Bioelectrochemistry (v.67, #1)

Polyelectrolyte multilayer (PEM) films containing polycationic osmium (Os) bipyridyl (bpy) complex-attached poly(4-vinylpyridine) (PVP) derivative [Os(bpy)2Cl]2+-PVP (Os-PVP) and polyanionic calf thymus DNA (CT-DNA) on the surface of gold (Au) electrodes were prepared using a layer-by-layer self-assembly method, and their redox properties were studied. Os complex shows different redox behavior with CT-DNA film in comparison with other PEM film which is composed of ordinary polymers. A cyclic voltammetric study suggested that the outermost polyanionic DNA layer does not hinder the redox reaction of Os complex within the Os-PVP/CT-DNA multilayer film, which may be helpful to understand the electron transfer mechanism with the DNA film. For all the Os-complex-containing PEM layers studied, a diffusion-free electron transfer from the Os complex moieties in these films to the electrode surface was observed. An electrocatalytic oxidation of ascorbic acid (AA) by this DNA-containing PEM film-covered electrode was also proposed.
Keywords: Polyelectrolyte multilayer (PEM); Calf thymus DNA; PVP-[Os(bpy)2Cl]2+; Redox properties; Electron transfer; Ascorbic acid;

Electrochemical oxidation of 2-thiouracil at pyrolytic graphite electrode by Rajendra N. Goyal; Udai P. Singh; Adil A. Abdullah (7-13).
2-Thiouracil has been studied in phosphate buffers of pH 1.95–11.08 using linear and cyclic sweep voltammetry, coulometry, controlled potential electrolysis and spectral studies. One well-defined oxidation peak Ia in the pH range 1.95–11.08 was noticed. The number of electrons involved in peak Ia was found to be four in a thin layer cell whereas under exhaustive electrolysis condition oxidation was found to involve six electrons. A reduction peak IIc (2e, 2H+) is noticed in the reverse sweep. Spectral studies during oxidation were carried out at different pH. Kinetic studies indicated that the decay of the UV-absorbing intermediate is a first order reaction. The products of the electrooxidation have been characterized and a tentative EC mechanism has been suggested for the oxidation of 2-thiouracil.
Keywords: Electrochemical oxidation; 2-Thiouracil; Electron;

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme (Γ E 0) on the basis of kinetic models reported in literature. The Γ E 0 on proposed electrode was high to 4.1×10−12 mol·cm−2, which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0×10−5–5.7×10−3 M with a sensitivity of 8.8 μA·mM−1·cm−2 and a detection limit of 8.2 μM. The apparent Michaelis–Menten constant (K m app) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.
Keywords: Glucose oxidase; Gold nanoparticles; Covalent attachment; Biosensors;

The metal complexes [Os(byp)2(pic)]+ and [Ru(byp)2(pic)]+ where byp is 2,2′-bipyridine and HPic is o-picolinic acid were synthesised and characterised using spectroscopic and electrochemical techniques. These complexes were then evaluated as mediators for a glucose oxidase (GOx)-based biosensor. Results demonstrate the electrocatalytic behaviour of both metal couples towards regeneration of the flavoprotein GOx (FADH2) group, when co-immobilised with glucose oxidase. Surface immobilisation was achieved by potential cycling in aqueous solutions of the metal complexes at a glucose oxidase (GOx)/Nafion modified electrode. This proved successful in terms of catalytic efficiency and stability of redox sites. Kinetic parameters associated with both enzymatic and mediator reactions were estimated and the stability/performance properties of the sensor were tested.
Keywords: Mediator; Biosensor; Electrocatalysis;

Electroporation of skin is accompanied by local heating, such that thermally induced structural changes of the stratum corneum (SC) accompany the field effect. Comparing on the time scale, the local changes in structure, temperature and conductance of the SC, during and after the pulse, it is seen that Joule heating also facilitates the subsequent molecular transport. It is found that the transport of medium-sized, ionic molecules occurs through localized transport regions (LTR). The size of a LTR increases with the pulse length, whereas the density of the LTRs increases with increasing voltage, for instance at U SC=80 V, the LTR cover approximately 0.02–1% of the surface area. The state of low resistance within the LTR is long-lived. During high voltage application, the center of the LTR is heated above the phase transition temperature of the SC lipids (70 °C) and the heat front propagates outwards.Inside the SC, the pulse causes aggregates of small-sized vesicles. At a higher temperature, the aggregate formation and their disappearance are delayed. Multiple pulses with the applied voltage of U appl=80 V induce the formation of long-lasting vesicle aggregates with a diameter of =1–30 μm, covering 0.05–0.5% of the total sample area.The electric energy dissipated within the LTR during high voltage application is apparently sufficient to raise the temperature well above the phase transition temperature of the lipids of the SC, accounting for the conformational changes from the multi-lamella to the vesicular structures.
Keywords: Stratum corneum; Electric field; Electroporation; Skin; Joule heating; SC;

A novel amperometric sensor system is presented which directly reflects the metabolic activity of prokaryotic and eukaryotic cells during cultivation. The principle of an externally mounted sensor is current measurement using a three-electrode system. Only living cells are detected since the current signal is based on a redox mediator. Added to a culture sample in its oxidized form, the mediator is reduced by cellular metabolism and subsequently re-oxidized at the anode.The spontaneous immobilisation of the cells in the reaction vessel of the sensor by swelling dextrane polymers (Sephadex™) prior to measurement is the key to a fast, consistent signal. Even metabolically less active mammalian cells produce a reliable signal within a few minutes; this may open up future applications of the electrochemical sensor in closed loop process control not only for bacterial and fungal bioprocesses, but also in cell culture technology.
Keywords: Activity sensor; Bioprocess control; Fermentation control; Mediator; Amperometry;

We present analytical equations for the transmembrane voltage (ΔΨ) induced by an alternating field on spherical cells arranged in orderly suspensions. For physiologically normal cells, the cell membrane was assumed to be nonconductive. With increasing alternating field frequency, capacitive property of the cytoplasm and the external medium become increasingly important and thus must be accounted for. Considering the symmetry of the arrayed cells and the tiny volume of the unit cell compared to that of the suspensions, the influence exerted on a unit cell by other cells was posited to be approximately symmetrical with respect to the symmetrical axis. This implies that the shape of the equipotential plane of spherical cells in the suspensions is similar to that of the single cell exposed to the external field, though the value of the potential of both cells should be different. Therefore, the internal field of the cytoplasm and the equivalent body of a unit cell in the suspensions should be approximately constant. This allows for calculating the effective average field in the equivalent body with Maxwell–Wagner and Bruggeman–Hanai equations for low and high cell concentrations, respectively. We investigated the conditions, under which the local electric field of a unit cells in suspension is approximately equal to that of a single cell. Under these conditions the analytical solution for ΔΨ induced by alternating fields on cells in suspensions can be derived from that of the single cell.
Keywords: Alternating field; Cell suspensions; Transmembrane voltage;

Electrical cell uncoupling via gap junction closure is assumed to cause characteristic changes of the passive dielectric spectrum of ischemic heart tissue. In order to find an independent evidence for this assumption, we analysed heart tissue during ischemia, measured the open state of gap junctions by means of dye transfer and correlated this parameter with the time course of the dielectric permittivity.The hearts were preischemically arrested by perfusion with Ringer solution containing 20 mmol/L of potassium (group KCL, n=10). This solution was also used with the addition of two gap junction blockers, either 3 mmol/L heptanol (group HEP, n=4) or 20 μmol/L palmitoleic acid (group PA, n=7). During subsequent ischemia at 21.0±0.5 °C, we monitored the passive dielectric permittivity spectrum and the spread of dye.After a sigmoidal increase the dielectric permittivity reached an upper plateau at 61±22 min of ischemia in KCL, at 45±7 min in PA, and already during perfusion at 2±1 min in group HEP. At the beginning of ischemia, dye migrated to neighbouring cells in groups KCL and PA but not in HEP. In KCL and PA, the intercellular diffusion of dye stopped after 64±26 and 40±11 min of ischemia, respectively.Our results suggest that the sigmoidal increase in dielectric permittivity and the reduction of dye diffusion depend on a common mechanism, namely gap junction closure.
Keywords: Gap junctions; Dielectric spectroscopy; Fluorescence microscopy; Dye transfer;

In 0.1 M phosphate buffer (pH 7.2), the interaction of chromium(VI) with cysteine in the presence and absence of UV irradiation was studied by cyclic voltammetry and electronic spectroscopy techniques. The reduction of Cr(VI) by cysteine takes place through the formation of Cr(VI)-thioester intermediate. On the cyclic voltammograms of cysteine and Cr(VI) mixture, the peaks at −0.315 and −0.800 V were observed, and these peaks are corresponding to the reduction of Cr(VI)-thioester and thiyl radical, respectively. In the cysteine solution exposed to UV irradiation, the formation of free cystine was observed at −0.792 V. In the cysteine and Cr(VI) mixture exposed to UV irradiation, the peak current of thiyl radical increases while the peak current of Cr(VI)-thioester reaches a maximum at 15 min and then decreases by increasing UV irradiation time. The formation of the thioester in the reaction between Cr(VI) and cysteine in aqueous media has been studied by monitoring the decrease of Cr(VI) at 370 nm. It was observed that the reaction is catalyzed by the UV irradiation of the Cr(VI) and cysteine mixture.
Keywords: Chromium(VI); Cysteine; Chromium(VI)-thioester; UV irradiation; Catalysis;

The effect of lipid phase order of isolated thylakoid membranes on fluorescent characteristics of both photosystems during illumination with high light intensity at 22 °C and 4 °C was investigated. For artificial modification of membrane fluidity two membrane perturbing agents were applied—cholesterol and benzyl alcohol. 77 K fluorescence emission and excitation spectra of control, cholesterol- and benzyl alcohol-treated thylakoid membranes were analysed in order to determine the high light-induced changes of emission bands attributed to different chlorophyll–protein complexes—F735, emitted by photosystem I—light-harvesting complex I; and F685 and F695, emitted by photosystem II—light-harvesting complex II. Analysis of emission bands showed that high light treatment leads to a decrease of the area of band at 695 nm and a concomitant increase of intensity of the band at 735 nm. The involvement of different pigment pools (chlorophyll a and chlorophyll b) in the energy supply of both photosystems before and after photoinhibitory treatment was estimated on the basis of excitation fluorescence spectra. The dependence of the ratios F735/F685 and the band areas at 685 and 695 nm on the illumination time was studied at both temperatures. Data presented indicate that cholesterol incorporation stabilized the intersystem structure in respect to light-induced changes of fluorescence emission of PSI and PSII. It was shown that the effect of fluid properties of thylakoid membranes on the 77 K fluorescence characteristics of main pigment protein complexes of pea thyalkoid membranes depends on the temperature during high light treatment.
Keywords: Benzyl alcohol; Chlorophyll fluorescence; Cholesterol; Excitation energy; Photoinactivation; Thylakoid membrane fluidity;

Excluded volume driven counterion condensation inside nanotubes in a concave electrical double layer model by Klemen Bohinc; Jan Gimsa; Veronika Kralj-Iglič; Tomaž Slivnik; Aleš Iglič (91-99).
The physical properties of organic nanotubes attract increasing attention due to their potential benefit in technology, biology and medicine. We study the effect of ion size on the electrical properties of cylindrical nanotubes filled with electrolyte solution within a modified Poisson–Boltzmann (PB) approach. For comparison purposes, small hollow nanospheres filled with electrolyte solution are considered. The finite size of the particles in the inner electrolyte solution is described by the excluded volume effect within a lattice statistics approach. We found that an increased ion size reduces the number of counterions near the charged inner surface of the nanotube, leading to an enlarged electrostatic surface potential. The concentration of counterions close to the inner surface saturates for higher surface charge densities and larger ions. In the case of saturation, the closest counterion packing is achieved, all lattice sites near the surface are occupied and an actual counterion condensation is observed. By contrast, the counterion concentration at the axis of the nanotube steadily increases with increasing surface charge density. This growth is more pronounced for smaller nanotube radii and larger ions. At larger nanotube radii for small ion size counterion condensation may also be observed according to the Tsao criterion, i.e. the counterion concentration at the centre is independent of the number of counterions in the system. With decreasing radius the Tsao condensation effect is shifted towards physiologically unrealistic surface charge densities.
Keywords: Nanotubes; Reverse osmosis; Excluded volume effect; Lattice statistics;

Changes of the solution pH due to exposure by high-voltage electric pulses by Gintautas Saulis; Remigijus Lapė; Rita Pranevičiūtė; Donatas Mickevičius (101-108).
The change of the pH of a NaCl solution (139–149 mM NaCl) buffered with 5–15 mM sodium phosphates (pH 7.4) during electromanipulation was studied. It has been determined that an increase in the pH value of electroporation solution of a whole chamber volume, caused by the application of electric field pulses, commonly used in cell electromanipulation procedures, can exceed 1–2 pH units.Several materials for the cathode were tested. In all cases a stainless steel anode was utilized. The aluminum cathode gave a two-fold greater ΔpH in comparison with platinum, copper or stainless steel cathodes. In addition, a substantial release of aluminum (up to 1 mg/l) from the cathode was observed. It has also been found that the shift in pH depended on the medium conductivity: ΔpH of the solution, in which sucrose was substituted for NaCl, was about 5 times less.On the basis of the results obtained here, to avoid the plausible undesirable consequences of the cathodic electrolysis processes, in particular under the conditions of strong electric treatment, it could be recommended that chambers with aluminum electrodes not be utilized and one should use strongly buffered solutions of low conductivity and alternating current (sine or square wave) bipolar electric pulses.
Keywords: Cell electromanipulation; Electroporation; pH shift; Electrolysis reactions; Electrodes;

The acid-treated multi-walled carbon nanotubes (MWNTs), which were modified on the surface of gold electrode, offers substantial improvements in voltammetric sensitivity and selectivity towards the determination of dopamine (DA). It can inhibit the voltammetric response of ascorbic acid (AA) while the redox reaction of dopamine is promoted. When a differential pulse voltammetric (DPV) technique was used, the peak separation between DAs and AAs was 244 mV. Based on this, a selective method could be constructed to detect DA in the presence of 1000 times higher concentration of AA. The effect of various experimental parameters on the voltammetric response of dopamine was investigated. Under the chosen conditions, the peak currents are correspondent linearly to the concentrations of DA in the range of 5×10−7∼4×10−4 mol L−1 with a limit of detection of 2×10−7 mol L−1. The proposed method can be applied to detect DA in real samples.
Keywords: MWNTs; Modified electrode; Dopamine; Selective response;

Direct electron transfer reactions of laccases from different origins on carbon electrodes by Sergey Shleev; Anna Jarosz-Wilkolazka; Anna Khalunina; Olga Morozova; Alexander Yaropolov; Tautgirdas Ruzgas; Lo Gorton (115-124).
Electrochemical studies of laccases from basidiomycetes, i.e., Trametes hirsuta, Trametes ochracea, Coriolopsis fulvocinerea, Cerrena maxima, and Cerrena unicolor, have been performed. Direct (mediatorless) electrochemistry of laccases on graphite electrodes has been investigated with cyclic voltammetry, square wave voltammetry as well as potentiometry. For all mentioned high potential laccases direct electron transfer (DET) has been registered at spectrographic graphite and highly ordered pyrolytic graphite electrodes. The characteristics of DET reactions of the enzymes were analysed under aerobic and anaerobic conditions. It is shown that the T1 site of the laccase is the primary electron acceptor, both in solution (homogenous case) and at surface of the graphite electrode (heterogeneous case). A mechanism of ET for the process of the electroreduction of oxygen at the laccase-modified graphite electrodes is proposed and the similarity of this heterogeneous process to the laccase catalysed oxygen reduction homogeneous reaction is concluded.
Keywords: Laccase; Redox potential; T1 site; Carbon electrode;