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The role of the anion on the ionophore properties of valinomycin was studied in a model floating bilayer lipid membrane (fBLM) using supporting electrolytes containing K+ with four different counter anion species (ClO4-, H2PO4-, Cl-, and F-). The electrochemical impedance spectra indicate that the membrane resistance of the bilayer decreases with the decrease of Gibbs free energy of anion solvation. The IR spectra demonstrate that valinomycin does not readily bind to K+ in the KH2PO4, KCl, and KF electrolyte solutions, but in the presence of KClO4, valinomycin readily binds to K+, forming a valinomycin-K+ complex. The results in the present paper reveal the role of the counter anion on the transport of cations by valinomycin across the lipid bilayer. The valinomycin-cation complex creates an ion pair with the anion, and this ion pair can enter the hydrophobic region of the bilayer transporting the cation across the membrane. Anions with low solvation energies facilitate the formation of the ion pair improving the ion conductivity of valinomycin-incorporated bilayers. This paper sheds new light on the transport mechanism of valinomycin ionophores and provides new information about the bioactivity of this molecule.

The inhibition effect of amiloride on alamethicin ion channels was studied in a model zwitterionic floating bilayer lipid membrane (fBLM). The EIS studies indicated that amiloride prevents the transport of ions through the alamethicin channels leading to an overall increase in membrane resistance. The PM-IRRAS data demonstrated that amiloride has no influence on the secondary structure of alamethicin but restricts the insertion of the peptides into the bilayer and blocks ion transport through preformed alamethicin channels. The effect of amiloride on ion channel formation in the floating bilayer formed by a zwitterionic lipid was compared to those of previous studies involving negatively charged fBLMs and tethered zwitterionic lipid bilayers. The findings from these studies show that the effects of amiloride on ion channel formation strongly depend on the mobility and charge of the membrane lipids.

The electrochemical impedance spectroscopy (EIS) and polarization-modulation infrared reflection absorption spectroscopy (PM-IRRAS) techniques were employed to study the ionophore properties of valinomycin in a model floating bilayer lipid membrane (fBLM) in perchlorate supporting electrolytes with potassium and sodium cations. Valinomycin decreases the membrane resistance of the fBLM in KClO4 solution by about 180 times, while it has a negligible effect on the membrane resistivity in NaClO4 solution. The IR spectra indicate that valinomycin forms a complex with K+, but not with Na+. The valinomycin-K+ complex adopts a small tilt angle with respect to the electrode surface normal and is well interdigitated between the acyl chains of the bilayer. The EIS and PM-IRRAS results indicate that valinomycin forms complexes with K+ and transports K+ across the lipid bilayer. This transport is potential independent and hence has a passive character.

The knowledge of the structure and properties of water adsorbed on metal surfaces is relevant for understanding electrocatalytic reactions. A monolayer of Pd deposited at a gold electrode surface i...

Neurodegeneration in Alzheimer's disease is associated with disruption of the neuronal cell membrane by the amyloid β (Aβ) peptide. However, the disruption mechanism and the resulting changes in membrane properties remain to be elucidated. To address this issue, herein the interaction of amyloid β monomers (AβMs) and amyloid β oligomers (AβOs) with a floating bilayer lipid membrane (fBLM) was studied using electrochemical and IR spectroscopy techniques. IR measurements showed that both Aβ forms interacted similarly with the hydrophobic membrane core (lipid acyl chains), causing conformational and orientational changes of the lipid acyl chains, thus decreasing acyl chain mobility and altering the lipid packing unit cell. In the presence of AβOs, these changes were more significant than those in the presence of AβMs. However, respective interactions of AβMs and AβOs with the membrane hydrophilic exterior (lipid heads) were quite different. AβMs dehydrated lipid heads without affecting their orientation while AβOs changed the orientation of lipid heads keeping their hydration level intact. Electrochemical measurements showed that only AβOs porated the fBLM, thus significantly changing the fBLM electrical properties. The present results provide new molecular-level insight into the mechanism of membrane destruction by AβOs and changes in the membrane properties.

The structure of water in the submembrane region of the bilayer of DPhPC floating (fBLM) on a monolayer of 1-thio-β-d-glucose (β-Tg)-modified gold nanoparticle film was studied by the surface-enhanced infrared absorption spectroscopy (SEIRAS). SEIRAS employs surface enhancement of the mean square electric field of the photon, which is acting on a few molecular layers above the film of gold nanoparticles. Therefore, it is uniquely suited to probe water molecules in the submembrane region and provides unique information concerning the structure of the hydrogen bond network of water surrounding the lipid bilayer. The IR spectra indicated that water with a strong hydrogen network is separating the membrane from the gold surface. This water is more ordered than the water in the bulk. When alamethicin, a peptide forming ion channels, is inserted into the membrane, the network is only slightly loosened. The addition of amiloride, an ion channel blocker, results in a significant decrease in the amount of water in the submembrane region. The remaining water has a significantly distorted hydrogen bond network. This study provides unique information about the effect of the ion channel on water transport across the bilayer. The electrode potential has a relatively small effect on water structure in the submembrane region. However, the IR studies demonstrated that water is less ordered at positive transmembrane potentials. The present results provide significant insight into the nature of hydration of a floating lipid bilayer on the gold electrode surface.

Valinomycin, a cyclic peptide, was incorporated into a biomimetic lipid membrane tethered to the surface of a gold (111) electrode. Electrochemical impedance spectroscopy was used to study the ionophore properties of the peptide, and polarization modulation infrared reflection absorption spectroscopy was employed to determine the conformation and orientation of valinomycin in the membrane. The combination of these two techniques provided unique information about the ionophore mechanism where valinomycin transports ions across the membrane by creating a complex with potassium ions and forming an ion pair with a counter anion. The ion pair resides within the hydrophobic fragment of the membrane and adopts a small angle of ∼22° with respect to the surface normal. This novel study provides new insights explaining the valinomycin ion transport mechanism in model biological membranes.

Monolayers of 1,2-dipalmitoyl-sn-glycero-3-cytidine were incubated with guanine in a 0.1 M NaF electrolyte at the surface of a Langmuir trough and transferred to gold (111) electrodes using the Lan...

The effects of amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl- sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged amiloride molecules and the negative charge on the membrane.

Chronocoulometry was applied to determine charge in a monolayer of nucleolipid (1,2-dipalmitoyl-sn‑glycero-3-(cytidine diphosphate)) deposited at a gold electrode surface. The immersion method was used to measure the potential of zero charge of the interface (Epzci), which is a sum of charge on the monolayer of the nucleolipid and charge on the gold surface. Photon polarization infrared reflection absorption spectroscopy (PM-IRRAS) was used to determine formation of the Watson-Crick complex between terminal cytidine moiety of the nucleolipid and guanine (its complementary base) added to the solution. The combination of electrochemical and spectroscopic studies allowed one to demonstrate that the Watson-Crick complex is formed when the interface is positively charged. The potential applied to the electrode affects not only the complex formation but also orientation of the cytosine moiety. The complex is formed when the cytosine moiety is oriented assuming a small angle with respect to the electrode surface. Ministerio de Ciencia e Innovación CTQ2014-57515-C2-1-R, RED2018-102412-T Junta de Andalucía FQM202 Natural Sciences and Engineering Research Council of Canada (NSERC) RG-03958

Summary Planar solid-supported lipid bilayers have been developed as simplified biological membranes to model the physical properties of cell membrane processes. Lipid bilayer membranes supported at conductive metal substrates provide a unique opportunity to investigate the effect of the static electric field on the membrane structure and function. The insights gained from this research can be used to develop novel biosensors and biomedical devices. This review summarizes the recent developments in metal-supported biomimetic membrane systems. It provides an overview of the various models, such as metal-supported monolayers and bilayers, hybrid bilayers, tethered bilayers, and floating bilayers, used to study membrane processes at electrode surfaces, such as metal-supported monolayers and bilayers, hybrid, tethered, and floating bilayers. The paper discusses the recent advancements in these biomimetic models and describes the fundamental knowledge about membrane processes that has been extracted from these different platforms. The potential for the design and improvement of biomedical devices using metal-supported bilayers is also discussed. Metal-supported bilayers allow for the application of a plethora of spectroscopic and surface imaging techniques to obtain information about the voltage-dependent properties of biomolecules at the molecular level. The underlying methodology of these analytical techniques and the structural, chemical and kinetic information extracted are reviewed.

Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-β-d-glucose (β-Tg). The EIS data showed that the presence of alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is assisted by the transmembrane potential. Molecular resolution AFM images provided visual evidence that alamethicin molecules aggregate forming hexagonal porous 2D lattices with periodicities of 2.0 ± 0.2 nm. The pore formation by alamethicin in the negatively charged membrane was compared with the interaction of this peptide with a bilayer formed by zwitterionic lipids. The comparison of these results showed that alamethicin preferentially forms ion translocating pores in negatively charged phospholipid membranes. Natural Sciences and Engineering Research Council of Canada (NSERC)

Alamethicin was reconstituted into 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DPhPC) bilayer supported at the gold (111) electrode surface using a combination of Langmuir-Blodgett/Langmuir-Schaefer deposition techniques. Chronocoulometry was employed to describe the behavior of the bilayer as a function of the electrode potential. Electrochemical impedance spectroscopy (EIS) was used to determine the membrane resistivity and capacitance. The photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements provided complementary information about the conformation and orientation of the alamethicin and DPhPC molecules in the adsorbed bilayer. The electrochemistry results demonstrated that the bilayer is stable at the electrode surface at potentials between 0.0 V and − 0.5 V vs Epzc. At more negative potentials, a progressive electro-dewetting of the membrane from the electrode surface takes place. The membrane is detached from the electrode surface when (E-Epzc) is more negative than − 1.1 V. The PM-IRRAS results illustrate that alamethicin peptides assume a surface state where their helices assume a large angle with respect to the surface normal at potentials close to Epzc. When negative potentials are applied, the alamethicin molecules are inserted into the membrane. Peptide insertion is complete at (E-Epzc) ~ − 0.5 V. The membrane resistivity determined from the ESI data indicates that alamethicin molecules adopt an open channel state when the helices are inserted into the membrane and a closed channel state (non-conductive) at E close to Epzc where they assume a flat orientation on the surface. The IR spectra collected during the negative and positive potential scans demonstrated that the open and closed ion channel states are potential controlled and reversible. In contrast to the significant changes in alamethicin orientation, the tilt angle of the acyl chains of DPhPC molecules show little to no change with the electrode potential. The data presented in this paper provides insight on the voltage-gating behavior of the alamethicin ion channels incorporated into phospholipid bilayers.

Alamethicin was reconstituted into DPhPC/DPTL tethered bilayer lipid membranes (tBLMs) to gain a better understanding of ion pore formation. The properties of the ion-conductive alamethicin pores were investigated by electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) as a function of the peptide concentration within the membrane. At low peptide concentrations (≤ 5%), the alamethicin-incorporated tBLM displays the highest membrane resistance since the alamethicin molecules adopt a surface state where the tilt angles of the helices are large with respect to the surface normal. At alamethicin concentrations ranging from 10 to 20 mol%, the tilt angles of the helices decrease to lower values signifying that the alamethicin molecules are inserting into the tBLM. Consequently, the membrane resistivity is also lowered indicating that the peptides are forming ion conductive channels. The results show an excellent correlation between molecular information concerning the orientation and conformation of alamethicin molecules obtained from the PM-IRRAS measurements and electrochemical properties of the membranes measured by EIS. These findings reveal that the alamethicin concentration within the film is directly related to the ion conductance across membrane.

In situ ATR-SEIRAS experiments were carried out to describe water structure at the surface of multilayers of Pd modified nanopatterned gold film electrodes in contact with aqueous 0.1 M HClO4 solution. The results show that the surface water becomes more ordered when the thickness of the Pd film increases. The “ice-like water” band becomes stronger and for the electrode with 5 ML Pd@Au, the ratio of “ice-like water” to “liquid-like water” becomes larger than that in bulk water. Concomitantly, the population of “monomers” or small water clusters diminishes. All surface water bands increase when hydrogen adsorption takes place. The bending band corresponding to “monomers” or “multimers” water is observed chiefly at the potentials of hydrogen adsorption.

A mixed monolayer of 1,2-dipalmitoyl-sn-glycero-3-(cytidine diphosphate) nucleolipid (DG-CDP) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was transferred from the air–water interface onto an gold (1 1 1) electrode surface using the Langmuir-Schaeffer (horizontal touch) technique. Compression isotherms were recorded for the monolayer spread at the air–water interface. These measurements demonstrated that the mixture composition 7:3 of DG-CDP:DPPC displayed reduced repulsive interactions between polar heads and ideal packing of acyl chains. Chronocoulometric experiments were performed to demonstrate stability of this film at the gold–solution interface. Photon polarization modulation infrared reflection absorption spectroscopy showed that the tilt angle of the acyl chains is slightly higher, and the chains are less twisted in the mixed than in the pure nucleolipid monolayer. In the mixed film, the tilt of the cytosine moiety (the sensing element of the monolayer) assumes a large angle with respect to the surface normal, even when the monolayer becomes detached from the gold surface. The mixed monolayer has improved properties for future applications for detection of guanine, its complementary base. Ministerio de Ciencia e Innovación CTQ2014-57515-C2-1-R, RED2018-102412-T Junta de Andalucía FQM202 Natural Sciences and Engineering Research Council of Canada (NSERC) RG-03958

The charge number per adsorbed molecule and surface concentration of 2,3-di- O -phytanyl- sn -glycero-1-octaethyleneglycol-D,L-α-lipoic acid ester (DPOL) at a Au(111) electrode surface was investigated by chronocoulometry using two different film deposition methods. First, a known amount of the thiolipid was transferred from the air-water interface of a Langmuir trough onto the gold electrode surface via a Langmuir-Blodgett (LB) deposition. The charge density measurements for this monolayer system were used to determine the charge number per adsorbed DPOL molecule (electrosorption valency). The observed electrosorption valency values of the adsorbed DPOL film were much lower than the expected number of transferred electrons for a simple reductive desorption process. In the second deposition method, charge densities were measured for the electrode covered by a self-assembled DPOL monolayer. The electrosorption valency values determined from the LB DPOL film were used to calculate the packing density of the DPOL molecules within the self-assembled monolayer. The surface concentration of the molecules within the thiol monolayer with octaethylene glycol chains gave similar results to a related thiolipid with tetraethylene glycol chains (DPTL). This new finding indicates that both of these molecules (DPTL and DPOL) assume a brush conformation in densely packed self-assembled monolayers.

Valinomycin, a cyclic peptide, was incorporated into a biomimetic lipid membrane tethered to the surface of a gold (111) electrode. Electrochemical impedance spectroscopy was used to study the ionophore properties of the peptide, and polarization modulation infrared reflection absorption spectroscopy was employed to determine the conformation and orientation of valinomycin in the membrane. The combination of these two techniques provided unique information about the ionophore mechanism where valinomycin transports ions across the membrane by creating a complex with potassium ions and forming an ion pair with a counter anion. The ion pair resides within the hydrophobic fragment of the membrane and adopts a small angle of ∼22° with respect to the surface normal. This novel study provides new insights explaining the valinomycin ion transport mechanism in model biological membranes.

Colicin E1 is a channel-forming bacteriocin produced by certain Escherichia coli cells in an effort to reduce competition from other bacterial strains. The colicin E1 channel domain was incorporated into a 1,2-diphytanoyl- sn-glycero-3-phosphocholine floating bilayer situated on a 1-thio-?-d-glucose-modified gold (111) surface. The electrochemical properties of the colicin E1 channel in the floating bilayer were measured by electrochemical impedance spectroscopy; the configuration and orientation of colicin E1 in the bilayer were determined by polarization-modulation-infrared-reflection absorption spectroscopy. The EIS and IR results indicate that colicin E1 adopts a closed-channel state at the positive transmembrane potential, leading to high membrane resistance and a large tilt angle of ?-helices. When the transmembrane potential becomes negative, colicin E1 begins to insert into the lipid bilayer, corresponding to low membrane resistance and a low tilt angle of ?-helices. The insertion of colicin E1 into the lipid bilayer is driven by the negative transmembrane potential, and the ion-channel open and closed states are potential reversible. The data in this report provide new insights into the voltage-gated mechanism of colicin E1 ion channels in phospholipid bilayers and illustrate that the floating bilayer lipid membrane at the metal electrode surface is a robust platform to study membrane-active proteins and peptides in a quasi-natural environment.

Two 4-thio pseudo-glycolipids, a disulfide and a thiol, were synthesized as candidates to build tethered bilayer lipid membranes (tBLMs) on gold (111) surface. Monolayers of the disulfide and thiol were built on gold surfaces using the self-assembly and Langmuir-Blodgett (LB) transfer methods. Monolayers were prepared in several solvents and at various temperatures; their quality was assessed by differential capacitance. Best monolayers were achieved when the disulfide was self-assembled in ethanol. The quality of such monolayers was further improved by allowing the assembly to proceed under stirring of the solution at increased temperatures. The charge number per adsorbed molecule and surface concentration of disulfide on gold (111) surface were determined by chronocoulometry. A DPhPC/disulfide tBLM was then built by vesicle fusion of 1,2-diphytanyl-sn-glycero-3-phosphocholine (DPhPC) on top of the disulfide monolayer. The minimum capacitance of the gold electrode with tBLM (1.3 mF cm 2) was close to the value of a real cell membrane and the AFM force spectroscopy measurements showed that the DPhPC/disulfide tBLM had a thickness of 6.2 ± 0.6 nm consistent with the thickness expected for a bilayer. Finally, the potential of the tBLM to study transmembrane proteins was assessed by investigating the reconstitution of gramicidin A into the membrane by polarization modulation infrared absorption spectroscopy (PM-IRRAS). These results demonstrate that the disulfide is a good candidate to construct tBLMs on gold surface and to study transmembrane proteins. Natural Sciences and Engineering Research Council of Canada 2020-12-12

The effect of the electrode potential on the orientation and conformation of the 1,2-dipalmitoyl- sn-glycero-3-cytidine monolayer deposited on a gold (111) electrode surface was described. The potential of zero free charge ( Epzc) for the monolayer-covered electrode was determined to be -0.2 V vs SCE. The differential capacitance and charge density data indicated that the monolayer is stable at the electrode surface when ( E - Epzc) > 0.0 V. At negative rational potentials, a progressive detachment (electrodewetting) of the monolayer occurs. The monolayer is fully detached from the electrode surface at ( E - Epzc) 0.0 V, the plane of the cytosine moiety assumes a small angle of ∼20° with respect to the surface. At negative potentials, the tilt angle of the cytosine fragment increases and rotates. With the help of DFT calculations, these changes were explained by the repulsion of the positive pole of the cytosine permanent dipole moment by the positively charged gold surface and its attraction to the metal surface at negative electrode potentials. This work provides unique information for the future development of sensors based on the molecular recognition of nucleoside targets.

Reduction of oxygen catalyzed by laccase was studied by surface-enhanced infrared absorption spectroscopy (SEIRAS) combined with electrochemical techniques. Laccase molecules were adsorbed on modified carbon nanotubes (CNTs). SEIRAS provided spectra of Amide I band of adsorbed laccase. Fourier self-deconvolution (FSD) and two-dimensional correlation spectroscopy (2D COS) techniques were employed to reveal the sub-band structure of the Amide I band. The analysis demonstrated that laccase adsorbed on CNTs remains its native state both in the oxidized and reduced states. The detailed analysis showed that change of the oxidation state leads to a small change of the secondary structure of the protein. This change is comparable to the change observed for similar blue-copper oxidases in solution during redox titration. The results of this study demonstrated that CNT's immobilized laccase is an excellent catalyst of oxygen reduction. This study illustrates power of quantitative analysis of IR data to provide information about three-dimensional structure of surface immobilized proteins.

A hybrid bilayer lipid membrane (hBLM), constructed with a 1-hexadecanethiol self-assembled interior leaflet and a 1,2-dipalmitoyl-sn-glycero-3-cytidine nucleolipid exterior leaflet, was deposited at the surface of a gold (111) electrode. This system was used to investigate the molecular recognition reaction between the cytosine moieties of the lipid head group with guanine molecules in the bulk electrolyte solution. Electrochemical measurements and photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) were employed to characterize the system and determine the extent of the molecular recognition reaction. The capacitance of the hBLM-covered gold electrode was very low (~1 μF cm−2), therefore the charge density at the gold surface was small. Changing the electrode potential had a minimal effect on the complexation between the cytosine moieties and guanine molecules due to small changes in the static electric field across the membrane. This behavior favored the formation of the guanine–cytosine complex.

The insertion and ion-conducting channel properties of alamethicin reconstituted into a 1,2-di- O-phytanyl- sn-glycero-3-phosphocholine bilayer floating on the surface of a gold (111) electrode modified with a 1-thio-β-d-glucose (β-Tg) self-assembled monolayer were investigated using a combination of electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). The hydrophilic β-Tg monolayer separated the bilayer from the gold substrate and created a water-rich spacer region, which better represents natural cell membranes. The EIS measurements acquired information about the membrane resistivity (a measure of membrane porosity), and the PM-IRRAS experiments provided insight into the conformation and orientation of the membrane constituents as a function of the transmembrane potential. The results showed that the presence of alamethicin had a small effect on the conformation and orientation of phospholipid molecules within the bilayer for all studied potentials. In contrast, the alamethicin peptides assumed a surface state, where the helical axes adopted a large tilt angle with respect to the surface normal, at small transmembrane potentials, and inserted into the bilayer at sufficiently negative transmembrane potentials forming pores, which behaved as barrel-stave ion channels for ionic transport across the membrane. The results indicated that insertion of alamethincin peptides into the bilayer was driven by the dipole-field interactions and that the transitions between the inserted and surface states were electrochemically reversible. Additionally, the EIS measurements performed on phospholipid bilayers without alamethicin also showed that the application of negative transmembrane potentials introduces defects into the bilayer. The membrane resistances measured in both the absence and presence of alamethicin show similar dependencies on the electrode potential, suggesting that the insertion of the peptide may also be assisted by the electroporation of the membrane. The findings in this study provide new insights into the mechanism of alamethicin insertion into phospholipid bilayers.

Unilamellar DMPC/DMPG vesicles in the absence and presence of alamethicin were fused onto the surface of a gold electrode modified with a 1-thio-β-d-glucose self-assembled monolayer. The resulting floating bilayer lipid membranes (fBLMs) were investigated using atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). A corrugated film structure was observed for the pure DMPC/DMPG fBLMs due to surface stress between the tightly packed lipids. These corrugations are removed by the addition of alamethicin suggesting the lipid-peptide interactions alleviate the overall surface stress creating a more uniform bilayer. Both DMPC/DMPG films in the absence and presence of alamethicin had thickness of 5.5 ± 0.9 nm demonstrating that alamethicin has a minimal effect on the overall bilayer thickness. However, a significant decrease in membrane resistivity was observed when alamethicin was inserted into the fBLM indicating that the peptides are forming ion conducting pores. A direct visualization of the alamethicin pores was obtained by molecular resolution AFM images revealing that the pores are not randomly dispersed throughout the bilayer, but instead form hexagonal aggregates. The diameter of an individual pore within the aggregates is equal to 2.3 ± 0.3 nm, which is consistent with the size of a hexameric pore predicted by molecular dynamics simulations. Additionally, the image revealed a broad size distribution of alamethicin aggregates, which explains the origin of multiple conductivity states observed for the incorporation of alamethicin into free standing bilayer lipid membranes. Natural Sciences and Engineering Research Council of Canada

The synthesis of the three 6”-deoxy-6”-thio glycolipid analogues β-D-Gal-(1→6)-β-D-Gal-(1→4)-β-D-Glu-(1→OCH2)-[1,2,3]-triazole-1-dodecane, β-D-Gal-(1→4)-β-D-Glu-(1→4)- β-D-Glu-(1→OCH2)-[1,2,3]-triazole-1-dodecane and β-D-Gal-(1→4)-β-D-Glu-(1→4)-β-D-Glu-(1→OCH2)-[1,2,3]-triazole-1-octadecane is presented. Glycosylation at position O-4’ of a propargyl cellobioside glycosyl acceptor and position O-6’ of a propargyl lactoside glycosyl acceptor with a 6-deoxy-6-thio galactosyl donor gave rise to two unique trisaccharides that in turn underwent copper-catalyzed azide-alkyne cycloadditions with either 1-azidododecane or 1-azidooctadecane. The potential for each of these analogues to function as tethers of lipid bilayers to Au(111) surface was assessed by differential capacitance experiments. A monolayer of the previously described monosaccharide 1-octadecane-4-(6-thio-β-D-galacto-pyranosyloxymethyl)-[1,2,3]-triazole either self-assembled or prepared by Langmuir-Blodgett (LB) transfer was found to support an outer leaflet monolayer (DMPC/cholesterol, 70:30) deposited by Langmuir-Schaefer (LS) touch. The bilayers obtained with this monosaccharide analogue had minimum differential capacitances of 1.0 and 0.9 µF/cm2 when the inner monolayer was prepared by self-assembly and LS touch, respectively. Attempts to produce bilayers using the trisaccharides synthesized here were unsuccessful; we are attributing these unsuccessful results mostly to the high water solubility of trisaccharides combined with the relatively short length of the hydrocarbon chains used in this study. Natural Sciences and Engineering Research Council of Canada

The effect of amiloride, an acid-sensing ion channel (ASIC) blocker, on the formation of alamethicin ion channels in tethered zwitterionic phospholipid bilayers was investigated using electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). EIS measurements show a decrease in conductivity indicating that ion transported across the alamethicin-rich phospholipid membrane is inhibited by the presence of amiloride. The PM-IRRAS spectra indicate that amiloride has no effect on the secondary structure of the alamethicin peptide. The IR data suggests that amiloride does not block ion translocation through the alamethicin ion pore but prevents the insertion of alamethicin peptides into the biomembrane suppressing the formation of ion channels. The mechanism responsible for amiloride inhibition is explained in terms of the electrostatic interactions between the molecule and membrane surface. Amiloride is positively charged at physiological pH levels invoking a repulsive force on the positive pole of the alamethicin molecular dipole. These repulsive interactions prevent the insertion of the alamethicin peptides into the hydrophobic core of the lipid bilayer. The present paper offers a molecular view of the interactions that occur between amiloride, an ion channel inhibitor, with the lipids and ion channel forming peptides in model tBLMs.

Natural Sciences and Engineering Research Council of Canada; National Natural Science Foundation of China [20873116]

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.langmuir.6b00635 Quantitative subtractively normalized interfacial Fourier transform infrared reflection spectroscopy (SNIFTIRS) was used to determine the molecular orientation and identify the metal–molecular interactions responsible for the adsorption of adenine from the bulk electrolyte solution onto the surface of the Au(111) electrode. The recorded p-polarized IR spectra of the adsorbed species were subtracted from the collected s-polarized IR spectra to remove the IR contributions of the vibrational bands of the desorbed molecules that are located within the thin layer cavity of the spectroelectrochemical cell. The intense IR band around 1640 cm–1, which is assigned to the pyrimidine ring stretching vibrations of the C5–C6 and C6–N10 bonds, and the IR band at 1380 cm–1, which results from a combination of the ring stretching vibration of the C5–C7 bond and the in-plane CH bending vibration, were selected for the quantitative analysis measurements. The transition dipoles of these bands were evaluated by DFT calculations. Their orientations differed by 85 ± 5°. The tilt angles of adsorbed adenine molecules were calculated from the intensity of these two vibrations at different potentials. The results indicate that the molecular plane is tilted at an angle of 40° with respect to the surface normal of the electrode and rotates by 16° around its normal axis with increasing electrode potential. This orientation results from the chemical interaction between the N10 and gold atoms coupled with the π–π parallel stacking interactions between the adjacent adsorbed molecules. Furthermore, the changes in the molecular plane rotation with the electric field suggests that the N1 atom of adenine must also participate in the interaction between the molecule and metal. Spanish Ministry of Science and Technology (CTQ2010- 19823), Spanish Ministry of Economy and Competitiveness (CTQ2014-57515-C2-1-R), Junta de Andalucia (PAI FQM202) and Discovery Grant from Natural Sciences and Engineering Council of Canada

The behavior, secondary structure, and orientation of a recently discovered bacteriocin-like peptide BacSp222 in a lipid model system supported at a gold electrode was investigated by chronocoulometry, polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and attenuated total reflectance infrared (ATR-IR) spectroscopy. The IR spectra show that the secondary structure of BacSp222 is predominantly α-helical. Analysis of the spectra in the amide I region shows that the α-helical fragment of the peptide is inserted into bilayer at the potential range at which the bilayer is stable and attached to the Au(111) surface, i.e., from -0.5 to 0.3 V vs Ag/AgCl. Insertion of BacSp222 to the membrane significantly changes the conformation of the acyl chains of lipid molecules, from all-trans to partially melted; however, the chains become less tilted. Based on these results, we propose that BacSp222 interacts with the DMPC bilayer through the barrel-stave pore formation. In this model, α-helix of BacSp222 inserts into the membrane with an angle between the α-helix axis and membrane normal equal to ∼18°. The changes in orientation of the α-helical fragment of the peptide indicate that the orientation of BacSp222 with respect to the bilayer surface is potential-dependent. The peptide is inserted into the membrane driven by the electrostatic field generated by negative charge at the metal surface. It is not inserted at negative potentials where the membrane is detached from the metal and no longer exposed to the electrostatic field of the metal.

The immersion method was used to measure the potential of zero free charge (E pzfc) of a bare Au(111) electrode and the electrode modified by self-assembled monolayers (SAMs) of 1-thio-β-D-glucose (β-Tg) and 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L-α-lipoic acid ester (DPTL). The measurements were performed in three electrolyte solutions: 0.1 M NaH2PO4, 0.1 MKClO4 and 0.1 M NaF. The E pzc of the Au(111) electrode has different values in these electrolyte solutions due to the specific adsorption of phosphate and fluoride anions on the Au(111) surface. In contrast, when the gold surface is covered by a SAM of β-Tg, the adsorption of anions is suppressed and similar values of E pzfc were measured in the three electrolytes. Additional chronocoulometric experiments were performed to determine the potentials of the zero total charge (E pztc) for the thiol covered electrode. Significant differences between numerical values of potentials of the zero free charge E pzfc and potentials of the zero total charge E pztc were observed.

In this study, a Au(111) electrode is functionalized with a monolayer of 1-thio-β-D-glucose (β-Tg), producing a hydrophilic surface. A monolayer of β-Tg was formed on a Au(111) surface by either (1) potential-assisted deposition with the thiol in a supporting electrolyte or (2) passive incubation of a gold substrate in a thiol-containing solution. For each method, the properties of the β-Tg monolayer were investigated using cyclic voltammetry (CV), differential capacitance (DC), and chronocoulometry. In addition, electrochemical scanning tunneling microscopy (EC-STM) was used to obtain images of the self-assembled monolayer with molecular resolution. Potential-assisted assembly of β-Tg onto a Au(111) electrode surface was found to be complicated by oxidation of β-Tg molecules. The EC-STM images revealed formation of a passive layer containing honeycomb-like domains characteristic of a formation of S(8) rings, indicating the S-C bond may have been cleaved. In contrast, passive self-assembly of thioglucose from a methanol solution was found to produce a stable, disordered monolayer of β-Tg. Since the passive assembly method was not complicated by the presence of a faradaic process, it is the method of choice for modifying the gold surface with a hydrophilic monolayer.

Subtractively normalized interfacial Fourier transform infrared reflection spectroscopy (SNIFTIRS) was applied to study (bi)sulfate adsorption on a Pt(111) surface in solutions of variable pH while maintaining a constant total bisulfate/sulfate ((bi)sulfate) concentration without the addition of an inert supporting electrolyte. The spectra were recorded for both the p- and s-polarizations of the IR radiation in order to differentiate between the IR bands of the (bi)sulfate species adsorbed on the electrode surface from those species located in the thin layer of electrolyte. The spectra recorded with p-polarized light consist of the IR bands from both the species adsorbed at the electrode surface and those present in the thin layer of electrolyte between the electrode surface and ZnSe window whereas the s-polarized spectra contain only the IR bands from the species located in the thin layer of electrolyte. A new procedure was developed to calculate the angle of incidence and thickness of the electrolyte between the Pt(111) electrode surface and the ZnSe IR transparent window. By combining these values with the knowledge of the optical constants for Pt, H(2)O and ZnSe, the mean square electric field strength (MSEFS) at the Pt(111) electrode surface and for thin layer of solution were accurately calculated. The spectra recorded using s-polarization were multiplied by the ratio of the average MSEFS for p- and s-polarizations and subtracted from the spectra recorded using p-polarization in order to remove the IR bands that arise from the species present within the thin layer cavity. In this manner, the resulting IR spectra contain only the IR bands for the anions adsorbed on the Pt(111) electrode surface. The spectra of adsorbed anions show little change with respect to the pH ranging from 1 to 5.6. This behavior indicates that the same species is predominantly adsorbed on the metal surface for this broad range of pH values and the results suggest that sulfate is the most likely candidate for this adsorbate.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.langmuir.5b04052 A phospholipid bilayer composed of 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (d54-DMPC) was deposited onto the Au(111) electrode modified with a self-assembled monolayer of 1-thio-β-d-glucose (β-Tg) via the Langmuir–Blodgett and Langmuir–Schaefer (LB-LS) techniques. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize structural and orientational changes in this model biological membrane on a hydrophilic surface modified gold electrode. The results of the spectroscopic measurements showed that the tilt angle of acyl chains obtained for deuterated DMPC bilayers supported on the β-Tg-modified gold is significantly lower than that reported previously for DMPC bilayers deposited directly on Au(111) electrodes. Moreover, tilt angles of ∼18° were obtained for d54-DMPC bilayers on β-Tg self-assembled monolayers (SAMs) at positive potentials, which are similar to the values calculated for h-DMPC deposited on bare gold in the desorbed state and to those observed for a stack of hydrated DMPC bilayers. This data confirms that the β-thioglucose SAM promotes the formation of a water cushion that separates the phospholipid bilayer from the metal surface. As a result, the DMPC polar heads are not in direct contact with the electrode and can adopt a zigzag configuration, which strengthens the chain–chain interactions and allows for an overall decrease in the tilt of the acyl chains. These novel supported model membranes may be especially useful in studies pertaining to the incorporation of peptides and proteins into phospholipid bilayers. Sinergia project no. CRSII2_154451/⁠1 financed by the Swiss National Science Foundation and Discovery grant from the Natural Sciences and Engineering Research Council of Canada