sub-nanometer roughness

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and iv) to their robust anchoring on glass, we were able to conduct X-ray reflectivity experiments on the hybrid coatings before and after membrane deposition. To illustrate such experiments a 25.5nm thick chromium film was characterized before and after functionalization to obtain sparse Cr-Ar-SO2-Brij58 groups. The true specular X-ray reflectivity curves already show clear differences once plotted on the same graph (Figure S-3) and could all be well fitted using a slab model that describes the electron density profile in depth 19. For that each layer came with three fitting parameters: its thickness (d in Å); its top root mean square roughness ( in Å) and its electronic density (e in electrons/Å3 that translates in a scattering length density Re(b) after multiplication by the Thomson factor r0 = 2.81794.10-5 Å). The layers linear attenuation coefficient (that translates in the imaginary part of the scattering length density Im(b), was fixed here to calculated values and we checked that it didn’t affected noticeably the fits quality). Best fitting parameters are reported in Table S-1 of the SI and main conclusions are discussed here on the bases of the plotted electron density profile in depth (Figure S-3). After drying, a surfactant coating thickness of 3.7nm with a roughness of 1.5nm with Cr and 0.4nm at air was found, with a fitted electron density being the mixture of the materials that compose the coating plus water molecules associated to the PEO blocks as expected41. After a long contact with a solution of SUVs, formation of a stBLM and after flushing with water plus drying in air, the reflectivity curve changed so that a best fit was obtained with addition of a supplementary slab describing the whole lipid membrane restructured in air. In the case of a fluid homogeneous membrane in full immersion, we expect a mean electronic density of echains 0.303 for the lipid chains and of eheads 0.47 e-/Å3 for the head groups region (or equivalently: bchains (8.55+i 0.12) 10-6 Å -2 and bheads (13.26+i 3.58) 10-6 Å -2 for 2 nm and less than 0.5 nm, respectively). Similar densities and thicknesses from our best fit were obtained (Table S-1), however we can’t reach a more detailed profile of the lipid bilayer in air, because the membrane stabilized in water unfolded when dried in air giving an inhomogeneous and incomplete surface coverage, as pictured with AFM (Figure S-2). Part of the top alkyl chains turn over to air42 and this complicates the electron density profile. Nonetheless, this laboratory experiment shows unambiguously the advantage of working with chromium sensors. Indeed, gold and metals with a higher electron density are commonly used for biosensors (SPR, QCM, EIS, ...) and it forbids the use of X-ray reflectivity to probe so thin organic coating profiles (eAu 4.66 e-/ Å3, so 2.3 times eCr). Due to the huge contrast between gold and organic materials, the reflectivity signal is generally dominated by the metal layer features instead of the thin organic coating structure19, 43. An alternative to these difficulties is to study the membrane in full immersion through a silicium or quartz substrate with neutrons reflectivity at the solvent interface where the membrane stands42, 44-45. Even for neutrons, thin chromium films present advantages over gold in terms of building a simpler scattering length density profile enabling to study the 4 nm membrane tethered on the electrode34. It is also to be noted that to anchor gold on silicates, chromium is generally evaporated prior to gold deposition.


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ACS Sensors Fluorescence microscopy through the membrane sensors. Thin chromium electrodes deposited on classical glass microscopy slides also offer a good conductivity and transparency to visible light, so that optical measurements can be carried out through the substrate. As a proof of concept, FRAP experiments (fluorescence recovery after photo-bleaching, see the SI IV)) were carried out on BLM using a confocal laser scanning microscope (CLSM) through the thin chromium layer. A fluorescent chain-labelled 18:1-12:0 phosphatidylcholine (NDB-PC46) was perfectly mixed with DOPC at 6 % weight/weight before the formation of SUVs. In a first control experiment, designed to check that the expected diffusion coefficient was obtained for DOPC fluid membranes, we formed a thick multi-layer by dipcasting47 the lipid solution on a cleaned glass slide and checked membrane alignment from X-rays48. Zones were obtained with different numbers of stacked bilayers thus giving different levels of fluorescence. In full immersion (NaCl 0.15M), the highest diffusion coefficient we obtained after fitting the FRAP signal (SI) is DS = 1.62 µm²/s, similar to the coefficients found in the literature for supported fluid PC bilayers 49-50. The excitation and emission spectra were measured for the NDB-PC probe giving a peak at 464 nm and 531nm, respectively, on all zones. On thinnest regions exhibiting lowest fluorescence intensities, we measured much smaller values for D (from 0.03 to 0.65 µm²/s). The lower coefficients correspond to bilayers with a reduced fluidity due to strong interactions with bare glass or due to the presence of non-fused SUVs, as discussed in the literature51. By increasing the laser power from 1% to 20%, we obtained the same level of fluorescence signal measured through the chromium thin film (30nm thick) than previously through glass for similar samples. Control experiments carried out without membrane confirmed that the bare chromium itself and sensor’s organic coatings made no contribution to the fluorescence observed at this wavelength (530-600 nm) in the FRAP measurement conditions. The functionalized sensors (Cr-Ar-SO2Brij58) were then used after contact with vesicles and FRAP signal was recorded, thereby validating the use of fluorescent techniques through the substrate with such sensors. A membrane tethered to the pure Brij-58 coating here gave low diffusion coefficients as expected for a strongly bounded membrane (0.01 µm²/s on pure Brij58). Experiments on mixed coatings with additional PEO molecules to increase membrane fluidity 52 are part of the polymer coating optimisation that will be the object of future studies.

CONCLUSION We developed sensors coatings to tether lipid bilayer membranes (stBLM) from the covalent grafting of commercial surfactants often considered as non-reactive. Here, Brij (and CiEj) were sparsely grafted via their hydrophilic block (anchor) and formed an “anchor-harpoon” system with their hydrophobic moiety ready to assemble with analyte’s hydrophobic groups. Hybrid coatings obtained by co-grafting PEO chains provided an excellent environment to stabilize a single stBLM. We could measure the kinetics of formation from DOPC vesicles fusion using QCM and SPR on coated gold standard substrates. The EIS signal measured in the non-faradaic mode drastically changed with the stBLM formation on all coatings containing Brij58 and remained unchanged on the pure PEO which does not harpoon membranes (even though vesicle might come to the interface). PEO forms a hydration cushion and is moreover one

of the best proteins antifouling layer. Low densities of grafted Brij58 in a PEGylated environment ensure a highly hydrophilic environment at the sensor surface and stabilized membranes. Values of capacitance a tenfold higher than the one obtained on tailor made PEG-lipid monolayers was measured on the sensor with resistances to ions Rci of the order of 100 k cm² while values of 1M cm² are reported for membranes considered as high insulating barriers with no ion permeation. Andersson et al. have shown the strong interest for membranes with possible permeation of ions and solvents to the sub-layer, as a screening platform for potential antimicrobial agents (embedding transmembrane proteins); while non-permeable sealed bilayers are better when probing ionic transport of inserted channel proteins40. The electrochemical sensors designed here are well adapted to probe biomimetic or biological membranes under electric fields and dynamics of surrounding molecules and ions. Membrane sensors built on flat thin chromium electrodes open the route to surface techniques dedicated to membranes structural investigation. Chromium allows one to envision Xray reflectivity experiments to probe the thin organic sample that are not possible on classical surface sensors made of high electron density materials (Au, Ag, Pt, …). Likewise, neutron reflectivity through the electrode in full immersion can be used to reach a quantitative assessment of the molecular architecture, as well as the level of hydration of the sub-membrane layer33-34, 42-43 . The good optical transparency of the sensors was used to carry out microscopy and FRAP experiments to check for the membrane’s large scale homogeneity and lipids 2D-fluidity in full immersion. Coupling several investigation techniques on the same sensor is a major goal in the field of membrane sensors. Because this coating strategy is: i) cheap since it avoids cumbersome chemistry of tailor-made anchor-harpoon molecules, ii) works on many materials, iii) and works with a versatile catalogue of commercial molecules, it opens the path to large scale production of membrane sensors and other analytical platforms suitable to surface science techniques.

ASSOCIATED CONTENT Supporting Information. Sensors surface functionalization and electrochemical characteristics; Measuring the kinetics of SUVs fusion at sensors surface: QCM analysis; SPR analysis; Control experiments on dried sensors with an anchored membrane: CA measurements; Dried sensors surfaces; Structure and Dynamics of membranes on flat and transparent Cr sensors: XRR measurements of a dry stBLM built on functionalized chromium thin films; NR Simulations of a stBLM built on gold, chromium and silicium; FRAP. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +33 617276236.

Author Contributions All authors have given approval to the final version of the manuscript. OS and GB worked jointly on all aspects of the work. CE and J.F.P participated to the design of EIS experiments and provided the electrochemistry facilities.

Funding Sources


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The French government financially supported this work. OS received her Ph.D. grant from the French ministry of research and higher education (via Ecole Doctorale 3MPL, ED 500).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT X-ray experiments were performed on the scattering platform of IMMM (Le Mans Université, Le Mans, France) and Cr electrodes on the Thin Film Elaboration Platform with help of M. Edely. We greatly thank R. Perrot for the confocal microscopy experiments presented in the SI (SCIAM, University of Angers) as well as J.C. Gimel (MINT laboratory, INSERM/CNRS/University of Angers, France).

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Electrodes for membrane surface science. Bilayer lipid membranes

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