Bioelectrochemistry (v.69, #1)

DNA-based electrochemical biosensors for monitoring of bis-indoles as potential antitumoral agents, chemistry, X-ray crystallography by Dorota Maciejewska; Iwona Szpakowska; Irena Wolska; Maria Niemyjska; Marco Mascini; Magdalena Maj-Żurawska (1-9).
Facile and practical electrochemical DNA bioassay, X-ray diffraction analysis, synthesis and 1H and 13C NMR data of the 5,5′-disubstituted-3,3′-methanediyl-bis-indoles are reported. On the basis of electrochemical measurements we have hypothesized that the analyzed bis-indoles have an effect on human tumor cells due to DNA binding at adenine-thymidine deoxynucleotides rich region in a concentration/substituent dependent manner. Interesting N–H⋯π and hydrogen-bonding intermolecular interactions were observed which may differentiate their biological features. The 5,5′-dimethoxy-3,3′-methanediyl-bis-indole (2) was found to reduce considerably the growth of cancer cell lines HOP-92 (lung), A498 (renal) and MDA-MB-231/1TCC (breast). The results indicate that title compounds could be interesting as potential antitumoral chemotherapeutics.
Keywords: Electrochemical DNA biosensors; 5,5′-Disubstituted-3,3′-methanediyl-bis-indoles; X-ray crystallography; Cytotoxicity;

By vapor deposition method, both hemoglobin (Hb) and colloidal silver nanoparticles (CSNs) were entrapped in a titania sol–gel matrix on the surface of a glassy carbon electrode (GCE). CSNs could greatly enhance the electron transfer reactivity of Hb and its catalytic ability toward nitrite. Direct fast electron transfer between Hb and the GCE was achieved, and a pair of well-defined, quasi-reversible redox peaks was observed. The anodic and cathodic peak potentials are located at − 0.298 V and − 0.364 V (vs. Ag/AgCl), respectively. The dependence of the formal potential on solution pH indicated that the direct electron transfer reaction of Hb was a one-electron transfer coupled with a one-proton transfer reaction process. Meanwhile, the catalytic ability of Hb toward the reduction of NO2 was also studied. Accordingly, a NO2 biosensor was prepared, with a linear range from 0.2 mM to 6.0 mM and a detection limit of 34.0 μM. The apparent Michaelis–Menten constant was calculated to be 7.48 mM. Moreover, the biosensor had good long-term stability.
Keywords: Hemoglobin; Colloidal silver nanoparticles; Titania sol–gel; Direct electrochemistry; Electrocatalysis;

Electrochemistry and kinetics of fungal laccase mediators by G.P. Shumakovich; S.V. Shleev; O.V. Morozova; P.S. Khohlov; I.G. Gazaryan; A.I. Yaropolov (16-24).
The screening of potential redox mediators for laccase was performed using homogeneous Trametes hirsuta laccase. Heterogeneous (electrochemical) and homogeneous (oxidation by laccase) reactions of the different types of the enhancers (mediators) of the enzyme were investigated. It was discovered that derivatives of phenyl-methyl-pyrazolones and benzoic acid, as well as N-hydroxynaphthalimide were efficient substrates for the laccase. The characterization of several representatives from each class was carried out using electrochemical and enzyme kinetics methods. The kinetic parameters for the oxidation of phenyl-methyl-pyrazolones and 3-(6-hylroxy)-aminobenzoic acid were comparable to those for 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by the laccase, whereas the rate of enzymatic oxidation of N-hydroxynaphthalimide was sufficiently lower. Electrochemical experiments demonstrated that only oxidation of phenyl-methyl-pyrazolones and N-hydroxynaphthalimide yielded several high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, whereas derivatives of benzoic acid showed low-potential intermediate, which was not able to oxidized lignin model compound. Phenyl-methyl-pyrazolones was about 50% as effective in degrading veratryl alcohol compared to ABTS as judged from HPLC kinetic studies, whereas N-hydroxynaphthalimide showed the same efficiency as ABTS. Phenyl-methyl-pyrazolones and hydroxynaphthalimides may be of commercial interest for oxidoreductase-catalyzed biodegradation of different xenobiotics.
Keywords: Laccase; Mediator; Enhancer; Phenyl-methyl-pyrazolone; Benzoic acid; Hydroxynaphthalimide;

Remote NADH imaging through an ordered array of electrochemiluminescent nanoapertures by Arnaud Chovin; Patrick Garrigue; Neso Sojic (25-33).
In this report, we present an ordered array comprising thousands of nanoapertures for the electrochemiluminescent (ECL) detection of NADH. It was fabricated on the distal face of a coherent optical fiber bundle. Such a high-density array of nanoapertures combines optical, imaging and electrochemical properties. Indeed, each nanoaperture is surrounded by a gold nanoring, which acts as an electrode material. The behavior of the array was characterized by cyclic voltammetry and it shows excellent electrochemical performances. NADH is the analyte, which is measured in presence of Ru(bpy)3 2+. The ruthenium complex mediates the NADH oxidation and this coenzyme acts as a co-reactant in the ECL mechanism. ECL light is generated at the distal face of the array by each gold ring electrode. A fraction of this ECL light is collected by the corresponding nanoaperture, transmitted through the optical fiber bundle and finally imaged on the proximal face with a CCD camera. In this work, we show that NADH concentration is remotely detected by an oxidative-reductive ECL mechanism. We present also some preliminary results about the ECL process of NADH with Ru(bpy)3 2+. The ECL behavior of NADH on gold surface is reported. The influence of the applied potential on the collected light intensity was investigated. The variation of the ECL intensity measured through the nanoaperture array with NADH concentration is linear. Remote ECL detection of NADH is spatially resolved over a large area with a micrometer resolution through the array. Therefore, such array integrates several complementary functions: ECL light generation, collection, transmission and remote imaging in an array format.
Keywords: Electrochemiluminescence; NADH; Array; Nanoaperture; Optical fiber bundle;

Electrochemical study of photovoltaic effect of nano titanium dioxide on hemoglobin by Hui Zhou; Xin Gan; Tao Liu; Qianlu Yang; Genxi Li (34-40).
Nano titanium dioxide (TiO2) and hemoglobin (Hb) were co-modified on pyrolytic graphite (PG) electrode to study the photovoltaic effect of TiO2 nanoparticles (NPs) on the electron transfer reactivity and catalytic activity of the protein. By means of cyclic voltammetry (CV) and FTIR measurements, the study was characterized in both aerobic and anaerobic environments. Experimental results revealed that the factors which mainly interacted with Hb were electron/hole pairs and reactive oxygen species (ROS) generated by the photovoltaic effect when TiO2 NPs were irradiated under ultraviolet (UV) light. The electron/hole pairs generated on the surface of TiO2 would influence the structure of Hb gently, so the electron transfer reactivity and catalytic ability of the protein slightly changed. In contrast, ROS interacted with Hb intensively, which brought in much conformational change to Hb and its active centers, and even cause some damage. Consequently, the electron transfer reactivity and catalytic activity of Hb changed with a process of increasing initially and decreasing afterwards.
Keywords: Titanium dioxide; Photovoltaic effect; Hemoglobin; Electrochemistry;

A conducting, polymeric film of poly(indole-5 carboxylic acid) has been prepared by electrochemical polymerization for covalent immobilization of an enzyme belonging to the family of phenoloxidases-tyrosinase. The polymer was characterized by cyclic voltammetry, UV–VIS and Raman spectroscopy in a buffer solution. As the polymer contains pendant carboxylic groups one-step carbodiimide method was used to immobilize tyrosinase on the polymer matrix. Immobilization of tyrosinase was confirmed by surface enhanced resonance Raman scattering spectra (SERRS) and by cyclic voltammetry as well. Tyrosinase was shown to retain its biological activity when being immobilized on the polymer surface. As proved by the electrochemical and spectroelectrochemical (UV–VIS) experiments, tyrosinase covalently bonded to the polymer matrix effectively catalyzes oxidation of catechol. The reduction current of o-quinones was measured as a function of catechol concentration. The linear dependence was found to be 15 μM of catechol with sensitivity of 250 mA/M cm2.
Keywords: Tyrosinase; Immobilization; Poly(indole-5 carboxylic acid); Raman spectra;

Dielectric spectroscopy of Anabaena 7120 protoplast suspensions by Kongshuang Zhao; Wei Bai; Hualing Mi (49-57).
The dielectric spectroscopy of Anabaena 7120 protoplast suspensions has been investigated over the frequency range of 40 Hz–110 MHz. The protoplast suspensions showed a complicated dielectric dispersion consisting of at least four distinct sub-dispersions with the increasing frequencies due to the Maxwell–Wagner interfacial polarization. The double-shell model, in which an equivalent shell of thylakoid was assumed inside the cytoplasm, was adopted to describe the special morphology of the protoplast. Under the assumption that the conductivity of plasmalemma was negligible and the conductivity of the equivalent shell was 0.1 μS/cm, we attempted to estimate the dielectric properties of various protoplast components by fitting theoretical curve to experimental data. The relative permittivity of the plasmalemma ε mem was estimated to be 6.5 ± 0.5, and the permittivity of the equivalent shell of thylakoid ε thy was estimated to be about 3.2 ± 0.2. The permittivity ε cyt and conductivity κ cyt of the cytoplasm were estimated to be 60 and 0.88 ± 0.11 mS/cm, respectively. The permittivity ε nuc and conductivity κ nuc of the nucleoplasmic region were determined to be 100 and 0.13 ± 0.02 mS/cm, respectively.
Keywords: Dielectric spectroscopy; Anabaena; Protoplast; Thylakoid;

Electrochemical detection of scDNA cleavage in the presence of macrocyclic hexaaza–copper(II) complex by Jingwan Kang; Shuqing Dong; Xiaoquan Lu; Biquan Su; Haixia Wu; Kun Sun (58-64).
The hexaaza macrocyclic copper(II) complex Cu(II)L(L=1,8-Dihydroxyethyl-1,3,6,8,10,13-hexaazacyclotetradecane), which has octahedral structure similar to some natural complexes, is synthesized and purified. In this study, oxidative breakage DNA by the reaction of Cu(II)L with H2O2 and ascorbate has been investigated by gel electrophoresis experiments. In electrochemical experiments, the on scDNA-modified glassy carbon electrode(GCE) is cleaved by the Cu(II)L and redox changing of the metal catalyst without adding any other reagents. Above all, the need for concentration of scDNA is much lower than that of gel electrophoresis experiments and the process of the performance is easy. Furthermore, Cyclic Voltammetry (CV) and A.C. Impedance, which are performed to monitor scDNA cleavage at the scDNA-modified glassy carbon electrode (GCE), are fast, simple and highly efficient. The mechanism of the damage can be suggested: Fenton.
Keywords: pBR322 supercoiled DNA; scDNA-modified GCE; Cu(II)L; Cleave; Cyclic Voltammetry; A.C. Impedance;

Electrocatalytical properties of polymethylferrocenyl dendrimers and their applications in biosensing by M. Pilar García Armada; José Losada; Magdalena Zamora; Beatriz Alonso; Isabel Cuadrado; Carmen M. Casado (65-73).
The electrochemical characterization of polymethylferrocenyl dendrimers deposited onto a platinum electrode and their applications as hydrogen peroxide and glucose sensor are described. The redox dendrimers consist of flexible poly(propileneimine) dendrimer cores functionalised with octamethylferrocenyl units. Amperometric biosensors for glucose were prepared by immobilization of glucose oxidase onto these modified electrodes. The influence of the dendrimer generation and the thickness of the dendrimer layer, the effect of the substrate concentration, and the interferences and reproducibility on the response of the sensors were investigated.
Keywords: Polymethylferrocene; Dendrimers; Modified electrodes; Glucose oxidase; Amperometric biosensor;

Escherichia coli-catalyzed bioelectrochemical oxidation of acetate in the presence of mediators by Yung-Fu Wang; Sheng-Shung Cheng; Seiya Tsujimura; Tokuji Ikeda; Kenji Kano (74-81).
Bioelectrocatalytic oxidation of acetate was investigated under anaerobic conditions by using Escherichia coli K-12 (IFO 3301) cells cultured on aerobic media containing poly-peptone, glucose or acetate as the sole carbon source. It was found that all E. coli cells cultured on the three media work as good catalysts of the electrochemical oxidation of acetate as well as glucose with Fe(CN)6 3−, 2,3-dimethoxy-5-methyl-1,4-benzo-quinone (Q0), 2,6-dichloro-indophenol, or 2-methyl-1,4-naphthoquinone as artificial electron acceptors (mediators). Acetate-grown E. coli cells exhibited the highest relative activity of the acetate oxidation against the glucose oxidation. On the other hand, all the artificial electron acceptors used work as inhibitors for the catalytic oxidation of acetate at increased concentrations. The inhibition phenomenon can be interpreted in terms of competitive substrate inhibition as a whole. Apparent values of Michaelis constant, catalytic constant, and inhibition constant were evaluated by amperometric methods. Q0 is an effective artificial mediator as evidenced by a large reaction rate constant between the cell and Q0 at least at low concentrations (< 50 μM). However, Fe(CN)6 3− is a promising mediator in biosensor applications because the inhibition constant is very large and it works as an electron acceptor even under aerobic conditions.
Keywords: Escherichia coli; Acetate oxidation; Mediated bioelectrocatalysis; Inhibition;

Kinetics of the surface redox reactions of adriamycin (doxorubicin hydrochloride) adsorbed on paraffin-impregnated graphite electrode (PIGE) and on mercury electrode is measured by square-wave voltammetry. In 0.9 mol/L KNO3 buffered to pH 4.65, the standard electrode reaction rate constants of the first quinone/hydroquinone redox couple (see Scheme 2) on PIGE and mercury are k s1  = 49 ± 12 s− 1 and k s1  = 147 ± 36 s− 1, respectively. Under the same conditions, the standard rate constant of the second redox couple on the PIGE is smaller than 4 s− 1 and the electron transfer coefficient of the reduction is α 2  = 0.35.
Keywords: Adriamycin; Doxorubicin; Kinetics; Redox reaction; Surface; Graphite electrode; Mercury electrode; Square-wave voltammetry;

Conditions that stimulate action potentials in one or more nerves is of widespread interest. Axon and nerve models are usually based on two dimensional pre-specified lumped equivalents that assume where currents will flow. In contrast, here we illustrate creation of three dimensional (3D) system models with a transport lattice of interconnected local models for external and internal electrolyte and axon membrane. The transport lattice solves Laplace's equation in the extracellular medium and is coupled to the Hodgkin–Huxley model at local membrane sites. These space-filling models incorporate the geometric scale, which allows explicit representation of confined axons and external electrodes. The present results demonstrate feasibility of the basic approach. These models are spatially coarse and approximate, but can be straightforwardly improved. The transport lattice system models are modular and multiscale (spatial scales ranging from the membrane thickness of 5 nm to the axon segment length of 2 cm).
Keywords: Transport lattice model; Axon; Action potential; External electrodes; Three dimensional model;

In this study we present novel evidence that strengthens the paradigm of selective transfer of energy mediated by a random gating of ion channels. Specifically, we investigated the spectral response of a noisy artificial biomembrane whose electrical properties were largely dictated by embedded alamethicin oligomers. In this respect, we first evaluated experimentally the linear transfer function of the system via the white-noise analysis method. We prove that such a system displays specific ranges of frequency over which input signals pass preferentially, depending on their spectral content and the holding potential across the artificial bilayer which contains alamethicin. By employing voltage-driven periodic stimulation of alamethicin oligomers, we demonstrate that overall response of the system obeys qualitatively the predictions inferred from the transfer function analysis of it. These results emphasize the exquisite ability of excitable membranes to behave as band-limited filters and allow for maximal transfer of energy from an external stimulus over well-defined frequency ranges.
Keywords: Spectral analysis; Alamethicin; Admittance; Lipid membranes; Excitability; Voltage-clamp;

Synthesis of DnaK and GroEL in Escherichia coli cells exposed to different magnetic field signals by Brunella Del Re; Ferdinando Bersani; Pietro Mesirca; Gianfranco Giorgi (99-103).
The effects of extremely low frequency magnetic field (ELF-MF)(1 mT, 50 Hz) on the heat shock protein (HSP) synthesis in Escherichia coli were investigated. Two magnetic field signals were studied: sinusoidal (SMF) and pulsed square wave (PMF). It was found that bacteria exposed to SMF showed a significantly higher level of DnaK and GroEL proteins as compared to sham-exposed bacteria as revealed by Western blot, whereas a lower level was observed after PMF exposure. Similar results were obtained when bacterial cells were exposed to heat shock (HS) after ELF-MF exposure: again SMF and PMF resulted in an increase and in a reduction of HSP amount in comparison with sham control, respectively. In conclusion, the MF influences the synthesis of HSPs in E. coli in a way that critically depends on the signal characteristics.
Keywords: Escherichia coli; Heat shock protein; Extremely-low-frequency magnetic field; Sinusoidal magnetic field; Square magnetic field;

Three new nitrofuryl substituted 1,4-dihydropyridine derivatives were electrochemically tested in the scope of newly found compounds useful as chemotherapeutic alternative to the Chagas' disease.All the compounds were capable to produce nitro radical anions sufficiently stabilized in the time window of the cyclic voltammetric experiment. In order to quantify the stability of the nitro radical anion we have calculated the decay constant, k 2. Furthermore, from the voltammetric results, some parameters of biological significance as E 7 1 (indicative of in vivo nitro radical anion formation) and K O2 (thermodynamic indicator of oxygen redox cycling) have been calculated. From the comparison of E 7 1, K O2 and k 2 values between the studied nitrofuryl 1,4-DHP derivatives and well-known current drugs an auspicious activity for one of the studied compounds i.e. FDHP2, can be expected.
Keywords: Nitro radical anion; Nitrofuryl, 1,4-dihydropyridine; Chagas' disease; Decay constants;

Real-time assay of immobilized tannase with a stopped-flow conductometric device by Fu-Shiang Chang; Po-Chung Chen; Richie L.C. Chen; Fu-Ming Lu; Tzong-Jih Cheng (113-116).
A stopped-flow manifold was developed to assay and characterize immobilized tannase (EC The immobilized enzyme reactor was inserted within the tube-type electrode pair (cell constant = 103.2 cm− 1) for a real-time conductometric measurement. Tris buffer (2 mM, pH = 7.0) was used as the carrier for sensitivity improvement. The activities and kinetic parameters (K m values) for propyl gallate, methyl gallate and tannic acid were investigated.
Keywords: Conductometric; Enzyme kinetics; Tannic acid; Stopped-flow;

Microfluidic electroporation of robust 10-μm vesicles for manipulation of picoliter volumes by Eunice S. Lee; David Robinson; Judith L. Rognlien; Cindy K. Harnett; Blake A. Simmons; C.R. Bowe Ellis; Rafael V. Davalos (117-125).
We present a new way to transport and handle picoliter volumes of analytes in a microfluidic context through electrically monitored electroporation of 10–25 μm vesicles. In this method, giant vesicles are used to isolate analytes in a microfluidic environment. Once encapsulated inside a vesicle, contents will not diffuse and become diluted when exposed to pressure-driven flow. Two vesicle compositions have been developed that are robust enough to withstand electrical and mechanical manipulation in a microfluidic context. These vesicles can be guided and trapped, with controllable transfer of material into or out of their confined environment. Through electroporation, vesicles can serve as containers that can be opened when mixing and diffusion are desired, and closed during transport and analysis. Both vesicle compositions contain lecithin, an ethoxylated phospholipid, and a polyelectrolyte. Their performance is compared using a prototype microfluidic device and a simple circuit model. It was observed that the energy density threshold required to induce breakdown was statistically equivalent between compositions, 10.2 ± 5.0 mJ/m2 for the first composition and 10.5 ± 1.8 mJ/m2 for the second. This work demonstrates the feasibility of using giant, robust vesicles with microfluidic electroporation technology to manipulate picoliter volumes on-chip.
Keywords: Electroporation; Electropermeabilization; Giant vesicle; Giant liposome; Sample management;

Decrease of luminol chemiluminescence upon exposure of human blood serum to 50 Hz electric fields by Violeta Calota; Simona Dragoiu; Aurelia Meghea; Maria Giurginca (126-127).
The chemiluminescence of luminol, after 1 and 2 h in vitro exposure of human serum to 50 Hz electric fields of different intensities, decreases as compared to the controls. This indicates a field-induced decrease in the concentration of the free radicals. The report is limited to the key kinetic and field data, inviting independent kinetic analysis of the data in terms of reaction moments or reaction susceptibilities for the various normal modes indicated by the data.
Keywords: Electric field exposure; Free radicals; Chemiluminescence; Human serum;

Graphite epoxy composite electrodes modified with bacterial cells by Ülkü Anık Kırgöz; Dilek Odacı; Suna Timur; Arben Merkoçi; Nurdan Pazarlıoğlu; Azmi Telefoncu; Salvador Alegret (128-131).
The modification of a graphite–epoxy composite electrode (GECE) with bacterial cells along with an analytical application are presented. Pseudomonas putida DSM 50026 was used as a biological component and the measurement was based on the respiratory activity of the cells. The optimization of working conditions of resulting biosensor (including pH and temperature) was conducted and the limit of detection was calculated as 7 μM phenol based on the signal to noise ratio. Then the system was applied for xenobiotic detection. Resulting sample signals were found to be very similar with the standard solutions having the same concentration while the recoveries of the spiked samples were close to 100%.
Keywords: Microbial biosensor; Graphite–epoxy composite electrode; Pseudomonas putida;