Bioelectrochemistry (v.68, #1)

Contents (v-vi).

Electrocatalytic activity of DNA on electrodes as an indication of hybridisation by Zheng-liang Zhi; Viktor Drazan; Otto S. Wolfbeis; Vladimir M. Mirsky (1-6).
Electron transfer between metal electrodes and ferro/ferricyanide is completely suppressed at low ionic concentration. We describe here a new phenomenon related to this reaction: an immobilisation of thiolated single-stranded DNA on gold electrodes retains this activity at low ionic strength up to the level corresponding to the high ionic strength. In contrast, a hybridisation of the complementary DNA with the thiolated single-stranded DNA followed by a binding onto the electrodes, attenuated the electrocatalytic effect. These effects can be used for discrimination between single-stranded DNA and double-stranded DNA and for semi-quantitative measurement of complementary DNA in a sample.
Keywords: DNA; DNA-sensor; Biosensor; Electrocatalysis; Hybridisation; Olygonucleotide; Gene analysis;

Degradation kinetics of ascorbic acid was determined in pH 5.7 buffer solution using an isothermal batch ohmic heater with stainless steel electrodes. Variables included in this study were temperature (40, 60 and 80 °C); power (0, 100,150 and 300 W); and electrical conductivity (varied using 0.25%, 0.5% and 1.0% NaCl). Ascorbic acid concentration was detected by using a HPLC technique. The results indicate that ascorbic acid degradation can be described successfully by a first order model during both conventional and ohmic heating. The Arrhenius relation showed negative values for temperature coefficient (E T) during most ohmic treatments, due to a combination of factors that may alter the reaction mechanism. In particular, it appears that at a given power level, higher electric field strengths are conducive to increased incidence of faradaic reactions. Increasing NaCl concentration appears to significantly influence reaction rates via its influence on dissolved oxygen, and through its participation in electrolytic reactions. Contrary to expectations, increasing temperature tended to significantly reduce reaction rate, likely due to decreased dissolved oxygen concentration at high temperature. The results indicate the importance of using inert electrodes in ohmic heating processes.
Keywords: Ohmic heating; Ascorbic acid; Degradation kinetics; Stainless steel electrodes;

Direct current voltammetry and differential pulse voltammetry have been used to investigate the electrochemical behaviour of two phytochelatins: heptapeptide (γ-Glu–Cys)3–Gly and pentapeptide (γ-Glu–Cys)2–Gly, tripeptide glutathione γ-Glu–Cys–Gly and its fragments: dipeptides Cys–Gly and γ-Glu–Cys at the hanging mercury drop electrode in the presence of cobalt(II) ions. Most interesting results were obtained with direct current voltammetry in the potential region of − 0.80 V up to − 1.80 V. Differential pulse voltammetry of the same solutions of Co(II) with peptides gives more complicated voltammograms with overlapping peaks, probably in connection with the influence of adsorption at slow scan rates necessarily used in this method. However, in using Brdička catalytic currents for analytical purposes, differential pulse voltammograms seem to be more helpful. Presented investigations have shown that particularly the prewave of cobalt(II) allows distinguishing among phytochelatins, glutathione, and its fragments.
Keywords: Phytochelatins; Cysteine peptides; Voltammetry; Cobalt(II) prewave; Brdička reaction; Hydrogen catalysis;

Oxygen is electroreduced to water on the “wired” bilirubin oxidase (w-BOD) catalyst at a considerably lesser potential than on pure platinum. The w-BOD catalyst could be of value in an implantable glucose–O2 biofuel cell, operating living tissue, if it were stable in serum. We found, however, that w-BOD loses its activity in a few hours in the combined presence of the urate and O2, both of which are normal serum constituents (Bioelectrochemistry, 2004, 65, 83–88). Here we report a second major instability: When the disconnected w-BOD cathode is allowed, in the absence of urate, to poise itself at the potential of the O2/H2O half cell at pH 7.2, it loses its activity rapidly. Unlike the urate/O2 caused loss, this loss can be avoided either by applying a potential that is reducing relative to the O2/H2O half-cell potential, or by excluding O2 and adding a mildly reducing reagent, such as urate. The w-BOD cathode can be stored, therefore, in deoxygenated serum, which contains urate.
Keywords: w-BOD cathode; Serum; Urate;

ATP synthesis in irreversibly electropermeabilized yeast Kluyveromyces lactis was studied by using different respiratory substrates. The permeabilization itself provoked a dramatic decrease of the total ATP level and the cells lost their ability to synthesize ATP via glycolysis. The addition of exogenous NADH supported ATP synthesis in irreversibly permeabilized cells for up to 4–6 h after substrate addition when the total ATP level became twice that of intact cells incubated for the same period with lactose.
Keywords: Irreversible electropermeabilization; Yeast; ATP synthesis; Kluyveromyces lactis;

Electrochemical formation of H2O2 and the subsequent ferryl porphyrin were examined by measuring luminol chemiluminescence and absorption spectrum using flow-injection method. Emission was observed under the cathodic potential (0.05 V at pH 2.0 and − 0.3V at pH 11.0) by the electrochemical reduction of buffer electrolytes solution but no emission was observed at anodic potentials. FeIIITMPyP solution was added at the down stream of the working electrode and was essential for the emission. Removal of dissolved O2 resulted in the decrease of emission intensity by more than 70%. In order to examine the lifetime of reduced active species, delay tubes were used in between working electrode and FeIIITMPyP inlet. Experimental results suggested the active species were stable for quite long. The emission was quenched considerably (> 90%) when hydroperoxy catalase was added at the down stream of the working electrode whereas SOD had little effect. Significant inhibition of the emission by the addition of alkene at the down stream of the FeIIITMPyP inlet was considered as evidence of oxo-ferryl formation. The spectra at reduction potential under aerated condition were shifted to the longer wavelength (> 430 nm) compared to the original spectrum of FeIIITMPyP (422 nm). All the spectra were perfectly reproduced by a combination of FeIIITMPyP and O=FeIVTMPyP (438 nm) spectra. These observations lead to the conclusion that H2O2 was produced first by electrochemical reduction of O2, which then converted FeIIITMPyP into O=FeIVTMPyP to activate luminol. The current efficiencies for the formation of H2O2 were estimated as about 30–65% in all over the pH.
Keywords: Iron porphyrin; H2O2 formation; Luminol chemiluminescence; Electrochemical reduction;

Effect of Tris on catalytic activity of MP-11 by Zhaopeng Qi; Xi Li; Dongmei Sun; Cun Li; Tianhong Lu; Xiaolan Ding; Xiaohua Huang (40-47).
The effect of tris(hydroxymethyl)aminomethane (Tris) on the catalytic activity and microstructure of heme undecapeptide, microperoxidase-11 (MP-11) in the aqueous solution was investigated using cyclic voltammetry, circular dichroism (CD) spectroscopy, UV–vis absorption spectroscopy and X-ray photoelectron spectroscopy (XPS). It was found for the first time that Tris would inhibit the catalytic activity and electrochemical reaction of MP-11 at the glassy carbon (GC) electrode. This is mainly due to the fact that Tris would induce more α-helix and β-turn conformations from the random coil conformation of MP-11, cause the asymmetric split-up in the Soret band region of MP-11, increase the non-planarity of the heme of MP-11, and change the electron densities of N, O and S atoms of MP-11. Meanwhile, It was found that the electrochemical reaction of MP-11 with Tris at GC electrode is diffusion-controlled, and the diffusion coefficient of MP-11 and the rate constant for the heterogeneous electron transfer of MP-11 in the presence of Tris are decreased by 19% and 16%, respectively. Further experiments showed that the electrocatalytic current of MP-11 on the reduction of H2O2 is decreased by about 25% after the addition of Tris to the MP-11 solution.
Keywords: Tris; MP-11; Catalytic activity; Secondary structure; Asymmetric split-up;

Differential pulse voltammetric studies of ethidium bromide binding to DNA by S.H. Minasyan; L.A. Tavadyan; A.P. Antonyan; H.G. Davtyan; M.A. Parsadanyan; P.O. Vardevanyan (48-55).
The interaction of ethidium bromide (EtBr) with calf thymus DNA is investigated electrochemically with the use of differential pulse voltammetry (DPV) at two different ionic strengths of a solution (0.154 M and 0.02 M [Na+], pH 7.0). It is revealed that EtBr binds with DNA in more than one way. The appropriate values of constants (K) and number site sizes (n) of EtBr binding to DNA are determined. The values of binding constants are equal to 1.9 · 106 and 5.6 · 105 M1, and number site sizes to 9 and 3.6 for strong interactions at ionic strengths of solutions 0.02 and 0.154 M Na+ at 28 °C, respectively. For a weaker interaction, these parameters are equal to 7 · 104 and 8 · 104 M1 and 1.5 and 1 at the mentioned ionic strengths of solutions, respectively. Thus, EtBr interacts with DNA in more than one way—intercalative and electrostatic at low ionic strength, and semi-intercalative and electrostatic at a higher strength of the solution. These results are in good accordance with the ones obtained by spectroscopic (absorption and fluorimetric) methods.
Keywords: DNA; EtBr; Differential pulse voltammetry; Binding constant; Number site sizes; Intercalation;

Effect of dendrimers on pure acetylcholinesterase activity and structure by D. Shcharbin; M. Jokiel; B. Klajnert; M. Bryszewska (56-59).
The effect of polyamidoamine (PAMAM) dendrimers on activity and fluorescence of pure acetylcholinesterase (EC 3.1.1.7.) was studied. It has been shown that all dendrimers studied decreased the enzymatic activity of acetylcholinesterase. This effect depended on the type of dendrimers. The data on the intrinsic fluorescence have shown that the dendrimers changed acetylcholinesterase conformation and the strongest effect was induced by PAMAM G3.5 dendrimer.
Keywords: Acetylcholinesterase; Enzyme activity; Conformation; PAMAM dendrimer; Fluorescence;

An electrochemical investigation on the interaction of acid chrome blue K (ACBK) with protein on the mercury electrode with different electrochemical methods such as cyclic voltammetry and linear sweep voltammetry was reported in this paper. In pH 3.0 Britton–Robinson (B–R) buffer solution, ACBK has an irrevisible voltammetric reductive peak at − 0.23 V (vs. SCE). The addition of human serum albumin (HSA) into the ACBK solution resulted in the decrease of reductive peak currents without the change of the peak potential and no new peaks appeared on the cyclic voltammogram. In the absence and presence of HSA, the electrochemical parameters such as the formal potential E 0, the electrode reaction standard rate constant k s and the charge transfer coefficient α of the interaction system were calculated and the results showed that there were no significant changes between each other. Thus, the interaction of ACBK with protein forms an electro-inactive supramolecular bio-complex, which induces the decrease of the free concentration of ACBK in the reaction solution, and the decrease of the reductive peak current of ACBK. The binding constant and the binding ratio are calculated as 1.29 × 108 and 1:2, respectively, and the interaction mechanism is discussed. Based on the binding reaction, this new electrochemical method is further applied to the determination of HSA with the linear range from 3.0–20.0 mg/L and the linear regression equation as ΔIpʺ(nA) = 10.08 + 19.90 C (mg/L). This method was further applied to determinate the content of protein in the healthy human serum samples with the results in good agreement with the traditional Coomassie brilliant blue G-250 spectrophotometric method.
Keywords: Acid chrome blue K; Serum albumin; Voltammetry; Interaction; Protein assay;

Methoxy-resorufin ether as an electrochemically active biological probe for cytochrome P450 O-demethylation by A. Toby A. Jenkins; Hayley-Ann Dash; Sam Boundy; Catherine M. Halliwell; Richard H. ffrench-Constant (67-71).
This paper describes the utilisation of methoxy-resorufin ether as an electrochemical probe for studying cytochrome P450 CYP6G1. Methoxy-resorufin ether is well established as a versatile substrate for cytochrome P450, as its demethylated product, resorufin, is a fluorophore. We show that in addition to these established properties, methoxy-resorufin ether also exhibits reversible two electron transfer on glassy carbon and edge plane graphite electrodes. Cyclic voltammetry measurements and differential pulse voltammetry measurements show that methoxy-resorufin ether can be easily detected at low concentrations (down to 200 nM) in a conventional three electrode electrochemical cell. These properties of methoxy-resorufin ether mean that it could be used as an electrochemical probe, to follow the rate of its demethylation by CYP6G1. We show that electrochemical measurements could discriminate between the enzyme activity of protein microsomes taken from two strains of Drosophila melanogaster (fruit fly).
Keywords: Cytochrome P450; CYP6G1; Electrochemistry; MROD; Resorufin;

This study investigated characteristics of a chitosan membrane from the carapace of the soldier crab Mictyris brevidactylus intended to construct an amperometric biosensor. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used in this study to characterize these chitosan membranes intended for constructing enzymatic biosensors. Chitosan membranes suffering various durations (> 10 min) of deacetylation had small charge-transfer resistances (< 7.88 kΩ) but large double-layer capacitances (> 0.55 μF). They were found in EIS where both the solution resistance and Warburg impedance upon electrode interface were almost independent of the durations and degree of deacetylation. The degree of deacetylation and the thickness of chitosan membranes were also determined. Membrane thickness was slightly dependent with the duration but degree of deacetylation was slightly dependent on the duration. Chitosan membranes with various thicknesses suffered various durations of deacetylation, but this did not influence their electrochemical characteristics. The chitinous membrane was covalently immobilized with glucose oxidase (EC 1.3.4.3) and then attached onto the platinum electrode of a homemade amperometric flow cell. Sensor signal was linearly related to glucose concentration (r  = 0.999 for glucose up to 1.0 mM). The system was sensitive (S  /  N  > 5 for 10 μM glucose) and reproducible (CV < 1.3% for 50 μM glucose, n  = 5).
Keywords: Chitosan; Chitin; Soldier crab; Electrochemical impedance spectroscopy; Enzyme immobilization; Biosensor;

A set of expressions is derived which quantifies the chlorophyll fluorescence yield in terms of rate constants of primary light reactions of PSII, the fraction of open and semi-open RCs and of the electric field sensed by the RC in the thylakoid membrane. The decay kinetics of the chlorophyll fluorescence yield after a single turnover excitation in the presence of DCMU show at least two components, one reversible within approx. 1 s and one with a dark reversion lasting more than 30 s. The latter is attributed to photochemical quenching; the fast component is interpreted to be associated at least partially with photo-electrochemical control. It will be illustrated that (i) the sub-maximal fluorescence yield in single turnover excitation is associated with semi-closure of RCs, (ii) the trapping efficiency of semi-closed centers is less than 50% of that of open centers and (iii) the fluorescence yield of antennas with semi-closed RCs has the highest sensitivity to changes in strength of photo-electric fields.
Keywords: Chlorophyll fluorescence yield; Single turnover excitation; Photochemical quenching; Electric field effect; Double hit trapping mechanism;

Micro pulsed radio-frequency electroporation chips by Huiqi He; Donald C. Chang; Yi-Kuen Lee (89-97).
Electroporation (EP) is one of the most important physical methods in biotechnology, which employs electrical pulses to transiently permeabilize cell membranes. In this study, a new micro pulsed radio-frequency electroporation cell (μPREP) chip was fabricated using a lift-off technique and SU-8 photolithography. The biological tests were carried out using three different plant protoplasts (cabbage, spinach and oil rape) on the micro EP chip and a pulsed RF electric field was applied to the microchip. The variations of fluorescent intensity and cell viability as functions of the electric pulse amplitude and duration time during the electroporation process were studied in detail at the single-cell level. Using such chip design and test method, one can easily optimize the efficiency and cell viability. Also, a large amount of statistical data can be quickly obtained. Finally, results of this parametric study were presented in the “phase diagram”, from which the critical electric field for inducing single-cell electroporation under different conditions can be clearly determined.
Keywords: Micro electroporation; Protoplast cells; SU-8; Propidium iodide; Membrane permeability;

Horseradish peroxidase (HRP) was incorporated in dipalmitoylphosphatidic acid (DPPA) to form a film and the film was modified on pyrolytic graphite electrode. UV–Vis spectra suggested that HRP in the film could keep its secondary structure similar to the native state. A pair of stable, well-defined, and quasi-reversible cyclic voltammetric peaks was observed with the formal potential at − 276.2 mV (vs. saturated calomel electrode), characteristic of heme FeIII/FeII redox couple of HRP. The apparent heterogeneous electron transfer rate constant and other electrochemical parameters were presented. The catalytic activity of HRP in DPPA film toward oxygen, hydrogen peroxide and nitric oxide were also examined.
Keywords: Horseradish peroxidase; Dipalmitoylphosphatidic acid; Hydrogen peroxide; Nitric oxide; Cyclic voltammetry;

The direct, reversible electrochemistry of horse-heart cytochrome c (cyt. c) was realized on a self-assembled glutathione (GSH) monolayer modified Au electrode. The voltammetric responses of cyt. c on GSH/Au electrode were found to be affected by pH during the electrode modification, metal ions and surfactants. Using potassium ferricyanide [K4Fe(CN)6] as a probe, these effects on the voltammetric responses of cyt. c were characterized by electrochemical methods. It was found that the pH during the electrode modification, metallic ions and surfactants changed GSH monolayer's charge state and the conformation on the electrode surface, and resulted in the influence on the voltammetric responses of cyt. c. The experimental results provided us information to understand the mechanism of the interfacial electron transfer of electrode-protein, as well as the electron transfer of cyt. c in life system.
Keywords: Cytochrome c; Glutathione; Self-assembled monolayers; Direct electrochemistry;

The electrochemical behaviors of a marmatite – carbon paste electrode with the chemical leaching of Fe3+ ions, or the microbial leaching using Acidithiobacillus ferrooxidans, were compared. The cyclic voltammograms of the electrode in the presence and absence of bacterial strains showed that the leaching process of marmatite was carried out by the different reactions occurring in the interface of the marmatite electrode — leach liquid. The polarization currents of the electrode under the differently applied potentials suggested that the microbial leaching of marmatite could be accelerated by the applied potential. The SEM observations indicated that the corrosion pits formed in the electrode surface were similar to the attached bacterial cells in shape and size, other than that by the chemical leaching of Fe3+ ions. The contact leaching of the attached cells on the mineral substrate played an important role on the dissolution of marmatite in addition to the chemical leaching of Fe3+ ions.
Keywords: Marmatite; Carbon paste electrode; Bacterial strains; Electrochemical behavior;