Bioelectrochemistry (v.77, #2)

Nanostructured zinc oxide platform for mycotoxin detection by Anees A. Ansari; Ajeet Kaushik; Pratima R. Solanki; B.D. Malhotra (75-81).
Nanostructured zinc oxide (Nano-ZnO) film has been deposited onto indium–tin–oxide (ITO) glass plate for co-immobilization of rabbit-immunoglubin antibodies (r-IgGs) and bovine serum albumin (BSA) for ochratoxin-A (OTA) detection. The results of X-ray diffraction (XRD) studies reveal the formation of Nano-ZnO with average particle size as ~5.0 nm. Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) techniques have been used to characterize Nano-ZnO/ITO electrode and BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode. Electrochemical impedimetric response of BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode obtained as a function of OTA concentration exhibits linearity as 0.006–0.01 nM/dm3, detection limit of 0.006 nM/dm3, response time as 25 s and sensitivity of 189 Ω/nM/dm3cm− 2 with a regression coefficient of 0.997.
Keywords: Sol–gel; Zinc oxide; Impedimetric immunosensor; Mycotoxin; Ochratoxin-A;

Electroreduction of nitrite to nitrogen oxide by a copper-containing nitrite reductase model complex incorporated into collagen film by Naoko Isoda; Hiroshi Yokoyama; Masaki Nojiri; Shinnichiro Suzuki; Kazuya Yamaguchi (82-88).
The electrocatalytic reduction of nitrite to NO by CuMe2bpaCl2, which is a model for the active site of copper-containing nitrite reductase, incorporated into collagen film was investigated. The 77-K EPR spectrum of CuMe2bpaCl2 in the collagen matrix revealed the typical axial signals (g //  = 2.26, g  = 2.05, A //  = 16.4 mT) of a tetragonal Cu2+ chromophore. The redox potential, which is related to the Cu+/Cu2+ couple, was − 63 mV (∆E  = 72 mV) at pH 5.5. In the presence of nitrite, an increase in the cathodic current was observed in the cyclic voltammogram of CuMe2bpaCl2 in the collagen matrix. Upon reaching − 300 mV, a linear generation of NO was observed for the CuMe2bpaCl2/collagen film-coated electrode. The relationship between the rate of NO generation and the nitrite concentration in solution was analyzed using the Michaelis–Menten equation, where V max  = 3.16 nM s− 1 and K m  = 1.1 mM at pH 5.5. The current increase and the reaction rate were dependent on the pH of the solution. The mechanism of nitrite reduction by the copper complex in the collagen matrix was the same mechanism as that of the enzyme in aqueous solution.
Keywords: Collagen; Copper-containing nitrite reductase; Electrocatalytic reduction of nitrite; NO; Model complex;

Electrochemical current rectification at bio-functionalized electrodes by Yaqing Liu; Andreas Offenhäusser; Dirk Mayer (89-93).
In the present paper, we demonstrate the electrochemical rectification of a redox current which is transferred between redox probes (ferricyanide) in solution and a gold electrode functionalized with the biomolecular redox mediator microperoxidase-11 (MP-11). MP-11 is the redox active, heme-containing domain of the biological electron shuttle cytochrome c (cyt c). In our system, a unidirectional current develops due to selective electron transport from the bio-functionalized electrode to ferricyanide such that MP-11 controls the read-out of our coupled redox system. The electrode was functionalized by adding a monolayer of undecanethiol (UDT) to promote the physisorption of MP-11 and inhibit the direct electron transfer between redox probe and electrode. The relative position of redox donator, mediator, and acceptor equilibrium potentials defines the charge transport and a potential-dependent electrochemical current rectification. The results of our investigations demonstrate that functional building blocks of proteins can be reassembled into new conceptual devices with operation modes deviating from their native function, which could prove highly useful in future design of biosensors and bioelectronic systems.
Keywords: Bio-diode; MP-11; Gold electrode; Bioelectrochemical current rectification; Bio-ECR;

This paper reports the development of an electrochemical biosensor for the detection of formaldehyde in aqueous solution, based on the coupling of the enzyme formaldehyde dehydrogenase and a carbon nanotubes (CNT)-modified screen-printed electrode (SPE). We monitored the amperometric response to formaldehyde released from U251 human glioblastoma cells situated in the biosensor chamber in response to treatment with various anticancer prodrugs of formaldehyde and butyric acid. The current response was higher for prodrugs that release two molecules of formaldehyde (AN-193) than for prodrugs that release only one molecule of formaldehyde (AN-1, AN-7). Homologous prodrugs that release one (AN-88) or two (AN-191) molecules of acetaldehyde, showed no signal. The sensor is rapid, sensitive, selective, inexpensive and disposable, as well as simple to manufacture and operate.
Keywords: Formaldehyde; Prodrugs; Glioblastoma; Butyric acid; Carbon nanotubes; Screen printed electrodes;

An electrochemical biosensor, constructed by immobilization of Zr(IV) on the topside of gold–mercaptopropionic acid self-assembled monolayer (Au–MPA–Zr SAM), is developed for the sensitive quantification of calf thymus DNA (ct-DNA). The sensor is based on ionic adsorption of ct-DNA from its phosphate backbone onto the Au–MPA–Zr(IV) SAM electrode. Preparation, characterization, and application of the sensor for determination of ct-DNA are described by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and Osteryoung square wave voltammetry (OSWV) in the presence of an appropriate redox reaction probe. Parameters influencing the method have been tested. A linear range calibration curve from 1.0 × 10− 4 to 5.0 × 10− 7 g mL− 1 ct-DNA with a detection limit of 9.5 × 10− 8 g mL− 1 and mean of relative standard deviations (R.S.D) of 2.5% for n  = 4 at each point was observed in the best conditions by EIS. Regeneration of the surface was carried out successfully by 5 min sonication in 0.1 M KOH solution and then 1 min incubation in 1.0 × 10− 3 M Zr(IV) with a good reproducibility, R.S.D = 1.5% for n  = 4 as detected by EIS. The long-term storage stability of the electrode was also studied.
Keywords: Zirconium; Calf thymus DNA; Electrochemical impedance spectroscopy; Self-assembled monolayers; DNA sensor;

To examine the amplification of the effects of low-frequency electric fields due to the junction between the cells, the amplitude of transmembrane potential P TMP0, the time-averaged normal stress σ n and the outward force density σ out on the shell-phase, and the squared intensity I ee of electric fields in the inner and the outer phases induced by uniform external ac fields were calculated with the boundary-element method for doublet-shaped cell models consisting of two spheres of the same size connected by the junction; the results were compared with those in a spherical model. When the external fields were parallel to the long axis of the doublet-shaped models, P TMP0, σ n and σ out at the pole were greater than those in the spherical model, and σ out and I ee at the junction increased with the decrease in the junction-radius. The external fields perpendicular to the long axis caused I ee greater than that at the center of the spherical model and negative σ out, at the junction. The amplification of P TMP0, σ n, σ out and I ee took place within restricted frequency-regions that could be specified by the characteristic frequencies for the frequency-dependence of the polarization factor of the models.
Keywords: Dielectric relaxation; Electric field; Maxwell stress tensor; Simulation; Transmembrane potential;

Approaching intelligent infection diagnostics: Carbon fibre sensor for electrochemical pyocyanin detection by Duncan Sharp; Patience Gladstone; Robert B. Smith; Stephen Forsythe; James Davis (114-119).
Pyocyanin is produced by Ps. aeruginosa as a result of quorum sensing during wound colonisation increasing bacterial virulence and damaging host physiology, both of which contribute to an increased risk of infection. The use of carbon fibre tow as an electrochemical sensing matrix for assessing pyocyanin production is evaluated. Prototype sensor assemblies have been developed and response characteristics towards pyocyanin are detailed. The sensitive and linear quantification of pyocyanin is presented (r 2  = 0.998) across the biomedically relevant concentration range (1–100 µM). Precise electrochemical measurements of pyocyanin by square wave voltammetry are established using carbon fibre assemblies (coefficient of variance = 1.2 and 1.4% for 10 and 50 µM pyocyanin, respectively). Further testing of the sensors in bacterial cultures shows the ability to monitor pyocyanin production by Ps. aeruginosa in agreement with the chloroform-acid/photometric method and in the presence of other bacterially derived pigments and metabolites. The proposed small and inexpensive sensor assembly is suggested for use in monitoring Ps. aeruginosa growth.
Keywords: Carbon fibre; Smart bandage; Pyocyanin; Pseudomonas aeruginosa; Infection;

Electrochemical oxidation of glucose on a glassy carbon disc electrode modified with multi-walled carbon nanotubes and nickel(II) oxide (GC/MWCNT/NiO) was examined by cyclic voltammetry and chronoamperometry in alkaline aqueous solutions. The results were compared with those obtained on a nickel(II) oxide modified glassy carbon electrode (GC/NiO). Both electrodes conditioned by potential cycling in a limited potential range (0.1–0.6 V vs. Ag/AgCl) in 0.10 M NaOH solution. It was found that the multi-walled carbon nanotubes improve remarkably the reactivity of nickel(II) oxide for glucose oxidation. The GC/MWCNT/NiO electrode exhibited good linear behavior in the concentration range from 2.0 × 10− 4 mol/L to 1.2 × 10− 2 mol/L for the quantitative analysis of glucose with a limit of detection of 1.6 × 10− 4 mol/L (3σ). The prepared electrode exhibits satisfactory stability and long life if stored at ambient conditions. Finally, it has been demonstrated that the proposed modified electrode can be successfully used for the assay of glucose in serum samples.
Keywords: Glucose; Electrooxidation; Multi-walled carbon nanotubes; Nickel oxide;

Performance of microbial fuel cell (MFC) was evaluated with the function of phosphatase and dehydrogenase activities at increasing organic loading rate (OLR) (0.195 kg chemical oxygen demand (COD)/m3-day; 0.458 kg COD/m3-day; 0.911 kg COD/m3-day; 1.589 kg COD/m3-day). Variation in enzyme activities along with power generation and substrate degradation was observed during MFC operation with the function of organic loading rate (OLR). Phosphatase activity showed a decreasing trend with time from 24 to 36th hour depending on OLR which is a good sign of substrate utilization. Dehydrogenase activity was observed to be high at the 12th hour irrespective of the OLR. However, the activity was increased with increasing OLR. Higher dehydrogenase activity was observed at 1.589 kg COD/m3-day representing the possibility of higher redox reactions. Higher power output was recorded at the 12th hour with 53.58 mW/m2 (0.195 kg COD/m3-day) and 24th hour with 60.29 mW/m2 (0.458 kg COD/m3-day) and 76.17 mW/m2 (0.911 kg COD/m3-day). At higher OLR studied (1.589 kg COD/m3-day), maximum power generation (49.86 mW/m2) was observed at 12th hour indicating decreased performance. Electron discharge and recovery properties observed during MFC operation were supporting higher performance at 0.911 kg COD/m3-day. Increase in OLR showed improvement in substrate degradation [OLR1, 56.32% (0.11 kg COD/m3-day); OLR2, 56.42% (0.26 kg COD/m3-day); OLR3, 59.53% (0.54 kg COD/m3-day); OLR4, 64.40% (1.78 kg COD/m3-day)].
Keywords: Anaerobic; Enzyme; Electron discharge; Mixed culture; Wastewater treatment;

Determination of isoniazid in human urine using screen-printed carbon electrode modified with poly-l-histidine by Márcio F. Bergamini; Daniela P. Santos; Maria Valnice B. Zanoni (133-138).
A sensitive voltammetric method for trace measurements of isoniazid (INZ) in synthetic human urine sample is described. The method is based on its electroreduction at − 0.98 V vs. Ag/AgCl on screen-printed carbon electrode (SPCE) modified with poly-l-histidine (PH). A film of good adherence on SPCE and electrocatalytic properties was obtained coating the electrode by histidine monomer electropolymerization process (SPCE/EPH). The electrochemical behavior of the modified SPCE was investigated by cyclic, linear sweep (LSV), differential pulse (DPV), square-wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). Limits of detection of 5.0 × 10− 7 mol L− 1, 1.7 × 10− 7 mol L− 1 and 2.5 × 10− 7 mol L− 1 were estimated from LSV, DPV and SWV determinations, respectively. The method was successfully applied to the determination of INZ in human urine samples.
Keywords: Screen-printed electrode; Modified electrode; Poly-l-histidine; Isoniazid; Determination;

In this paper, an acetylcholinesterase (AChE)/dendrimers polyamidoamine (PAMAM)-Au/Carbon nanotubes (CNTs) multilayer modified electrode based on LbL self-assembled technique was employed in the detection of carbofuran in samples. The configuration of the nanostructure on the electrode provided a favorable environment to the immobilization of AChE. The modified films also improved the electrocatalytic characteristics and electron transfer speed between the films and the surface of electrode. The PAMAM-Au nanoparticles were characterized by SEM and UV–VIS methods. A set of experimental conditions were also optimized for the detection of the pesticides. A linear response over carbofuran concentration in the range of 4.8 × 10− 9  M to 0.9 × 10− 7  M was exhibited with a detection limit of 4.0 × 10− 9  M. The biosensor showed high sensitivity, good stability and reproducibility with promising application.
Keywords: PAMAM-Au; Inhibition; Carbofuran; Sensor;

Electrochemical behaviour of dimethyl-2-oxoglutarate on glassy carbon electrode by Afzal Shah; Victor C. Diculescu; Rumana Qureshi; Ana Maria Oliveira-Brett (145-150).
The electrochemical behaviour of dimethyl-2-oxoglutarate (MOG), a key intermediate in the Krebs cycle and an important nitrogen transporter in the metabolic pathways in biological processes, was investigated by cyclic voltammetry, square wave voltammetry and differential pulse voltammetry using a glassy carbon electrode. The reduction of MOG is an irreversible diffusion-controlled process that occurs in a cascade mechanism. For electrolytes with pH <3.0 and pH >7.0 one peak occurred and for 3.0 < pH < 8.0 two peaks corresponding to consecutive charge transfer reactions were observed. The effects of scan rate, concentration and pH of the electrolyte solution were monitored, and both peaks were found to shift cathodically with the increase in pH. DPV measurements allowed the determination of the number of electrons and protons i.e., one electron and one proton, involved in the reduction mechanism of MOG. Based upon the results obtained a reduction mechanism was proposed and the observed waves were attributed to the hydroxylation of the keto group of MOG to form dimethyl-2-hydroxyglutarate. Furthermore, two methodologies for the electroanalytical determination of MOG were also compared.
Keywords: Dimethyl-2-oxoglutarate; Voltammetry; Adsorption; Electro-reduction;

The alterations of the passive electrical parameters (the permittivity ε and the electrical conductivity σ) of human erythrocyte cell membrane induced by the presence of glucose in the extracellular medium have been investigated by means of dielectric spectroscopy measurements. The membrane permittivity ε s and the permittivity ε p and electrical conductivity σ p of the cytosol have been evaluated on the basis of a recent analytical model proposed by Prodan et al., 1983 [16], that takes into account the whole dielectric spectrum of a cell suspension, consisting of both the low-frequency α-dispersion and the high-frequency β-dispersion. Our results show a marked increase of the membrane permittivity ε s close to a glucose concentration of 20 mM. On the contrary, the electrical properties of the cytosol do not change appreciably. This finding strengthens the hypothesis that glucose interactions involve primarily the cell membrane and the mechanism of the transport is briefly discussed.
Keywords: Human erythrocytes; Dielectric spectroscopy technique; Glucose transport across the membrane;

Analysis of radiofrequency energy stored in the altered shapes: Stomatocyte–echinocyte of human erythrocytes by Sagrario Muñoz; José Luis Sebastián; Miguel Sancho; Genoveva Martínez (158-161).
The aim of this study is to analyze the electromagnetic energy stored in stomatocyte, erythrocyte and echinocyte cells exposed to a linearly polarized electromagnetic plane wave at 900, 1800 and 2450 MHz radiofrequency signals. This analysis can provide a better understanding of the order of appearance of altered shapes of erythrocytes (RBC) in the stomatocyte–echinocyte transition under radiofrequency exposure in terms of the deposited electromagnetic energy. For this purpose we use a realistic geometrical cell model based on parametric equations that allow for continuous transformations between normal erythrocytes and three stomatocyte subclasses with different degree of invagination and also between normal erythrocytes and echinocytes with an arbitrary number of spicules. We use a finite element technique with adaptive meshing for calculating the electromagnetic energy deposited on the different regions of the cell models. It is found that the echinocyte cell stores the minimum electromagnetic energy and therefore from an energetic point of view it would be the most stable and preferred cell state when this electromagnetic energy is the predominant energy component.
Keywords: Red blood cells; Stomatocyte; Echinocyte; Deformability of cells; Erythrocyte shape;