Bioelectrochemistry (v.102, #C)

A new uric acid biosensor was constructed using ferrocene (Fc) induced electro-activated uricase (UOx) deposited within Nafion (Naf) on glassy carbon electrode (GCE). Electro-activation of UOx was successfully achieved by cyclic voltammetry through the electrostatic interaction of Fc with Trp residues within the hydrophobic pockets in UOx. The Naf/UOx/Fc composite was characterised by AFM, FTIR and EDX to ensure proper immobilisation. The interaction of Fc with the enzyme was analysed by Trp fluorescence spectroscopy and the α-helicity of the protein was measured by CD spectropolarimetry. The charge transfer resistance (Rct), calculated from electrochemical impedance spectroscopy, for the modified sensor was lowered (1.39 kΩ) and the enzyme efficiency was enhanced by more than two fold as a result of Fc incorporation. Cyclic voltammetry, differential pulse voltammetry and amperometry all demonstrated the excellent response of the Naf/UOx/Fc/GCE biosensor to uric acid. The sensor system generated a linear response over a range of 500 nM to 600 μM UA, with a sensitivity and limit of detection of 1.78 μA μM− 1 and 230 nM, respectively. The heterogeneous rate constant (ks) for UA oxidation was much higher for Naf/UOx/Fc/GCE (1.0 × 10− 4  cm s− 1) than for Naf/UOx/GCE (8.2 × 10− 5  cm s− 1). Real samples, i.e. human blood, were tested for serum UA and the sensor yielded accurate results at a 95% confidence limit.Display Omitted
Keywords: Uricase; Ferrocene; Uric acid; Biosensor; Cyclic voltammetry; Differential pulse voltammetry;

A mathematical model for electrochemically active filamentous sulfide-oxidising bacteria by Keelan M. Fischer; Damien J. Batstone; Mark C.M. van Loosdrecht; Cristian Picioreanu (10-20).
Oxygen and sulfide in ocean sediments can be consumed biologically over long spatial distances by way of filamentous bacteria in electron-conducting sheaths. To analyse observations, a mathematical model of these filamentous sulfur-oxidising bacteria was developed, including electrical conduction between reactive zones. Mechanisms include Nernst–Planck diffusion and migration of ions coupled with Ohm's law for conduction along filaments, and metabolic activity throughout the filaments. Simulations predict outward biomass growth toward the boundaries of the sediment floor and top surface, resulting in two distinct zones with anode (sulfide consumption) and cathode (oxygen consumption) reactions enabled by electron conduction. Results show inward fluxes of 4.6 mmol O2/m2/d and 2.5 mmol S/m2/d, with consumption increasing with growth to final fluxes of 8.2 mmol O2/m2/d and 4.34 mmol S/m2/d. Qualitatively, the effect of varying cell conductivity and substrate affinity is evaluated. Controlling mechanisms are identified to shift from biomass limitation, to substrate limitation, and to conductivity limitations as the lengths of the filaments increase. While most observed data are reflected in the simulation results, a key discrepancy is the lower growth rates, which are largely fixed by thermodynamics, indicating that microbes may utilise secondary substrates or an alternative metabolism.Display Omitted
Keywords: Sulfur-oxidising bacteria; Filaments; Current conduction; Desulfobulba; Mathematical model;

Topotecan (TPT) is a semisynthetic, water soluble analog of the plant alkaloid camptothecin which has been widely used for the treatment of ovarian and cervical cancers. To obtain better understanding on how it can affect DNA structure, electrochemical biosensor platforms for the investigation of TPT-double stranded DNA (dsDNA) interaction were developed for the first time in this study. The electrochemical detection of TPT, and TPT–dsDNA interaction were investigated at the surface of pencil graphite electrodes (PGEs) and single-walled carbon nanotube (SWCNT) modified PGEs by using differential pulse voltammetry (DPV). The changes at the oxidation signals of TPT and guanine were evaluated before/after each modification/immobilization step. An enhanced sensor response was obtained by using SWCNT–PGEs compared to unmodified PGEs with resulting limits of detection (LODs) for TPT as 0.51 μg/mL, 0.45 μg/mL, 0.37 μg/mL (130 pmol, 117 pmol, 96.5 pmol in a 110 μL sample, respectively) by using electrochemically pretreated PGE, unmodified PGE and SWCNT modified PGE. In addition, electrochemical impedance spectroscopy (EIS) measurements were performed for the purpose of modification of PGEs by using SWCNTs and the interaction process at the surface of SWCNT–PGEs by evaluating the changes at the charge transfer resistance (R ct).Display Omitted
Keywords: Topotecan; Drug–DNA interactions; Pencil graphite electrode; Single walled carbon nanotubes; Differential pulse voltammetry; Electrochemical impedance spectroscopy;

Bacteria-based bioelectrochemical systems (BESs) are promising technologies used for alternative energy generation, wastewater treatment, and environmental monitoring. However, their practical application is limited by the bioelectrode performance, mainly resulting from low extracellular electron transfer (EET) efficiency. In this study, a carbon-coated hematite (C/Hematite) electrode was successfully obtained by a green and solvent-free route, that is, heat treatment in an oxygen-rich environment using solid ferrocene as the precursor. The as-prepared C/Hematite electrode was evaluated as a high-performance electrode material in a Shewanella oneidensis-inoculated BES. The maximum biocurrent density of the Shewanella-attached C/Hematite electrode reached 0.22 ± 0.01 mA cm− 2, which is nearly 6-times higher than that of a bare carbon cloth (CC) electrode (0.036 ± 0.005 mA cm− 2). Electrochemical measurements revealed that the enhanced conductivity and better energy matching between the outer membrane c-type cytochromes of S. oneidensis and the electrode contributed to the improved EET efficiency. The results of this study demonstrated that the semiconductive properties of iron oxides play important roles for the involved bacterial extracellular respiration activities.
Keywords: Carbon-coated hematite; Extracellular electron transfer; Conduction-band edge; Semiconductor; Bioelectrochemical system;

Cell electroporation with a three-dimensional microelectrode array on a printed circuit board by Youchun Xu; Shisheng Su; Changcheng Zhou; Ying Lu; Wanli Xing (35-41).
Electroporation is a commonly used approach to rapidly introduce exogenous molecules into cells without permanent damage. Compared to classical electroporation protocols, microchip-based electroporation approaches have the advantages of high transfection efficiency and low consumption, but they also commonly rely on costly and tedious microfabrication technology. Hence, it is desirable to develop a novel, more affordable, and effective approach to facilitate cell electroporation. In this study, we utilized a standard printed circuit board (PCB) technology to fabricate a chip with an interdigitated array of electrodes for electroporation of suspended cells. The electrodes (thickness ~ 35 μm) fabricated by PCB technology are much thicker than the two-dimensional (2D) planar electrodes (thickness < 1 μm) fabricated by conventional microfabrication techniques and possess a smooth corner edge. Numerical simulations showed that the three-dimensional (3D) electrodes fabricated by PCB technology can provide a more uniformly distributed electric field compared to 2D planar electrodes, which is beneficial for reducing the electrolysis of water and improving cell transfection efficiency. The chip constructed here is composed of 18 individually addressable wells for high throughput cell electroporation. HeLa, MCF7, COS7, Jurkat, and 3T3-L1 cells were efficiently transfected with the pEGFP-N1 plasmid using individually optimal electroporation parameters. This work provides a novel method for convenient and rapid cell transfection and thus holds promise for use as a low-cost disposable device in biomedical research.
Keywords: Electroporation; PCB; Three-dimensional; Transfection;

The current provided by oxygen-reducing microbial cathodes is related to the composition of their bacterial community by Mickaël Rimboud; Elie Desmond-Le Quemener; Benjamin Erable; Théodore Bouchez; Alain Bergel (42-49).
Oxygen reducing biocathodes were formed from sludge under constant polarization at − 0.2 and + 0.4 V/SCE. Under chronoamperometry at pH 10.3 ± 0.3, current densities of 0.21 ± 0.03 and 0.12 ± 0.01 A m− 2 were displayed at − 0.2 V/SCE by the biocathodes formed at − 0.2 and 0.4 V/SCE, respectively. Voltammetry revealed similar general characteristics for all biocathodes and higher diffusion-limited current densities (0.84 ± 0.26 A m− 2) than chronoamperometry. Up to 3.7 A m− 2 was reached under air bubbling. A theoretical model was proposed to show the consistency of the chronoamperometric and voltammetric data.The biocathodes formed at − 0.2 V/ECS that gave the highest electrochemical performance showed a homogeneous selection of Deinococcus–Thermus and Gemmatimonadetes, while the biocathodes formed at 0.4 V/SCE were enriched in different bacteria. The biocathode that led to the worst electrochemical characteristics, while formed at − 0.2 V/SCE, showed the largest bacterial diversity. The biocathode performance was consequently related to the enrichment in specific microbial phyla. Moreover, the strong presence of bacteria parented to Deinococci may also have some interest in biotechnology.
Keywords: Oxygen reduction; Microbial cathode; Biocathode; Deinococci; Microbial fuel cell;

Selective microbial electrosynthesis of methane by a pure culture of a marine lithoautotrophic archaeon by Pascal F. Beese-Vasbender; Jan-Philipp Grote; Julia Garrelfs; Martin Stratmann; Karl J.J. Mayrhofer (50-55).
Reduction of carbon dioxide to methane by microorganisms attached to electrodes is a promising process in terms of renewable energy storage strategies. However the efficient and specific electrosynthesis of methane by methanogenic archaea on cathodes needs fundamental investigations of the electron transfer mechanisms at the microbe–electrode interface without the addition of artificial electron mediators. Using well-defined electrochemical techniques directly coupled to gas chromatography and surface analysis by scanning electron microscopy, it is shown that a pure culture of the marine lithoautotrophic Methanobacterium-like archaeon strain IM1 is capable to utilize electrons from graphite cathodes for a highly selective production of methane, without hydrogen serving as a cathode-generated electron carrier. Microbial electrosynthesis of methane with cultures of strain IM1 is achieved at a set potential of − 0.4 V vs. SHE and is characterized by a coulomb efficiency of 80%, with rates reaching 350 nmol d− 1  cm− 2 after 23 days of incubation. Moreover, potential step measurements reveal a biologically catalyzed hydrogen production at potentials more positive than abiotic hydrogen evolution on graphite, indicating that an excessive supply of electrons to strain IM1 results in proton reduction rather than in a further increase of methane production.Display Omitted
Keywords: Biocatalysis; Bioelectrochemistry; Carbon dioxide reduction; Extracellular electron transfer; Methanogenesis; Microbial electrosynthesis;

Autotrophic hydrogen-producing biofilm growth sustained by a cathode as the sole electron and energy source by Ludovic Jourdin; Stefano Freguia; Bogdan C. Donose; Jurg Keller (56-63).
It is still unclear whether autotrophic microbial biocathode biofilms are able to self-regenerate under purely cathodic conditions without any external electron or organic carbon sources. Here we report on the successful development and long-term operation of an autotrophic biocathode whereby an electroactive biofilm was able to grow and sustain itself with CO2 as a sole carbon source and using the cathode as electron source, with H2 as sole product. From a small inoculum of 15 mgCOD (in 250 mL), containing 30.3% Archaea, the bioelectrochemical system operating at − 0.5 V vs. SHE enabled an estimated biofilm growth of 300 mg as COD over a period of 276 days. A dramatic change in the microbial population was observed during this period with Archaea disappearing completely (< 0.1% of population). The predominant phyla enriched were Proteobacteria (57.3%), Firmicutes (12.4%), Bacteroidetes (11.6%) and Actinobacteria (1.1%). Up to 9.2 L H2  m− 2  day− 1 (1.88 A m− 2) was achieved when the cathode potential was decreased to − 0.75 V vs. SHE. This study demonstrates that purely autotrophic biofilm growth coupled to proton reduction to hydrogen alone can be sustained with a cathode as the sole electron source, while avoiding the development of H2-consuming microorganisms such as methanogens and acetogens.
Keywords: Bioelectrochemical systems; Biocathode; Autotrophic biofilm growth; Hydrogen;

Theoretical Analyses of Cellular Transmembrane Voltage in Suspensions Induced by High-frequency Fields by Yong Zou; Changzhen Wang; Ruiyun Peng; Lifeng Wang; Xiangjun Hu (64-72).
A change of the transmembrane voltage is considered to cause biophysical and biochemical responses in cells. The present study focuses on the cellular transmembrane voltage (Δφ) induced by external fields. We detail analytical equations for the transmembrane voltage induced by external high-frequency (above the relaxation frequency of the cell membrane) fields on cells of a spherical shape in suspensions and layers. At direct current (DC) and low frequencies, the cell membrane was assumed to be non-conductive under physiologic conditions. However, with increasing frequency, the permittivity of the cytoplasm/extracellular medium and conductivity of the membrane must be accounted for. Our main work is to extend application of the analytical solution of Δφ to the high-frequency range. We first introduce the transmembrane voltage generated by DC and low-frequency exposures on a single cell. Then, we focus on cell suspensions exposed to high-frequency fields. Using the effective medium theory and the reasonable assumption, the approximate analytical solution of Δφ on cells in suspensions and layers can be derived. Phenomenological effective medium theory equations cannot be used to calculate the local electric field of cell suspensions, so we raised a possible solution based on the Bergman theory.
Keywords: Cell suspension; High-frequency field; Transmembrane voltage; Effective medium theory; Bergman theory;