Bioelectrochemistry (v.70, #2)

Contents of Volume 70 (XIII-XVII).

Contents (iv-vi).

Electroenzymatic reactions with oxygen on laccase-modified electrodes in anhydrous (pure) organic solvent by A. Yaropolov; S. Shleev; E. Zaitseva; J. Emnéus; G. Marko-Varga; L. Gorton (199-204).
The electroenzymatic reactions of Trametes hirsuta laccase in the pure organic solvent dimethyl sulfoxide (DMSO) have been investigated within the framework for potential use as a catalytic reaction scheme for oxygen reduction. The bioelectrochemical characteristics of laccase were investigated in two different ways: (i) by studying the electroreduction of oxygen in anhydrous DMSO via a direct electron transfer mechanism without proton donors and (ii) by doing the same experiments in the presence of laccase substrates, which display in pure organic solvents both the properties of electron donors as well as the properties of weak acids. The results obtained with laccase in anhydrous DMSO were compared with those obtained previously in aqueous buffer. It was shown that in the absence of proton donors under oxygenated conditions, formation of superoxide anion radicals is prevented at bare glassy carbon and graphite electrodes with adsorbed laccase. The influence of the time for drying the laccase solution at the electrode surface on the electroreduction of oxygen was studied. Investigating the electroenzymatic oxidation reaction of catechol and hydroquinone in DMSO reveals the formation of various intermediates of the substrates with different electrochemical activity under oxygenated conditions. The influence of the content of aqueous buffer in the organic solvent on the electrochemical behaviour of hydroquinone/1,4-benzoquinone couple was also studied.
Keywords: Laccase; Electroenzymatic reaction; Anhydrous organic solvent; Dimethyl sulfoxide;

Electrochemical screening of anti-microbial peptide LL-37 interaction with phospholipids by Frances Neville; David Gidalevitz; Girish Kale; Andrew Nelson (205-213).
LL-37 is an α-helical antimicrobial peptide of human origin. It is a 37 residue cathelicidin peptide. This paper explores the use of electrochemical methods to investigate the interaction of LL-37 with phospholipid and lipid A monolayers on a mercury drop electrode. Experiments were carried out in Dulbecco's phosphate buffered saline at pH ∼ 7.6. The capacity-potential curves of the coated electrode in the presence and absence of LL-37 were measured using out-of-phase ac voltammetry. The frequency dependence of the complex impedance of the coated electrode in the presence and absence of LL-37 was estimated at − 0.4 V versus Ag/AgCl 3.5 mol dm− 3 KCl. The monolayer permeability to ions was studied by following the reduction of Tl(I) to Tl(Hg) at the coated electrode. LL-37 shows no significant interaction with DOPC. However, LL-37 shows a small interaction with DOPG and lipid A within a DOPC monolayer where the monolayer permeability is marginally increased and the zero frequency capacitance (ZFC) is marginally decreased in both cases. LL-37 shows a significant interaction with a lipid A monolayer thereby decreasing the ZFC by 30%. The results concur with the known membrane active properties of LL-37 and establish this electrochemical approach as a key technique for screening peptides.
Keywords: Antimicrobial; LL-37; Phospholipid; Lipid A; Impedance; Mercury;

In the present study we compared the proliferation behavior, the ultrastructural morphology and the glycolitic metabolism of K562 cells irradiated by low-power wide-band millimeter waves, with those of sham-exposed K562 cells (control), maintained in the same culture conditions. The gigaHertz radiation treatments, performed between 53–78 109 Hz, induced a noticeable inhibition of the cell proliferation that could be related to relevant ultrastructural changes. Such effects brought the irradiated cell system to lose the homeostasis and to trigger defense/reparatory mechanisms in order to reestablish a new steady state. 13C-Nuclear magnetic resonance data on the kinetic of glucose metabolism demonstrated that the irradiated cells enhanced the glycolitic aerobic pathway, indicating that such system need to produce an extra-bioenergy. Most of the ATP synthesized served probably to perform the above processes resulting in a significant decrease of the proliferation rate without significant cell death increment.
Keywords: Millimeter radiation; K562 cells; Cell proliferation; Cell ultrastructure; Glucose metabolism;

The effects of conductivity and electrochemical doping on the reduction of methemoglobin immobilized in nanoparticulate TiO2 films by Elizabeth V. Milsom; Hayley A. Dash; Toby A. Jenkins; Marcin Opallo; Frank Marken (221-227).
Methemoglobin (bovine) is immobilized from aqueous phosphate buffer (pH 5.5) solution into thin porous TiO2 (anatase) films at ITO electrode surfaces. Films of TiO2 are produced in a deposition process employing 40 nm diameter TiO2 nanoparticles suspended in dry methanol followed by calcination. The pore size in these films is sufficient for methemoglobin (ca. 6 nm diameter) to diffuse into the porous structure (over several hours) and to remain immobilized in electrochemically active form.The electrochemical reduction of methemoglobin immobilized in TiO2 and immersed in aqueous phosphate buffer at pH 5.5 is observed in two steps with (i) a small quasi-reversible voltammetric response at − 0.16 V vs. SCE (Process 1) and (ii) an irreversible reduction peak at ca. − 0.5 V vs. SCE (Process 2). The irreversible response is recovered only after slow chemical re-oxidation of hemoglobin to methemoglobin. At sufficiently negative applied potential “electrochemical doping” of the TiO2 host is observed to lead to a considerably enhanced reduction Process 1. TiO2 can be temporarily switched from a non-conducting (irreversible electron transfer) into a conducting (reversible electron transfer) state.
Keywords: Hemoglobin; Nanoparticle; Doping; Catalysis; Voltammetry; Sensor;

DNA biosensor based on the electrochemiluminescence of Ru(bpy)3 2+ with DNA-binding intercalators by Jeong-Gun Lee; Kyusik Yun; Guei-Sam Lim; Sang Eun Lee; Suhyeon Kim; Je-Kyun Park (228-234).
This paper reports a novel detection method for DNA hybridization based on the electrochemiluminescence (ECL) of Ru(bpy)3 2+ with a DNA-binding intercalator as a reductant of Ru(bpy)3 3+. Some ECL-inducible intercalators have been screened in this study using electrochemical methods combined with a chemiluminescent technique. The double-stranded DNA intercalated by doxorubicin, daunorubicin, or 4′,6-diamidino-2-phenylindole (DAPI) shows a good ECL with Ru(bpy)3 2+ at + 1.19 V (versus Ag/AgCl), while the non-intercalated single-stranded DNA does not. In order to stabilize the self-assembled DNA molecules during ECL reaction, we constructed the ECL DNA biosensor separating the ECL working electrode with an immobilized DNA probe. A gold electrode array on a plastic plate was assembled with a thru-hole array where oligonucleotide probes were immobilized in the side wall of thru-hole array. The fabricated ECL DNA biosensor was used to detect several pathogens using ECL technique. A good specificity of single point mutations for hepatitis disease was obtained by using the DAPI-intercalated Ru(bpy)3 2+ ECL.
Keywords: Electrochemiluminescence (ECL); Tris-(2,2′-bipyridyl)ruthenium(II); DNA-binding intercalator; 4′,6-diamidino-2-phenylindole (DAPI); DNA biosensor; DNA hybridization detection;

A nano-composite of DNA/poly(p-aminobenzensulfonic acid) bi-layer modified glassy carbon electrode as a biosensor was fabricated by electro-deposition method. The DNA layer was electrochemically deposited on the top of electropolymerized layer of poly(p-aminobenzensulfonic acid) (Pp-ABSA). Scanning electron microscopy, X-ray photoelectron spectroscopy and electrochemical impedance spectrum were used for characterization. It demonstrated that the deposited Pp-ABSA formed a 2-D fractal patterned nano-structure on the electrode surface, and which was further covered by a uniform thin DNA layer. Cyclic voltammetry and electrochemical impedance spectrum were used to characterize the deposition, and demonstrated the conductivity of the Pp-ABSA layer. The biosensor was applied to the detection of dopamine (DA) and uric acid (UA) in the presence of ascorbic acid (AA). In comparison with DNA and Pp-ABSA single layer modified electrodes, the composite bi-layer modification provided superior electrocatalytic actively towards the oxidation of DA, UA and AA, and separated the originally overlapped differential pulse voltammetric signals of UA, DA and AA oxidation at the bare electrode into three well-defined peaks at pH 7 solution. The peak separation between AA and DA, AA and UA was 176 mV and 312 mV, respectively. In the presence of 1.0 mM AA, the anodic peak current was a linear function of the concentration of DA in the range 0.19–13 μM. The detection limit was 88 nM DA (s  /  n  = 3). The anodic peak current of UA was also a linear function of concentration in the range 0.4–23 μM with a detection limit of 0.19 μM in the presence of 0.5 mM AA. The superior sensing ability was attributed to the composite nano-structure. An interaction mechanism was proposed.
Keywords: DNA; Poly(p-aminobenzensulfonic acid); Dopamine; Uric acid; Nanostructure;

Electrochemical detection of short sequences related to the hepatitis B virus using MB on chitosan-modified CPE by Guo Mandong; Li Yanqing; Guo Hongxia; Wu Xiaoqin; Fan Lifang (245-249).
A novel electrochemical DNA biosensor based on methylene blue (MB) and chitosan-modified carbon paste electrode (CCPE) for short DNA sequences and polymerase chain reaction (PCR) amplified real samples related to the hepatitis B virus (HBV) hybridization detection is presented. Differential pulse voltammetry (DPV) was used to investigate the surface coverage and hybridization event. The decrease in the peak current of MB, an electroactive label, was observed upon hybridization of probe with the target. Numerous factors affecting the target hybridization and indicator binding reaction are optimized to maximize the sensitivity.
Keywords: DNA biosensor; Hybridization; Chitosan; MB; HBV;

The direct electrochemistry of glucose oxidase (GOD) immobilized on a hexagonal mesoporous silica modified glassy carbon electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of − 417 mV in 0.1 M pH 6.1 phosphate buffer solution (PBS). The response showed a diffusion-controlled electrode process with a two-electron transfer coupled with a two-proton transfer reaction process. GOD immobilized on a hexagonal mesoporous silica retained its bioactivity and stability. In addition, the immobilized GOD could electrocatalyze the oxidation of glucose to gluconlactone by taking ferrocene monocarboxylic acid (FMCA) as a mediator in N2 saturated solutions, indicating that the electrode may have the potential application in biosensors to analyze glucose. The sensor could exclude the interference of commonly coexisted uric acid, p-acetaminophenol and ascorbic acid and diagnose diabetes very fast and sensitively. This work demonstrated that the mesoporous silica provided a novel matrix for protein immobilization and the construction of biosensors.
Keywords: Biosensors; Glucose oxidase; Hexagonal mesoporous silica; Chemically modified electrode; Direct electrochemistry; Glucose;

The interactions between the recombinant dust mite allergen Der f2 and murine monoclonal antibody were monitored by electrochemical impedance spectroscopy (EIS). Allergen Der f2 were immobilized through the nanogold formed by electrodeposition of gold on planar glassy carbon electrode. A 30-s gold electrodeposition provided a desirable substrate for the immobilization of allergen. Electrochemical deposition of gold on a glassy carbon electrode showed significant improvement in allergen immobilization. The impedance measurements were based on the charge-transfer kinetics of the [Fe(CN)6]3−/4− redox pair. The interactions between allergen and antibody occurred on electrode surface altered the interfacial electron transfer resistance, R CT, by preventing the redox species approaching the electrode. The results showed that R CT increased with increasing concentration of monoclonal antibodies.
Keywords: Gold electrodeposition; Recombinant allergen; Antibody; Electron transfer resistance (R CT); Electrochemical impedance spectroscopy (EIS);

Electrochemical behaviors of ethinylestradiol at a carbon paste electrode (CPE) in the presence of cetyl pyridine bromide (CPB) are investigated by electrochemical techniques. Compared with that at a CPE without CPB, the oxidation peak potential of ethinylestradiol shifts negatively and the peak current is increased significantly, due to the enhanced accumulation of ethinylestradiol via electrostatic interaction with CPB at the hydrophobic electrode surface. It is verified by the influences of different kinds of surfactants on the electrochemical signals of ethinylestradiol. Some parameters such as pH, scan rate, accumulation potential and accumulation time on the oxidation of ethinylestradiol are optimized. Under optimal conditions, the oxidation peak current is proportional to ethinylestradiol concentration in the range of 5.0 × 10− 8 to 2.0 × 10− 5 mol L− 1 with a detection limit of 3.0 × 10− 8 mol L− 1 for 150 s accumulation by linear sweep voltammetry (LSV). The proposed procedure is successfully applied to determine ethinylestradiol in pharmaceutical formulation (Levonorgestrel and Etinylestradiol tablets) and the results are satisfying compared with that of high-performance liquid chromatography (HPLC).
Keywords: Ethinylestradiol; Cetyl pyridine bromide; Carbon paste electrode; Electroanalysis;

A single HBsAg DNA vaccination in combination with electroporation elicits long-term antibody responses in sheep by Shawn Babiuk; Cemaine Tsang; Sylvia van Drunen Littel-van den Hurk; Lorne A. Babiuk; Philip J. Griebel (269-274).
Vaccines continue to be the most cost effective method to reduce the burden of disease in both human and animal health. However, there is a need to improve the duration of immunity following vaccination, since maintenance of protective levels of antibody in serum or the ability to rapidly respond upon re-exposure (memory) is critical if vaccines are to provide long-term protective immunity. The purpose of this experiment was to test the duration of antibody responses and the ability to generate anamnestic responses following a single immunization with a DNA vaccine encoding hepatitis B surface antigen (HBsAg) delivered by a variety of routes. Sheep immunized with the conventional HBsAg subunit vaccine (Engerix-B) as well as sheep immunized with a HBsAg DNA vaccine, combined with electroporation, generated significant antibody responses that were sustained for 25 weeks after primary immunization. At 25 weeks, all experimental groups received a secondary immunization with the HBsAg subunit vaccine. Sheep that received a primary DNA immunization, in combination with electroporation, mounted an anamnestic response similar to the cohort immunized with the HBsAg subunit vaccine. In contrast, animals immunized with DNA vaccines administered without electroporation elicited no detectable memory response. The presence of immune memory was significantly correlated with the induction of a prolonged primary immune response. Thus, a single DNA vaccination, in combination with electroporation, approached the efficacy of the commercial subunit vaccine in the maintenance of long-term protective serum antibody titres and immune memory.
Keywords: DNA vaccine; Electroporation; HBsAg; Immune memory; Sheep;

High electrical field effects on cell membranes by U. Pliquett; R.P. Joshi; V. Sridhara; K.H. Schoenbach (275-282).
Electrical charging of lipid membranes causes electroporation with sharp membrane conductance increases. Several recent observations, especially at very high field strength, are not compatible with the simple electroporation picture. Here we present several relevant experiments on cell electrical responses to very high external voltages. We hypothesize that, not only are aqueous pores created within the lipid membranes, but that nanoscale membrane fragmentation occurs, possibly with micelle formation. This effect would produce conductivity increases beyond simple electroporation and display a relatively fast turn-off with external voltage. In addition, material loss can be expected at the anode side of cells, in agreement with published experimental reports at high fields. Our hypothesis is qualitatively supported by molecular dynamics simulations. Finally, such cellular responses might temporarily inactivate voltage-gated and ion-pump activity, while not necessarily causing cell death. This hypothesis also supports observations on electrofusion.
Keywords: Electroporation; High Field; Electrical Conductivity; Lipid Membrane; Molecular Dynamics;

Electrochemotherapy of Mycosis fungoides by interferon-α by E. Peycheva; I. Daskalov; I. Tsoneva (283-286).
Eight patients with 29 lesions of histologically verified 1st stage of Mycosis fungoides were successfully treated by electrochemotherapy with interferon-α. For this purpose 8 biphasic pulses were used, each of 50 + 50 μs duration with 900 μs interpulse intervals, resulting in a burst of 7.1 ms total duration. Compared to the traditional monoimmunotherapy with interferon-α applied three times weekly for a total of 4 weeks, the electrochemotherapy was very efficient. Complete response (CR) was observed in 25 (86%) of the 29 treated lesions by single-act electrochemotherapy with interferon-α. At the end of the 12-month period, all 29 lesions showed 100% complete response (CR). New lesions for a period of 12 months were not observed.The expected mechanism involved in multiple cytotoxic action of interferon-α could be the local increased concentration in the tumour and prolongation of the time of its action after the application of pulses.
Keywords: Electrochemotherapy; Mycosis fungoides; Interferon-α;

Electroporation is used for in vivo gene therapy, drug therapy and minimally invasive tissue ablation. Applying electrical pulses across cells can have a variety of outcomes; from no effect to reversible electroporation to irreversible electroporation. Recently, it has been proposed that measuring the passive electrical properties of electroporated tissues could provide real time feedback on the outcome of the treatment. Here we describe the results from the impedance characterization (single dispersion Cole model) for up to 30 min of the electroporation process in in vivo rat livers (n  = 8). The electroporation sequence consisted of 8 pulses of 100 μs with a period of 100 ms. Half of the animals were subjected to field magnitudes considered to have reversible effects (R group, E  = 450 V/cm) whereas for the other half irreversible field amplitudes were applied (I group, E  = 1500 V/cm). As expected, there was an immediate increase of conductivity (R group Δσ  /  σ t=0  = 9 ± 3%; I group Δσ  /  σ t=0  = 43 ± 1%). However, the overall long term pattern of change in conductivity after electroporation is complex and different between reversible and irreversible groups. This suggests the superposition of different phenomena which together affect the electrical properties.
Keywords: Electroporation; Electrical impedance; Electrical conductivity; Liver;

In this paper, some electrochemical parameters of ethamsylate at a multi-walled carbon nanotube modified glassy carbon electrode, such as the charge number, exchange current density, standard heterogeneous rate constant and diffusion coefficient, were measured by cyclic voltammetry, chronoamperometry and chronocoulometry. The modified electrode exhibits good promotion of the electrochemical reaction of ethamsylate and increases the standard heterogeneous rate constant of ethamsylate greatly. The differential pulse voltammetry responses of ethamsylate were linearly dependent on its concentrations in a range from 2.0 × 10− 6 to 6.0 × 10− 5 mol L− 1, with a detection limit of 4.0 × 10− 7 mol L− 1.
Keywords: Carbon nanotube; Modified electrode; Ethamsylate; Electrochemistry; Determination;

The interactions between double-stranded DNA (dsDNA) and three different kinds of surfactants, i.e., cationic, anionic, and nonionic surfactants, were investigated by cyclic voltammetry, electrochemical impedance spectroscopy and UV–vis spectroscopy. Multilayer films composed of DNA and surfactants were prepared at gold electrode by electrostatic or hydrophobic interactions. It was found that the cationic surfactant, CTAB, can bind to DNA by electrostatic interaction, and the electron transfer resistance of CTAB–DNA complex film increases first and then decreases with CTAB concentration. The anionic surfactant, LAS, can bind to DNA but by hydrophobic interaction, and the electron transfer resistance of the complex film keeps decreasing with LAS concentration. Nonionic surfactants can also directly bind to DNA by hydrophobic interaction. All the three different kinds of surfactants can form multilayer films with DNA on the electrode surface. The chemical structure of DNA keeps unchanged during interacting with these surfactants. The binding modes of DNA with these three different kinds of surfactants were also deduced.
Keywords: Electrochemical study; Cationic surfactant; Anionic surfactant; Nonionic surfactant; dsDNA; Multilayer film;

Although the physiological role of endogenous monoamine oxidase (MAO) inhibitor still remains unclear, the present study examined whether or not immobilization stress (IMMO) induce MAO inhibitor. An endogenous inhibitor of MAO was separated by gel filtration from 105,000 g supernate in rat liver cytosol following IMMO. The molecular weight of this inhibitor was estimated to be 500–600 by gel filtration. This inhibitor was proved to be heat-stable resistant to protease treatment. IMMO for 2 h significantly decreased MAO. These results suggest that this inhibitor is induced by IMMO. MAO activity in rat liver might be regulated by the level of this inhibitor.
Keywords: Monoamine oxidase (MAO); Immobilization stress (IMMO); Endogenous MAO inhibitor; Benzylamine; Rat liver;

Positively charged hemoglobin (Hb) or myoglobin (Mb) at pH 5.0 in solutions and negatively charged zeolite particles in dispersions were alternately adsorbed onto solid surfaces forming {zeolite/protein} n layer-by-layer films, which was confirmed by quartz crystal microbalance (QCM) and cyclic voltammetry (CV). The protein films assembled on pyrolytic graphite (PG) electrodes exhibited a pair of well-defined, nearly reversible CV peaks at about − 0.35 V vs. SCE at pH 7.0, characteristic of the heme Fe(III)/Fe(II) redox couples. Hydrogen peroxide (H2O2) and nitrite (NO2 ) in solution were catalytically reduced at {zeolite/protein}7 film modified electrodes, and could be quantitatively determined by CV and amperometry. The shape and position of infrared amide I and II bands of Hb or Mb in {zeolite/protein}7 films suggest that the proteins retain their near-native structure in the films. The penetration experiments of Fe(CN)6 3− as the electroactive probe into these films and scanning electron microscopy (SEM) results indicate that the films possess a great amount of pores or channels. The porous structure of {zeolite/protein} n films is beneficial to counterion transport, which is crucial for protein electrochemistry in films controlled by the charge-hopping mechanism, and is also helpful for the diffusion of catalysis substrates into the films. The proteins with negatively charged net surface charges at pH 9.0 were also successfully assembled with like-charged zeolite particles into layer-by-layer films, although the adsorption amount was less than that assembled at pH 5.0. The possible reasons for this were discussed, and the driving forces were explored.
Keywords: Layer-by-layer assembly; Zeolite; Hemoglobin; Myoglobin; Direct electrochemistry; Electrochemical catalysis;

Electrostatic properties and macroscopic electrodiffusion in OmpF porin and mutants by Marcel Aguilella-Arzo; Juan J. García-Celma; Javier Cervera; Antonio Alcaraz; Vicente M. Aguilella (320-327).
The bacterial porin OmpF found in the outer membrane of E. coli is a wide channel, characterized by its poor selectivity and almost no ion specificity. It has an asymmetric structure, with relatively large entrances and a narrow constriction. By applying continuum electrostatic methods we determine the ionization states of titratable amino acid residues in the protein and calculate self-consistently the electric potential 3-D distribution within the channel. The average electrostatic properties are then represented by an effective fixed charge distribution along the pore which is the input for a macroscopic electrodiffusion model. The theoretical predictions agree with measurements performed under different salt gradients and pH. The sensitivity of reversal potential and conductance to the direction of the salt gradient and the solution pH is captured by the model. The theory is also able to explain the influence of the lipid membrane charge. The same methodology is satisfactorily applied to some OmpF mutants involving slight structural changes but a large number of net charges. The correlation found between atomic structure and ionic selectivity shows that the transport characteristics of wide channels like OmpF and its mutants are mainly regulated by the collective action of a large number of residues, rather than by the specific interactions of residues at particular locations.
Keywords: Electrodiffusion; OmpF; Bacterial porin; Channel selectivity;

The dielectric spectra of aqueous suspensions of unilamellar liposomial vesicles built up by zwitterionic phospholipids (dipalmitoylphosphatidyl-choline, DPPC) were measured over the frequency range extending from 1 kHz to 10 MHz, where the interfacial polarization effects, due to the highly heterogeneous properties of the system, prevail. The dielectric parameters, i.e., the permittivity ε′(ω) and the electrical conductivity σ(ω), have been analyzed in terms of dielectric models based on the effective medium approximation theory, considering the contribution associated with the bulk ion diffusion on both sides of the aqueous interfaces. The zwitterionic character of the lipidic bilayer has been modeled by introducing an “apparent” surface charge density at both the inner and outer aqueous interface, which causes a tangential ion diffusion similar to the one occurring in charged colloidal particle suspensions. A good agreement with the experimental results has been found for all the liposomes investigated, with size ranging from 100 to 1000 nm in diameter, and the most relevant parameters have briefly discussed in the light of the effective medium approximation theory.
Keywords: Zwitterionic liposome; Dielectric spectra; Dielectric model; Dynamic light scattering; Liposome suspension;

Colloid Au (Aunano) with a diameter of about 10 nm was prepared and used in combination with dihexadecylphosphate (DHP) to immobilize glucose oxidase (GOD) onto the surface of a graphite electrode (GE). The direct electrochemistry of GOD confined in the composite film was investigated. The immobilized GOD displayed a pair of redox peaks with a formal potential of − 0.475 mV in pH 7.0 O2-free phosphate buffers at scan rate of 150 mV s− 1. The GOD in the composite film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of glucose, the reduction peak current of dissolved oxygen decreased, which could be developed for glucose determination. A calibration linear range of glucose was 0.5–9.3 mM with a detection limit of 0.1 mM and a sensitivity of 1.14 μA mM− 1. The glucose biosensor showed good reproducibility and stability. The general interferences that coexisted in human serum sample such as ascorbic acid and uric acid did not affect glucose determination.
Keywords: Glucose; Glucose oxidase; Colloid Au; Dihexadecylphosphate; Biosensor;

A novel film of chitosan-gold nanoparticles is fabricated by a direct and facile electrochemical deposition method and its application in glucose biosensor is investigated. HAuCl4 solution is mixed with chitosan and electrochemically reduced to gold nanoparticles, which can be stabilized by chitosan and electrodeposited onto glassy carbon electrode surfaces along with the electrodeposition of chitosan. Then a model enzyme, glucose oxidase (GOD) is immobilized onto the resulting film to construct a glucose biosensor through self-assembly. The resulting modified electrode surfaces are characterized with both AFM and cyclic voltammetry. Effects of chitosan and HAuCl4 concentration in the mixture together with the deposition time and the applied voltage on the amperometric response of the biosensor are also investigated. The linear range of the glucose biosensor is from 5.0 × 10− 5  ∼ 1.30 × 10− 3 M with a Michaelis–Menten constant of 3.5 mM and a detection limit of about 13 μM.
Keywords: Chitosan; Gold nanoparticles; Electrochemical deposition; Glucose oxidase; Biosensor;

The carbohydrate research has emerged as a “new frontier” in chemical/biological field. The binding of lectin with carbohydrate is one of the important courses of life activities. The report studies concanavalin A (Con A)–glycogen interaction on gold electrode surfaces by electrochemical piezoelectric quartz crystal impedance (EPQCI) method. The piezoelectric quartz crystal (PQC) parameters, resonant frequency shift (Δf 0) and the motional resistance change (ΔR 1), and the electrochemical impedance (EI) parameters, electrolyte resistance change (ΔR s) and the double layer capacitance change (ΔC s), were measured and discussed simultaneously. Two methods were adopted for measuring the Con A–glycogen association. Based on EPQCI measurement during Con A reaction with glycogen adsorbed on Au electrode, association constant K a and the amount of the binding sites s calculated are 1.48 × 106 M− 1 and 4.09, respectively. Based on single PQC measurement of glycogen reaction with Con A assembled on Au electrode, K a was estimated to be 1.26 × 106 M− 1.
Keywords: Concanavalin A; Glycogen; Electrochemical piezoelectric quartz crystal impedance; The association constant; Au electrode;

Electrochemical oxidation of morin and interaction with DNA by Fang Wang; Yanxia Xu; Jia Zhao; Shengshui Hu (356-362).
A poly (tetrafluroethylene)-deoxyribonucleate acid (PTFE-DNA) film-modified glassy carbon electrode (GCE) has been fabricated. The electrochemical oxidation behaviors of morin as well as its interaction with DNA have been studied at PTFE-DNA film-modified GCE and bare GCE by electrochemical methods. This modified electrode shows an enhanced effectiveness towards the oxidation of morin. Importantly, as to the interaction between morin and DNA in solution, characteristic parameters such as the binding stoichiometry and association equilibrium constant according to the Hill model for cooperative binding have been determined on the basis of linear sweep voltammetry and chronocoulometry.
Keywords: Morin binding; Deoxyribonucleate acid (DNA); Glassy carbon electrode (GCE); Chemical analytics;

In this study, a new micro electroporation (EP) cell chip with three-dimensional (3D) electrodes was fabricated by means of MEMS technology, and tested on cervical cancer (HeLa) cells. Extensive statistical data of the threshold electric field and pulse duration were determined to construct an EP “phase diagram”, which delineates the boundaries for 1) effective EP of five different size molecules and 2) electric cell lysis at the single-cell level. In addition, these boundary curves (i.e., electric field versus pulse duration) were fitted successfully with an exponential function with three constants. We found that, when the molecular size increases, the corresponding electroporation boundary becomes closer to the electric cell lysis boundary. Based on more than 2000 single-cell measurements on five different size molecules, the critical size of molecule was found to be approximately 40 kDa. Comparing to the traditional instrument, MEMS-based micro electroporation chip can greatly shorten the experimental time.
Keywords: Micro electroporation; MEMS; 3D electrode; HeLa cells; Membrane permeability; Phase diagram; Size effect;

A sensitive electrochemical method was developed for the differential pulse voltammetric determination of rhein at a glassy carbon electrode (GCE) modified with a nanoparticle composite film. In the present paper, multi-wall carbon nanotube (MWNT) was dispersed into dihexadecyl phosphate (DHP) to give a homogeneous suspension. After the solvent evaporation, a uniform film of MWNT-DHP composite film was obtained on the GCE surface. The MWNT-DHP composite film-modified GCE exhibited excellent electrocatalytic behavior toward the redox of rhein. Compared with an irreversible reduction of rhein at the bare GCE, a reversible redox behavior of rhein was observed at the MWNT-DHP composite film-modified GCE and the redox current was also enhanced greatly. Based on this, a cathodic differential pulse voltammetry (DPV) was applied for the determination of rhein. The experimental parameters, which influence the current of rhein, were optimized. Under optimal conditions, the cathodic DPV measurements were performed and a linear response of rhein was obtained in the range from 1.0 × 10− 8 to 5.0 × 10− 6 mol L− 1 and with a limit of detect (LOD) of 5.0 × 10− 9 mol L− 1. The proposed procedure was successfully applied to assay rhein in real samples with satisfactory results.
Keywords: Rhein; Electrocatalysis; Differential pulse voltammetry; Nanoparticle composite film-modified electrode;

The development of a cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOX) on oxygen electrode is described. The electrode consists of gold cathode and Ag/AgCl anode. The enzymes were immobilized by cross-linking with glutaraldehyde and Bovine Serum Albumin (BSA). The immobilized enzymatic membrane was attached to the tip of the electrode by a push cap system. The optimum pH and temperature of the sensor was determined, these are 6 and 25 °C respectively. The developed sensor was calibrated from 1–75 mg/dl of cholesterol palmiate and found linear in the range of 2–50 mg/dL. The calibration curve was drawn with V i (ppm/min)(initial velocity) vs different concentrations of cholesterol palmiate (mg/dL). The application of the sensor to determine the total cholesterol in different real food samples such as egg, meat was investigated. The immobilized enzymatic layer can be reused over 30 times and the stability of the enzymatic layer was studied up to 9 weeks.
Keywords: ChEt; Cholesterol biosensor; ChOX; Food sample analysis; Stability;

Alamethicin forms voltage-gated ion channels that have moderate cation-selectivity. The enhancement of the cation-selectivity by introducing negatively charged residues at positions 7 and 18 has been studied using the tethered homodimers of alamethicin with Q7 and E18 (di-alm-Q7E18) and its analog with E7 and Q18 (di-alm-E7Q18). In the dimeric peptides, monomer peptides are linked at the N-termini by a disulfide bond. Both the peptides formed long lasting ion channels at cis-positive voltages when added to the cis-side membrane. Their long open duration enabled us to obtain current–voltage (IV m) relations and reversal potentials at the single-channel level by applying a voltage ramp during the channel opening. The reversal potentials measured in asymmetric KCl solutions indicated that ionized E7 provided strong cation-selectivity, whereas ionized E18 little influenced the charge selectivity. This was also the case for the macroscopic charge selectivity determined from the reversal potentials obtained by the macroscopic IV m measurements. The results are accounted for by stronger electrostatic interactions between permeant ions and negatively charged residues at the narrowest part of the pore than at the pore mouth.
Keywords: Alamethicin; Ion channel; Planar BLM; Charge selectivity; Reversal potential;

Self assembled monolayers and bilayers are produced on a flat glass surface, bound by a thiolipid onto bare gold. 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol (DPPTE) is used as the molecule binding to the electrode surface. The lipid λ-α-Phosphatidyl-Choline-β-Oleoyl-g-Palmitoyl (POPC) and the lipid mixture eggphosphatadiylcholine (EPC) are used as spacer lipids with the aim of achieving solid-supported artificial lipid membranes. With the aim of creating and investigating more natural systems, ion carrier proteins such as valinomycin are introduced into the DPPTE/EPC system. The direct influence on the membranes as well as the effects of different ionic solutions on the proteins is shown.
Keywords: Solid supported biomimetic lipid bilayer membranes; Thiolipids; Peptides; Ionophores; Valinomycin; POPC; DPPTE; EPC; Egg-PC; Electrochemical impedance spectroscopy;

Myoglobin modified electrodes as anchors for d metal cationic complexes by María F. Cerdá; Gonzalo Obal; Jorge S. Gancheff; Carlos Kremer; Ana M. Castro Luna; André M. Braun; Michael Woerner; Eduardo Méndez (394-400).
The capability of adsorption of different electroactive cationic Re(V)–amine complexes onto myoglobin-containing electrodes has been investigated. The goal of this work was the development of an Au/thiol/myo electrode and, after incubation of such ensemble in the presence of three different Re(V)–amine complexes, the evaluation of the extent of surface coverage by the complexes (as a way to evaluate the interaction complex-protein) using electrochemical techniques. Our results showed that a protein-containing electrode could therefore be used for the detection of the interaction of small electroactive cationic complexes and the biomolecule. The extent of the coverage of the myoglobin electrode by the complex depends on the number of free tails from the ligands and the total charge of the complex.
Keywords: SAM; Myoglobin; Modified electrodes; Protein–metal interaction;

Electrical probing of endothelial cell behaviour on a fibronectin/polystyrene/thiol/gold electrode by Faradaic electrochemical impedance spectroscopy (EIS) by Amira Bouafsoun; Saloua Helali; Saida Mebarek; Caroline Zeiller; Annie-France Prigent; Ali Othmane; Abdelhamid Kerkeni; Nicole Jaffrézic-Renault; Laurence Ponsonnet (401-407).
The electrochemical impedance spectroscopy (EIS) technique has been shown to be an effective tool for monitoring endothelial cell behaviour on a multilayer functionalised gold electrode. Polystyrene, a reproducible model substrate, is deposited as a thin layer on a thiol functionalised gold electrode. Fibronectin, a protein promoting endothelial cell adhesion, is then adsorbed on the polystyrene surface. The different steps of this multilayer assembly are characterized by Faradaic impedance. The charge transfer resistance and the capacitance for the total layer are modified at each step according to the electrical properties of each layer. This gives the endothelial cells' electrical state in terms of its resistive and capacitive properties. In this study, the endothelial cell layer presents a specific charge transfer resistance equal to 1.55 kΩ cm2 with no large defects in the cell layer, and a specific capacitance equal to few μF cm− 2 explained by the existence of pseudopods. These electrical properties are correlated to the endothelial cell viability, adhesion and cytoskeleton organization.
Keywords: Electrochemical Impedance Spectroscopy (EIS); Endothelial cell; Fibronectin; Polystyrene;

A carbon paste electrode (CPE) modified by a monolayer film of sodium dodecyl sulfate (SDS) was used for detection of dopamine (DA). Cyclic voltammetry demonstrated improved response of the DA sensor. This suggests the effectivity of surface modification of CPE by SDS. Impedance spectroscopy was used for the characterization of CPE surface properties. The effect of SDS concentration on the electrode quality also reveals that SDS formed a monolayer on CPE surface with a high density of negative-charged end directed outside the electrode. As a result, the carbon paste electrode modified with SDS (SDS/CPE) exerted discrimination against ascorbic acid in physiological circumstance. Thus, it can selectively determine dopamine even in the presence of 220-fold AA combined with differential pulse stripping voltammetry. In pH 7.40 phosphate buffer solution, the oxidation peak current on differential pulse voltammograms increases linearly with the concentration of DA in the range of 5.0 × 10− 7 to 8.0 × 10− 4 mol · L− 1 with a detection limit of 5.0 × 10− 8 mol · L− 1. Satisfying results are achieved when detecting the DA in injection and simulated biology sample.
Keywords: Dopamine; Modified carbon paste electrode; Sodium dodecyl sulfate; Cyclic voltammetry; Differential pulse voltammetry; Impedance spectroscopy;

Cytochrome c (Cyt c) was successfully immobilized on l-cysteine modified gold electrode by multicyclic voltammetry method. The electrochemical behavior of Cyt c on the l-cysteine modified electrode was explored. In 0.10 M, pH 7.0 phosphate buffer solution (PBS), Cyt c showed a quasi-reversible electrochemical redox behavior with E pc  = 0.180 V, E pa  = 0.208 V (versus Ag/AgCl). The Cyt c/l-cysteine modified electrode gave an excellent electrocatalytic activity towards the oxidation of nitric oxide, and the catalysis currents were proportional to the nitric oxide concentration in the range of 7.0 × 10− 7 to 1.0 × 10− 5 M, the linear regression equation is I (μA) = − 0.124–0.003C NO (μM), with a correlation coefficient 0.996, The detection limit was 3.0 × 10− 7 M (times the ratio of signal to noise, S/N  = 3).
Keywords: Cytochrome c; l-Cysteine; Nitric oxide; Electrocatalysis;

Probing organization and communication at layered interfaces by Monika Dominska; Maciej Mazur; Kelly P. Greenough; Monique M. Koan; Paweł G. Krysiński; G.J. Blanchard (421-434).
We have investigated the local organization intrinsic to a variety of interfacial structures, by both electrochemical and spectroscopic means. Our focus has been on the design and construction of biomimetic interfaces, where a lipid bilayer or a hybrid bilayer membrane can be bound to an interface. The goal of this work is ultimately to create an interface on a transducer surface that can support an enzyme in its active form. To this point, we have examined the extent of organization that is achievable in monolayers that will be used to bind bilayer structures to a transducer surface. Our electrochemical data point to the important role of the substrate surface in determining adlayer organization. We have also investigated the fluidity and structural heterogeneity of lipid bilayers using time-resolved and steady state fluorescence spectroscopy. Our data point to the highly interactive nature of lipid bilayer constituents, where perturbations introduced to one region have significant consequences on other regions of the bilayer. Such information is directly relevant to the existence and properties of lipid raft structures in both model and biological bilayers.
Keywords: Surface-modification; Interfacial electron transfer; Unilamellar vesicles; Fluorescence anisotropy;

The electrochemical behaviors of native and thermally denatured fish DNA was investigated using boron-doped diamond (BDD) film electrode by cyclic voltammetry. The BDD electrode afforded us to measure weak current less than μA for the DNA solution in 100 μl. The mixture of acetic acid and sodium acetate solution (0.2 M) was used as a supporting electrolyte. Two oxidation peaks were observed at about + 1.1 V and + 1.3 V at pH 4.6 for thermally denatured fish DNA. This is due to the oxidation of guanine and adenine in the denatured fish DNA, respectively. In contrast, the native fish DNA showed ill-defined peaks at + 1.1 V. Furthermore, the electrochemical behaviors of thermally denatured fish DNA were studied in the presence of cytosine, cytidine, cytidine-5-monophosphate, tetrakis(1-methypyridinium-4-yl)porphyrin (H2(TMPyP)4+) and RuII(TMPyP)4+. The oxidation peak intensity at + 1.1 V gradually decreased with the increase of the concentrations of the above compounds. Based on the above studies, electrochemical behaviors of the thermally denatured fish DNA at BDD electrode is discussed.
Keywords: Cyclic voltammetry; Boron doped diamond electrode; Denatured DNA;

Electrooxidation of dissolved dsDNA backed by in situ UV–Vis spectroscopy by Anna M. Nowicka; Ewelina Zabost; Mikolaj Donten; Zofia Mazerska; Zbigniew Stojek (440-445).
The electrooxidation of double-stranded DNA (dsDNA) from calf thymus was carried by using cyclic voltammetry. A glassy carbon disk-, a platinum disk-, a platinum mesh- and a carbon vapor-deposited platinum mesh electrodes were used. It is shown that the appropriate chemical and biological (steam treatment) purification of the complete cell allows, for the graphite electrode, formation of a wide anodic dsDNA signal with two visible anodic peaks. There was no necessity of preaccumulation of dsDNA on the electrode surface and of use of mediators to get well defined voltammetric signals. These peaks apparently reflect electrooxidation of the DNA's guanine and adenine. The spectrophotometric data obtained during the electrooxidation indicate that the absorbance increases with an increase in potential and electrooxidation current of dsDNA. However, the absorption band maximum either does or does not change its position depending on the mesh material. This different spectroscopic behavior may mean that the changes in the dsDNA structure upon electrooxidation are different in the case of Pt and C electrodes.
Keywords: dsDNA; Voltammetry; In-situ UV–Vis spectroelectrochemistry; Electrooxidation;

The interactions between cobalt polypyridyl coordination compounds Co(L)3 3+(L  = 1,10-phenanthroline(phen), and bipyridine(bpy)),6-mercaptopurine and calf thymus DNA have been investigated using electrochemical methods(cyclic voltammetry, differential pulse voltammetry), electronic absorption spectroscopy and viscosity measurements. Results indicate that there is an obvious interaction equilibrium between Co(L)3 3+, 6-mercaptopurine and DNA. The phenomena are investigated for the first time, and believed to be helpful to use the anticancer drugs more efficiently.
Keywords: Cyclic voltammetry; Cobalt polypyridyl complexes; DNA; Antitumor;

The electrocatalytic oxidations of guanine, adenine, guanosine-5′-monophosphate(GMP) and ssDNA were performed in the presence of Fe(II) bis(2,2′:6′,2″-terpyridine) and Fe(II) tris(1,10-phenanthroline) complexes as homogeneous catalysts by cyclic voltammetric methods. The Fe(II/III) redox couple of these compounds is responsible for their catalytic properties. The electrocatalytic oxidation current of above substrates were developed from the anodic peak currents of Fe(II) bis(2,2′:6′,2″-terpyridine) and Fe(II) tris(1,10-phenanthroline) complexes at about + 0.93 V and 0.97 V, respectively. The electrocatalytic oxidative properties of guanine by Fe(II) bis(2,2′:6′,2″-terpyridine) complex was measured by amperometry method using the rotating disk electrodes. Electropolymerization of Fe(II) tris(5-amino-1,10-phenanthroline) complex produced thin polymer films on gold and glassy carbon electrodes. The electrochemical quartz crystal microbalance (EQCM) and cyclic voltammetry were used to study the in situ growth of the polymer. The poly(FeII(5-NH2-1,10-phen)3) exhibited a good electrocatalytic oxidation towards guanine and also for the mixture of guanine and adenine too.
Keywords: DNA; Guanine; Adenine; Film-modified electrodes; Electrocatalysis;

The passage of water through the aquaporin-1 (AQP1) transmembrane channel protein of the human erythrocyte is known to be inhibited by organic mercurials such as p-chloromercuribenzoate (pCMB), which react with the free SH-group of the critical cysteine (Cys189) located near the constriction of the AQP1 water-specific channel. Sodium nitroprusside (SNP), which is known as a nitric oxide (NO) donor in interactions with SH-containing molecules, is shown here to suppress the diffusional water permeability (P d) of the erythrocyte membrane, presumably as a result of reaction with the Cys189 of the human erythrocyte AQP1 water channels. Further, treatment of erythrocytes with HgCl2 is found to result in a cell volume decrease that can be related to activation of membrane K+-selective Gárdos channels and subsequent loss of intracellular K+ and cell shrinkage. The variations in P d and volume of the erythrocyte were deduced from induced variations in the measured proton (1H) nuclear magnetic resonance (NMR) transverse (T 2) relaxation functions of water exchanging between diamagnetic intracellular and paramagnetic extracellular compartments of the 20–25% hematocrit samples. The extracellular solvent contained 10 mM membrane-impermeable paramagnetic Mn2+ ions. The 1H-T 2 NMR technique allows determination of the time constant τ exch (for exchange of the erythrocyte intracellular water) that is inversely proportional to the permeability coefficient P d when the intracellular water volume is left unmodified, as in the case of SNP-treated erythrocytes. However, for HgCl2-treated erythrocytes, this technique showed simultaneous variation of both τ exch and the volume ratio V in/V out of intracellular and extracellular water in proportions suggesting that P d was left unmodified. The HgCl2 effect has been found to be partly reversible by the reducing activity of added mercaptoethanol.
Keywords: Human erythrocyte; Water permeability coefficient; Cell volume; Na nitroprusside; HgCl2; Proton NMR;

Maxwell displacement current allows to study structural changes of gramicidin A in monolayers at the air–water interface by Pavol Vitovič; Martin Weis; Pavol Tomčík; Július Cirák; Tibor Hianik (469-480).
We applied methods of measurement Maxwell displacement current (MDC) pressure–area isotherms and dipole potential for analysis of the properties of gramicidin A (gA) and mixed gA/DMPC monolayers at an air–water interface. The MDC method allowed us to observe the kinetics of formation of secondary structure of gA in monolayers at an air–water interface. We showed, that secondary structure starts to form at rather low area per molecule at which gA monolayers are in gaseous state. Changes of the MDC during compression can be attributed to the reorientation of dipole moments in a gA double helix at area 7 nm2/molecule, followed by the formation of intertwined double helix of gA. The properties of gA in mixed monolayers depend on the molar fraction of gA/DMPC. At higher molar fractions of gA (around 0.5) the shape of the changes of dipole moment of mixed monolayer was similar to that for pure gA.The analysis of excess free energy in a gel (18 °C) and in a liquid-crystalline phase (28 °C) allowed us to show influence of the monolayer structural state on the interaction between gA and the phospholipids. In a gel state and at the gA/DMPC molar ratio below 0.17 the aggregates of gA were formed, while above this molar ratio gA interacts favorably with DMPC. In contrast, for DMPC in a liquid-crystalline state aggregation of gA was observed for all molar fractions studied. The effect of formation ordered structures between gA and DMPC is more pronounced at low temperatures.
Keywords: Gramicidin A; Maxwell displacement currents; Area-pressure isotherms; Surface potential; Lipid monolayers; Aggregation;

Electrochemical detection of E. coli 16S rDNA sequence using air-plasma-activated fullerene-impregnated screen printed electrodes by Haruki Shiraishi; Tomohito Itoh; Hiroki Hayashi; Kazuyoshi Takagi; Masayasu Sakane; Tatsuhiko Mori; Joseph Wang (481-487).
A new DNA modified electrode for the electrochemical detection of 16S rDNA extracted from Escherichia coli (JCM1649) is proposed. The electrodes were fabricated by screen printing a fullerene-impregnated carbon ink onto a poly(methylmethacrylate) substrate and immobilizing a probe DNA on the surface after activating the electrode with air plasma. The results indicated a dramatic improvement in the surface coverage of the immobilized probe DNA, and of the reduction peak of the redox indicator (Co(phen)3 3+) due to the incorporation of fullerene. By immobilizing the probe onto the fullerene-impregnated screen-printed electrodes, the PCR product of the 16S rDNA extracted from E. coli was directly detected without any pretreatment. A well defined signal difference was observed between the perfectly matching oligonucleotide and the mismatching one, and it was possible to detect the target at the modified electrode. This method enabled us to clearly detect the two base mismatches in the ca. 1500-bases long 16S rDNA sequence.
Keywords: Air plasma activation; Fullerene impregnated screen printed electrodes; E. coli 16S rDNA; Electrochemical hybridization detection;

Electrochemical response of oligonucleotides on carbon paste electrode by I. Stempkowska; M. Ligaj; J. Jasnowska; J. Langer; M. Filipiak (488-494).
Electrochemical response of synthetic oligonucleotides with different DNA bases sequences was investigated to find relationships between a chain composition and a signal. All DNA mononucleotides present electroactivity at a carbon paste electrode yielding anodic peaks at potentials: 1.00 (GMP), 1.28 (AMP), 1.47 (TMP) and 1.53 V (CMP). Also 15-mer homooligonucleotides show respective anodic peaks. Electrochemical response of 15- and 19-mer oligonucleotides consisting of all four DNA bases in different amounts was determined by the composition of oligonucleotide chain. When the contribution of different bases in oligonucleotide was balanced two anodic peaks were obtained that can be attributed to guanine and adenine moieties. Thymine residue is shown as a separate peak in voltammogram when its content in oligonucleotide chain is close to 50% of the total number of bases. Cytosine also yields a peak at its significant contribution in oligonucleotide chain and both pyrimidinic moieties produce catalytic waves easier when one of them is dominating or when only one pyrimidine derivative is present in a chain. Guanine is the easiest oxidized base and it produces a peak even at its minimal contribution (one guanine residue in 19-mer oligonucleotide). Guanine peak potential is dependent on oligonucleotide concentration and oligonucleotide composition. The lowest oligonucleotide concentration detected by guanine peak was 12.5 nM whereas detected by thymine peak was 90 nM.
Keywords: Oligonucleotides; Square wave voltammetry; Structure/signal relationship;

The electrochemical reduction of coenzyme CoQ10 and CoQ0 on glassy carbon (GC) has been investigated in mixed solvent containing 80 vol.% acetic acid and 20 vol.% acetonitrile. A combination of cyclic voltammetry (CV) and rotating disk electrode technique (RDE) was employed to elucidate the mechanism of electrode processes. The results obtained were interpreted in terms of an E r E q mechanism involving the inverted ordering of formal potentials, i.e. E 2 0′  >  E 1 0′. The cathodic processes of both compounds consist of two successive one-electron one-proton steps, whereas the second electron transfer is thermodynamically more facile than the first. The processes occur with the generation of unstable semiquinone radicals as primary products. The results presented can help in explanation of the biochemical properties of CoQ10 in the living cell.
Keywords: Coenzymes Q10 and Q0; Electroreduction; Cyclic voltammetry; Potential inversion; Digital simulation;

Real time electroporation control for accurate and safe in vivo non-viral gene therapy by David Cukjati; Danute Batiuskaite; Franck André; Damijan Miklavčič; Lluis M. Mir (501-507).
In vivo cell electroporation is the basis of DNA electrotransfer, an efficient method for non-viral gene therapy using naked DNA. The electric pulses have two roles, to permeabilize the target cell plasma membrane and to transport the DNA towards or across the permeabilized membrane by electrophoresis. For efficient electrotransfer, reversible undamaging target cell permeabilization is mandatory. We report the possibility to monitor in vivo cell electroporation during pulse delivery, and to adjust the electric field strength on real time, within a few microseconds after the beginning of the pulse, to ensure efficacy and safety of the procedure. A control algorithm was elaborated, implemented in a prototype device and tested in luciferase gene electrotransfer to mice muscles. Controlled pulses resulted in protection of the tissue and high levels of luciferase in gene transfer experiments where uncorrected excessive applied voltages lead to intense muscle damage and consecutive loss of luciferase gene expression.
Keywords: DNA electrotransfer; Gene therapy; Electropermeabilization; Electroporation; Electrochemotherapy; Finite element modeling;

Electrochemical determination of Ag-ions in environment waters and their action on plant embryos by Radka Mikelova; Jiri Baloun; Jitka Petrlova; Vojtech Adam; Ladislav Havel; Jiri Petrek; Ales Horna; Rene Kizek (508-518).
We utilized liquid chromatography coupled with electrochemical detector (HPLC–ED) for analyzing of silver ions. The optimization of basic chromatographic parameters has been done. The detection limit (3 S/N) obtained were 20 nmol/dm3. Influence of different interferences (anions and cations) on current response of silver ions has been described. Moreover, we used HPLC–ED to analyze waters of different purity including photographic emulsion, which naturally contained silver ions. We found out that content of silver ions in the emulsion was 1.57 × 0.03 mmol/dm3. Moreover, we investigated influence of silver ions on early somatic embryos of Blue Spruce. We were interested in the issue how much silver ions can embryos uptake during four days long treatment. For this purpose, we used optimized HPLC–ED technique. The content increased with increasing treatment time and applied concentration. We also studied how silver ions can influence thiols content in the treated embryos. For these purposes we used adsorptive transfer stripping voltammetry in connection with differential pulse voltammetry — Brdicka reaction. It clearly follows from the obtained results that content of thiols increased with increasing treatment time and applied concentration.
Keywords: Glassy carbon electrode; Carbon paste electrode; Silver ions; Somatic embryos; Liquid chromatography; Brdicka reaction;

The changes of the stainless-steel electrode surface morphology occurring due to dissolution of the anode under the action of electric pulses which are commonly utilized in cell electromanipulation procedures, have been studied by using atomic force microscopy. The surface of the polished electrode was rather smooth — the average roughness was 13–17 nm and the total roughness 140–180 nm. After the treatment of the chamber filled with 154 mM NaCl solution to a series of short (about 20 μs), high-voltage (4 kV) pulses, the roughness of the surface of the anode has increased, depending on the total amount of the electric charge that has passed through the unit area of the electrode, and exceeded 400 nm for the dissolution charge of 0.24 A s/cm2. No changes of the cathode surface were detected. Well-defined peaks with the width of 1–2 μm and the height of over 400 nm have appeared. These peaks create local enhancements of the electric field at the interface between the solution and the electrode surface which can lead to the non-homogeneity treatment of cells by electric pulses and can facilitate the occurrence of the electrical breakdown of the liquid samples.
Keywords: Surface topography; Electrolysis; Electroporation; Arcing; PEF food processing;

Study of the interaction between oxygen and bile salts by Giorgio Feroci; Aldo Roda; Adamo Fini (524-531).
The interaction between molecular oxygen and bile salts, previously observed using chemiluminescence techniques, is studied in this paper by electrochemical techniques to further highlight the nature of the interaction. A shift of half-wave potential of the first polarographic wave for the reduction of molecular oxygen was observed in solutions in the presence of bile salts. The shift could be related to different phenomena, such as adsorption of bile salt molecules on the mercury electrode, irreversibility of the oxygen reduction reaction, pH of the solution. Experimental results suggest the exclusion of the above mentioned processes and outline the occurrence of a direct interaction between oxygen and bile salts, where the hydrophobic face of bile salt monomers and/or small aggregates are involved, enhancing so dismutation of superoxide ion produced at the electrode. The presence of bile salts in solutions containing triphenylphosphine oxide, a hydrophobic surfactant, increases also the wave of reduction of molecular oxygen. As a consequence bile salts, beside the well-assessed physiological roles, can behave as oxygen carrier and as antioxidant, preventing the oxidation of biological compounds by superoxide ion.
Keywords: Oxygen; Bile salts; Polarography; Electro-generated superoxide ion; Antioxidant;

In order to investigate the origin of large intensity the α-relaxation in skeletal muscles observed in dielectric measurements with extracellular electrode methods, effects of the interfacial polarization in the T-tubules on dielectric spectra were evaluated with the boundary-element method using two-dimensional models in which the structure of the T-tubules were represented explicitly. Each model consisted of a circular inclusion surrounded by a thin shell corresponding to the sarcolemma. The T-tubules were represented by simplified two types of invagination of the shell: straight invagination along the radial directions, and branched one. Each of the models was subjected to two kinds of calculations relevant to experiments with the extracellular and the intracellular electrode methods. Electrical interactions between the cells were omitted in the calculations. Both calculations showed that the dielectric spectra of the models contained two relaxation terms. The low-frequency relaxation term assigned to the α-relaxation depended on the structure of the T-tubules. Values of the relaxation frequency of the α-relaxation obtained from the two types of calculations agreed with each other. At the low-frequency limit, the permittivity obtained from the extracellular-electrode-type calculations varied in proportion to the capacitance obtained from the intracellular-electrode-type ones. These results were consistent with conventional lumped and distributed circuit models for the T-tubules. This confirms that the interfacial polarization in the T-tubules in a single muscle cell is not sufficient to explain the experimental results in which the intensity of the α-relaxation in the extracellular-electrode-type experiments exceeded the intensity expected from the results of the intracellular-electrode-type experiments. The high-frequency relaxation term that was assigned to the β-relaxation was also affected by the T-tubule structure in the calculations relevant to the extracellular-electrode-type experiments.
Keywords: Boundary element method; Complex permittivity; Dielectric relaxation; Interfacial polarization; Muscle cell; T-tubule;

We have investigated the origin of the dielectric response of the plasma membrane of living yeast cells (Saccharomyces cerevisiae) by using radiofrequency dielectric spectroscopy. The cells were genetically engineered to overexpress in the membrane of yeast cells a G protein-coupled receptor – the Sterile2-α factor receptor protein (Ste2p) – fused to the green fluorescent protein (GFP). Presence of the Ste2-GFP proteins in the plasma membrane was confirmed by exciting the cells at 476 nm and observing with a confocal microscope the emission characteristic of the GFP from individual cells. The dielectric behavior of cells suspended in KCl solution was analyzed over the frequency range 40 Hz–110 MHz and compared to the behavior of control cells that lacked the ability to express Ste2p. A two-shell electrical cell model was used to fit the data starting from known structural parameters and adjustable electrical phase parameters. The best-fit value for the relative permittivity of the plasma membrane showed no significant difference between cells expressing Ste2p (1.63 ± 0.11) and the control cells (1.75 ± 0.16). This result confirmed earlier predictions that the dielectric properties of the plasma membrane in the radiofrequency range mostly reflect the properties of the hydrophobic layer of the membrane, which is populated by the hydrocarbon tails of the phospholipids and hydrophobic segments of integral membrane proteins. We discuss ways by which dielectric spectroscopy can be improved to be used for tag-free detection of proteins on the membrane.
Keywords: Permittivity; Conductivity; Yeast; Cell suspension; Tag-free detection; Fluorescence;

Electroporation, cell membrane permeabilization with short electrical field pulses, is used in tissue for in vivo gene therapy, drug therapy and minimally invasive tissue ablation. For the electroporation to be successful, the electrical field that develops during the application of the pulses needs to be precisely controlled. In this study we investigate the use of electrolytic and non-electrolytic gels to generate the precise electrical fields required for controlled electroporation, in heterogeneous and irregular tissues, in vivo. Finite element computer simulations are used to illustrate various applications, such as the treatment of irregularly shaped organs and interior cavities. The feasibility of the concept is demonstrated experimentally in vivo with a rat liver subjected to irreversible electroporation.
Keywords: Electroporation; Electropermeabilization; Electrolytic gel; Gene therapy; Irreversible electroporation;