Bioelectrochemistry (v.68, #2)

Contents (iii-iv).

Application of polymaleimidostyrene as a convenient immobilization reagent of enzyme in biosensor by Shunichi Uchiyama; Ryoichi Tomita; Naoyuki Sekioka; Emi Imaizumi; Hiroshi Hamana; Tokio Hagiwara (119-125).
Sulfhydryl groups of glucose oxidase (GOD) were reacted with maleimide groups of polymaleimidostyrene (PMS) which was coated onto the porous carbon sheet, and the carbon sheet immobilized by GOD was combined with an oxygen electrode to fabricate a glucose sensor. The activity of thiolated GOD immobilized to PMS is much larger than that of native GOD immobilized to PMS. The good linear relationship of glucose and oxygen current response was obtained in a concentration range from 0.1 to 2 mM and upper limit of linear range was found to be 3.0 mM. The immobilized GOD activity is highly dependent on pH at immobilization and the maximum activity was obtained at pH 5.5, probably because the SH groups of GOD that are indispensable for generation of enzyme activity is not exposed at this pH. It was found that PMS is very effective reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups.
Keywords: Polymaleimidestyrene; Glucose sensor; Thiolation of enzyme; Conformation change;

A novel biosensor for sterigmatocystin constructed by multi-walled carbon nanotubes (MWNT) modified with aflatoxin–detoxifizyme (ADTZ) by Dong-sheng Yao; Hong Cao; Shengmei Wen; Da-ling Liu; Yan Bai; Wen-jie Zheng (126-133).
Sterigmatocystin, ST, is carcinogenic mycotoxin with toxicity second to aflatoxins, contaminated in foods- and feeds-stuff widely. A three-electrode system was employed to examine the response character of the covalently united ADTZ–MWNTs electrode to ST, and the results indicated that an oxidation peak of ST was observed at about + 400 mv, the linear detection range of ST was from 4.16 × 10− 5 mg/ml (0.13 μM) to 1.33 × 10− 3 mg/ml (4.29 μM) with the detection limit at 0.13 μM. Compared to the corresponding results obtained from the MWNTs modified electrode that ADTZ was directly sediment (adsorbed) on it, the sensitivity of ours had been improved by two orders of magnitude, which could provide some important data to further research.
Keywords: Sterigmatocystin; Multi-walled carbon nanotubes; Aflatoxin–detoxifizyme; Biosensor;

Bioelectrocatalysis in ionic liquids. Examining specific cation and anion effects on electrode-immobilized cytochrome c by Cory M. DiCarlo; David L. Compton; Kervin O. Evans; Joseph A. Laszlo (134-143).
Cytochrome c immobilized on alkylthiol self-assembled monolayers exhibits a characteristic Fe(II)/Fe(III) redox signal that is lost when exposed to ionic liquids composed of a butylimidazolium cation combined with either hexafluorophosphate or bis(trifluoromethylsulfonyl)imide anion. In this study it was shown that exposure to the aqueous solubilized ionic liquid components, butyl-, hexyl-, and octyl-imidazolium cations and hexafluorophosphate, tetrafluoroborate, and bis(trifluoromethylsulfonyl)imide anions, resulted in partial electrochemical signal loss. Absorbance and fluorescence measurements showed that signal loss due to the cationic ionic liquid component followed a different mechanism than that of the anionic component. Although a portion of the signal was recoverable, irreversible signal loss also occurred in both cases. The source of the irreversible component is suggested to be the loss of protein secondary structure through complexation between the ionic liquid components and the protein surface residues. The reversible electrochemical signal loss is likely due to interfacial interactions imposed between the electrode and the cytochrome heme group. The influence of the amount of exposed surface residues was explored with a simplified model protein, microperoxidase-11.
Keywords: Cytochrome c; Ionic liquid; Cyclic voltammetry; Electrode immobilized protein; Self-assembled monolayer;

Electrochemistry and voltammetry of procaine using a carbon nanotube film coated electrode by Kangbing Wu; Hong Wang; Fang Chen; Shengshui Hu (144-149).
A new rapid, convenient and sensitive electrochemical method is described for the determination of procaine in pharmaceutical preparations, based on the unique properties of a multi-wall carbon nanotube (MWNT) thin film. The electrochemical behavior of procaine at the MWNT film-coated glassy carbon electrode (GCE) was investigated in detail, showing that the MWNT-coated GCE exhibits electrocatalytic activity to the oxidation of procaine because of the significant peak current enhancement and the lowering of oxidation overpotential. Furthermore, the mechanism for the oxidation of procaine at the MWNT-coated GCE was also studied. Finally, various experimental parameters such as solution pH value, the amount of MWNT, accumulation conditions and scan rate were optimized for the determination of procaine, and a new method with detection limit of 2 × 10− 7 mol/L was developed for procaine determination. This newly proposed method was successfully demonstrated with procaine hydrochloride injection.
Keywords: Procaine; Electrochemical determination; Carbon nanotube-modified electrode;

Studies on collective behaviour of gap junction channels by Paramita Ghosh; Subhendu Ghosh (150-157).
Gap junction provides low resistance pathways for cell-to-cell passive diffusion of ions, metabolites, second messengers etc. and thus, controls development, differentiation in embryonic tissues, and communication in adult tissues. It has been pointed out in our previous work that these passive diffusion channels behave cooperatively which in turn depends on the structural parameters and also membrane potentials. In the present paper, we have analyzed the multichannel bilayer electrophysiological data of rat liver gap junction Connexin 32 (Cx32) hemichannels. Through the measurements of relaxation time it has been demonstrated that one of the relaxation time constants follows a decay pattern with the number of channels open at various potentials applied across the bilayer membrane. This leads to the conclusion that the collective behaviour of rat liver gap junction hemichannels is cooperative in multichannel ensembles.
Keywords: Gap junction; Bilayer electrophysiology; Relaxation studies; Collective behaviour; Channel-to-channel cooperativity;

Interfacial ternary complex DNA/Ca/lipids at anionic vesicle surfaces by Alina Frantescu; Katja Tönsing; Eberhard Neumann (158-170).
The electroporative transfer of gene DNA and other bioactive substances into tissue cells by electric pulses gains increasing importance in the new disciplines of electrochemotherapy and electrogenetherapy. The efficiency of the electrotransfer depends crucially on the adsorption of the gene DNA and oligonucleotides to the plasma cell membranes. Here it is shown that the adsorption of larger oligonucleotides such as fragments (ca. 300 bp) of sonicated calf-thymus DNA, to anionic lipids of unilamellar vesicles (diameter Φ  = 300 ± 90 nm) is greatly enhanced by divalent cations such as Ca2+-ions. Applying centrifugation, bound and free DNA are monitored optically at the wavelength λ  = 260 nm. Using arsenazo III as a Ca2+-indicator and atomic absorption spectroscopy (AAS), Ca2+-titrations of DNA and vesicles yield the individual equilibrium constants of Ca2+- and DNA-binding not only for the binary complexes: Ca/lipids, Ca/DNA and DNA/lipids, respectively, but also for the various processes to form the ternary complex DNA/Ca/lipids. The data provide the basis for goal-directed optimization protocols for the adsorption and thus efficient electrotransfer of oligonucleotides and polynucleotides into cells.
Keywords: Polynucleotides; Oligonucleotides; Ca2+-binding constants; Electrified membrane interface; Monolayer adsorption; Complex thermodynamics;

The change in the energy barriers for the heterogeneous reduction of pyruvate decarboxylase (PDC) relative to its coenzyme, thiamin pyrophosphate (ThPP), was determined experimentally using square wave voltammetry (SWV) to be 5.3 kcal/mol. These results are in agreement with those of reaction rate acceleration provided by thiamin-dependent decarboxylases relative to their coenzyme as determined kinetically based on the pK a suppression by the enzyme environment.
Keywords: Pyruvate decarboxylase; Thiamin pyrrophosphate; Square wave voltammetry;

A novel amperometric method for antioxidant activity determination using DPPH free radical by Stjepan Milardović; Damir Iveković; Božidar S. Grabarić (175-180).
A new method for the determination of antioxidant activity based on the amperometric reduction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) at the glassy carbon electrode is proposed. All experiments were done in three-electrode electrochemical cell at 140 mV vs. Hg2Cl2 | 3 M KCl using ethanolic solution (φ  = 40%) and 0.033 M KCl in 0.033 M phosphate buffer, pH = 7.4. The linear range obtained for Trolox in 100 μM DPPH ethanol–water solution was up to 30 μM, with a limit of detection of 0.05 μM.The developed method was applied for the evaluation of antioxidant activity of some water or ethanol soluble pure compounds of antioxidants and of the samples of tea, wine and some other beverages. The good correlation of measurements (R 2  = 0.9993) expressed as Trolox equivalent was obtained between the proposed amperometric method and classic spectroscopic method.
Keywords: Amperometric detection; Antioxidant; DPPH; Trolox equivalent;

Model of a confined spherical cell in uniform and heterogeneous applied electric fields by T.R. Gowrishankar; Donald A. Stewart; James C. Weaver (181-190).
Cells exposed to electric fields are often confined to a small volume within a solid tissue or within or near a device. Here we report on an approach to describing the frequency and time domain electrical responses of a spatially confined spherical cell by using a transport lattice system model. Two cases are considered: (1) a uniform applied field created by parallel plane electrodes, and (2) a heterogeneous applied field created by a planar electrode and a sharp microelectrode. Here fixed conductivities and dielectric permittivities of the extra- and intracellular media and of the membrane are used to create local transport models that are interconnected to create the system model. Consistent with traditional analytical solutions for spherical cells in an electrolyte of infinite extent, in the frequency domain the field amplification, G m (f) is large at low frequencies, f  < 1 MHz. G m (f) gradually decreases above 1 MHz and reaches a lower plateau at about 300 MHz, with the cell becoming almost “electrically invisible”. In the time domain the application of a field pulse can result in altered localized transmembrane voltage changes due to a single microelectrode. The transport lattice approach provides modular, multiscale modeling capability that here ranges from cell membranes (5 nm scale) to the cell confinement volume (∼40 μm scale).
Keywords: Three dimensional cell model; Transport lattice; Electric field; Spherical cell; Cell membrane; Confined cell; Microelectrode;

Carbon paste electrodes modified with baker's yeast Saccharomyces cerevisiae (a source of flavocytochrome b2) were investigated as amperometric biosensors for lactic acid. Phenazine methosulphate was used as a mediator. The optimal operational conditions of the electrodes were: an operating potential 0.0 V, solution pH 7.2, concentration of phenazine methosulphate in solution 0.2 mM. A linear range in the dependence of the current responses on the concentration of lactic acid was up to 1 mM. The suitability of the electrodes for determination of lactic acid in milk and dairy products such as kefir and yoghurt was tested. The yeast cells in the paste remained viable at least for 1 month.
Keywords: Biosensor; Saccharomyces cerevisiae; Flavocytochrome b2; Phenazine methosulphate; Lactic acid; Carbon paste;

The electrochemical behavior of colchicine at an acetylene black-dihexadecyl hydrogen phosphate (denoted as AB-DHP) composite film coated glassy carbon electrode (GCE) was investigated using cyclic voltammetry (CV), linear sweep voltammetry (LSV) and differential pulse voltammetry (DPV). Compared with the poor electrochemical signal at the unmodified GCE, the electrochemical response of colchicine at the AB-DHP film modified GCE was greatly improved, as confirmed from the significant peak current enhancement. The remarkable peak current enhancement indicates that the AB-DHP modified GCE has great potential in the sensitive determination of colchicine. Thus, all the experimental conditions, which influence the electrochemical response of colchicine, were studied and the optimum conditions were achieved. Finally, a sensitive and simple voltammetric method with a good linear relationship in the range of 1.0 × 10− 7∼4.0 × 10− 5 mol/L, was developed for the determination of colchicine. The detection limit of colchicine was also examined and a low value of 4.0 × 10− 8 mol/L for 4-min accumulation was obtained (S  /  N  = 3). This electrode was successfully applied to detect colchicine in human urine samples.
Keywords: Colchicine; Voltammetry; Acetylene black; Modified electrodes; Urine sample;

Responses of Bruguiera gymnorrhiza to saltwater monitored by miniature electrodes by Satoshi Sasaki; Yosuke Chiba; Nobutaka Hanagata (202-205).
We measured the voltage between two Ag/AgCl electrodes, one inserted into the seedling of the salt-tolerant plant Bruguiera gymnorrhiza and the other into the vermiculite in which the seedling was potted. Four seedlings were placed in different environments, in saltwater or pure water, with light or alternating light/dark conditions. We have found that (1) the voltage profiles showed periodical oscillatory behavior; (2) seedlings in saltwater showed higher voltage compared to the ones in pure water; (3) in the light environment, the voltage was higher compared to the one in the dark environment; (4) in the dark environment, a voltage wave was hardly observable; and (5) electrodes inserted into the propagule cortex, stem cortex, and petiole showed different voltage wave amplitudes. The voltage profiles will provide an effective way to evaluate the movement of salt water inside the salt-tolerant plant.
Keywords: Bruguiera gymnorrhiza; Vigna angularis; Safranin; Ag/AgCl electrode;

Voltammetric methods were used to probe the interaction of antimicrobial drug metronidazole (MTZ) with calf thymus DNA. Binding constants (K) and binding site sizes (s) were determined from the voltammetric data, i.e., shifts in potential and changes in limiting current with the addition of DNA. MTZ showed appreciable electrostatic binding to DNA in solution with K  = 2.2(± 1.3) × 104 M− 1 and s  = 0.34 bp. One reduction peak of MTZ at the bare glassy carbon electrode (GCE) split into two peaks at the DNA modified GCE (DNA/GCE). These changes in the cyclic voltammogram can only be due to the interaction of MTZ with the surface-confined DNA. In addition, the peak current of MTZ at the DNA/GCE was nearly 8-fold of the response at the bare GCE. The low detection limit of 2.0 × 10− 8 M made the DNA/GCE a promising biosensor for MTZ determination. And this method was successfully applied with high precision and accuracy compared with spectroscopic methods (relative error < 6%) for estimation of the total MTZ drug content in pharmaceutical dosage forms.
Keywords: Drug analysis; Metronidazole; Interaction; DNA; Voltammetry; Biosensor;

The spatial distribution of electrical potential and current in a suspension of spherical cells under an applied electric field was numerically obtained using the equivalent circuit method (ECM). The effect of the proximity of the cells was studied in a set of simulations where the volumetric fraction varied from 0.24 to 0.66. The results show that the transmembrane potential for cells in the suspension is lower than the theoretically predicted value for a single dielectric membrane under a uniform electric field. It was also observed that as the volumetric fraction is increased, the transmembrane potential on the pole of the cells decreases linearly. Furthermore, the conductivity of the suspension was also observed to be a function of the volumetric fraction and this result is in a good agreement with the Maxwell's model for spherical particles suspended in a volume conductor.
Keywords: Numerical simulation; Electrical polarization; Spherical cells polarization; Electromagnetic modeling;

A reagentless lactate biosensor is described, based on an electropolymerized copolymer film poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-3-acetic acid-1,4-naphthoquinone). The quinone group, as part of the polymer backbone, is electroactive and very stable in neutral aqueous medium. It can therefore act as an immobilized mediator for the enzyme recycling, at a working potential much lower than those commonly reported in the literature for other mediators. Experimental conditions for amperometric measurements (temperature, pH) are studied, especially the interference between quinone and molecular oxygen to investigate the enzyme/quinone recycling kinetic. Some well-known interferents are shown to have no measurable effect on the amperometric curves.
Keywords: Conducting polymer; Lactate oxidase; Reagentless detection; Quinone; Interferents;

A simply prepared DNA immobilized on a carbon nanotube paste electrode (CNTPE) was utilized to monitor dopamine ion concentration using the cyclic voltammetry (CV) and square-wave (SW) stripping voltammetry methods. The optimum analytical conditions were sought. The result obtained was a very low detection limit compared to other common voltammetry methods. The optimal parameters were found to be as follows: 3.5 pH, 0.48 V SW amplitude, 71 Hz frequency, 5 s accumulation time, 0.01 V increment potential, and − 1.3 V (anodic-•-) and 1.2 V (cathodic-○-) accumulation potentials. Given these conditions, the linear working range was observed to be within 0.01–0.11 μg L− 1 (SW anodic and CV). The analytical detection limit was determined to be SW anodic and CV: 4.0 μg L− 1 (2.1 × 10− 11 mol L− 1) dopamin, and the relative standard deviation at the dopamine concentration of SW anodic 0.05 μg L− 1 was 0.02% (n  = 15) at the optimum conditions.
Keywords: Dopamin; Carbon nano tube; DNA; Paste electrode; Voltammetry;