Bioelectrochemistry (v.79, #2)

Application of peptide nucleic acid towards development of nanobiosensor arrays by Ravindra P. Singh; Byung-Keun Oh; Jeong-Woo Choi (153-161).
Peptide nucleic acid (PNA) is the modified DNA or DNA analogue with a neutral peptide backbone instead of a negatively charged sugar phosphate. PNA exhibits chemical stability, resistant to enzymatic degradation inside living cell, recognizing specific sequences of nucleic acid, formation of stable hybrid complexes like PNA/DNA/PNA triplex, strand invasion, extraordinary thermal stability and ionic strength, and unique hybridization relative to nucleic acids. These unique physicobiochemical properties of PNA enable a new mode of detection, which is a faster and more reliable analytical process and finds applications in the molecular diagnostics and pharmaceutical fields. Besides, a variety of unique characteristic features, PNAs replace DNA as a probe for biomolecular tool in the molecular genetic diagnostics, cytogenetics, and various pharmaceutical potentials as well as for the development of sensors/arrays/chips and many more investigation purposes. This review paper discusses the various current aspects related with PNAs, making a new hot device in the commercial applications like nanobiosensor arrays.
Keywords: PNA; DNA; Biosensor array; Microarrays; Biochips;

Electrochemistry of interaction of 2-(2-nitrophenyl)-benzimidazole derivatives with DNA by Mabel Catalán; Alejandro Álvarez-Lueje; Soledad Bollo (162-167).
In this study the interaction between new benzimidazole molecules, 2-(2-nitrophenyl)-1H-benzimidazole (NB) and N-benzoyl-2-(2-nitrophenyl)-benzimidazole (BNB), with dsDNA and ssDNA was assessed at pH 7.4. Using differential pulse voltammetry at glassy carbon electrode, both molecules were electrochemically reduced due to the presence of a nitro group in their structures. When DNA was added to the solution, the electrochemical signal of NB and BNB decreased and shifted to more negative potentials. The interaction mode was electrostatic when ionic strength was low. Under this condition DNA–nitro complexes were characterized and binding constant values of 8.22 × 104  M− 1 and 3.08 × 106  M− 1 for NB and BNB with dsDNA were determined. On the other hand, only NB was able to interact when a high concentration of NaCl was used. Finally, a glassy carbon electrode modified with carbon nanotubes and DNA was tested in order to determine the nitrocompound in solution. The electrochemical reduction of the nitrocompound adsorbed on GCE/CHIT-CNT/DNA was used as an analytical signal. Using 10 min as accumulation time, a linear dependence was observed between 20 and 80 µM nitrocompound concentrations and the electrode response. Detection and quantification limits in the range of µM were determined.
Keywords: DNA; Nitrophenyl-benzimidazole; Interaction; Voltammetry;

We report the selective determination of homocysteine (HCY) in the presence of one of the very important interferents, ascorbic acid (AA) using electropolymerized film of 2-amino-1,3,4-thiadiazole (ATD) modified glassy carbon electrode (GCE) at physiological pH for the first time. An atomic force microscopic image showed that the electropolymerized film of ATD (p-ATD) formed a spherical like structure with a thickness of 25 nm. This nanostructured film oxidized HCY at 0.55 V while bare GCE failed to oxidize it at physiological pH. Further, p-ATD modified electrode successfully separated the voltammetric signals of AA and HCY with a peak separation of 490 mV. The amperometric current was increased linearly from 100 to 1400 nM HCY and achieved the detection limit of 51 pM (S/N  = 3). The present modified electrode showed better recoveries for spiked HCY in human blood serum samples.
Keywords: Electropolymerization; 2-Amino-1,3,4-thiadiazole; AFM; Homocysteine; Ascorbic acid; Amperometry;

Electrochemical reduction mechanism of camptothecin at glassy carbon electrode by Afzal Shah; Victor C. Diculescu; Rumana Qureshi; Ana Maria Oliveira-Brett (173-178).
Camptothecin (CPT) is a cytotoxic quinoline alkaloid endowed with the inhibition of topoisomerase I, an essential enzyme for the normal functioning of DNA. The redox behaviour of CPT was investigated at a glassy carbon electrode using cyclic, differential pulse and square wave voltammetry. It was shown that CPT can undergo reduction in a pH-dependent mechanism. In acid media only one irreversible charge transfer reaction was observed whereas by increasing the pH of the supporting electrolyte, two reduction peaks occurred. The diffusion coefficient of CPT was calculated in pH 4.5 0.1 M acetate buffer using cyclic voltammetry to be D CPT  = 5.77 × 10− 6  cm2  s− 1. Differential pulse voltammetry measurements were carried out over a wide pH range and allowed the determination of the number of electrons and protons transferred during each step in the CPT reduction mechanism, one electron and one proton. The use of square wave voltammetry proved the quasi-reversibility of CPT reduction as a function of the pH of the supporting electrolyte. Based upon the results obtained a reduction mechanism was proposed and the observed waves were attributed to the hydroxylation of the lactone ring of CPT to a lactol ring.
Keywords: Camptothecin (CPT); Voltammetry; Adsorption; Reduction; Lactone ring;

A simple, bistable rate-equation based model is used to predict trends of cellular apoptosis following electric pulsing. The caspase-8 extrinsic pathway with inherent delays in its activation, cytochrome c release, and an internal feedback mechanism between caspase-3 and cleavage of Bid are incorporated. Results obtained were roughly in keeping with the experimental cell-survival data and include an electrical pulse-number threshold followed by a near-exponential fall-off. The extrinsic caspase-8 mechanism is predicted to be more sensitive than the mitochondrial intrinsic pathway for electric pulse induced cell apoptosis. Also, delays of about an hour are predicted for detectable molecular concentration increases following electrical pulsing. Finally, our results suggest that multi-needle electrode systems with adjustable field orientations would likely enhance apoptosis in the context of pulsed voltage-induced inactivation of tumor cells.
Keywords: Electrical pulsing; Apoptosis; Mechanisms; Numerical model; Cell survival;

Layer-by-layer assembly of collagen and electroactive myoglobin by Xin Miao; Yi Liu; Wenchao Gao; Naifei Hu (187-192).
In this work, an electrochemically inert protein collagen was successfully assembled with electroactive myoglobin (Mb) into {collagen/Mb} n layer-by-layer (LbL) films on solid surfaces. UV–vis spectroscopy and cyclic voltammetry (CV) were used to confirm the film growth and characterize the films. The collagen films provided a biocompatible microenvironment for Mb, and Mb in the stable {collagen/Mb} n films showed a nearly reversible CV response for its heme Fe(III)/Fe(II) redox couple. The direct electrochemistry of Mb in the films was used to electrocatalyze the reduction of oxygen and hydrogen peroxide. The interaction between collagen and Mb under different pH conditions was also explored. Not only the positively charged Mb at pH 5.0 but also the negatively charged Mb at pH 9.0 could be assembled with collagen into {collagen/Mb} n films. This work provides a novel example to assemble LbL films with two different proteins, and may establish a foundation for fabricating the new type of biosensors based on the direct electron transfer of redox proteins with underlying electrodes.
Keywords: Myoglobin; Collagen; Layer-by-layer assembly; Direct electrochemistry; Electrocatalysis; Driving force;

Dielectric properties of human ovary follicular fluid at 9.2 GHz by Olga А. Gorobchenko; Anna G. Gerodes; Oleg A. Nardid; Oleg T. Nikolov (193-197).
The influence of the follicle size, rapid freezing to −196 °С and cryopreservation in liquid nitrogen within a period of one month of the human ovary follicular fluid (FF) on its dielectric properties is studied by the microwave dielectric method. The FF was obtained from dominant follicles of patients who received treatment for infertility by extracorporal fertilization. We have measured the real part (ε′) of the complex permittivity of the native and frozen follicular fluids at the room temperature. A resonator type ultra high frequency (UHF) dielectrometer at the frequency of 9.2 GHz has been used. We have also obtained the values of the total protein, hormones and glucose concentration in the FF. It was found that rapid freezing reduces ε′ of the FF. It can result from the bound water increase in the system. It was also found the rise in permittivity and the total protein concentration with the increase of a follicle size, which could be explained by protein dehydration as a result of its clustering and aggregation.
Keywords: Follicular fluid; Permittivity; UHF-dielectrometry; Freezing;

Synchronization of Dictyostelium discoideum adhesion and spreading using electrostatic forces by Marius Socol; Christine Lefrou; Franz Bruckert; Didier Delabouglise; Marianne Weidenhaupt (198-210).
Synchronization of cell spreading is valuable for the study of molecular events involved in the formation of adhesive contacts with the substrate. At a low ionic concentration (0.17 mM) Dictyostelium discoideum cells levitate over negatively charged surfaces due to electrostatic repulsion. First, a two-chamber device, divided by a porous membrane, allows to quickly increase the ionic concentration around the levitating cells. In this way, a good synchronization was obtained, the onsets of cell spreading being separated by less than 5 s. Secondly applying a high potential pulse (2.5 V/Ref, 0.1 s) to an Indium Tin Oxide surface attracts the cells toward the surface where they synchronously spread as monitored by LimE Δcoil-GFP. During spreading, actin polymerizes in series of active spots. On average, the first spot appears 8–11 s after the electric pulse and the next ones appear regularly, separated by about 10 s. Synchronized actin-polymerization activity continues for 40 s. Using an electric pulse to control the exact time point at which cells contact the surface has allowed for the first time to quantify the cellular response time for actin polymerization. Electrochemical synchronization is therefore a valuable tool to study intracellular responses to contact.
Keywords: Dictyostelium discoideum; Cell adhesion; Electrostatic forces; Indium Tin Oxide; Reflection Interference Contrast Microscopy; LimE Δcoil fluorescence;

In situ synthesis of biomolecule encapsulated gold-cross-linked poly(ethylene glycol) nanocomposite as biosensing platform: A model study by Dilek Odaci; Muhammet U. Kahveci; Elif L. Sahkulubey; Caglar Ozdemir; Tamer Uyar; Suna Timur; Yusuf Yagci (211-217).
In situ synthesis of poly(ethylene glycol) (PEG) hydrogels containing gold nanoparticles (AuNPs) and glucose oxidase (GOx) enzyme by photo-induced electron transfer process was reported here and applied in electrochemical glucose biosensing as the model system. Newly designed bionanocomposite matrix by simple one-step fabrication offered a good contact between the active site of the enzyme and AuNPs inside the network that caused the promotion in the electron transfer properties that was evidenced by cyclic voltammetry as well as higher amperometric biosensing responses in comparing with response signals obtained from the matrix without AuNPs. As well as some parameters important in the optimization studies such as optimum pH, enzyme loading and AuNP amount, the analytical characteristics of the biosensor (AuNP/GOx) were examined by the monitoring of chronoamperometric response due to the oxygen consumption through the enzymatic reaction at − 0.7 V under optimized conditions at sodium acetate buffer (50 mM, pH 4.0) and the linear graph was obtained in the range of 0.1–1.0 mM glucose. The detection limit (LOD) of the biosensor was calculated as 0.06 mM by using the signal to noise ratio of 3. Moreover, the presence of AuNPs was visualized by TEM. Finally, the biosensor was applied for glucose analysis for some beverages and obtained data were compared with HPLC as the reference method to test the possible matrix effect due to the nature of the samples.
Keywords: Biosensor; Bionanocomposite; Hydrogel; Gold nanoparticles; Photopolymerization;

Nano-structured bismuth oxide (nano-BiO x ) is a suitable material for enzyme immobilization owing to its attractive properties, such as large specific surface area, suitable permeability of the resulting film, the high biocompatibility, and as well as photovoltaic effect from semiconductor nanoparticles. Thus, a new type of amperometric glucose biosensor based on nano-BiO x was constructed. The amperometric detection of glucose was assayed by potentiostating the GOD/nano-BiO x electrode at 0.5 V to oxidize the enzymatically generated hydrogen peroxide. The proposed biosensor provided a linear response to glucose over a concentration range of 1 × 10− 6  M to 1.5 × 10− 3  M with a sensitivity of 51.0 ± 0.4 mA/(M cm2) and a detection limit of 4 × 10− 7  M based on S/N = 3. The apparent Michaelis–Menten constant was calculated to be 2.9 × 10− 3  M. In addition, characterization of nano-BiO x and modified electrode was performed by FT-IR spectroscopy, Raman spectroscopy, scanning electron microscope (SEM) and rotating-disk electrode (RDE) voltammetry.
Keywords: Bismuth oxidase; Inorganic matrix; Glucose oxidase; Biosensor;

The influence of a finite volume of ions and orientational ordering of water Langevin dipoles on the dielectric permittivity profile in the vicinity of charged surface is studied theoretically via a numerical solution of the modified Poisson–Boltzmann equation. It is shown that the dielectric permittivity profile close to the charged surface is mainly determined by two mechanisms; specifically, the depletion of dipoles at the charged surface due to accumulated counterions and the increased orientational ordering of the water dipoles.
Keywords: Membrane electrochemistry; Langevin dipoles; Excluded volume effect; Dielectric permittivity; Orientational ordering;

Performance of microbial fuel cells (MFCs), fabricated using an earthen pot (MFC-1) and a proton exchange membrane (MFC-2), was evaluated while treating rice mill wastewater at feed pH of 8.0, 7.0 and 6.0. A third MFC (MFC-3), fabricated using a proton exchange membrane (PEM), was operated as control without pH adjustment of the acidic raw wastewater. Maximum chemical oxygen demand (COD) removal efficiencies of 96.5% and 92.6% were obtained in MFC-1 and MFC-2, respectively, at feed pH of 8.0. MFC-3 showed maximum COD removal of 87%. The lignin removal was 84%, 79%, and 77% and the phenol removal was 81%, 77%, and 76% in MFC-1, MFC-2, and MFC-3, respectively. Maximum sustainable volumetric power was obtained at feed pH of 8.0, and it was 2.3 W/m3 and 0.53 W/m3, with 100 Ω external resistance, in MFC-1 and MFC-2, respectively. The power was lower at lower feed pH. MFC-3 generated lowest volumetric power (0.27 W/m3) as compared to MFC-1 and MFC-2. More effective treatment of rice mill wastewater and higher energy recovery was demonstrated by earthen pot MFC as compared to MFC incorporated with PEM.
Keywords: Rice mill wastewater treatment; Microbial fuel cell; Earthen pot MFC; Power density; Coulombic efficiency;

Electrochemical determination of amlodipine besylate (ADB) using single and multi-walled carbon nanotubes modified edge plane pyrolytic graphite electrodes (SWNT/EPPGE and MWNT/EPPGE) is described by using cyclic and square wave voltammetries at physiological pH 7.2. An increased peak current with a shift of peak potential to less positive value was observed using carbon nanotubes modified EPPGE as compared to bare electrode. The effect of pH, scan rate and analyte concentration has been examined. Under the optimum conditions the peak current was linear to the concentration of amlodipine in the range 5.0 × 10− 9 to 1.0 × 10− 6  mol L− 1 for SWNT/EPPGE and the detection limit was found to be 1.0 × 10− 9  mol L− 1 whereas, for MWNT/EPPGE the detection limit was found to be 5.0 × 10− 9  mol L− 1. The method was successfully used to determine the content of amlodipine in the pharmaceutical preparations and human urine samples of angina patients undergoing treatment with amlodipine. A comparison of electrocatalytic activities of SWNT and MWNT modified electrodes indicated that SWNT modified EPPGE is ∼ 1.8 times more sensitive in comparison to MWNT/EPPGE. Biological relevance of the developed method has been described by the determination of amlodipine in human body fluids. Amlodipine can be determined without any interference from common urine metabolites such as uric acid, ascorbic acid and xanthine.
Keywords: Amlodipine besylate; Voltammetry; Carbon nanotube; Pyrolytic graphite; Human urine;

Utilizing the fascinating strong adsorptive ability, high chemical and mechanical stability properties of montmorillonite-calcium (MMT-Ca) clay, a MMT-Ca modified carbon paste electrode was developed for the sensitive determination of methocarbamol. Methocarbamol has been oxidized in buffered solutions at the developed MMT-Ca-modified CPE in a single 2-electron irreversible anodic peak. A simple and sensitive square-wave adsorptive anodic stripping voltammetric method was optimized for determination of methocarbamol in bulk form, pharmaceutical formulation and in spiked human serum using the developed modified CPE. This was carried out without the necessity for samples pretreatment and/or time-consuming liquid–liquid or solid-phase extraction steps prior to the analysis. The developed electrode exhibited an excellent sensitivity and selectivity towards methocarbamol even in the presence of 102–104-fold of its active ingredient “ibuprofen,” common excipients, common metal ions or co-administrated drugs. Limits of detection of 3 × 10 9 and 1.2 × 10 8  M and limits of quantitation of 1 × 10 8, and 4 × 10 8  M methocarbamol were achieved in the bulk form and in spiked human serum, respectively. Moreover, the developed method was successfully applied for determination of methocarbamol in human plasma of subjects following administration of an oral dose of Ibuflex® tablets.
Keywords: Methocarbamol; Montmorillonite-CPE; Voltammetry; Tablets; Human blood;

Electrochemistry and electrocatalytic of hemoglobin immobilized on FDU-15-Pt mesoporous materials by Dongxia Nie; Ying Liang; Tianshu Zhou; Xiaohong Li; Guoyue Shi; Litong Jin (248-253).
In this paper, the composite films of Nafion/Hb/FDU-Pt-15/PDDA were constructed by layer-by-layer assembly technique. The FDU-15-Pt mesoporous materials were synthesized and characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The resulting films were verified by electrochemical impedance spectroscopy (EIS), cyclic voltammetry and UV–vis spectroscopy. The direct electrochemical and electrocatalytic properties of hemoglobin (Hb) in the Nafion/Hb/FDU-15-Pt/PDDA films were also investigated. A pair of stable and well-defined reversible redox peaks was observed and the formal potential of Hb FeIII/FeII redox couple was found to be − 0.324 V (vs. SCE). Based on a series of optimized conditions, the films modified electrode displayed a good electrocatalytic activity to the reduction of hydrogen peroxide (H2O2), which had linear current response from 2.0 × 10− 6  M to 6.0 × 10− 2  M with the detection limit of 1.0 × 10− 6  M (S/N  = 3). The apparent Michaelis–Menten constant (K m app) was determined to be 0.15 mM in the range of lower concentration of H2O2. The apparent heterogeneous electron transfer rate constant (k s) was 1.05 ± 0.03 s− 1. The good sensitivity, reproducibility, excellent stability and wide linear range make Nafion/Hb/FDU-15-Pt/PDDA a promising application of the preparation of third-generation biosensor.
Keywords: Hemoglobin; FDU-Pt-15; Cyclic voltammetry; Hydrogen peroxide;

Two reliable methods have been combined: i) the electroporation of the cell membranes for facilitating the sensitizer incorporation into hystiocytic human lymphoma cells U-937 and (ii) the photodynamics applied by excitation of natural and synthetic sensitizers for cancer therapy. In the case of cytostatic sensitizers as daunomycin or actinomycin their photooxidation of guanine in DNA was added to their known dark medical efficacy for the first time. Several applications of single d.c. pulses and continuous visible light irradiations were performed, which resulted in about five times higher efficacy by 14 min of irradiation after the electroporation than the ordinary photodynamic effect itself on intact cell membranes. Yielding about 90% killing rate by a combination of electroporation and photooxidation this synergism will be the basis of an extended electrochemotherapy by light irradiation according to the photodynamic mechanism type 1 for the treatment of malignant cells and tissues. Analogous results – including the first synergistic treatment of tumor mice according to photodynamic mechanism type 2 – were discussed, too.
Keywords: Electroporation; Photooxidation; Cancer cells; Synergism; Cytostatic sensitizers;

In order to increase the permeability of cell membranes for low doses of cytostatic drugs, two bioelectrochemical methods have been compared: (a) electric pore formation in the plasma membranes by single electric impulses (electroporation), and (b) reordering of membrane structure by alternating currents (capacitively coupled). These treatments were applied to human leukemic K-562 cells and human lymphoma U-937 cells, yielding apoptotic and necrotic effects, determinated by flow cytometry. Additional cell death occurs after exposure to light irradiation at wavelengths λ  > 600 nm, of cells which were electroporated and had incorporated actinomycin-C or daunomycin (daunorubicine). It is observed that drug uptake after an exponentially decaying electroporation pulse of the initial field strength Eo = 1.4 kV/cm and pulse time constants in the time range 0.5–3 ms is faster than during PEMF-treatment, i.e., application of an alternating current of 16 kHz, voltage U  < 100 V, I  = 55 mA, and exposure time 20 min. However, at the low a.c. voltage of this treatment, more apoptotic and necrotic cells are produced as compared to the electroporation treatment with one exponentially decaying voltage pulse. Thus, additional photodynamic action appears to be more effective than solely drugs and electroporation as applied in clinical electrochemotherapy, and more effective than the noninvasive pulsed electromagnetic fields (PEMFs), for cancer cells in general and animals bearing tumors in particular.
Keywords: Cancer cells; Electroporation; Photodynamics; Capacitive coupling; Apoptosis; Necrosis; Actinomycin-C; Daunomycin;

The performance of tubular microbial fuel cells (MFC) with and without Nafion solution as binding agent for the cathode catalyst preparation was investigated using different electrochemical techniques. The current output of both types of MFCs was monitored as a function of time using an external resistor. The current did not change much with time and was higher for the water cell (WC) than for the Nafion cell (NC). Cell voltage (U c)–current (I) curves were recorded using a potentiodynamic technique. From the U cI curves power concentration (P)–I and PU c curves were constructed. The water cell (without Nafion) also achieved a higher maximum power output. The internal resistance that was determined from the cell voltage at which the power concentration reached its maximum value was higher for the NC than that for the WC, possibly due to the higher cathodic polarization resistance of the NC cell. The impedance for the cathodes decreased with exposure time for both cells due to increased porosity of the surface layers covering the cathode materials. No changes of the impedance were observed for the WC anode. For the NC anode the impedance spectra changed from a one-time constant system to a two-time constant system at the longer exposure time.
Keywords: Microbial fuel cell (MFC); Nafion; Electrochemical impedance spectroscopy (EIS); Internal resistance; Electricity production;

Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization by Saša Haberl; Damijan Miklavčič; Mojca Pavlin (265-271).
Gene electrotransfer is a promising nonviral method that enables DNA to be transferred into living cells with electric pulses. However, there are many parameters that determine gene electrotransfer efficiency. One of the steps involved in gene electrotransfer is interaction of DNA with the cell membrane. Divalent cations in the electroporative media can influence the anchoring of DNA to the cell membrane and by that gene electrotransfer efficiency. Here we report the effect of different concentrations of Mg2+ on electropermeabilization for small molecule (propidium iodide), gene electrotransfer and viability of the cells. We also used TOTO-1 dye to visualize DNA-cell membrane interaction for different [Mg]. For this purpose, we used different electroporative media with increasing [Mg]. Our study shows that higher [Mg] lead to higher electropermeabilization for propidium iodide and higher viability, while causing lower gene electrotransfer efficiency. Because we observed higher TOTO-1 labeled DNA at cell surface when using higher [Mg], we suggest that Mg2+ ions can bind DNA at cell surface at such strength that cannot pass into the cell during application of electric pulses, which can lead to lower gene transfection. There may also be other mechanisms involved, since there are many steps of gene electrotransfer on which Mg2+ ions can have an effect on. Our results also imply that membrane permeability changes are not sufficient for an efficient gene electrotransfer.
Keywords: Mg2+ ions; Gene electrotransfer; Electropermeabilization; Divalent cations; TOTO-1;

In this paper we explored the use of an electrochemical quartz crystal microbalance (QCM) to follow the development of electrochemically active biofilms on electrodes. With this technique it should be possible to monitor simultaneously the increase in biomass and the current generated by the electrogenic bacteria in the biofilm. We monitored the adsorption and the subsequent growth of bacteria that are used in microbial electrolysis cells, on a gold electrode (anode). After attachment it took about 3 h for the bacteria to start to grow and develop a biofilm. Although the current was still relatively low, there is a clear correlation with the increase in biomass. The method is promising for the further investigation of the development of biofilms on electrodes (bioelectrodes).
Keywords: Bioelectrode; Microbial electrolysis; Biofilm growth; Electrogenic bacteria; QCM;