Bioelectrochemistry (v.74, #1)

Contents (vii-ix).

by Justin Teissie; Lluis M. Mir (1).

The positively charged polyene molecule amphotericin B 3-dimethylaminopropylamide (AMA) is an efficient agent for the delivery of antisense oligodeoxyribonucleotides (ODN) into target cells. In the present study, bilayer lipid membrane (BLM) conductance, elasticity modulus perpendicular to the membrane plane, surface potential and electrical capacitance were measured by conductance and electrostriction methods in the presence of AMA, pure or complexed to 20-mer single stranded ODN at different ratios. Pure AMA did not induce changes in conductance of cholesterol-containing BLM, but did induce an increase in elasticity modulus and surface potential. ODN/AMA complexes changed BLM properties depending on the charge ratio. The most pronounced effect on membrane conductance was observed for positively charged ODN/AMA complexes (charge ratio ρ−/+ = 0.1), while for negatively charged complexes these changes were less marked/apparent, correlating to substantially lower binding constants. The effect of ODN/AMA complexes on elasticity modulus and charge potential was biphasic. After an increase in both values, a decrease was observed for higher incubation times and ODN/AMA concentrations. These results are interpreted as indicating that the membrane property changes result from the large AMA aggregates induced by the presence of the negatively charged ODN, which condensate on these aggregates. It is suggested that the decrease of elasticity modulus and surface potential in the presence of increasing incubation time and AMA concentration result from desorption of the complexes in the complex-free compartment of the BLM cell, or appearance of a non-linear conductance of the lipid bilayer. The first alternative would explain the AMA-induced transmembrane transfer of ODN.
Keywords: Oligonucleotide; Amphotericin; Bilayer lipid membranes; Conductance; Electrostriction; Compressibility; Surface potential;

Microscopic observation of living cells during their exposure to modulated electromagnetic fields by Mihaela G. Moisescu; Philippe Leveque; Jean-Rémi Bertrand; Eugenia Kovacs; Lluis M. Mir (9-15).
Studying cell behaviour under irradiation with radiofrequency electromagnetic fields (RF-EMF) is often impeded by the difficulty to monitor cell characteristics during irradiation. Here we report the design and the application of a complete device for continuous microscopic observation of cells exposed to modulated EMF similar to mobile phones signals. The system allows the follow up of cell progression into mitosis under controlled temperature and CO2 environment. Protocols are proposed in which the same cells are the controls before and after the EMF exposure and we demonstrate the interest of the “before exposure” controls. The exposure system was validated by cell endocytosis measurements. While the endocytosis rate was increased, no alteration of mitosis progression and mitosis duration was observed in cells exposed to 900 MHz modulated EMF for 1 h, at 30 °C and at a Specific Absorption Rate of 2.2 W/kg.
Keywords: Mitosis; Fluid phase endocytosis; Phase contrast microscopy; TEM cell; GSM-EMF;

Charge induced closing of Dionaea muscipula Ellis trap by Alexander G. Volkov; Tejumade Adesina; Emil Jovanov (16-21).
In terms of bioelectrochemistry, Venus flytrap responses can be considered in three stages: stimulus perception, electrical signal transmission, and induction of mechanical and biochemical responses. When an insect touches the trigger hairs, these mechanosensors generate receptor potentials, which induce solitary waves activating the motor cells. We found that the electrical charge injected between a midrib and a lobe closes the Venus flytrap leaf by activating motor cells without mechanical stimulation of trigger hairs. The mean electrical charge required for the closure of the Venus flytrap leaf is 13.6 μC. To close the trap, electrical charge can be submitted as a single charge or applied cumulatively by small portions during a short period of time. Ion channel blocker such as Zn2+ as well as an uncoupler CCCP, dramatically decreases the speed of the trap closing a few hours after treatment of the soil. This effect is reversible. After soil washing by distilled water, the closing time of Venus flytrap treated by CCCP or ZnCl2 decreases back from 2–5 s to 0.3 s, but higher electrical charge is needed for trap closure. The mechanism behind closing the upper leaf of Venus flytrap is discussed.
Keywords: Plant electrophysiology; Electrical signaling; Venus flytrap; Motor cells; Ion channels;

Formation of bilayer lipid membrane (BLM) by Montal–Mueller technique across a small aperture in a partition film traditionally requires coating of the aperture with a hydrophobic substance, often just an organic solvent. However, we demonstrate here that the most effective coating is not strictly hydrophobic but rather provides water/oil repellent properties. BLM were formed from diphytanoylphosphatidylcholine (DPhPC) on small 0.1–0.8 mm apertures made in specially prepared alkylated glass coverslips. The coverslips were either fluorosiliconized by 3,3,3-Trifluoropropyl-trimethoxysilane, which reduces adsorption of DPhPC in addition to creation of hydrophobic surface, or silanized, which promote adsorption of DPhPC. At fluorosiliconized surfaces stable BLM were formed. Specific capacitance of these BLM was 0.86 μF/cm2  ± 5%, while their lateral tension was estimated as 4.3 ± 0.4 mN/m. BLM were stable for hours under moderate voltage applied. At silanized surfaces stable BLM were formed only in acidic medium (3 < pH < 4), while at higher pH the membranes could cover the aperture only partially. Thus, apertures in fluorosiliconized glass can be robustly used for formation of model lipid membranes under physiological conditions.
Keywords: Solvent-free bilayer lipid membranes; BLM; Alkylation;

Electrochemotherapy is a treatment based on combination of chemotherapeutic drug and electroporation. It is used in clinics for treatment of solid tumours. For electrochemotherapy of larger tumours multiple needle electrodes were already suggested. We developed and tested electrode commutation circuit, which controls up to 19 electrodes independently. Each electrode can be in one of three possible states: on positive or negative potential or in the state of high impedance. In addition, we tested a pulse sequence using seven electrodes for which we also calculated electric field distribution in tumour tissue by means of finite-elements method. Electrochemotherapy, performed by multiple needle electrodes and tested pulse sequence on large subcutaneous murine tumour model resulted in tumour growth delay and 57% complete responses, thus demonstrating that the tested electrode commutation sequence is efficient.
Keywords: Electrode commutation sequence; Electroporation; Finite-elements method; Multiple electrodes;

An electrochemical biosensor for the detection of bar gene coding phosphinothricin herbicide resistance is presented. The detection was based on hybridization reaction between the specific to bar gene 19-mer probe immobilized on the electrode surface and complementary DNA in a sample. Single-stranded DNA probe specific to bar gene was covalently attached by 5′-phosphate end to the surface of carbon paste electrode. Outer layer of a conventional CPE was provided with carboxyl groups of stearic acid. ssDNA was coupled to the electrode through ethylenediamine with the use of water-soluble 1-ethyl-3(3′-dimethylaminopropyl)-carbodiimide and N-hydroxy-sulfosuccinimide as activating reagents. Hybridization reaction at the electrode surface was detected via Co(bpy)3 3+, which posses a much higher affinity to the resulting DNA duplex compared to ssDNA probe. Detection limit of the sensor was 0.1 μM of target DNA fragments and its response was linear from 5 to 20 μM. Hybridization event was also detected by measuring guanine peak but this approach presented distinctly higher detection limit (1 μM) and lower reproducibility. Complete time of one measurement with the use of the biosensor including covalent attachment of ethylenediamine (linker) and ssDNA probe to the electrode, hybridization with target and interaction with electroactive indicator was about 70 min.
Keywords: bar gene; GM food; Electrochemical biosensor; Modified CPE; Hybridization; Co(bpy)3 3+;

Electroporation is usually described as a formation of transient pores in the cell membrane in the presence of a strong electric field, which enables transport of molecules and ions across the cell membrane. Several experimental studies of electroporation showed a complex dependence of the transport on pulse parameters. In only few studies, however, the actual transport across the membrane was quantified. Current theoretical studies can describe pore formation in artificial lipid membranes but still cannot explain mechanisms of formation and properties of long-lived pores which are formed during cell electroporation. The focus of our study is to connect theoretical description of pore formation during the electric pulses with experimental observation of increased transport after the pulses. By analyzing transient increase in conductivity during the pulses in parallel with ion efflux after the pulses the relation between short-lived and long-lived pores was investigated. We present a simple model that incorporates an increase in the fraction of long-lived pores with higher electric field due to larger area of the cell membrane exposed to above-critical voltage and due to higher energy which is available for pore formation. We also show that each consecutive pulse increases the probability for the formation of long--lived pores.
Keywords: Electroporation; Electrogene transfer; Electrochemotherapy; Theory; Pore formation; Cell suspension; Ion diffusion;

The immobilization of nitrate reductase (NR) was performed by entrapment in a LAPONITE® clay gel and cross-linking by glutaraldehyde. In presence of nitrate and methyl viologen, a catalytic current appeared at − 0.60 V illustrating the enzymatic reduction of nitrate into nitrite via the reduced form of the freely diffusing methyl viologen. The electropolymerization of a water-soluble pyrrole viologen derivative within the interlamellar spaces and channels of the host clay matrix successfully carried out the electrical wiring of the entrapped NR. Rotating disk measurements led to the determination of kinetic constants, namely k 2  = 10.7 s1 and K M  = 7 μM. These parameters reflect the efficiency of the electro-enzymatic reduction of nitrate and the substrate affinity for the immobilized enzyme.
Keywords: Nitrate reductase; Enzyme wiring; Clay electrode; Viologen Biosensor;

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1.While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 μs and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 μl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 °C) to 4 °C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.
Keywords: Electropermeabilization; Membrane order parameter; Temperature; In vitro;

Phototoxicity and cytotoxicity of chlorophyll a/cyclodextrins complexes on Jurkat cells by Pinalysa Cosma; Paola Fini; Sergio Rochira; Lucia Catucci; M. Castagnolo; Angela Agostiano; Roberto Gristina; Marina Nardulli (58-61).
The aggregation status of chlorophyll a (Chl a) and the ability of four cyclodextrins, hydroxypropyl-β-cyclodextrin (HP-β-CD), hydroxypropyl-γ-cyclodextrin (HP-γ-CD), heptakis(2,6-di-O-methyl)-β-cyclodextrin (DIMEB), and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB), to solubilize the pigment in the complete cellular medium RPMI 1640 was estimated by means of UV–Vis absorption and static resonance light scattering (RLS) measurements. The results indicate that the pigment interacts with cyclodextrins in the cellular medium differently to that observed in water. The cytotoxic and phototoxic activity of these complexes towards human leukemia T-lymphocytes (Jurkat cells) was tested by means of experiments aimed to discriminate between the intrinsic toxicity and the toxicity induced by light.The overall data indicate that the HP-β-CD is the cyclodextrins having the best characteristics to form with Chl a a potential supramolecular system for the photodynamic therapy.
Keywords: PDT; Chlorophyll a; Cyclodextrins; Jurkat cells;

A Gold–Platinum nanoparticle-decorated titania nanotubular electrode is fabricated by electrochemically depositing Au and Pt nanoparticles onto a highly-oriented titania nanotube array. The prepared electrode, characterized by SEM and EDX, shows remarkably improved catalytic activities in the oxidation of hydrogen peroxide. By modifying the electrode with glucose oxidase (GOx) the resultant glucose biosensor exhibits a high sensitivity to glucose in the range of 0 to 1.8 mM with a response time of 3 s and detection limit of 0.1 mM.
Keywords: Catalytic; Titania; Nanotube array; Nanoparticle; Electrochemical;

A carbon paper electrode was modified with the conducting copolymer of 3-methylthiopene and thiophene-3-acetic acid prepared electrochemically on the electrode, and an enzyme electrode was fabricated by covalent immobilization of glucose oxidase on the modified electrode. The modification with the conducting copolymer increased the surface area of the electrode and the amount of the immobilized enzyme. As a result, the enzyme electrode showed a high catalytic activity. Moreover, it was found that the increased surface area led to a high rate of electron transfer reaction between the electrode and p-benzoquinone employed as an electron mediator. The enzyme electrode fabricated with the modified carbon paper gave a larger glucose oxidation current than that fabricated with the bare one. In addition, the glucose oxidation current was found to increase with increasing content of the conducting copolymer in the modified carbon paper. Corresponding to the large glucose oxidation current, high performance was confirmed for the glucose fuel cell constructed with the enzyme electrode based on the modified carbon paper.
Keywords: Enzyme electrode; Glucose oxidase; Polythiophene derivative; Carbon paper; Glucose fuel cell;

This research details the isolation and purification of a new type of lactate dehydrogenase that is dependent upon the coenzyme pyrroloquinoline quinone (PQQ). PQQ-dependent enzymes have been of interest in the literature over the last decade due to the fact that many of them can undergo direct electron transfer (DET) at electrode surfaces which is of interest for biosensor and biofuel cell applications. In the paper, we detail the isolation of PQQ-dependent lactate dehydrogenase (PQQ-LDH) from two sources of Gluconobacter (Gluconobacter sp. 33 and Gluconobacter suboxydans). This paper also shows the first evidence that PQQ-LDH can undergo direct electron transfer at gold and carbon electrode surfaces for future use in biosensors and biofuel cells.
Keywords: Lactate dehydrogenase; Pyrroloquinoline quinone; Direct electron transfer; Biofuel cells;

The measurement of electricity generation from an air-cathode microbial fuel cell (MFC) with a mixed bacteria culture at different pH showed that this MFC could tolerate an initial (feed solution) pH as high as 10. The optimal initial pH was between 8 and 10 with higher current generation compared to lower or higher pH. The bacterial metabolism exhibited a buffer effect and changed the electrolyte pH. The impedance spectra of the anode and cathode of the MFC at the open-circuit potential (OCP) revealed that the anodic microbial process preferred a neutral pH and microbial activities decreased at higher or lower pH; while the cathodic reaction was improved with increasing pH.
Keywords: Microbial fuel cell; Air cathode; pH; Electrochemical impedance spectroscopy;

Direct electrochemical response was first time observed for the redox centers of Desulfovibrio gigas [NiFe]-Hase, in non-turnover conditions, by cyclic voltammetry, in solution at glassy carbon electrode. The activation of the enzyme was achieved by reduction with H2 and by electrochemical control and electrocatalytic activity was observed. The inactivation of the [NiFe]-Hase was also attained through potential control. All electrochemical data was obtained in the absence of enzyme inhibitors. The results are discussed in the context of the proposed mechanism currently accepted for activation/inactivation of [NiFe]-Hases.
Keywords: Hydrogenases; Biocatalysis; Electrochemistry; Activation;

α-zirconium phosphate nanosheets (ZrPNS) derived via the delamination of layered α-zirconium phosphate (α-ZrP) have been proven to be efficient support matrixes for the immobilization of horseradish peroxidase (HRP). X-ray powder diffraction (XRD) results revealed that ZrPNS in HRP–ZrPNS film remained unorderly structured for the effect of HRP. Fourier transform infrared (FTIR) spectra results revealed that HRP remained the secondary structure in HRP–ZrPNS film. The direct electrochemistry of HRP was realized in HRP–ZrPNS film on a glassy carbon electrode (GCE), showing a pair of well-defined, nearly reversible cyclic voltammetry (CV) peaks for the HRP heme FeIII/FeII redox couple. The average surface concentration (Γ ) of electroactive HRP in HRP–ZrPNS film was estimated to be 1.35 × 10− 10 mol cm− 2, which indicated a high loading of enzyme molecules in HRP–ZrPNS film. Based on these, a third generation reagentless biosensor was constructed for the determination of hydrogen peroxide (H2O2). The response time of the biosensor was less than 3 s, and the linear response range of the biosensor for H2O2 was from 1.3 × 10− 6 to 1.6 × 10− 2 M with a correlation coefficient of 0.9997.
Keywords: Biosensor; Horseradish peroxidase; α-Zirconium phosphate nanosheets; Direct electrochemistry; Electrocatalysis;

Two methods for the determination of vitamin K3 have been developed. Vitamin K3 in its oxidized form is determined by direct and indirect methods. Its standard solution was prepared by the indirect method using Ti(III) as reducing agent. For this purpose vitamin K3 (menadion) in a clinical injection solution, which is in its hydroquinone form in the presence of sulfite, is oxidized with oxygen. In 0.2 M HAc and 0.02 M HCl electrolyte vitamin K3 and Ti(IV) have reduction peaks at − 0.58 V at − 0.82 V respectively. The reaction between Ti(III) and vitamin takes place quantitatively in a medium of 0.2 M HAc and 0.002 M HCl. After the reduction, the reaction product Ti(IV) is followed from its polarographic peak at about − 0.82 V. The most important result in this work is that, with this method vitamin K3 can be standardized and after standardization this solution can be used for the direct determination in routine analysis with a very simple and fast method, using only the peak at − 0.71 V in 0.2 M HAc medium. Both direct and indirect methods have been used for the determination of Vitamin K3 in a clinical injection solution. The limit of quantification (LOQ) was 1.5 × 10− 6 M and in both methods the detection limit found was 7 × 10− 7 M.
Keywords: Vitamin K3; Determination; Titan (III) chloride; Differential pulse polarography;

The key characteristics of multicopper oxidases are redox potentials of Type 1, Type 2 and Type 3 copper centers of enzymes. However, there is still a challenge to obtain a value of the redox “signature” of the enzymes. In this study, the electrochemical behavior of T1 and T2/T3 redox copper centers of bilirubin oxidase (BOD) from the fungi Myrothecium verrucaria was studied based on direct bioelectrocatalysis. Two distinct redox peaks corresponding to reduction and oxidation of T1 and T2/T3 redox centers of enzymes have been clearly detected in anaerobic conditions. The bioelectrocatalytic activity of the enzyme was studied in the presence of oxygen and redox mediators. The electron-transfer rate constant for BOD immobilized on carbon electrode (CE) is 1.5 s− 1. The mechanism of enzyme inactivation by ABTS has been proposed. The physical architecture of BOD layers immobilized on the electrode surface, including elemental and chemical composition, relative thickness and assembly of layers was investigated by Angle Resolved X-ray photoelectron spectroscopy. Unique peaks of BOD at 288.5 eV and of CE at 284.6 eV were used in a substrate over layer model for estimation of the thickness of the of BOD film on the carbon electrode surface.
Keywords: Bilirubin oxidase; Fungus Myrothecium verrucaria; Redox potentials of T1 and T2/T3 copper centers; Direct electron transfer; Angle-resolved XPS;

A universal, site-addressable DNA linking strategy is deployed for the programmable assembly of multifunctional, long-lasting redox protein nanoelectronic devices. This addressable linker, the first incorporated into a redox enzyme-nanoelectronic system, promotes versatility and renewability by allowing the reconfiguration and replacement of enzymes at will. The linker is transferable to all redox proteins due to the simple conjugation chemistry involved. The efficacy of this linking strategy is assessed using two model enzymes, glucose oxidase (GOx) and alcohol dehydrogenase (ADH), self-assembled onto separate nanoelectrode regions comprised of a highly ordered carbon nanotube (CNT) array. The sequence-specificity of DNA hybridization provides the means of encoding spatial address to the self-assembling process that conjugates enzymes tagged with single-stranded DNA (ssDNA) to the tips of designated CNTs functionalized with the complementary strands. In this study, we demonstrate the feasibility of multiplexed, scalable, reconfigurable and renewable transduction of redox protein signals by virtue of DNA addressing.
Keywords: Biosensor; Carbon nanotube; Enzyme; Direct electron transfer; Self-assembly;

Amyloid-β detection with saccharide immobilized gold nanoparticle on carbon electrode by Miyuki Chikae; Tomohiro Fukuda; Kagan Kerman; Koutarou Idegami; Yoshiko Miura; Eiichi Tamiya (118-123).
The electrochemical sensing of saccharide–protein interactions using a couple of sialic acid derivatives and Alzheimer's amyloid-beta (Aβ) is described. The densely-packed saccharide area for recognition of protein was fabricated onto a carbon electrode by three steps, which were electrochemical deposition of Au nanoparticles on a screen printed strip, self-assembled monolayer (SAM) formation of the acetylenyl group on Au nanoparticles, and the cycloaddition reaction of an azide-terminated sialic acid to the acetylenyl group. The attachment of Aβ peptides to the sialic acid layer was confirmed by electrochemistry and atomic force microscopy imaging. The intrinsic oxidation signal of the captured Aβ(1-40) and (1-42) peptides, containing a single tyrosine (Tyr) residues, was monitored at a peak potential of 0.6 V (vs Ag/AgCl within this sensor) in connection with differential pulse voltammetry. The peak current intensities were concentration dependent. The proposed process provides new routes for analysis of saccharide–protein interactions and electrochemical biosensor development.
Keywords: Sialic acid; Biosensor; Alzheimer's amyloid-β peptide; Gold nanoparticle; Self-assembled monolayer; Voltammetry;

Optimization of bulk cell electrofusion in vitro for production of human–mouse heterohybridoma cells by Katja Trontelj; Matej Reberšek; Maša Kandušer; Vladka Čurin Šerbec; Marjana Šprohar; Damijan Miklavčič (124-129).
Cell electrofusion is a phenomenon that occurs, when cells are in close contact and exposed to short high-voltage electric pulses. The consequence of exposure to pulses is transient and nonselective permeabilization of cell membranes. Cell electrofusion and permeabilization depend on the values of electric field parameters including amplitude, duration and number of electric pulses and direction of the electric field. In our study, we first investigated the influence of the direction of the electric field on cell fusion in two cell lines. In both cell lines, applications of pulses in two directions perpendicular to each other were the most successful. Cell electrofusion was finally used for production of human–mouse heterohybridoma cells with modified Koehler and Milstein hybridoma technology, which was not done previously. The results, obtained by cell electrofusion, are comparable to usually used polyethylene glycol mediated fusion on the same type of cells.
Keywords: Electrofusion; Electropermeabilization; Electric field direction; Heterohybridoma;

The transmembrane potential (Δϕ) induced by external electric fields is important both in biotech applications and in new medical therapies. We analyzed the effects of AC field frequency and cell orientation for cells of a general ellipsoidal shape. Simplified equations were derived for the membrane surface points where the maximum Δϕ is induced. The theoretical results were confirmed in experiments with three-axial chicken red blood cells (a:b:c  = 6.66 μm:4.17 μm:1.43 µm). Propidium iodide (PI) staining and cell lysis were detected after an AC electropermeabilization (EP) pulse. The critical field strength for both effects increased when the shorter axis of a cell was parallel to the field, as well as at higher field frequency and for shorter pulse durations. Nevertheless, data analysis based on our theoretical description revealed that the Δϕ required is lower for the shorter axis, i.e. for smaller membrane curvatures. The critical Δϕ was independent of the field frequency for a given axis, i.e. the field strength had to be increased with frequency to compensate for the membrane dispersion effect. Comparison of the critical field strengths of PI staining in a linear field aligned along semi-axis a (142 kV m− 1) and a field rotating in the ab plane (115 kV m− 1) revealed the higher EP efficiency of rotating fields.
Keywords: Induced transmembrane potential; Electroporation; Electropermeabilization; Lab-on-chip; Chicken red blood cells;

Interfacial electron transfer on cytochrome-c sensitised conformally coated mesoporous TiO2 films by Emmanuel Topoglidis; Thierry Lutz; James R. Durrant; Emilio Palomares (142-148).
Hybrid protein films incorporating Cyt-c immobilized on TiO2 films were prepared and characterised optically with UV–visible spectroscopy and electrochemically with cyclic voltammetry, and their conductivity properties were studied in detail. In addition the effects of a thin overlayer coating of a second metal oxide such as SiO2, Al2O3, ZrO2 and MgO2 were studied and the effects over the electrochemical properties of the hybrid working electrodes were discussed.
Keywords: Cytochrome-c; Metal oxide coatings; Bioelectrochemistry; Conductivity; TiO2 films;

The dependence of membrane properties on their composition was studied by following the adhesion and spreading of unilamellar and multilamellar liposomes on static mercury electrodes with the help of chronoamperometry. The analysis of the peak-shaped signals allows determining the kinetic parameters of the three-step adhesion-spreading process. The presence of cholesterol in the membrane stabilizes the bilayer in the liquid-crystalline phase, and destabilizes the gel phase. The kinetic parameters also show the effect of superlattice formation in the DMPC–cholesterol system. The detergent triton X-100 is only incorporated in the liquid-crystalline DMPC membranes, and it is expelled to the solution when the membrane is transformed to the gel phase. In the liquid-crystalline membrane, it enhances the adhesion-spreading of liposomes on mercury. The lytic peptides mastoparan X and melittin affect the adhesion-spreading in a similar manner. For the rupture-spreading step, their effect is explained by pore formation. The results obtained with lecithins of different length suggest that the bilayer opening process has much in common with flip-flop translocations. For this process the activation energies were found to be independent of the chain length of the lecithin molecules, while the preexponential factor in the Arrhenius equation decreases drastically for longer chains.
Keywords: Liposomes; Adhesion; Mercury electrode; Chronoamperometry; Triton X-100; Lytic peptides;

TiO2 nanoparticles were homogeneously coated on multiwalled carbon nanotubes (MWCNTs) by hydrothermal deposition, and this nanocomposite might be a promising material for myoglobin (Mb) immobilization in view of its high biocompatibility and large surface. The glassy carbon (GC) electrode modified with Mb–TiO2/MWCNTs films exhibited a pair of well-defined, stable and nearly reversible cycle voltammetric peaks. The formal potential of Mb in TiO2/MWCNTs film was linearly varied in the range of pH 3–10 with a slope of 48.65 mV/pH, indicating that the electron transfer was accompanied by single proton transportation. The electron transfer between Mb and electrode surface, k s of 3.08 s− 1, was greatly facilitated in the TiO2/MWCNTs film. The electrocatalytic reductions of hydrogen peroxide were also studied, and the apparent Michaelis–Menten constant is calculated to be 83.10 μM, which shows a large catalytic activity of Mb in the TiO2/MWCNTs film to H2O2. The modified GC electrode shows good analytical performance for amperometric determination of hydrogen peroxide. The resultant Mb–TiO2/MWCNTs modified glassy carbon electrode exhibited fast amperometric response to hydrogen peroxide reduction, long term life and excellent stability. Finally the activity of the sensor for nitric oxide reduction was also investigated.
Keywords: Myoglobin; Multiwalled carbon nanotubes; TiO2; Electrocatalysis; Electrochemistry;

Studies on the interaction of DNA with vitamin B6 were carried out with a DNA-modified electrode by electrochemistry and Ultraviolet–visible spectroscopy. The results showed that there exists the supra-molecule interaction between base groups on DNA and vitamin B6 by forming hydrogen binding, the binding equilibrium constant of the interaction is equal to 115.3 M−1, the binding ratio of nucleotide to vitamin B6 is 5:1. Based on the electrochemical and Ultraviolet–visible spectrum studies the interaction mode of DNA with vitamin B6 was explored.
Keywords: Vitamin B6; DNA; Supra-molecule; Interaction; Cyclic voltammetry;

A glucose biosensor based on polyvinylpyrrolidone (PVP) protected Prussian blue nanoparticles (PBNPs)-polyaniline/multi-walled carbon nanotubes hybrid composites was fabricated by electrochemical method. A novel route for PBNPs preparation was applied in the fabrication with the help of PVP, and from scanning electron microscope images, Prussian blue particles on the electrode were found nanoscaled. The biosensor exhibits fast current response (<   6 s) and a linearity in the range from 6.7 × 10− 6 to 1.9 × 10− 3 M with a high sensitivity of 6.28 μA mM− 1 and a detection limit of 6 × 10− 7 M (S/N = 3) for the detection of glucose. The apparent activation energy of enzyme-catalyzed reaction and the apparent Michaelis–Menten constant are 23.9 kJ mol− 1 and 1.9 mM respectively, which suggests a high affinity of the enzyme-substrate. This easy and controllable construction method of glucose biosensor combines the characteristics of the components of the hybrid composites, which favors the fast and sensitive detection of glucose with improved analytical capabilities. In addition, the biosensor was examined in human serum samples for glucose determination with a recovery between 95.0 and 104.5%.
Keywords: Prussian blue; Polyaniline; Carbon nanotubes; Hybrid composites; Biosensor;

The enzymatic hydrolysis reaction of urea by urease is optimized in this work by the chemometric response surface methodology (RSM), based on an initial rate potentiometric measurement using an NH4 + ion-selective electrode (ISE). In this investigation, the ranges of critical variables determined by a preliminary “one at a time” (OVAT) procedure were used as input for the subsequent RSM chemometric analysis. The RSM quadratic response was found to be quite appropriate for modeling and optimization of the hydrolysis reaction as illustrated by the relatively high value of the determination coefficient (R 2  = 90.1%), along with the satisfactory results obtained by the analysis of variance (ANOVA). All the evaluated analytical characteristics of the optimized method such as: the linear calibration curve, the upper and lower detection limits, the within-day precisions at low and at high levels, the assay recovery in pool serum media, along with the activation kinetic parameters, were also reported. Further, in order to check the quality of the optimization and the validity of the model, the assay of urea, both in aqueous laboratory and human serum samples, were performed. It has to be noted that the kinetic initial rate measurement method used in this work, permitted to overcome the general problem of NH4 + ISE low selectivity against Na+ and K+ interfering ions in real samples.
Keywords: Urea; Urease; NH4 + ISE; Initial rate method; Response surface methodology;

Direct electrochemistry of horseradish peroxidase immobilized in a chitosan–[C4mim][BF4] film: Determination of electrode kinetic parameters by Jenny S. Long; Debbie S. Silvester; Gregory G. Wildgoose; Annette-E. Surkus; Gerd-Uwe Flechsig; Richard G. Compton (183-187).
The direct electrochemistry of a HRP–chi–[C4mim][BF4] film (where HRP = horseradish peroxidase, chi = chitosan, and [C4mim][BF4] = the room temperature ionic liquid (RTIL) 1-butyl-3-methylimidazolium tetrafluoroborate) has been studied by cyclic voltammetry on a glassy carbon electrode. The mechanism for the electrochemical reaction of HRP is suggested to be EC for the reduction, and CE for the following re-oxidation, as the oxidative peak potential remained approximately unchanged across the scan rate range. The half wave potential of HRP reduction was found to be pH dependent, suggesting that a concomitant proton and electron transfer is occurring. Using theoretical simulations of the experimentally obtained peak positions, the standard electron transfer rate constant, k 0, was found to be 98 (± 16) s− 1 at 295 K in pH 7 phosphate buffer solution, which is very close to the value reported in the absence of ionic liquid. This suggests that the ionic liquid used here in the HRP–chi–[C4mim][BF4]/GC electrode does not enhance the rate of electron transfer. k 0 was found to increase systematically with increasing temperature and followed a linear Arrhenius relation, giving an activation energy of 14.20 kJ mol− 1. The electrode kinetics and activation energies obtained are identical to those reported for HRP films in aqueous media. This leads us to question if the use of RTIL films provide any unique benefits for enzyme/protein voltammetry. Rather the films may likely contain aqueous zones in which the enzymes are located and undergo electron transfer.
Keywords: Horseradish peroxidase; Enzymes; Cyclic voltammetry; Room temperature ionic liquids; Electrochemical rate constants;

Carbon paste electrode modified with baker' and wine yeast Saccharomyces cerevisiae (a source of flavocytochrome b 2) were investigated as amperometric biosensors for l-lactic acid. Before immobilization on the electrode surface, yeast cells were pretreated with various electrolytes, alcohols and weak organic acids. Electrode responses to l-lactic acid were tested in the presence of various mediators (potassium ferricyanide, phenazine methosulfate, 2,6-dichlorophenolindophenol sodium salt hydrate, 1,2-naphthoquinone-4-sulfonic acid sodium salt). The highest (144 ± 7 nA per 0.2 mM l-lactic acid) and the most stable responses were obtained after yeast pretreatment with 30% ethanol using potassium ferricyanide as a mediator. Different electrode sensitivities with mediator phenazine methosulphate probably reflected diverse changes in yeast membrane (and/or cell wall).
Keywords: Saccharomyces cerevisiae; Biosensor; L-lactic acid; Permeabilization;

Electrochemical time-of-flight was used to measure the diffusion coefficients of 2,2,6,6-tetramethylpiperidine 1-oxyl derivatives, C n TPA, (3 to 7 CH2 groups), in tropocollagen I solution, as a function of the chain length and the cross-linking with glutaraldehyde. The values of the diffusion coefficient of C n TPA in pure aqueous electrolyte follow the Stokes–Einstein law, i.e. the diffusion coefficient is inversely proportional to the size of the redox probe. Different behavior is observed in 0.5% (w/v) tropocollagen solution where the molecules with longer alkyl chains show larger diffusion coefficients than the smaller molecules. This behavior is explained in terms of electrostatic interactions between tropocollagen chains and the C n TPA molecules. The measurements of the diffusion coefficients of C n TPA in 0.5% tropocollagen cross-linked with glutaraldehyde indicate that while the C7TPA and C5TPA probes exhibit lower diffusion coefficients upon addition of 0.05% GA and 0.1% (v/v) GA respectively, the other C n TPA molecules exhibit either unchanged or increased diffusion coefficients under the same conditions thus indicating the presence of hydrophobic pockets selectively interacting with C n TPAs. These results demonstrate the utility of electrochemical time-of-flight in measurements of diffusion coefficients in complex biopolymeric media.
Keywords: Collagen; Cross-linking; Diffusion; Hydrophobicity;

The homogeneous reaction between glucose oxidase and osmium bipyridine–pyridine carboxylic acid in the presence of glucose has been studied in detail by cyclic voltammetry and digital simulation.Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate–enzyme reaction: K MS  = 10.8 mM, k cat  = 254 s− 1 and for the redox mediator–enzyme reaction, k  = 2.2 × 105 M− 1 s− 1.The accurate determination of the kinetic parameters at low substrate concentrations (< 7 mM) is limited by depletion of the substrate close to the electrode surface. At high substrate concentrations (> 20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.
Keywords: Enzyme electrode; Mediator; Kinetics; Glucose oxidase; Osmium bipyridine;

The adhesion and spreading of thrombocyte vesicles on electrode surfaces by Victor Agmo Hernández; Juliane Niessen; Falk Harnisch; Stephan Block; Andreas Greinacher; Heyo K. Kroemer; Christiane A. Helm; Fritz Scholz (210-216).
The interaction of thrombocyte vesicles with the surface of metal electrodes, i.e., mercury, gold and gold electrodes modified with self assembled monolayers (SAM), was studied with the help of chronoamperometry, atomic force microscopy, and quartz crystal microbalance measurements. The experimental results show that the interaction of the thrombocyte vesicles with the surface of the electrodes depends on the hydrophobicity of the latter: whereas on very hydrophobic surfaces (mercury and gold functionalized with SAM) the thrombocyte vesicles disintegrate and form a monolayer of lipids, on the less hydrophobic gold surface a bilayer is formed. The chronoamperometric measurements indicate the possibility of future applications to probe membrane properties of thrombocytes.
Keywords: Thrombocyte vesicles; Chronoamperometry; Quartz crystal microbalance; Adhesion; Mercury electrode;

The flower-like gold nanoparticles together with spherical and convex polyhedron gold nanoparticles were fabricated on boron-doped diamond (BDD) surface by one-step and simple electrochemical method through easily controlling the applied potential and the concentration of HAuCl4. The recorded X-ray diffraction (XRD) patterns confirmed that these three shapes of gold nanoparticles were dominated by different crystal facets. The cyclic voltammetric results indicated that the morphology of gold nanoparticles plays big role in their electrochemical behaviors. The direct electrochemistry of hemoglobin (Hb) was realized on all the three different shapes of nanogold-attached BDD surface without the aid of any electron mediator. In pH 4.5 acetate buffer solutions (ABS), Hb showed a pair of well defined and quasi-reversible redox peaks. However, the results obtained demonstrated that the redox peak potential, the average surface concentration of electroactive heme, and the electron transfer rates of Hb are greatly dependent upon the surface morphology of gold nanoparticles. The electron transfer rate constant of hemoglobin over flower-like nanogold/BDD electrode was more than two times higher than that over spherical and convex polyhedron nanogold. The observed differences may be ascribed to the difference in gold particle characteristics including surface roughness, exposed surface area, and crystal structure.
Keywords: Flower-like gold nanoparticles; Electrochemical property; Electron transfer-bridge; Different electrocatalytical activity;