Bioelectrochemistry (v.73, #1)

Nanosecond electric pulse-induced calcium entry into chromaffin cells by P. Thomas Vernier; Yinghua Sun; Meng-Tse Chen; Martin A. Gundersen; Gale L. Craviso (1-4).
Electrically excitable bovine adrenal chromaffin cells were exposed to nanosecond duration electric pulses at field intensities ranging from 2 MV/m to 8 MV/m and intracellular calcium levels ([Ca2+]i) monitored in real time by fluorescence imaging of cells loaded with Calcium Green. A single 4 ns, 8 MV/m pulse produced a rapid, short-lived increase in [Ca2+]i, with the magnitude of the calcium response depending on the intensity of the electric field. Multiple pulses failed to produce a greater calcium response than a single pulse, and a short refractory period was required between pulses before another maximal increase in [Ca2+]i could be triggered. The pulse-induced rise in [Ca2+]i was not affected by depleting intracellular calcium stores with caffeine or thapsigargin but was completely prevented by the presence of EGTA, Co2+, or the L-type calcium channel blocker nitrendipine in the extracellular medium. Thus, a single nanosecond pulse is sufficient to elicit a rise in [Ca2+]i that involves entry of calcium via L-type calcium channels.
Keywords: Chromaffin cell; Nanosecond electric pulses; Calcium influx; Intracellular calcium; Calcium Green; L-type calcium channel;

Determination of selected drugs in human urine by differential pulse voltammetry technique by Irena Baranowska; Piotr Markowski; Anna Gerle; Jacek Baranowski (5-10).
A new, simple and selective differential pulse voltammetry (DPV) method for the simultaneous determination of selected drugs in model solutions and spiked human urine samples with prior extraction was developed and validated. The objects of analysis were paracetamol, furosemide, dipyrone, cefazolin and dexamethasone belonging to four different therapeutic groups (antibiotics, analgesic, demulcent and diuretic). Analytical methods for the preparation of urine samples for voltammetric analysis (liquid–liquid extraction — LLE and solid-phase extraction — SPE) were worked out and optimized. Hanging mercury drop electrode (HMDE) and graphite electrode were used as working electrodes. Reference electrode was Ag|AgCl|KCl(sat.), whereas auxiliary electrode — platinum electrode. The optimal conditions for quantitative determination were obtained in a Britton–Robinson (BR) buffer at pH 2.4. Quantification was performed by means of calibration curve and standard addition methods. The calibration curves of analysed drugs are linear within the range of concentration: 6.61–66.10, 6.05–54.42, 6.00–65.00, 4.20–33.58 and 0.51–3.06 μM for paracetamol, furosemide, dipyrone, cefazolin and dexamethasone, respectively. The levels of analysed compounds in human urine can be successfully determined using this developed method with no matrix effect.
Keywords: Drugs; Differential pulse voltammetry; Liquid–liquid extraction; Solid-phase extraction; Human urine;

Simultaneous determination of dopamine, ascorbic acid and uric acid at poly (Evans Blue) modified glassy carbon electrode by Liqing Lin; Jinghua Chen; Hong Yao; Yuanzhong Chen; Yanjie Zheng; Xinhua Lin (11-17).
A sensitive and selective electrochemical method for the determination of dopamine using an Evans Blue polymer film modified on glassy carbon electrode was developed. The Evans blue polymer film modified electrode shows excellent electrocatalytic activity toward the oxidation of dopamine in phosphate buffer solution (pH 4.5). The linear range of 1.0 × 10− 6–3.0 × 10− 5 M and detection limit of 2.5 × 10− 7 M were observed in pH 4.5 phosphate buffer solutions. The interference studies showed that the modified electrode exhibits excellent selectivity in the presence of large excess of ascorbic acid and uric acid. The separation of the oxidation peak potentials for dopamine–ascorbic acid and dopamine–uric acid were about 182 mV and 180 mV, respectively. The differences are large enough to determine AA, DA and UA individually and simultaneously. This work provides a simple and easy approach to selectively detect dopamine in the presence of ascorbic acid and uric acid in physiological samples.
Keywords: Poly (Evans blue) modified electrode; Electrocatalysis; Dopamine; Uric acid; Ascorbic acid;

The interaction of anticancer herbal drug berberine with double-strand DNA (dsDNA) and single-strand DNA (ssDNA) in solution, dsDNA immobilized on the glassy carbon electrode prepared by Langmuir–Blodgett technique, were investigated by electrochemical techniques (cyclic voltammetry, differential pulse voltammetry) and UV spectroscopy. The presence of DNA results in a decrease of the currents and a negative shift of the electrode potentials from the DPV curves of berberine, indicating the dominance of electrostatic interactions. The spectroscopy data confirmed that the predominant interaction between berberine and DNA is electrostatic. The binding of berberine with DNA, when analyzed in terms of the cooperative Hill model, yields the binding constant K a  = 2.2(± 0.2) × 104 M− 1, corresponding to the dissociation equilibrium constant K d  = 4.6(± 0.3) × 10− 5 M, which in the range of the applied concentrations of DNA (bp) and berberine, and a Hill coefficient m  = 1.82(± 0.08) in Britton–Robinson buffer solution (0.05 M, pH 5.72) at T  = 298 K (25 °C). Apparently, at least two molecules of berberine have to bind as a couple to cause, e.g., the “elementary event” of current change. The results are suggestive for further fruitful applications of this anticancer herbal drug and DNA-modified electrodes.
Keywords: Berberine; DNA; Anticancer herbal drug; Hill parameters; Langmuir–Blodgett film;

Effects of glycerol and sorbitol on the thermal dependence of the lysis of human erythrocytes by ethanol by N. Penha-Silva; L.R. Arvelos; C.C. Cunha; T.A. Aversi-Ferreira; L.F. Gouvêa-e-Silva; M.S. Garrote-Filho; C.J. Finotti; M. Bernardino-Neto; F.G. de Freitas Reis (23-29).
In this work, the effects of 1 mol/L glycerol or sorbitol on the thermal dependence (27−47 °C) of the lysis of human erythrocytes by ethanol in saline solution (0.154 mol/L NaCl) have been evaluated. Lysis was monitored by measurement of the absorbance at 540 nm. Ethanol produced either lysis or protection against lysis depending on the conditions. These antagonistic effects are attributed to the existence of expanded (R) and compacted (T) erythrocytes, present under conditions of low and high osmolarity, respectively. The transitions of lysis of the R state and formation and lysis of the T state were all found to be sigmoidally defined. The ethanol concentration at the midpoint of the lysis transition of the R state (D 50R) was found to decrease with increasing temperature and osmolarity. In the presence of glycerol or sorbitol, an increase in temperature led to smaller decreases in D 50R and osmotic protection against lysis. The ethanol concentration at the midpoint of formation (S 50T) and lysis (D 50T) of the T state also decreased with increasing temperature and osmolarity. Lysis of R state erythrocytes is determined by the chaotropic action of ethanol, but the formation and lysis of T state erythrocytes are determined by osmotic pressure effects.
Keywords: Erythrocytes; Ethanol; Membrane stability; Osmolytes; Temperature;

A simple and rapid voltammetric method has been developed for the quantitative determination of coenzyme Q10 (CoQ10) in pharmaceutical preparations. Studies with differential pulse voltammetry (DPV) were carried out using a glassy carbon electrode (GCE) in a mixed solvent containing 80 vol.% acetic acid and 20 vol.% acetonitrile. A well-defined reduction peak of CoQ10 was obtained at − 20 mV vs. Ag/AgCl. The voltammetric technique applied provides a precise determination of CoQ10 using the multiple standard addition method. The statistical parameters and the recovery study data clearly indicate good reproducibility and accuracy of the method. The accuracy of the results assessed by recovery trials was observed to be within the range of 101.1% to 102.5%. The detection and quantification limits were found to be 0.014 mM (12 mg L− 1) and 0.046 mM (40 mg L− 1), respectively. An analysis of real samples containing CoQ10 showed no interferences with common additives and excipients, such as unsaturated fatty acids and soya lecitine. The method proposed does not require any pretreatment of the pharmaceutical dosage forms. A spectrophotometric determination of CoQ10 in real samples diluted in mixtures containing ethanol and n-hexane was also performed for comparison.
Keywords: Coenzyme Q10; Acetic acid; Acetonitrile; Differential pulse voltammetry; Determination;

Poly(amidosulfonic acid) modified glassy carbon electrode for determination of isoniazid in pharmaceuticals by Gongjun Yang; Cunxiao Wang; Rui Zhang; Chenying Wang; Qishu Qu; Xiaoya Hu (37-42).
Amidosulfonic acid was electropolymerized by cyclic voltammetry onto the surface of glassy carbon electrode (GCE) to fabricate the chemically modified electrode, which showed high stability, good selectivity and reproducibility for determination of isoniazid. The modified electrode showed an excellent electrocatalytical effect on the oxidation of isoniazid. Under the optimum conditions, there was a good linear relationship between anodic peak current and isoniazid concentration in the range of 5.0 × 10−8– 1.0 × 10−5 M, and a detection limit of 1.0 × 10−8 M (S/N = 3) was obtained after 120 s at the accumulation potential of − 0.2 V (vs. SCE). This developed method had been applied to the direct determination of isoniazid in injection and tablet samples with satisfactory results.
Keywords: Isoniazid; Poly(amidosulfonic acid); Electrocatalysis; Differential pulse voltammetry;

Photosystem II (PSII) complex activity is known to decrease under strong white light illumination, and this photoinhibition phenomenon is connected to the photobleaching of the PSII photosynthetic pigments. In this work the pigment photobleaching has been studied on PSII core complexes, by observing the effects of different factors such as the aggregation state (PSII monomers and dimers were used), temperature (20 °C and 10 °C temperatures were tested) and the presence of the exogenous phospholipids (cardiolipin and phosphatidylglycerol). In particular, PSII resistance against white light stress was studied by means of UV/VIS Absorption and Fluorescence Emission measurements. It was found that PSII dimers resulted more resistant against photobleaching and that lower temperature reduces the pigment photodestruction. Moreover, the presence of phosphatidylglycerol or cardiolipin enhanced the PSII resistance to the photobleaching phenomenon, mainly at lower temperatures.
Keywords: Photobleaching; Photosystem II; Phosphatidylglycerol; Cardiolipin; Aggregation state;

Modulation of RBC volume distributions by oxidants (phenazine methosulfate and tert-butyl hydroperoxide): Role of Gardos channel activation by Irina L. Lisovskaya; Irina M. Shcherbachenko; Rimma I. Volkova; Vladimir P. Tikhonov (49-54).
A study was made comparing the effects of two oxidants – phenazine methosulfate (50–1500 µM) + 10 mM ascorbate and t-butyl hydroperoxide (1–3 mM) – on the volume-related parameters of normal human red blood cells. Incubation with either oxidative system for 20–30 min resulted in red blood cell density and osmotic resistance distribution shifts. Treatment with the phenazine methosulfate + ascorbate system in the presence of Ca2+ led to cell shrinking, with the maximum effect being more than 20%. In contrast, under the same conditions, t-BHP caused cell swelling by up to 15%. Modification of the suspending medium (Ca2+ removing, clotrimazole addition, or enrichment with K+) modulated the redistribution effects, suggesting that they were mediated to some extent by Gardos channel activation. These findings are important for understanding how oxidants modulate RBC cation channels.
Keywords: Red blood cell; Erythrocyte; Gardos channel; Phenazine methosulfate; Tert-butyl-hydroperoxide;

The thermodynamics and kinetics of the reaction DeoxyHb-Fe2+  ↔ MetHb-Fe3+ for human hemoglobin A (HbA), alpha- and beta-fumarate crosslinked hemoglobins were investigated by spectroelectrochemistry. Information from this study is used to determine what structural features and experimental conditions stabilize ferrous vs. ferric form of hemoglobin, and what implications this stabilization may have on the autoxidation reaction. Alpha- and beta-fumarate crosslinked hemoglobins, αXL-HbA and βXL-HbA, were obtained by crosslinking deoxyhemoglobin and oxyhemoglobin, respectively, with bis(3,5-dibromosalicyl) fumarate (DBSF). Formal redox potentials, E 0, and reduction/oxidation rates were measured in the presence of mediator, hexammineruthenium(III) chloride. It was found that E 0 shifted positive for the alpha-, and negative for the beta-fumarate crosslinked hemoglobin compared to HbA for all experimental conditions investigated. This shift was consistent with stabilization of the tense (positive shift) or relaxed conformation (negative shift) conferred by crosslinking. Formal redox potentials shifted positive with addition of nitrate and chloride ions for αXL-HbA, indicating additional stabilization of the T quaternary. The slopes of the Nernst plots showed evidence of cooperativity as expressed by n max. The data points (E 0, n max) were fitted by the MWC model which states that the electron transfer and the addition/removal of water are concerted. The set of K R and c values, where the parameter c is the ratio K R/K T and K R and K T are the ligand (water molecule and an electron–hole) dissociation constants for the R and T states, for the beta-crosslinked hemoglobin compared to that of HbA and alpha-crosslinked hemoglobin indicated that crosslinking of oxyhemoglobin affected differently the inner-coordination sphere at the heme site. By modulating the electrolyte concentration the reduction rates were measured as a function of ΔE 0, the difference in E 0 between hemoglobin molecules and mediator. Linearization of the Marcus cross-relationship (based on the concerted water and electron transfer) was good for HbA, and poor for αXL-HbA and βXL-HbA, consistent with results obtained by the MWC analysis. This may imply that the reduction of HbA is controlled by the driving force, ΔE 0, whereas the reduction of αXL-HbA and βXL-HbA occurs by a non-concerted mechanism controlled by structural features brought about by crosslinking. The autoxidation reaction, conversion of oxygen-bound ferrous hemoglobin to ferric hemoglobin, was found independent of E 0. Alpha-fumarate crosslinked hemoglobin showed the highest autoxidation rate despite its positive shift in formal redox potential as compared to HbA, followed by beta-fumarate crosslinked hemoglobin, and by native hemoglobin. These data suggest that the chemical mechanism of oxygen dissociation and accessibility of water and oxygen radicals to heme site control autoxidation.
Keywords: Hemoglobin; Fumarate crosslinked hemoglobin; Spectroelectrochemistry; Formal redox potentials; Oxidation kinetics;

In this work, the complex cadmium(II)–morin was synthesized and its interaction with double-stranded salmon sperm DNA was studied by electrochemical methods on glassy carbon electrode (GCE). It was shown that Cd(II)–Morin with high electrochemical activity can intercalate into the double-helix DNA, and the binding stoichiometry and equilibrium dissociation constant according to the Hill model for cooperative binding were calculated to be 1.761 and 2.5 × 10− 5 M, respectively. Using Cd(II)–Morin as a novel hybridization indicator, the hybridization between the probe and its complementary and mismatched sequence was investigated by differential pulse voltammetry (DPV), which was to access the selectivity of the developed electrochemical DNA biosensor. The complementary target ssDNA could be quantified over the range from 2.69 × 10− 8 M to 9.16 × 10− 7 M with a linear correlation of 0.9971 and a detection limit of 9.30 × 10− 9 M. These results demonstrated that the Cd(II)–Morin indicator provides great promise for the rapid and selective measurement of the target DNA.
Keywords: Electrochemical DNA biosensor; Morin; Cadmium; DNA hybridization detection; Differential pulse voltammetry;

Insights into genes involved in electricity generation in Geobacter sulfurreducens via whole genome microarray analysis of the OmcF-deficient mutant by Byoung-Chan Kim; Bradley L. Postier; Raymond J. DiDonato; Swades K. Chaudhuri; Kelly P. Nevin; Derek R. Lovley (70-75).
Geobacter sulfurreducens effectively produces electricity in microbial fuel cells by oxidizing acetate with an electrode serving as the sole electron acceptor. Deletion of the gene encoding OmcF, a monoheme outer membrane c-type cytochrome, substantially decreased current production. Previous studies demonstrated that inhibition of Fe(III) reduction in the OmcF-deficient mutant could be attributed to poor transcription of the gene for OmcB, an outer membrane c-type cytochrome that is required for Fe(III) reduction. However, a mutant in which omcB was deleted produced electricity as well as wild type. Microarray analysis of the OmcF-deficient mutant versus the wild type revealed that many of the genes with the greatest decreases in transcript levels were genes whose expression was previously reported to be upregulated in cells grown with an electrode as the sole electron acceptor. These included genes with putative functions related to metal efflux and/or type I secretion and two hypothetical proteins. The outer membrane cytochromes, OmcS and OmcE, which previous studies have demonstrated are required for optimal current generation, were not detected on the outer surface of the OmcF-deficient mutant even though the omcS and omcE genes were still transcribed, suggesting that the putative secretion system could be involved in the export of outer membrane proteins necessary for electron transfer to the fuel cell anode. These results suggest that the requirement for OmcF for optimal current production is not because OmcF is directly involved in extracellular electron transfer but because OmcF is required for the appropriate transcription of other genes either directly or indirectly involved in electricity production.
Keywords: Geobacter sulfurreducens; Cytochrome; Microbial fuel cells; Microarray;

Electrochemical detection of thrombin by sandwich approach using antibody and aptamer by Yan Kang; Ke-Jun Feng; Ji-Wei Chen; Jian-Hui Jiang; Guo-Li Shen; Ru-Qin Yu (76-81).
The goal of this work was to introduce a modified electrochemical sandwich model for target protein detection, exploiting antibody as the capturing probe, aptamer as the detection probe and methylene blue as the electrochemical active marker intercalating in the probing aptamer without previous labeling. With appropriate design of the sequence of the aptamer, the aptamer was successfully utilized instead of antibody for obtaining the electrochemical detection. A special immobilization interface consisting of nanogold-chitosan composite film was used to improve the conductivity and performance characteristics of the electrode. The capturing antibody was linked to the glassy carbon electrodes modified with composite film via a linker of glutaraldehyde. Differential pulse voltammetry was performed to produce the response signal. Thrombin was taken as the model target analyte to demonstrate the feasibility of proposed methodology. The sensor shows the linear response for thrombin in the range 1–60 nM with a detection limit of 0.5 nM. The proposed approach provides an alternative approach for sandwich protein assay using aptamers.
Keywords: Protein; Electrochemical analysis; Nanoparticle; Aptamer;