Bioelectrochemistry (v.87, #C)

Table of Contents (vii-ix).

by Malgorzata Kotulska; Wlodzimierz Kutner (1-2).

Nucleoside analogue electrochemical behaviour and in situ evaluation of DNA–clofarabine interaction by H. Eda Satana; A. Dora R. Pontinha; Victor C. Diculescu; Ana Maria Oliveira-Brett (3-8).
Nucleoside analogues have had a substantial impact on the treatment of cancer, especially haematological malignancies. The electrochemical behaviour of the nucleoside analogue clofarabine (CLF) was investigated at a glassy carbon electrode using cyclic, differential pulse and square wave voltammetry in different pH supporting electrolytes. The oxidation process of CLF is irreversible and pH-dependent with transfer of two protons and two electrons, following a diffusion controlled mechanism. The oxidation mechanism of CLF involves deprotonation and leads to the formation of a hydroxylated species that undergoes reversible redox reactions. The interaction of DNA and the antileukemia drug CLF was investigated using a dsDNA–electrochemical biosensor in incubated solutions by differential pulse voltammetry. The CLF–DNA interaction leads to changes in the DNA morphological structure, confirmed using the purinic homo-polynucleotide single stranded sequences of guanosine and adenosine, poly[G] and poly[A]-electrochemical biosensors.► Clarify clofarabine electrochemical behaviour. ► Propose an oxidation mechanism for the nucleoside analogue. ► In situ evaluation of DNA-clofarabine interaction.
Keywords: Clofarabine; DNA; Oxidation; Voltammetry;

Investigation of the mediated electron transfer mechanism of cellobiose dehydrogenase at cytochrome c-modified gold electrodes by David Sarauli; Roland Ludwig; Dietmar Haltrich; Lo Gorton; Fred Lisdat (9-14).
The present study reports on the comparison of direct and mediated electron transfer pathways in the interaction of the fungal enzyme cellobiose dehydrogenase (CDH) with the redox protein cytochrome c (cyt c) immobilised at a modified gold electrode surface. Two types of CDHs were chosen for this investigation: a basidiomycete (white rot) CDH from Trametes villosa and a recently discovered ascomycete from the thermophilic fungus Corynascus thermophilus. The choice was based on the pH-dependent interaction of these enzymes with cyt c in solution containing the substrate cellobiose (CB). Both enzymes show rather similar catalytic behaviour at lower pH, dominated by a direct electron exchange with the electrode. With increasing pH, however, also cyt c-mediated electron transfer becomes possible. The pH-dependent behaviour in the presence and in the absence of cyt c is analysed and the potential reaction mechanism for the two enzymes with a different pH-behaviour is discussed.Display Omitted► MET and DET pathways of CDH from two different sources with have been investigated. ► At lower pH ( < 5) DET is the dominating electron transfer pathway for both enzymes. ► At pH 6 a mediation by surface-bound redox protein cyt c is verified. ► Signal chains from cellobiose in via CDH and immobilised cyt c are obtained.
Keywords: Cellobiose dehydrogenase; Direct electron transfer; Mediated electron transfer; Cytochrome c; Immobilised enzyme;

Electrochemical oxidation of berberine and mass spectrometric identification of its oxidation products by Jana Skopalová; Jan Vacek; Barbora Papoušková; David Jirovský; Vítězslav Maier; Václav Ranc (15-20).
Electrochemical oxidation of the isoquinoline alkaloid berberine in aqueous medium was studied by cyclic and differential pulse voltammetry at a glassy carbon electrode (GCE). Two anodic peaks of the quaternary form of berberine were observed at + 1.2 V and + 1.4 V (vs. SCE) in acidic and neutral solutions. When the anodic polarization exceeded the value of + 1.1 V, the redox active film is formed on the GCE surface. The formation of adsorbed film was well-documented by quasireversible redox couple at + 0.25 V which was studied in redox cycling experiments. In alkaline medium, a new anodic peak at + 0.5 V appeared due to oxidation of berberine pseudobase to 8-oxoberberine. Solutions of berberine at different pH were subjected to controlled potential electrolysis on platinum gauze electrode and analyzed using liquid chromatography (HPLC) equipped with electrospray ionization/quadrupole time-of-flight mass spectrometry. The main water soluble monomeric product of berberine oxidation under physiological-near experimental conditions, OP1, was identified as demethyleneberberine cation (2,3-dihydroxy-9,10-dimethoxy-5,6-dihydroisoquinolino[3,2-a]isoquinolin-7-ium).Display Omitted► Electrochemical oxidation of the isoquinoline alkaloid berberine in aqueous medium has been studied. ► In this study cyclic and differential pulse voltammetry at a glassy carbon and platinum electrode was used. ► The main water soluble product of berberine oxidation (OP1) was identified as demethyleneberberine cation. ► The electrochemical approaches presented here could be useful for the study of oxidation of berberine and its derivatives.
Keywords: Berberine; Oxidation; Demethylenation; Hydroxylation; Mass spectrometry;

We have investigated the efficiency of electron transmission through thiolated oligoproline derivatives of general formula: Cys–(Pro)n–CSA, where CSA is a cystamine linker and n = 1–4. The conductance measurements were performed using STM-based molecular junction approach. We have noted that the conductance of the oligoprolines decays exponentially with increasing length of the molecules and the decay constant was 4.3 nm− 1. This indicates that electron transfer is dominated by superexchange mechanism. Based on this observation, we have concluded that the height of the barrier is affected by the specific conformation of the peptide backbone. Such conclusion is supported by the fact that the oligoprolines do not form intramolecular hydrogen bonds, which could provide alternative electron transfer pathways.► Oligoprolines act as mediators for electron transfer (ET). ► The conductance decays exponentially with increasing length of the peptide. ► ET is efficient although oligoprolines do not form intramolecular hydrogen bonds. ► Conformation of the peptide backbone has primary effect on efficiency of ET.
Keywords: Electron transfer; Monolayer; Molecular junction; Scanning tunneling spectroscopy; Peptide;

Electron transfer mechanism in Shewanella loihica PV-4 biofilms formed at graphite electrode by Anand Jain; Xiaoming Zhang; Gabriele Pastorella; Jack O. Connolly; Niamh Barry; Robert Woolley; Satheesh Krishnamurthy; Enrico Marsili (28-32).
Electron transfer mechanisms in Shewanella loihica PV-4 viable biofilms formed at graphite electrodes were investigated in potentiostat-controlled electrochemical cells poised at oxidative potentials (0.2 V vs. Ag/AgCl). Chronoamperometry (CA) showed a repeatable biofilm growth of S. loihica PV-4 on graphite electrode. CA, cyclic voltammetry (CV) and its first derivative shows that both direct electron transfer (DET) mediated electron transfer (MET) mechanism contributes to the overall anodic (oxidation) current. The maximum anodic current density recorded on graphite was 90 μA cm− 2. Fluorescence emission spectra shows increased concentration of quinone derivatives and riboflavin in the cell-free supernatant as the biofilm grows. Differential pulse voltammetry (DPV) show accumulation of riboflavin at the graphite interface, with the increase in incubation period. This is the first study to observe a gradual shift from DET to MET mechanism in viable S. loihica PV-4 biofilms.► S. loihica PV- 4 forms uniform 2-3 µm thick biofilms on graphite electrode. ► Flavins accumulate at the interface S. loihica PV- 4 biofilm/graphite electrode. ► DET shift towards MET mechanism as S. loihica PV-4 biofilm grew on graphite.
Keywords: Extracellular electron transfer; Shewanella loihica PV-4; Electroactive biofilms; Graphite electrode; Mediated electron transfer;

Human sulfite oxidase electrochemistry on gold nanoparticles modified electrode by Stefano Frasca; Oscar Rojas; Johannes Salewski; Bettina Neumann; Konstanze Stiba; Inez M. Weidinger; Brigitte Tiersch; Silke Leimkühler; Joachim Koetz; Ulla Wollenberger (33-41).
The present study reports a facile approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase (hSO) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl4 with branched poly(ethyleneimine) in an ionic liquids resulting particles with a diameter less than 10 nm. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range between 0.5 and 5.4 μM with a high sensitivity (1.85 nA μM− 1). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.► Human sulfite oxidase immobilized on PEI covered gold nanoparticle modified electrode. ► Efficient and bioelectrocatalytic sulfite oxidation at zero potential. ► A facile approach for sulfite biosensing.Display Omitted
Keywords: Direct electron transfer; Gold nanoparticle; Human sulfite oxidase; Ionic liquid; Sulfite biosensor;

Influence of polymer–surfactant aggregates on fluid flow by Tadeusz Malcher; Barbara Gzyl-Malcher (42-49).
This paper describes the influence of interactions of poly(ethylene oxide) (PEO) with cationic cetyltrimethylammonium bromide (CTAB) micelles on drag reduction. Since the interactions between PEO and CTAB micelles alone are weak, salicylate ions were used as CTAB counterions. They facilitate formation of polymer–micelle aggregates by screening the electrostatic repulsions between the charged surfactant headgroups. The influence of polymer–surfactant interactions on drag reduction is of biomedical engineering importance. Drag reducing additives introduced to blood produce beneficial effects on blood circulation, representing a novel way to treat cardiovascular disorders. PEO is a blood-compatible polymer. However, it quickly mechanically degrades when subjected to high shear stresses. Thus, there is a need to search for other additives able to reduce drag, which would be more mechanically stable, e.g. polymer–surfactant aggregates. Numerical simulations of the flow were performed using the CFX software. Based on the internal structure of the polymer–surfactant solution, a hypothesis explaining the reason of increase of drag reduction and decrease in dynamic viscosity with increasing shear rate was proposed. It was suggested that the probable reason for the abrupt increase in friction factor, observed when the critical Reynolds number was exceeded, was the disappearance of the difference in the dynamic viscosity.► Numerical simulations of flow of polymer–surfactant solution were performed in CFX. ► A hypothesis explaining drag reduction properties of aggregates was presented. ► Distribution of dynamic viscosity in the longitudinal section of pipe was obtained. ► The increase in shear rate causes decrease in the dynamic viscosity. ► Abrupt increase in friction factor corresponds to the constant dynamic viscosity.
Keywords: Drag reduction; Polymer–surfactant aggregate; Dynamic viscosity; CFD; Numerical simulation;

Electrokinetic and bioactive properties of CuO∙SiO2 oxide composites by Magdalena Nowacka; Anna Modrzejewska-Sikorska; Łukasz Chrzanowski; Damian Ambrożewicz; Tomasz Rozmanowski; Kamila Myszka; Katarzyna Czaczyk; Karol Bula; Teofil Jesionowski (50-57).
CuO∙SiO2 hybrid oxide precipitated on a semi-technical scale was thoroughly characterised in terms of physicochemical properties. Its particle size distribution and SEM analysis were performed to establish dispersion and surface morphology. Chemical analysis provided information on the content of CuO and SiO2 oxides in the hybrid systems. The oxide systems were also subjected to elemental analysis. Zeta potential determinations were evaluated to obtain information regarding the interactions between colloidal particles. The stability of copper silicates' water dispersions was estimated on the basis of zeta potential measurements. The obtained oxide systems were used as components of polymer composites with polyester resins, which were subjected to mechanical tests and bactericidal tests against Pseudomonas aeruginosa, a well known biofilm-forming microorganism. The anti-adhesive activity of the CuO·SiO2 enriched polymers was assessed using a 9-degree scale of adhesion. A significant reduction in the P. aeruginosa biofilm development rate was achieved for Palatal A 400-01 resins enriched with both 2 and 8 phr of the filler. In the case of Aropol M 105 TB resins the introduction of CuO∙SiO2 caused inhibition of bacterial colonisation but to a smaller extent. These results strongly indicate that the biological activity of Cu was maintained. The release of copper ions into the local environment was examined by atomic absorption spectrometry (AAS). Maximum values of 1.621 and 5.934 mg/dm3 of released copper were detected. The surface composition of both resins studied by energy dispersive X-ray spectroscopy (EDS) contributed to the data suggesting homogenous distribution of Si; however copper seemed to form local aggregates. The presented results may be of great significance for those dealing with materials tailored for specific needs.► Synthesis of CuO∙SiO2 composite oxide with special surface properties and biactivity. ► Electrokinetic stability of copper silicates' water dispersions is estimated by zeta potential measurements. ► Bactericidal tests against Pseudomonas aeruginosa, a well known biofilm forming microorganism. ► Polymer composites with polyester resins and CuO∙SiO2 filler.
Keywords: CuO∙SiO2 oxide composites; Electrokinetic potential; Bioactive and structural properties;

Deconvolution of protein film voltammetric data by fitting multiple components (sigmoids, derivative peaks) often is ambiguous when features are partially overlapping, due to exchangeability between the width and the number of components. Here, a new method is presented to obtain the width of the components. This is based on the equivalence between the sigmoidal catalytic response as function of electrode potential, and the classical saturation curve obtained for the enzyme activity as function of the soluble substrate concentration, which is also sigmoidal when plotted versus log[S]. Thus, analysis of the catalytic voltammogram with Lineweaver–Burk, Eadie–Hofstee, and Hanes–Woolf plots is feasible. This provides a very sensitive measure of the cooperativity number (Hill coefficient), which for electrons equals the apparent (fractional) number of electrons that determine the width, and thereby the number of components (kinetic phases). This analysis is applied to the electrocatalytic oxygen reduction by Paracoccus denitrificans cytochrome aa 3 (cytochrome c oxidase). Four partially overlapping kinetic phases are observed that (stepwise) increase the catalytic efficiency with increasingly reductive potential. Translated to cell biology, the activity of the terminal oxidase stepwise adapts to metabolic demand for oxidative phosphorylation.Display Omitted► The applied potential is equivalent to log(substrate concentration). ► A catalytic voltammogram thus is equivalent to a Michaelis–Menten saturation curve. ► Voltammetric Lineweaver–Burk, Eadie–Hofstee, and Hanes–Woolf plots can be drawn. ► These provide new information on enzyme kinetics. ► These provide the electron cooperativity number needed for deconvolution.
Keywords: Protein film voltammetry; Cytochrome c oxidase; Deconvolution; Enzyme kinetics; Electrocatalysis;

Sorption of copper(II) ions in the biomass of alga Spirogyra sp. by Małgorzata Rajfur; Andrzej Kłos; Maria Wacławek (65-70).
Sorption of copper ions by the alga Spirogyra sp. was investigated to determine the influence of experimental conditions and the methods of sample preparation on the process. The experiments were carried out both under the static and the dynamic conditions. Kinetics and equilibrium parameters of the sorption were evaluated. In addition, the influence was studied of the algae preparation methods on the conductivity of demineralized water in which the algae samples were immersed. The static experiments showed that the sorption of Cu2+ ions reached equilibrium in about 30 min, with approximately 90% of the ions adsorbed in the initial 15 min. The sorption capacity determined from the Langmuir isotherms appeared highly uncertain (SD  = ± 0.027 mg/g dry mass or ± 11%, for the live algae). Under static conditions, the slopes of the Langmuir isotherms depended on the ratio of the alga mass to the volume of solution. The conductometric measurements were proven to be a simple and fast way to evaluate the quality of algae used for the experiments.► Kinetics of Cu2+ ions sorption was investigated, using fresh and preparated algae. ► Influence of algae preparation method on kinetics of Cu2+ sorption was investigated. ► During 15 min about 90% of Cu2+ ions were sorbed ► The slopes of isotherms depended on the ratio of alga mass to volume of solution. ► Conductometric measurements are simple and fast way of algae quality evaluation.
Keywords: Heavy metals; The alga Spirogyra sp.; Sorption kinetics; Equilibrium parameters;

Direct and mediated electrochemical response of the cytochrome P450 106A2 from Bacillus megaterium ATCC 13368 by Hélène Colas; Kerstin M. Ewen; Frank Hannemann; Nikitas Bistolas; Ulla Wollenberger; Rita Bernhardt; Pedro de Oliveira (71-77).
CYP106A2 is one of only a few known steroid hydroxylases of bacterial origin, which might be interesting for biotechnological applications. Despite the enzyme having been studied for more than 30 years, its physiological function remains elusive. To date, there have been no reports of the redox potential of CYP106A2, which was supposed to be unusually low for a cytochrome P450. In this work we show that cyclic voltammetry is not only suitable to determine the redox potential of challenging proteins such as CYP106A2, measured at − 128 mV vs. NHE, but also to study molecular interactions of the enzyme with different interaction partners via the respective electrochemical responses. The effect of small ligands, such as carbon monoxide and cyanide, was observed on the cyclic voltammograms of CYP106A2. Furthermore, we found that Tween 80 caused a positive shift of the redox potential of immobilised CYP106A2 indicative for water expulsion from the haem environment. Moreover, electron transfer mediation phenomena with biological redox partners (e.g. ferredoxins) were studied. Finally, the influence of two different kinds of substrates on the electrochemical response of CYP106A2 was assessed, aligning observations from spectral and electrochemical studies.► CYP106A2 entrapped in modified electrodes responds in cyclic voltammetry. ► The response is modulated by haem ligands and by protein partners. ► Ferredoxins render CYP106A2 easier to reduce and may facilitate the catalytic process. ► Some CYP106A2 substrates influence its redox potential.
Keywords: Cytochrome P450; Cyclic voltammetry; Modified electrode; Protein interaction; Substrate binding;

Electrochemical detection of DNA hybridization using metallacarborane unit by Robert Ziółkowski; Agnieszka B. Olejniczak; Łukasz Górski; Justyna Janusik; Zbigniew J. Leśnikowski; Elżbieta Malinowska (78-83).
The evaluation of novel electrochemically active label for electrochemical detection of DNA hybridization is presented. Metallacarborane units modified with iron, cobalt or chromium were investigated. The value of redox potential and relatively strong current signal facilitate usage of Fe-carborane as marker covalently attached to the ssDNA. In electrochemical genosensor the sequence complementary to UL55 gene was labeled and used as a target for biosensor device. Interactions were investigated using electrochemical and piezoelectric methods. Obtained results confirm usefulness of the designed label in electrochemical detection of DNA hybridization.► Complexes of carboranes with Co3 +, Cr3 + and Fe3 + metal cations were tested as electrochemical markers for DNA biosensors. ► Electrochemical signal can be observed only with the metallacarborane marker located away from the electrode. ► Fe-carboranes can be applied as useful markers for electrochemical DNA sensing.
Keywords: Electroanalytical methods; Biosensors; Nucleic acids; Redox labels;

We have shown that proteins produce at bare mercury electrodes a well-developed chronopotentiometric peak H. At sufficiently high current densities and low ionic strengths, this peak is sensitive to changes in protein structures. At higher ionic strengths this sensitivity can be lost but it can be restored, when instead of bare, thiol-modified Hg electrodes are used. Here we studied properties of the dithiothreitol (DTT) layer at the hanging mercury drop electrode and showed that at low concentrations (5 μM–200 μM) the DTT is adsorbed as a dithiol with both –SH groups attached to the surface. At higher DTT concentrations than 1 mM, a densely packed pinhole-free layer is formed with the DTT molecules bound to the electrode surface by a single –SH group, oriented perpendicularly to the surface. We found that, if a sufficiently high DTT concentration is used, preparation of the DTT-modified Hg electrodes can be omitted and proteins can be co-adsorbed with DTT on liquid Hg or solid amalgam electrodes without the loss of sensitivity for changes in protein structures. The newly observed properties of the DTT self assembled monolayers (SAMs) at Hg electrodes appear important for designing new types of solid amalgam electrode arrays for electrochemical analysis of proteins.Display Omitted► DTT-modified mercury and solid amalgam electrodes for protein structure-sensitive analysis. ► One step co-adsorption of DTT and protein at electrode surface. ► DTT molecules perpendicularly orientated to the surface form a densely packed pinhole-free layer.
Keywords: Protein electroanalysis; DTT-modified Hg electrodes; Electrocatalysis; Constant current chronopotentiometry; Protein structure;

Lysophosphatidic acid induced red blood cell aggregation in vitro by Lars Kaestner; Patrick Steffen; Duc Bach Nguyen; Jue Wang; Lisa Wagner-Britz; Achim Jung; Christian Wagner; Ingolf Bernhardt (89-95).
Under physiological conditions healthy RBCs do not adhere to each other. There are indications that RBCs display an intercellular adhesion under certain (pathophysiological) conditions. Therefore we investigated signaling steps starting with transmembrane calcium transport by means of calcium imaging. We found a lysophosphatidic acid (LPA) concentration dependent calcium influx with an EC50 of 5 μM LPA. Downstream signaling was investigated by flow cytometry as well as by video-imaging comparing LPA induced with “pure” calcium mediated phosphatidylserine exposure and concluded the coexistence of two branches of the signaling pathway. Finally we performed force measurements with holographic optical tweezers (HOT): The intercellular adhesion of RBCs (aggregation) exceeds a force of 25 pN. These results support (i) earlier data of a RBC associated component in thrombotic events under certain pathophysiological conditions and (ii) the concept to use RBCs in studies of cellular adhesion behavior, especially in combination with HOT. The latter paves the way to use RBCs as model cells to investigate molecular regulation of cellular adhesion processes.► Lysophosphatidic acid leads to concentration dependent Ca2+ entry in red blood cells. ► Lysophosphatidic acid induces phosphatydylserine exposure via 2 alternative pathways. ► Lysophosphatidic acid triggered intercellular red blood cell adhesion exceeds 25 pN. ► Red blood cells and laser tweezers are a good model system to investigate adhesion. ► Red blood cell adhesion/aggregation has most likely a pathophysiological relevance.
Keywords: Erythrocyte adhesion; Fluorescence imaging; Holographic optical tweezers; Calcium signaling; Phosphatydylserine exposure;

Interaction of prazosin with model membranes — A Langmuir monolayer study by Barbara Gzyl-Malcher; Jadwiga Handzlik; Ewelina Klekowska (96-103).
In this study, the effect of prazosin on the molecular interactions between cholesterol and 1,2-dipalmitoylphosphatidylcholine (DPPC) within a monolayer at an air–water interface was studied. A mixed cholesterol/DPPC monolayer was employed as a model lipid membrane.From a detailed analysis of surface pressure–area isotherms, it was concluded that DPPC and cholesterol were miscible and formed non-ideal monolayers on prazosin solution. The thermodynamic stability of the mixed monolayers was investigated by analyzing the free energy of mixing. It was found that the mixed monolayers were more stable than the single component monolayers. Monolayers spread over a subphase with prazosin were more compressible than those spread on pure water. To quantify the effect of prazosin on the monolayer stability, the Gibbs free energy due to the presence of prazosin in the water subphase was calculated. It was found that prazosin penetrated and destabilized mixed cholesterol/DPPC monolayers. However, a comparison of the drug penetration into the pure DPPC monolayer and the mixed cholesterol/DPPC monolayer showed that the presence of cholesterol in the DPPC monolayer considerably restricted the drug penetration.► Mixed cholesterol/DPPC monolayer was employed as a model lipid membrane. ► Prazosin penetration into the mixed cholesterol/DPPC monolayers was studied. ► Prazosin reduces attractive interactions between DPPC and cholesterol. ► Cholesterol condenses monolayer and reduces the extent of prazosin penetration
Keywords: Langmuir monolayer; The excess free energy of mixing; Phospholipid; Cholesterol; Prazosin;

Monitoring of beer fermentation based on hybrid electronic tongue by Anna Kutyła-Olesiuk; Michał Zaborowski; Piotr Prokaryn; Patrycja Ciosek (104-113).
Monitoring of biotechnological processes, including fermentation is extremely important because of the rapidly occurring changes in the composition of the samples during the production. In the case of beer, the analysis of physicochemical parameters allows for the determination of the stage of fermentation process and the control of its possible perturbations. As a tool to control the beer production process a sensor array can be used, composed of potentiometric and voltammetric sensors (so-called hybrid Electronic Tongue, h-ET). The aim of this study is to apply electronic tongue system to distinguish samples obtained during alcoholic fermentation. The samples originate from batch of homemade beer fermentation and from two stages of the process: fermentation reaction and maturation of beer. The applied sensor array consists of 10 miniaturized ion-selective electrodes (potentiometric ET) and silicon based 3-electrode voltammetric transducers (voltammetric ET). The obtained results were processed using Partial Least Squares (PLS) and Partial Least Squares-Discriminant Analysis (PLS-DA). For potentiometric data, voltammetric data, and combined potentiometric and voltammetric data, comparison of the classification ability was conducted based on Root Mean Squared Error (RMSE), sensitivity, specificity, and coefficient F calculation. It is shown, that in the contrast to the separately used techniques, the developed hybrid system allowed for a better characterization of the beer samples. Data fusion in hybrid ET enables to obtain better results both in qualitative analysis (RMSE, specificity, sensitivity) and in quantitative analysis (RMSE, R 2, a, b).► Hybrid electronic tongue based on potentiometric and voltammetric sensors is reported. ► The sensor array is applied to beer fermentation monitoring. ► Classification ability is reliably described with the use of objective methods.
Keywords: Sensor array; Hybrid electronic tongue; Beer fermentation;

Iron content (PIXE) in competent and incompetent veins is related to the vein wall morphology and tissue antioxidant enzymes by Wirginia Krzyściak; Joanna Kowalska; Mariusz Kózka; Monika A. Papież; Wojciech M. Kwiatek (114-123).
Impaired venous drainage of the lower extremities determines a cascade of pathologic events leading to chronic venous disease (CVD). It is believed that the one cause of CVD is red blood cell extravasation and local iron overload that could generate free radicals and iron-dependent inflammation. The aim of this study was to investigate the relationship between: the intracellular iron deposits in varicose veins and tissue oxidative state measured by: the Proton Induced X-ray Emission Spectroscopy (FePIXE), (tSOD), (tGPx), (tTBARs) and (boxDNA). Patients with diagnosed CVD were qualified for surgical procedure. Entire trunk of the great saphenous vein (GSV) was extracted. Part located near medial ankle was considered competent (C) in duplex ultrasonography (USG) examination. The incompetent (I) part was extracted from GSV where USG showed incompetent valves and massive venous reflux. The difference between local tFePIXE, tTBARS, boxDNA, tGPx, tSOD in incompetent and competent part of vein tissue was statistically significant. Intima/media ratio directly correlated with FePIXE C/I concentration. Iron deposition in competent vs incompetent part of vein was also related to the oxidative stress parameters (boxDNA). The findings from this pilot study suggest that FePIXE measurement may be useful for explaining the progression of chronic venous disease.► Tissue iron overload could generate free radicals in varicose veins. ► PIXE method can be used for iron content in venous vessels. ► The presence of free iron ions result in damage of DNA.
Keywords: Proton Induced X-ray Emission (PIXE); Reactive oxygen species (ROS); Chronic venous insufficiency (CVI); Tissue iron; Blood oxidized DNA (boxDNA);

Changing bioperformance of TiO2 amorphous nanotubes as an effect of inducing crystallinity by A. Mazare; M. Dilea; D. Ionita; I. Titorencu; V. Trusca; E. Vasile (124-131).
Controlled-diameter TiO2 nanotubes were obtained by electrochemical anodizing of two different substrates (Ti and Ti6Al7Nb) in an aqueous electrolyte. As-formed TiO2 nanotubes are amorphous and by subjecting to thermal treatments, the structure becomes crystalline. An optimal thermal treatment with a specific anatase/rutile ratio was chosen, determined from X-ray diffraction (XRD). The electrochemical behaviour of annealed and as-formed samples was followed with Tafel plots and Electrochemical impedance spectroscopy (EIS), while surface analysis involved scanning electron microscopy (SEM) and contact angle measurements (CA). Annealed samples have a more hydrophilic character than as-formed as well as a better stability in bioliquids. Such behaviour of annealed samples is connected with a better biocompatibility expressed in terms of cell morphology and gene expression of bone specific markers obtained from Reverse Transcription Polymerase Chain Reaction (RT-PCR).Display Omitted► TiO2 nanotubes are obtained on Ti and on Ti6Al7Nb, furthermore optimal thermal treatments are chosen. ► Annealed TiO2 nanotubes are more stable regarding corrosion. ► RT-PCR data present a two fold increase of osteocalcin expression in MG-63 cells grown on annealed TiO2 nanotubes. ► All these data suggest that annealed TiO2 nanotubes may represent a promising material for orthopaedic cellular therapy.
Keywords: TiO2 nanotubes; Ti6Al7Nb; Annealing; Biocompatibility; RT-PCR;

Electroporation-mediated genetic vaccination for antigen mapping: Application to Plasmodium falciparum VAR2CSA protein by Bita Bordbar; Sédami Gnidehou; Nicaise Tuikue Ndam; Justin Doritchamou; Azizath Moussiliou; Mickael Quiviger; Philippe Deloron; Daniel Scherman; Pascal Bigey (132-137).
Genetic vaccination, consisting in delivering a genetically engineered plasmid DNA by a non-viral vector or technique into a tissue, is currently of great interest. New delivery technique including DNA transfer by electroporation recently greatly improved the potency of this concept. Because it avoids the step of producing a recombinant protein, it is particularly of use in studying the immunogenic properties of large proteins. Here we describe the use of electroporation mediated DNA immunization to identify important protective epitopes from the large VAR2CSA protein from Plasmodium falciparum implicated in the pathology of placental malaria. Immunizing mice and rabbit with DNA plasmids encoding different fragments of VAR2CSA leads to high titer antisera. Moreover an N-terminal region of the protein was found to induce protective functional antibodies.► Genetic vaccination was used to rapidly identify antigenic fragments. ► Plasmid electroporation for genetic vaccination against Plasmodium falciparum protein. ► A VAR2CSA protein fragment against placental-malaria erythrocyte adhesion.
Keywords: Electrotransfer; Plasmid electroporation; Genetic immunization; Placental malaria;

The interactions between proteins and solid surfaces are important for the formation of biocompatible materials. In this study, the physicochemical properties of Bovin serum albumin (BSA) in solution and on a solid surface were studied. The zeta potential and number of uncompensated charges on BSA surfaces were determined from electrophoretic mobility measurements. The dynamic viscosity was also measured to determine BSA conformations in solution, and the data were converted to the effective length L ef of the BSA molecule. The length of a BSA molecule was measured to be 8.3 nm in the compact state (N form at pH 4–9) and 26.7 nm in the extended state (F-form). This study demonstrates that the relationship between the hydrodynamic radius, dynamic viscosity and electrophoretic mobility can provide information about the shape and conformation of biopolymer in solution.The contact angle measurements and deposition of fluorescent latex particles were used to characterise BSA monolayers on a mica surface, which were produced by controlled adsorption under diffusion transport. The results suggest that the distribution of charge across a BSA molecule is heterogeneous as evidenced by the presence of positive and negative patches. The maximum contact angle was observed under conditions in which both BSA and mica were oppositely charged. A higher positive zeta potential of BSA was observed to correlate with a higher contact angle. However, at a higher negative zeta potential, BSA exhibited a lower binding affinity. The charge distribution across BSA monolayers was also studied via the colloidal deposition method using negatively charged fluorescent latex particles. Unexpectedly, the fluorescent latex particles adsorbed onto BSA monolayers, even when the effective zeta potential of BSA was negative. This phenomenon may originate from the heterogeneous charge distribution across BSA molecules.Display Omitted► Zeta potential and uncompensated charges of BSA by electrophoretic mobility measurement. ► Dynamic viscosity converted to the effective length of the molecule. ► Contact angle of BSA monolayers using the sessile drop shape analysis. ► Number of latex particles captured by the protein monolayer on mica using fluorescent microscope. ► The heterogeneous charge distribution over the BSA molecule.
Keywords: Conformation of Bovine serum albumin (BSA); Intrinsic viscosity; BSA adsorption on mica surface;

Influence of dicephalic ionic surfactant interactions with oppositely charged polyelectrolyte upon the in vitro dye release from oil core nanocapsules by Urszula Bazylińska; Renata Skrzela; Marek Piotrowski; Krzysztof Szczepanowicz; Piotr Warszyński; Kazimiera A. Wilk (147-153).
The electrostatic interactions between dicephalic-type surfactants: cationic, N,N-bis[3,3′-(trimethylammonio)propyl]dodecanamide dimethylsulphate (C12(TAPAMS)2) and anionic, disodium N-dodecyliminodiacetate (C12(COONa)2) with oppositely charged polyelectrolytes, natural, λ-carrageenan (CAR) and synthetic, poly(diallyldimethylammonium chloride) (PDADMAC) were investigated at the oil/water interface by interfacial tension measurements to obtain the most stable anchor layer of polymeric shells of nanocapsules obtained via LbL method. All nanocarriers coated with further λ-carrageenan/poly-l-lysine (CAR/PLL) bilayers were created on the nanoemulsion templates loaded with hydrophobic cyanine-type photosensitizer, IR-786. To evaluate progress of the layer-by-layer deposition of polyelectrolytes on the liquid core and nanocapsules' stability, zeta potential measurements were used. Size (< 100 nm) and morphology of the obtained nanoproducts were examined by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM) techniques. The in vitro release profile features were studied spectrophotometrically and interpreted in terms of diffusion-controlled processes, proving that selection for the first shell layer of an appropriate ionic surfactant and polyelectrolyte type and their strong interactions are the most desirable features for fabrication of long sustained nanocapsules encapsulating a hydrophobic photosensitizer.► Diffusion-controlled release of IR-786 from multilayer long-sustained oil-core nanocapsules. ► Electrostatic interactions between dicephalic ionic surfactants with PEs at the oil/water interface. ► Fabrication of oil-core nanocapsules via layer-by-layer (LbL) adsorption. ► Stability, sizing, morphology and permeability of cyanine-loaded multilayer nanocarriers.
Keywords: Interfacial tension; Dicephalic surfactants; Polyelectrolytes; Multilayer nanocapsules; Nanoemulsions;

Hybrid biobattery based on arylated carbon nanotubes and laccase by Krzysztof Stolarczyk; Małgorzata Sepelowska; Dominika Lyp; Kamila Żelechowska; Jan F. Biernat; Jerzy Rogalski; Kevin D. Farmer; Ken N. Roberts; Renata Bilewicz (154-163).
Single-walled carbon nanotubes (SWCNT) were covalently modified with anthracene and anthraquinone and used for the construction of cathodes for biocatalytic reduction of dioxygen. The nanotubes with aromatic groups casted onto the electrode increased the working surface of the electrode and enabled efficient direct electron transfer (DET) between the enzyme and the electrode. The aryl groups enter the hydrophobic pocket of the T1 center of laccase responsible for exchanging electrons with the substrate. Glassy carbon electrode covered with arylated SWCNT and coated with a layer of neutralized Nafion containing laccase was found to be a very efficient cathode in the hybrid battery. Zn wire covered with a Nafion film served as the anode. The cell parameters were determined: power density was 2 mW/cm2 and the open circuit potential was 1.5 V.Display Omitted
Keywords: Carbon nanotubes; Bioelectrocatalysis; Oxygen reduction; Biofuel cell; Laccase;

Microbial analysis of anodic biofilm in a microbial fuel cell using slaughterhouse wastewater by Krishna P. Katuri; Ann-Marie Enright; Vincent O'Flaherty; Dónal Leech (164-171).
The ability of dual-chambered microbial fuel cell, fed with slaughterhouse wastewater with an anaerobic mixed-sludge as initial source of bacteria, to generate power is investigated. MFC voltage generation across a fixed 100 Ω load indicates power generation capability, with power production correlated to changes in anolyte VFA content. A maximum MFC power density of 578 mW/m2 is obtained for an MFC developed under 100 Ω load, compared to a maximum power density of 277 mW/m2 for an MFC developed under higher resistance (1 MΩ) control conditions. Voltammetry of the biofilm developed under 100 Ω load displays a current–voltage signal indicative of bioelectrocatalytic oxidation of feed at a potential of − 0.35 V vs. Ag/AgCl, compared to negligible signals for biofilms developed under control conditions. Denaturing gradient gel electrophoresis of PCR amplified 16S rRNA gene fragments reveals that the anodic bacterial communities in reactors operated under 100 Ω load result in communities of lower diversity than for the control condition, with Geovibrio ferrireducens dominant in the anodic biofilm community. These results indicate that in MFC reactors, functionally stable electroactive bacteria are enriched under 100 Ω load compared to high resistance control conditions, and were able to sustain higher power in MFCs.Display Omitted► A two-chambered microbial fuel cell generates electricity from slaughterhouse wastewater. ► Fuel cell voltage-time profile correlates with production of anolyte VFA content. ► Biofilm developed under 100 Ω load yields increased power compared to that developed under 1 MΩ. ► Bacterial community analysis indicates external load induces bacterial diversity.
Keywords: Microbial fuel cell; Anodic biofilm; Microbial ecology; Slaughterhouse waste; Electroanalysis;

A comparison of glucose oxidase and aldose dehydrogenase as mediated anodes in printed glucose/oxygen enzymatic fuel cells using ABTS/laccase cathodes by Peter Jenkins; Saara Tuurala; Anu Vaari; Matti Valkiainen; Maria Smolander; Dónal Leech (172-177).
Current generation by mediated enzyme electron transfer at electrode surfaces can be harnessed to provide biosensors and redox reactions in enzymatic fuel cells. A glucose/oxygen enzymatic fuel cell can provide power for portable and implantable electronic devices. High volume production of enzymatic fuel cell prototypes will likely require printing of electrode and catalytic materials. Here we report on preparation and performance of, completely enzymatic, printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks, enzyme and mediator. A comparison of cell performance using a range of mediators for either glucose oxidase (GOx) or aldose dehydrogenase (ALDH) oxidation of glucose at the anode and ABTS and a fungal laccase, for reduction of oxygen at the cathode, is reported. Highest power output, although of limited stability, is observed for ALDH anodes mediated by an osmium complex, providing a maximum power density of 3.5 μW cm− 2 at 0.34 V, when coupled to a laccase/ABTS cathode. The stability of cell voltage in a biobattery format, above a threshold of 200 mV under a moderate 75 kΩ load, is used to benchmark printed fuel cell performance. Highest stability is obtained for printed fuel cells using ALDH, providing cell voltages over the threshold for up to 74 h, compared to only 2 h for cells with anodes using GOx. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells.► Enzymatic fuel cells prepared by printing enzyme, mediator and carbon onto filter paper. ► Mediated glucose oxidase or aldose dehydrogenase anodes are coupled to a laccase/ABTS cathode. ► Highest power from cells based on aldose dehydrogenase using osmium complex as mediator. ► Best cell voltage stability using aldose dehyrogenase anodes with a TMPD mediator.
Keywords: Biofuel cell; Printing; Laccase; Glucose oxidase; Aldose dehydrogenase;

A new synthesis route for Os-complex modified redox polymers for potential biofuel cell applications by Sascha Pöller; Yvonne Beyl; Jeevanthi Vivekananthan; Dmitrii A. Guschin; Wolfgang Schuhmann (178-184).
A new synthesis route for Os-complex modified redox polymers was developed. Instead of ligand exchange reactions for coordinative binding of suitable precursor Os-complexes at the polymer, Os-complexes already exhibiting the final ligand shell containing a suitable functional group were bound to the polymer via an epoxide opening reaction. By separation of the polymer synthesis from the ligand exchange reaction at the Os-complex, the modification of the same polymer backbone with different Os-complexes or the binding of the same Os-complex to a number of different polymer backbones becomes feasible. In addition, the Os-complex can be purified and characterized prior to its binding to the polymer. In order to further understand and optimize suitable enzyme/redox polymer systems concerning their potential application in biosensors or biofuel cells, a series of redox polymers was synthesized and used as immobilization matrix for Trametes hirsuta laccase. The properties of the obtained biofuel cell cathodes were compared with similar biocatalytic interfaces derived from redox polymers obtained via ligand exchange reaction of the parent Os-complex with a ligand integrated into the polymer backbone during the polymer synthesis.
Keywords: Os-complex modified polymers; Bioelectrocatalysis; Laccase; Oxygen reduction; Biofuel cell;

In the present study we demonstrate for the first time the possibility for conversion of solar energy into electricity on the principles of Direct Photosynthetic Plant Fuel Cell (DPPFC) technology by using aquatic higher plants. Lemna minuta duckweed was grown autotrophically in specially constructed fuel cells under sunlight irradiation and laboratory lighting. Current and power density up to 1.62 ± 0.10 A.m− 2 and 380 ± 19 mW.m− 2, respectively, were achieved under sunlight conditions. The influence of the temperature, light intensity and day/night sequencing on the current generation was investigated. The importance of the light intensity was demonstrated by the higher values of generated current (at permanently connected resistance) during daytime than those through the nights, indicating the participation of light-dependent photosynthetic processes. The obtained DPPFC outputs in the night show the contribution of light-independent reactions (respiration). The electron transfer in the examined DPPFCs is associated with a production of endogenous mediator, secreted by the duckweed. The plants' adaptive response to the applied polarization is also connected with an enhanced metabolism resulting in an increase of the protein and carbohydrate intracellular content. Further investigations aiming at improvement of the DPPFC outputs and elucidation of the electron transfer mechanism are required for practical application.Display Omitted► Conversion of solar energy into electricity by Direct Photosynthetic Plant Fuel Cell. ► 1.62 ± 0.10 A.m– 2 and 380 ± 19 mW.m– 2 generated under sunlight irradiation. ► Established dependence of fuel cell electrical outputs on the light intensity. ► Enhanced metabolism of duckweed as an adaptive response to polarization. ► Extracellular electrochemically active metabolite as an electron transfer mediator.
Keywords: Direct Photosynthetic Plant Fuel Cell (DPPFC); Lemna minuta duckweed; Electricity generation; Day/night cycle; Electron transfer mediator;

Langmuir monolayer studies and electrochemical methods were employed to investigate the effect of model membrane organization on the interactions with persistent pollutants such as perfluorinated carboxylic acids (PFCAs). 1,2-dimyristoyl-sn-glycero-3-phosphoethanoloamine (DMPE) was employed to construct the model lipid membrane and perfluorooctanesulphonic acid (PFOS) was chosen as the representative of perfluorinated pollutants. We demonstrate that perfluorinated compounds penetrate a model membrane only when it is less condensed. Such liquid-expanded phase was achieved by compressing the Langmuir monolayer to lower surface pressures. PFOS incorporation into model DMPE membrane during membrane formation was observed in liquid-expanded region, while at higher surface pressures, in the well-organized monolayer the expulsion of perfluorinated compound occurred as the result of a strong attraction between the DMPE molecules. The DMPE monolayers prepared by the Langmuir–Blodgett/Langmuir–Schaefer method were transferred onto gold electrode under surface pressure of 3 mN/m and 20 mN/m. The model membrane organization depends on surface pressure during transfer and time of exposure to PFOS solution and is shown to affect the electrode accessibility by three electroactive compounds used as the probes of the blocking properties of the monolayer: menadione, potassium ferricyanide and hexaamineruthenium chloride, differing in the properties and kinetics of electron transfer.► Interactions with perfluorinated compound depend on membrane organization. ► Perfluorinated compounds penetrate a model membrane only when it is in the liquid-expanded phase. ► In well-organized lipid monolayers the expulsion of perfluorinated compound occurred. ► Electrode behavior of electroactive probes is sensitive to differences in the organization of DMPE bilayers. ► Exposure to PFOS solutions affects the transport efficiency of electroactive species across the bilayer.
Keywords: Perfluorooctanesulphonic acid (PFOS); 1,2-dimyristoyl-sn-glycero-3-phosphoethanoloamine (DMPE); menadione; Cyclic voltammetry; Langmuir monolayer;

The dipole moment of a water molecule in liquid water differs from that of an isolated one because each molecule is further polarized by the electric field of its neighbours. In this work a formula for the spatial dependence of the relative permittivity of an electrolyte near a highly charged surface is obtained in which the mutual influence of the water molecules is taken into account by means of the cavity field. The orientational ordering of water dipoles is considered in the saturation regime. It is predicted that the relative permittivity of an electrolyte solution near the highly charged surface (i.e. in saturation regime) may be substantially decreased due to orientational ordering of water (saturation effect) and depletion of water molecules (excluded volume effect) due to accumulation of counterions.► Theoretical consideration of an electrolyte solution near a highly charged surface. ► Orientational ordering of water dipoles in the saturation regime. ► Depletion of water near the charged surface due to accumulation of counterions. ► Analytical expression for spatial dependence of relative permittivity. ► Decrease of relative permittivity of the electrolyte solution near the charged surface.
Keywords: Relative permittivity; Orientational ordering of water; Excluded volume effect; Cavity field; Saturation effect;

The transport along membrane nanotubes driven by the spontaneous curvature of membrane components by Doron Kabaso; Nataliya Bobrovska; Wojciech Góźdź; Ekaterina Gongadze; Veronika Kralj-Iglič; Robert Zorec; Aleš Iglič (204-210).
Intercellular membrane nanotubes (ICNs) serve as a very specific transport system between neighboring cells. The underlying mechanisms responsible for the transport of membrane components and vesicular dilations along the ICNs are not clearly understood. The present study investigated the spatial distribution of anisotropic membrane components of tubular shapes and isotropic membrane components of spherical shapes. Experimental results revealed the preferential distribution of CTB (cholera toxin B)–GM1 complexes mainly on the spherical cell membrane, and cholesterol–sphingomyelin at the membrane leading edge and ICNs. In agreement with previous studies, we here propose that the spatial distribution of CTB–GM1 complexes and cholesterol–sphingomyelin rafts were due to their isotropic and anisotropic shapes, respectively. To elucidate the relationship between a membrane component shape and its spatial distribution, a two-component computational model was constructed. The minimization of the membrane bending (free) energy revealed the enrichment of the anisotropic component along the ICN and the isotropic component in the parent cell membrane, which was due to the curvature mismatch between the ICN curvature and the spontaneous curvature of the isotropic component. The equations of motion, derived from the differentiation of the membrane free energy, revealed a curvature-dependent flux of the isotropic component and a curvature-dependent force exerted on a vesicular dilation along the ICN. Finally, the effects of possible changes in the orientational ordering of the anisotropic component attendant to the transport of the vesicular dilation were discussed with connection to the propagation of electrical and chemical signals.
Keywords: Computational model; Membrane and cell biomechanics; Intercellular nanotubes; Transport mechanism; Nanotube dilations (gondolas);

Interaction of poly(N-isopropylacrylamide) (pNIPAM) based nanoparticles and their linear polymer precursor with phospholipid membrane models by Nerea Ormategui; Shengwen Zhang; Iraida Loinaz; Rik Brydson; Andrew Nelson; Alexander Vakurov (211-219).
Poly(N-isopropylacrylamide) (pNIPAM) is a thermoresponsive polymer which has promising applications in nanomedicine for drug delivery. The cross-linking of pNIPAM based copolymer using the chain collapse method leads to the synthesis of pNIPAM based polymer nanoparticles. This study looks at the interaction of pNIPAM polymers and pNIPAM nanoparticles with biomembrane models of, (i) a dioleoyl phosphatidylcholine (DOPC) monolayer on a mercury (Hg) electrode and (ii) DOPC and dimyristoyl phosphatidylcholine (DMPC) vesicles. The following techniques were used to follow the interactions: Dynamic light scattering (DLS), differential scanning calorimetry (DSC), rapid cyclic voltammetry (RCV) and electrochemical impedance spectroscopy (EIS). Results showed that the polymers interacted more extensively than the nanoparticles with the phospholipid. The interaction of the polymer was more rapid and led to a polymer-phospholipid conjugate whereas the nanoparticle adsorbed on the phospholipid monolayer surface and penetrated the monolayer at longer contact times. The association of the linear polymer with the phospholipid can be related to the larger molecular area available with the pendant − Cl groups and the inherent polymeric flexibility compared to the nanoparticle structure. The apparent dissociation constant for nanoparticles–DOPC complex was Kd,app  = 1.67 ∗ 10− 5  ± 1.2 ∗ 10− 6  mol dm− 3. The apparent kinetic constant of nanoparticle penetration through the DOPC monolayer was k2,app  = 1.054 ∗ 10− 2  ± 9.1 ∗ 10− 4  s− 1. It can be concluded therefore that the pNIPAM nanoparticle because of its lower affinity for phospholipids is more appropriate for medical applications.Display Omitted► Poly N-isopropylacrylamide (pNIPAM) nanoparticle derived from collapsed pNIPAM polymer. ► pNIPAM polymer has stronger/more rapid interaction with phospholipid membrane. ► pNIPAM polymer forms complex with phospholipid. ► pNIPAM nanoparticle adsorbs on and penetrates DOPC after longer times. ► Activities of pNIPAM polymer and pNIPAM nanoparticle can be related to their structures.
Keywords: Poly(N-isopropylacrylamide) polymers; Poly(N-isopropylacrylamide) nanoparticles; Chain collapse; Phospholipid monolayer; Polymer-phospholipid complex;

Complexation of cytochrome c with calixarenes incorporated into the lipid vesicles and supported membranes by Zuzana Garaiová; Muhammad Ali Mohsin; Veronika Vargová; Florinel-Gabriel Banica; Tibor Hianik (220-225).
We studied the interaction of cytochrome c (cyt c) with specific calixarenes (CX) incorporated into the large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) or supported lipid membranes (sBLM) and compared this with not specific adsorption of cyt c to the LUV containing DMPC and anionic phosphatidic acid (PA) or sBLM composed of a mixture of DMPC and dimyristoylphosphatidic acid (DMPA). We showed that with increasing concentration of CX the average size of LUV increased and zeta potential become more negative as it is suggested from dynamic light scattering experiments. For PA containing LUV the increase in vesicle diameter was less expressed, but zeta potential decreased similarly like that of LUV contained CX. Cyt c did not affect significantly the LUV size, but reduced the negative zeta potential both for CX and PA containing vesicles. Electrochemical impedance spectroscopy allowed us to determine binding of cyt c to sBLM contained CX or DMPA. In both cases we observed decrease of charge transfer resistance with increasing cyt c concentration. The analysis of binding process suggests that the main driving force for interaction of cyt c with sBLM is the negative surface charge.Display Omitted► Calixarenes (CX) affect the size and zeta potential of lipid vesicles. ► Cytochrome c (cyt c) reduces the negative charge of vesicles, but does not affect their size. ► Cyt c reduces the charge transfer resistance at electrodes coated with supported membranes containing CX or negatively charged lipids. ► Negative surface charge is the main driving force in interaction of cyt c with membranes.
Keywords: Calixarenes; Cytochrome c; Lipid vesicles; Zeta potential; Electrochemical Impedance Spectroscopy;

Effects of chronic kidney disease on blood cells membrane properties by Z. Kaderjakova; I. Lajdova; M. Horvathova; M. Morvova; L. Sikurova (226-229).
Chronic kidney disease (CKD) is progressive loss of renal function associated among others with increased intracellular calcium concentration. The purpose of this study was to identify the effects of CKD on cell membrane properties such as human red blood cell Ca2 + ATPase activity, lymphocyte plasma membrane P2X7 receptor expression and function. This could help us in elucidating the origin of increased calcium concentration in blood cells. We found out Ca2 + ATPase activity is decreased in early stage CKD patients resulting in altered calcium removal from cytoplasm. By means of flow cytometry we assessed that P2X7 receptor expression on lymphocyte membrane is 1.5 fold increased for CKD patients. Moreover, we detected an increased uptake of ethidium bromide through this receptor in CKD at basal conditions. It means CKD lymphocyte membranes contain more receptors which are more permeable thus allowing increased calcium influx from extracellular milieu. Finally, we can state alterations in blood cell membranes are closely linked to CKD and may be responsible for intracellular calcium accumulation.► Membrane alterations in early stages chronic kidney disease. ► Increased calcium influx through P2X7 receptor and increased P2X7 receptor expression. ► Decreased plasma membrane calcium ATPase activity. ► Multifactorial reasons of increased calcium level in peripheral mononuclear cells.
Keywords: Chronic kidney disease; P2X7 receptor; Ca2 + ATPase;

Interaction of gentamicin polycation with model and cell membranes by Eugenia Kovács; Tudor Savopol; Maria-Minodora Iordache; Lavinia Săplăcan; Iuliana Sobaru; Claudia Istrate; Marie-Paule Mingeot-Leclercq; Mihaela-Georgeta Moisescu (230-235).
The interaction of positively-charged antibiotic gentamicin with cell membranes was studied to determine if any changes in membrane organization were induced by the drug. Opossum kidney epithelia (OK) cells were used as models of eukaryotic cells.Two methods were used: laurdan fluorescence spectroscopy and fluorescence anisotropy recordings on 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) labeled cell suspensions. Both methods showed an altered membrane hydration and fluidity of gentamicin treated cells. Liposomes prepared from dimyristoyl-phosphatidylcholine (DMPC) mixed with cardiolipin, which mimics the heterogeneous charge composition of the natural cell membrane, were used to determine the effect of gentamicin on artificial bilayers. The membrane lipid packing as revealed by generalized polarization (GP) and fluorescence anizotropy variation with increasing temperature was studied. It was found that the generalized polarization of liposomal membranes containing a negatively charged lipid (cardiolipin) is higher in the presence of gentamicin; in the membrane of living cell (OK), gentamicin induces, on the contrary, a decrease of general polarization. Considering the role of membrane organization in the function of transmembrane channels and receptors, our findings suggest hypotheses that may explain the permeation of gentamicin through the living cell membrane by using these channels.►Effects of gentamicin on biophysical properties of membranes were studied. ►Gentamicin affects membranes only if they contain negatively charged phospholipids. ►In cells, gentamicin influences the thermal behavior of membranes. ►Results are explained in terms of an electrostatic interaction gentamicin-membrane.
Keywords: Gentamicin; Generalized polarization; Fluorescence anisotropy; OK cell lines; Liposomes;

A brief overview of electroporation pulse strength–duration space: A region where additional intracellular effects are expected by James C. Weaver; Kyle C. Smith; Axel T. Esser; Reuben S. Son; T.R. Gowrishankar (236-243).
Electroporation (EP) of outer cell membranes is widely used in research, biotechnology and medicine. Now intracellular effects by organelle EP are of growing interest, mainly due to nanosecond pulsed electric fields (nsPEF). For perspective, here we provide an approximate overview of EP pulse strength–duration space. This overview locates approximately some known effects and applications in strength–duration space, and includes a region where additional intracellular EP effects are expected. A feature of intracellular EP is direct, electrical redistribution of endogenous biochemicals among cellular compartments. For example, intracellular EP may initiate a multistep process for apoptosis. In this hypothesis, initial EP pulses release calcium from the endoplasmic reticulum, followed by calcium redistribution within the cytoplasm. With further EP pulses calcium penetrates mitochondrial membranes and causes changes that trigger release of cytochrome c and other death molecules. Apoptosis may therefore occur even in the presence of apoptotic inhibitors, using pulses that are smaller, but longer, than nsPEF.► We review electroporation pulse parameter space. ► A relatively unexplored range of pulse durations may be useful for maximizing intracellular effects. ► Cell system modeling can assist in the understanding and development of experimental electroporation applications.
Keywords: Electroporation; Cell system; Cell model; Intracellular electroporation; Apoptosis;

Modulation of intracellular Ca2+ levels in chromaffin cells by nanoelectropulses by Gale L. Craviso; Sophie Choe; Indira Chatterjee; P. Thomas Vernier (244-252).
Exposing chromaffin cells to a single 5 ns, 5 MV/m pulse causes Ca2+ influx and a rapid, transient rise in intracellular calcium concentration ([Ca2+]i). A comparison of responses at room temperature versus 37 °C revealed no effect of temperature on the magnitude of the increase in [Ca2+]i. The Ca2+ transient, however, was shortened in duration almost twofold at 37 °C, indicating that the rate of recovery was temperature-sensitive. Temperature also affected the interval required for a second pulse to elicit another maximal rise in [Ca2+]i, which was shorter at the higher temperature. In addition, a second pulse applied 5 s after the first pulse was sufficient to cause cells at room temperature to become refractory to subsequent stimulation. At 37 °C, cells became refractory after 5 pulses regardless of whether pulse delivery was at low (1 and 10 Hz) or high (1 kHz) rates. When refractory, cells showed no signs of swelling or uptake of the impermeant dye YO-PRO-1. These results demonstrate that temperature plays a role in determining how chromaffin cells respond to and become refractory to nanoelectropulses. They also indicate that despite the ultra-short duration of the pulses, pronounced effects on cell excitability result from the application of only very few pulses.► One 5 ns, 5 MV/m pulse causes a rise in [Ca2+]i in adrenal chromaffin cells. ► [Ca2+]i increases similarly at 25 °C and 37 °C, but declines faster at 37 °C. ► Recovery time before another maximal rise in [Ca2+]i is faster at 37 °C. ► More pulses are required at 37 °C to render the cells refractory to re-stimulation.
Keywords: Nanosecond pulses; Excitable cell; Temperature; Pulse number; Refractory period;

Giant lipid vesicles under electric field pulses assessed by non invasive imaging by Chloé Mauroy; Thomas Portet; Martin Winterhalder; Elisabeth Bellard; Marie-Claire Blache; Justin Teissié; Andreas Zumbusch; Marie-Pierre Rols (253-259).
We present experimental results regarding the effects of electric pulses on giant unilamellar vesicles (GUVs). We have used phase contrast and coherent anti-Stokes Raman scattering (CARS) microscopy as relevant optical approaches to gain insight into membrane changes under electropermeabilization. No addition of exogenous molecules (lipid analogue, fluorescent dye) was needed. Therefore, experiments were performed on pure lipid systems avoiding possible artefacts linked to their use. Structural membrane changes were assessed by loss of contrast inside the GUVs due to sucrose and glucose mixing. Our observations, performed at the single vesicle level, indicate these changes are under the control of the number of pulses and field intensity. Larger number of pulses enhances membrane alterations. A threshold value of the field intensity must be applied to allow exchange of molecules between GUVs and the external medium. This threshold depends on the size of the vesicles, the larger GUVs being affected at lower electric field strengths than the smaller ones. Our experimental data are well described by a simple model in which molecule entry is driven by direct exchange. The CARS microscopic study of the effect of pulse duration confirms that pulses, in the ms time range, induce loss of lipids and membrane deformations facing the electrodes.► Giant vesicles were used as membrane model to study electropermeabilization processes. ► Phase contrast and CARS microscopy are relevant approaches. ► No addition of exogenous molecules (lipid analogue, fluorescent dye) was needed. ► The CARS microscopy confirms that pulses induce loss of lipids.
Keywords: Pulsed electric fields; Electroporation; Imaging; Phospholipids;

In our study, we used bleomycin to evaluate the permeabilization caused by nanosecond duration electric pulses (nanopulses). Bleomycin is a non permeant molecule which can be used both as a sensitive and quantitative marker to evaluate cell electropermeabilization. Indeed, the penetration of as few as 500 molecules is sufficient to entail a major biological effect: cell death. We show that one single nanopulse with a duration of 10 ns and a field strength of 40 kV/cm is sufficient to allow the uptake of at least 500 molecules of bleomycin in 20% of the cells when the external bleomycin concentration is 3 μM. When the external bleomycin concentration is reduced by a 100 fold, the same levels of cytotoxicity require an increase of about 25 times in the number of pulses. These results are in favor of the fact that each nanopulse creates new pores or defects on the cell membrane even if most of these pores can reseal between two consecutive pulses. Results also suggest that the cell permeability observed with classical markers when a large number of pulses are delivered results from the large number of nanopores or defects of the cell membrane created by the train of nanopulses.► Bleomycin (Mr 1500) is a very sensitive cell permeabilization marker. ► Pulses of field strength E = 40 kV/cm and duration 10 ns induce permeabilization. ► One pulse is sufficient for cross-membrane transport of bleomycin. ►These pulses allow thus the penetration of molecules of 1500 Da. ► Each pulse creates new pores or defects on the cell membrane.
Keywords: Bleomycin; Nanopulse; Electroporation; Electropermeabilization; nsPEF;

Patient-specific treatment planning of electrochemotherapy: Procedure design and possible pitfalls by Denis Pavliha; Bor Kos; Anže Županič; Marija Marčan; Gregor Serša; Damijan Miklavčič (265-273).
Electrochemotherapy uses electroporation for enhancing chemotherapy. Electrochemotherapy can be performed using standard operating procedures with predefined electrode geometries, or using patient-specific treatment planning to predict electroporation. The latter relies on realistic computer models to provide optimal results (i.e. electric field distribution as well as electrodes' position and number) and is suitable for treatment of deep-seated tumors.Since treatment planning for deep-seated tumors has been used in radiotherapy, we expose parallelisms with radiotherapy in order to establish the procedure for electrochemotherapy of deep-seated tumors. We partitioned electrochemotherapy in the following phases: the mathematical model of electroporation, treatment planning, set-up verification, treatment delivery and monitoring, and response assessment. We developed a conceptual treatment planning software that incorporates mathematical models of electroporation. Preprocessing and segmentation of the patient's medical images are performed, and a 3D model is constructed which allows placement of electrodes and implementation of the mathematical model of electroporation.We demonstrated the feasibility of electrochemotherapy of deep-seated tumors treatment planning within a clinical study where treatment planning contributed to the effective electrochemotherapy treatment of deep-seated colorectal metastases in the liver. The described procedure can provide medical practitioners with information on using electrochemotherapy in the clinical setting. The main aims of this paper are: 1) to present the procedure for treating deep-seated tumors by electrochemotherapy based on patient-specific treatment planning, and 2) to identify gaps in knowledge and possible pitfalls of such procedure.► Electrochemotherapy is currently used for treatment of superficial tumor nodules. ► Treatment planning of electrochemotherapy is needed for treating deep-seated tumors. ► We developed a proof-of-principle treatment planning software for electrochemotherapy. ► We demonstrated the feasibility of treatment planning within a clinical study.
Keywords: Electrochemotherapy; Electroporation; Treatment planning; Medical imaging; Numerical modeling;