This short review describes our efforts to apply scanning tunneling microscopy and atomic force microscopy to provide an understanding of the mechanism of phospholipid bilayer formation at a gold electrode surface. We also show that these scanning probe microscopies provide unique information concerning the insertion and aggregation of antibiotic peptides and the mechanism of amyloid peptide aggregation at model biological membranes. We demonstrate that the electrochemical techniques such as electrochemical impedance spectroscopy and infrared reflection spectroscopy could be conveniently used in these systems to acquire complementary molecular level information concerning the membrane structure and properties of biomolecules embedded or adsorbed at the membrane surface. This information has strong relevance for the development of biosensors.

Protein misfolding diseases, like Alzheimer's, Parkinson's, and Huntington's disease, are associated with misfolded protein aggregation. Alzheimer's disease is related to a progressive neuronal death induced by small amyloid β oligomers. Here, we describe the procedure to prepare and identify different types of small toxic amyloid β oligomers by atomic force microscopy (AFM).

The ion channel properties of gramicidin A (gA) in a model floating bilayer lipid membrane were studied by electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). To determine if the transport of Na+ across the membrane is coupled with the transport of the counterion, fluoride and perchlorate anions were explored due to their large differences in hydration energy. The combined EIS and IR experiments demonstrated that in contrast to valinomycin, the mechanism of Na+ transfer through the gA channel is independent of the accompanying anion present in the supporting electrolyte. These studies also demonstrated that the conductivity of the gA sodium channel depends on the transmembrane potential and the IR measurements indicated that more gA molecules insert into the bilayer with the decrease of the transmembrane potential. The results of this present study demonstrate that the fBLM system is a suitable model bilayer for the in situ investigations of membrane-active peptides.

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 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 KClO 4 solution by about 180 times, while it has a negligible effect on the membrane resistivity in NaClO 4 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.

Amyloid β, Aβ(1-42), is a component of senile plaques present in the brain of Alzheimer's disease patients and one of the main suspects responsible for pathological consequences of the disease. Herein, we directly visualize the Aβ activity toward a brain-like model membrane and demonstrate that this activity strongly depends on the Aβ oligomer size. PeakForce quantitative nanomechanical mapping mode of atomic force microscopy imaging revealed that the interaction of large-size (LS) Aβ oligomers, corresponding to high-molecular-weight Aβ oligomers, with the brain total lipid extract (BTLE) membrane resulted in accelerated Aβ fibrillogenesis on the membrane surface. Importantly, the fibrillogenesis did not affect integrity of the membrane. In contrast, small-size (SS) Aβ oligomers, corresponding to low-molecular-weight Aβ oligomers, created pores and then disintegrated the BTLE membrane. Both forms of the Aβ oligomers changed nanomechanical properties of the membrane by decreasing its Young's modulus by ∼45%. Our results demonstrated that both forms of Aβ oligomers induce the neurotoxic effect on the brain cells but their action toward the membrane differs significantly.

Modern society relies on the constant flow of quality electricity. Various safety measures in the form of uninterruptible power supplies (UPS) combined with diesel generation systems are used to ensure permanent power delivery to strategic services during a power outage. Batteries UPS systems are held in buffer mode to avoid the self discharge process progressing. The impact of various buffer battery maintenance regimes is an important factor in ensuring the reliability of UPS systems. A series of tests on five battery pairs were performed to estimate the impact of five different buffer regimes on the batteries' state of health. The tests were carried out in the span of one year, at heightened temperature to accelerate the negative impact of the said regimes on the batteries' state of health. The test results showed that, contrary to widely-accepted belief, rippling of the buffer charging current does not have a significant negative impact on battery health. A comparison did indeed show that rippled charging current delivered lower total capacity loss than unrippled current. Then desulfation was applied to the batteries after testing to estimate the amount of capacity that was lost due to sulfation. It determined that rippling promotes more irreversible capacity loss (not caused by sulfation) than unrippled current with the same average voltage. The insights gained from these tests could inform attempts by industry to slow down the deterioration of lead-acid batteries in UPS applications.

Shell-isolated gold nanoparticles (SHINs) were employed to record shell-isolated nanoparticle-enhanced Raman spectra (SHINERS) of a passive layer formed at a gold surface during gold leaching from thiosulfate solutions. The (3-aminopropyl)triethoxysilane (APTES) and a sodium silicate solution were used to coat gold nanoparticles with a protective silica layer. This protective silica layer prevented interactions between the thiosulfate electrolyte and the gold core of the SHINs when the SHINs-modified gold electrode was immersed into the thiosulfate lixiviant. The SHINERS spectra of the passive layer, formed from thiosulfate decomposition, contained bands indicative of hydrolyzed APTES. We have demonstrated how to exploit the presence of these APTES bands as an internal standard to compensate for fluctuations of the surface enhancement of the electric field of the photon. We have also developed a procedure that allows for removal of the interfering APTES bands from the SHINERS spectra. These methodological advancements have enabled us to identify the species forming the passive layer and to determine that the formation of elemental sulfur, cyclo-S8, and polymeric sulfur chains is responsible for inhibition of gold dissolution in oxygen rich thiosulfate solutions.

Electrochemical methods are combined with shell-isolated nanoparticle-enhanced Raman spectroscopy (EC-SHINERS) for a comprehensive study of pyridine adsorption on Au(111), Au(100) and Au(110) single crystal electrode surfaces. The effects of crystallographic orientation, pyridine concentration, and applied potential are elucidated, and the formation of a second pyridine adlayer on Au(111) is observed spectroscopically for the first time. Electrochemical and SHINERS results correlate extremely well throughout this study, and we demonstrate the potential of EC-SHINERS for thorough characterization of processes occurring on single crystal surfaces. Our method is expected to open up many new possibilities in surface science, electrochemistry and catalysis. Analytical figures of merit are discussed.

To date, detailed studies of the thickness of coatings using surface plasmon resonance have been limited to samples that are very uniform in thickness, and this technique has not been applied quantitatively to samples that are inherently rough or undergo instabilities with time. Our manuscript describes a significant improvement to surface plasmon resonance imaging (SPRi) that allows this sensitive technique to be used for quantitative tracking of the thickness and roughness of surface coatings that are rough on the scale of tens of nanometers. We tested this approach by studying samples with an idealized, one-dimensional roughness: patterned channels in a thin polymer film. We find that a novel analysis of the SPRi data collected with the plane of incidence parallel to the patterned channels allows the determination of the thickness profile of the channels in the polymer film, which is in agreement with that measured using atomic force microscopy. We have further validated our approach by performing SPRi measurements perpendicular to the patterned channels, for which the measured SPR curve agrees well with the single SPR curve calculated using the average thickness determined from the thickness profile as determined using AFM. We applied this analysis technique to track the average thickness and RMS roughness of cellulose microfibrils upon exposure to cellulolytic enzymes, providing quantitative determinations of the times of action of the enzymes that are of direct interest to the cellulosic ethanol industry.

This lecture will review our research involving IR spectroscopy at the solid –liquid interface. After a brief review of basic principles of IR spectroscopy and the laws of reflection of light at interfaces, three methods of Infrared Reflection Absorption Spectroscopy (IRRAS) will be described. The first is subtractively normalized interfacial Fourier transform infrared spectroscopy (SNIFTIRS) or in short potential difference IR spectroscopy. This technique finds application to study reversible adsorption of soluble molecules at electrode surfaces. The methods of optimisation of the SNIFTIRS experiment will be discussed. Its application will be illustrated by the case of adsorption of a soluble surfactant such as sodium dodecyl sulphate (SDS) at a gold electrode surface.1 The second technique to measure IR spectra at interfaces is the photon polarization modulation infrared reflection absorption spectroscopy (PM IRRAS). This technique is used to study films of insoluble molecules a various interfaces. I will discuss application of this technique to study model biological membranes supported at a gold electrode surface.2 The third technique is the surface enhanced infrared reflection absorption spectroscopy (SEIRAS). It is performed using attenuated total internal reflection (ATR) element covered by nanoparticles of gold. The intensity of the IR signal is then enhanced by a factor of 100. In addition, since the film of metal nanoparticles is conductive it is used to study phenomena at electrified interfaces. I will describe application of this technique to study structure of water in model biological membrane supported at a gold electrode surface.3 I will emphasize complementarity of SEIRAS and PMIRRAS in application to bio-electrochemical research. Literature 1.Jay Leitch, John Collins, K. Andreas Friedrich, Ulrich Stimming, John R. Dutcher, Jacek Lipkowski “Infrared Studies of the Potential Controlled Adsorption of Sodium Dodecyl Sulfate at the Au(111) Electrode Surface”, Langmuir, 2012,28,2455-2464. 2. Jacek Lipkowski, “Building model biomimetic membrane at a gold electrode surface”, Phys. Chem. Chem. Phys., 2010,12,13874-13887. 3. Taro Uchida, Masatoshi Osawa and Jacek Lipkowski, “SEIRAS studies of water structure at the gold electrode surface in the presence of supported lipid bilayer”, J.Electroanal.Chem. DOI:10.1016/j.jelechem.2013.10.015

The anodic dissolution of two laboratory-made Ni samples obtained using the carbonyl method was investigated to understand the origin of residue formation in the anode basket in an electroplating tank. The first sample was obtained with 3 ppm addition of carbonyl sulfide to introduce a small amount of sulfur (CN-S sample). The second was obtained without sulfur impurities (CN sample). Linear sweep voltammetry and chronopotentiometry were applied to characterize the dissolution of these samples. The dissolution of the CN-S sample took place in the active region at low overpotentials. This behavior is determined by the presence of sulfur impurities that break down the passive layer and facilitate Ni dissolution. The CN sample without sulfur was dissolved at high overpotentials. The overpotential-time plots displayed regular large amplitude oscillations in which the overvoltage periodically moved between the transpassive and passive regimes. The anodic dissolution of this sample was controlled by two competing processes: breakdown and formation of the passive layer. Scanning electron microscopy and white light interference microscopy were applied to monitor the morphological changes of the two samples as a function of the dissolution time. The results of these studies showed that the CN-S sample dissolved uniformly across the surface. However, the roughness and the aspect ratio of the protruding features on the surface increased with time. This sample produced a fine residue due to detachment of small protruding crystallites. In contrast, the dissolution of the CN sample involved pit formation and took place predominantly from the bulk of the pits. The dissolution of this sample left a porous skeleton of more passivated Ni. The residue in this case consisted of large, porous chunks of the skeleton. (C) 2014 Elsevier Ltd. All rights reserved.

High-resolution atomic force microscopy (AFM) was used to image the real-time in situ degradation of crystalline by three types of T. reesei cellulolytic enzymes-TrCel6A, TrCel7A, and TrCel7B-and their mixtures. TrCel6A and TrCel7A are exo-acting cellobiohydrolases processing cellulose fibers from the nonreducing and reducing ends, respectively. TrCel7B is an endoglucanase that hydrolyzes amorphous cellulose within fibers. When acting alone on native cellulose fibers, each of the three enzymes is incapable of significant degradation. However, mixtures of two enzymes exhibited synergistic effects. The degradation effects of this synergism depended on the order in which the enzymes were added. Faster hydrolysis rates were observed when TrCel7A (exo) was added to fibers pretreated first with TrCel7B (endo) than when adding the enzymes in the opposite order. Endo-acting TrCel7B removed amorphous cellulose, softened and swelled the fibers, and exposed single microfibrils, facilitating the attack by the exo-acting enzymes. AFM images revealed that exo-acting enzymes processed the TrCel7B-pretreated fibers preferentially from one specific end (reducing or nonreducing). The most efficient (almost 100%) hydrolysis was observed with the mixture of the three enzymes. In this mixture, TrCel7B softened the fiber and TrCel6A and TrCel7A were directly observed to process it from the two opposing ends. This study provides high-resolution direct visualization of the nature of the synergistic relation between T. reesei exo- and endo-acting enzymes digesting native crystalline cellulose.

We present direct visualization of pores formed by alamethicin (Alm) in a matrix of phospholipids using electrochemical scanning tunneling microscopy (EC-STM). High-resolution EC-STM images show individual peptide molecules forming channels. The channels are not dispersed randomly in the monolayer but agglomerate forming 2D nanocrystals with a hexagonal lattice in which the average channel–channel distance is 1.90 ± 0.1 nm. The STM images suggest that each Alm is shared between the two adjacent channels. Every channel consists of six Alm molecules. Three or four of these molecules have the hydrophilic group oriented toward the center of the channel allowing for water column formation inside the channel. The dimensions of the central pore in the images are consistent with the dimension of the water column in a model of hexameric pore proposed in the literature. The images obtained in this work validate the barrel-stave model of the pore formed in phospholipid membranes by amphiphatic peptides. They also provide direct evidence for cluster formation by such pores.

Polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS) was employed to detect the chemical warfare agent (CWA) simulant triethyl phosphate (TEP) on gold, as well as on US military paint, i.e., chemical agent resistant coating (CARC). The targeted CWAs (G and V-series nerve agents) are characterized by phosphoric group vibrations present in the 1200 cm(-1) region. TEP displays two prominent peaks at 1268 cm(-1) and 1036 cm(-1) corresponding to P=O and (P)-O-C vibrations, respectively. A droplet of TEP solution in cyclohexane was deposited on gold and CARC substrates and after solvent evaporation PM-IRRAS spectra were collected in the 1200 cm(-1) region. The integrated peak area of the PO and (P)OC vibrations was used to construct calibration curves and to determine the experimental limit of detection (LoD). In the case of gold as the substrate the estimated LoD of ~0.48 μg and 1.23 μg was obtained for the P=O and (P)-O-C vibrations, respectively. In the case of CARC, a LoD of 24 μg was determined. These detection limits are at least 3 orders of magnitude lower than the typical lethal dose of G and V-series nerve agents, demonstrating potential of PM-IRRAS for non-contact detection of these CWAs.

Potentiometric titrations of the cytochrome c oxidase (CcO) immobilized in a biomimetic membrane system were followed by two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode. Direct electron transfer was employed to vary the redox state of the enzyme. The CcO was shown to undergo a conformational transition from a non-activated to an activated state after it was allowed to turnover in the presence of oxygen. Differences between the non-activated and activated state were revealed by 2D SEIRA spectra recorded as a function of potential. The activated state was characterized by a higher number of correlated transitions as well as a higher number of amino acids associated with electron transfer.

The first version of this article was published in January issue of the Canadian Chemical News in 2000. This issue featured Electrochemistry and coincided with the 200th anniversary of the famous. Volta pile experiment. It also coincided with the 50th anniversary of the International Society of Electrochemistry. In that article I reflected on the past and I pondered on the future of electrochemistry. It was written for general community of Canadian chemists. The present version has been updated. However most of my original thoughts have been retained. I have also retained the original language of an article written to a non-specialized readership.