Bioelectrochemistry (v.94, #C)

Gene electrotransfer (GET) enhances delivery of DNA vaccines by increasing both gene expression and immune responses. Our lab has developed the multi-electrode array (MEA) for DNA delivery to skin. The MEA was used at constant pulse duration (150 ms) and frequency (6.67 Hz). In this study, delivery parameters including applied voltage (5–45 V), amount of plasmid (100–300 μg), and number of treatments (2−3) were evaluated for delivery of a DNA vaccine. Mice were intradermally injected with plasmid expressing Bacillus anthracis protective antigen with or without GET and αPA serum titers measured. Within this experiment no significant differences were noted in antibody levels from varying dose or treatment number. However, significant differences were measured from applied voltages of 25 and 35 V. These voltages generated antibody levels between 20,000 and 25,000. Serum from animals vaccinated with these conditions also resulted in toxin neutralization in 40–60% of animals. Visual damage was noted at MEA conditions of 40 V. No damage was noted either visually or histologically from conditions of 35 V or below. These results reflect the importance of establishing appropriate electrical parameters and the potential for the MEA in non-invasive DNA vaccination against B. anthracis.
Keywords: Gene electrotransfer; DNA vaccination; Electroporation; Anthrax; Bacillus anthracis;

Nitric oxide synthase (NOS) plays a critical role in a number of key physiological and pathological processes. Investigation of electron-transfer reactions in NOS would contribute to a better understanding of the nitric oxide (NO) synthesis mechanism. Herein, we describe an electrochemically driven catalytic strategy, using a nanocomposite that consisted of the oxygenase domain of neuronal NOS (D290nNOSoxy), indium tin oxide (ITO) nanoparticles and polyvinyl alcohol (PVA). Fast direct electron transfer between electrodes and D290nNOSoxy was observed with the heterogeneous electron transfer rate constant (ket ) of 154.8 ± 0.1 s− 1 at the scan rate of 5 V s− 1. Moreover, the substrate Nω-hydroxy-l-arginine (NHA) was used to prove the concept of electrochemically driven biocatalysis of D290nNOSoxy. In the presence of the oxygen cosubstrate and tetrahydrobiopterin (BH4) cofactor, the addition of NHA caused the decreases of both oxidation current at + 0.1 V and reduction current at potentials ranging from − 0.149 V to − 0.549 V vs Ag/AgCl. Thereafter, a series of control experiments such as in the absence of BH4 or D290nNOSoxy were performed. All the results demonstrated that D290nNOSoxy biocatalysis was successfully driven by electrodes in the presence of BH4 and oxygen. This novel bioelectronic system showed potential for further investigation of NOS and biosensor applications.
Keywords: Nitric oxide synthase; Tetrahydrobiopterin; Nω-hydroxy-l-arginine; Indium tin oxide nanoparticles; Biocatalysis;

Bioanodes formed at an optimal potential of 200 mV vs. SHE and biocathodes developed at − 300 mV vs. SHE in bioelectrochemical cells (BECs) enhanced the subsequent performances of microbial fuel cells (MFCs) compared to the un-treated controls. While the startup times were reduced to 320 h (bioanodes) and 420–440 h (biocathodes), PCP degradation rates were improved by 28.5% (bioanodes) and 21.5% (biocathodes), and power production by 41.7% (bioanodes) and 44% (biocathodes). Accordingly, there were less accumulated products of PCP de-chlorination in the biocathodes whereas PCP in the bioanodes was more efficiently de-chlorinated, resulting in the formation of a new product of 3,4,5-trichlorophenol (24.3 ± 2.2 μM at 96 h). Charges were diverted to more generation of electricity in the bioanodes at 200 mV while oxygen in the biocathodes at − 300 mV acted as a primary electron acceptor. Dominant bacteria known as recalcitrant organic degraders and/or exoelectrogens/electrotrophs included Desulfovibrio carbinoliphilus and Dechlorospirillum sp. on the bioanodes at 200 mV, and Desulfovibrio marrakechensis, Comamonas testosteroni and Comamonas sp. on the biocathodes at − 300 mV. These results demonstrated that an optimal potential was a feasible approach for developing both bioanodes and biocathodes for efficient PCP degradation and power generation from MFCs.Display Omitted
Keywords: Pentachlorophenol; Microbial fuel cell; Bioanode; Biocathode; Bioelectrochemical cell;

Activation of intracellular phosphoinositide signaling after a single 600 nanosecond electric pulse by Gleb P. Tolstykh; Hope T. Beier; Caleb C. Roth; Gary L. Thompson; Jason A. Payne; Marjorie A. Kuipers; Bennett L. Ibey (23-29).
Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways — phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2 kV/cm, 600 ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions.
Keywords: Electric pulse; Plasma membrane nanopore; PKC and PLC; PIP2 depletion; Phosphoinositide signaling;

Human colon adenocarcinoma HT-29 cell: Electrochemistry and nicotine stimulation by S.C.B. Oliveira; I.B. Santarino; T.A. Enache; C. Nunes; J. Laranjinha; R.M. Barbosa; A.M. Oliveira-Brett (30-38).
Recently, it was demonstrated that colorectal cancer HT-29 cells can secrete epinephrine (adrenaline) in an autocrine manner to auto-stimulate cellular growth by adrenoreceptors activation, and that this secretion is enhanced by nicotine, showing an indirect relation between colorectal cancer and tobacco. The electrochemical behaviour of human colon adenocarcinoma HT-29 cells from a colorectal adenocarcinoma cell line, the hormone and neurotransmitter epinephrine, and nicotine, were investigated by cyclic voltammetry, using indium tin oxide (ITO), glassy carbon (GC) and screen printed carbon (SPC) electrodes. The oxidation of the HT-29 cells, previously grown onto ITO or SPC surfaces, followed an irreversible oxidation process that involved the formation of a main oxidation product that undergoes irreversible reduction, as in the epinephrine oxidation mechanism. The effect of nicotine stimulation of the HT-29 cells was also investigated. Nicotine, at different concentration levels 1, 2 and 15 mM, was introduced in the culture medium and an increase with incubation time, 0 to 3 h and 30 min, of the HT-29 cells oxidation and reduction peaks was observed. The interaction of nicotine with the HT-29 cells stimulated the epinephrine secretion causing an increase in epinephrine release concentration, and enabling the conclusion that epinephrine and nicotine play an important role in the colorectal tumour growth.Display Omitted
Keywords: Colon cancer HT-29 cells; Epinephrine; Nicotine; Carbon electrodes; ITO electrodes;

The direct electrochemistry of hemoglobin (Hb) immobilized by a fullerene–nitrogen doped carbon nanotubes and chitosan (C60–NCNTs/CHIT) composite matrix is demonstrated. The cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the modified electrode. In the deaerated buffer solution, the cyclic voltammogram of the Hb/C60–NCNTs/CHIT composite film modified electrode showed a pair of well-behaved redox peaks with the E°′ = − 0.335 (± 0.3) V (vs. SCE). The redox peaks are assigned to the redox reaction of Hb(FeIII/FeII) and confirm the effective immobilization of Hb on the composite film. The large value of k s  = 1.8 (± 0.2) s− 1 suggests that the immobilized Hb achieved a relative fast electron transfer process. The fast electron transfer interaction between protein and electrode surface suggested that the C60–NCNTs/CHIT composite film may mimic some physiological process and further elucidate the relationship between protein structures and biological functions. Moreover, the resulting electrode exhibited excellent electrocatalytic ability towards the reduction of hydrogen peroxide (H2O2) with the linear dynamic range of 2.0–225.0 μM. The linear regression equation was I p/μA = 7.35 (± 0.08) + 0.438 (± 0.007) C/μM with the correlation coefficient of 0.9993. The detection limit was estimated at about 1 μM (S/N = 3). The sensitivity was 438.0 (± 2.5) μA mM− 1. It is expected that the method presented here can not only be easily extended to other redox enzymes or proteins, but also be used as an electrochemical sensing devices for the determination of H2O2 in cell extracts or urine.A pair of well-defined and quasi-reversible redox peaks is observed at the Hb/C60–NCNT/CHIT/GCE (curve d) with the formal potential of − 0.335 V (vs. SCE) and the peak to peak separation was 76 mV.Display Omitted
Keywords: Hemoglobin; Fullerene; Nitrogen doped carbon nanotubes; Direct electrochemistry; Biosensor;

In spite of the promising applications of aptamers in the bioassays, the development of aptamer-based electrochemical biosensors with the improved limit of detection has remained a great challenge. A strategy for the amplification of signal, based on application of nanostructures as platforms for the construction of an electrochemical adenosine triphosphate (ATP) aptasensor, is introduced in the present manuscript. A sandwich assay is designed by immobilizing a fragment of aptamer on a nanoporous gold electrode (NPGE) and its association to second fragment in the presence of ATP. Consequently, 3, 4-diaminobenzoic acid (DABA), as a molecular reporter, is covalently attached to the amine-label of the second fragment, and the direct oxidation signal of DABA is followed as the analytical signal. The sensor can detect the concentrations of ATP as low as submicromolar scales. Furthermore, 3.2% decrease in signal is observed by keeping the aptasensor at 4 °C for a week in buffer solution, implying a desirable stability. Moreover, analog nucleotides, including GTP, UTP and CTP, do not show serious interferences and this sensor easily detects its target in deproteinized human blood plasma.
Keywords: Aptasensor; Aptamer; Adenosine triphosphate; Nanoporous gold electrode; 3, 4-Diaminobenzoic acid;

Voltage-controlled cellular viability of preosteoblasts on polarized cpTi with varying surface oxide thickness by Morteza Haeri; Torsten Wöllert; George M. Langford; Jeremy L. Gilbert (53-60).
Cathodic voltage shifts of metallic biomaterials were recently shown to induce cell apoptosis in-vitro. The details of the reduction-based physico-chemical phenomena have not yet been fully elucidated. This study shows how surface oxide thickness of commercially pure titanium affects the voltage viability range, and whether anodic oxidation can extend this range. Cell viability, cytoskeletal organization, and cellular adhesion on bare and anodized Ti, at − 500, − 400 mV(Ag/AgCl) and open circuit potential were assessed. Surfaces were characterized using contact angle measurement and atomic force microscopy, and the observed cellular response was related to the changes in electrochemical currents, and impedance of the samples. Results show that anodization at 9 V in phosphate buffer saline generates a compact surface oxide with comparable surface roughness and energy to the starting bare surface. The anodized surface extends the viability range at 24 h from − 400 mV(Ag/AgCl) by about − 100 mV, which corresponds to an increase in impedance of the surface from 58 kΩ cm2 to 29 MΩ cm2 at − 400 mV(Ag/AgCl) and results in low average current densities below 0.1 μA cm− 2. The results demonstrate that the voltage range for cell viability under cathodic polarization is expanded due to anodization of the surface oxide and lowering of cathodic currents.
Keywords: Titanium; Cathodic polarization; Cell viability; Anodization;

Alternating electric (AC) fields are known to activate tumor cell death, but the underlying cellular mechanisms are poorly understood. We thus combined live-cell imaging with computational modeling to investigate the dynamic interactions between AC fields and cultured mammalian cells. Our results showed extensive cell death activated via two distinct mechanisms. At frequency range of 100–300 kHz and 800–1000 kHz, AC fields triggered prolonged mitotic arrest followed by apoptosis, and the cell death kinetics showed linear dependence on both field frequency and intensity. However, at intermediate frequencies, from 300 kHz to 800 kHz, cells died as a result of field-induced surface detachment, and the process exhibited a resonance frequency. Based on models of induced dielectric polarization and charge oscillation, we simulated the functional dependence of cell death kinetics on field frequency and intensity for both the linear and resonance response regimes. By comparing the simulated and experimental results, we not only determined the crucial electrical properties of mammalian cells that govern their interaction with AC fields but also acquired novel mechanistic understanding of the resulting cell death processes, which provides important new insight for potentially utilizing AC fields as an alternative anti-tumor remedy.
Keywords: Bioelectric effect; Cell death dynamics; Apoptosis; Mitotic arrest; Live-cell imaging;

Oxygen biosensor based on bilirubin oxidase immobilized on a nanostructured gold electrode by Marcos Pita; Cristina Gutierrez-Sanchez; Miguel D. Toscano; Sergey Shleev; Antonio L. De Lacey (69-74).
Gold disk electrodes modified with gold nanoparticles have been used as a scaffold for the covalent immobilization of bilirubin oxidase. The nanostructured bioelectrodes were tested as mediator-less biosensors for oxygen in a buffer that mimics the content and the composition of human physiological fluids. Chronoamperometry measurements showed a detection limit towards oxygen of 6 ± 1 μM with a linear range of 6-300 μM, i.e. exceeding usual physiological ranges of oxygen in human tissues and fluids. The biosensor presented is the first ever-reported oxygen amperometric biosensor based on direct electron transfer of bilirubin oxidase.
Keywords: Biocatalysis; Bilirubin oxidase; Biosensor; Direct electron transfer; Gold nanoparticles;

The two-step electrochemical oxidation of alcohols using a novel recombinant PQQ alcohol dehydrogenase as a catalyst for a bioanode by Kouta Takeda; Hirotoshi Matsumura; Takuya Ishida; Masahiro Samejima; Kiyohiko Igarashi; Nobuhumi Nakamura; Hiroyuki Ohno (75-78).
A bioanode has been developed based on the oxidation of ethanol by the recombinant pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase from Pseudomonas putidaKT2440 heterologously expressed in Pichia pastoris. The apo form of the recombinant protein (PpADH) was purified and displayed catalytic activity for binding PQQ in the presence of Ca2 +. PpADH exhibited broad substrate specificity towards various alcohols and aldehydes. The K m values for the aldehydes of PpADH were increased compared to those for the alcohols, whereas the k cat values were unaltered. For instance, the K m values at T = 298.15 K (25 °C) for ethanol and acetaldehyde were 0.21 (± 0.02) mM and 5.8 (± 0.60) mM, respectively. The k cat values for ethanol and acetaldehyde were 24.8 (± 1.2) s− 1 and 31.1 (± 1.2) s− 1, respectively. The aminoferrocene was used as an electron transfer mediator between PpADH and the electrode during electrochemical experiments. The catalytic currents for the oxidation of alcohol and acetaldehyde by PpADH were also observed in this system. The electric charge for the oxidation of ethanol (Q = 2.09 × 10− 3·C) was increased two-fold compared to that for the oxidation of acetaldehyde (Q = 0.95 × 10− 3·C), as determined by chronoamperometric measurements. Thus, we have electrochemically demonstrated the two-step oxidation of ethanol to acetate using only PpADH.Display Omitted
Keywords: Enzymatic biofuel cell; Electrocatalysis; Quinoprotein; Pyrroloquinoline quinone; Mediated electron transfer;

Electrostatic discharges occur naturally as lightning strokes, and artificially in light sources and in materials processing. When an electrostatic discharge interacts with living matter, the basic physical effects can be accompanied by biophysical and biochemical phenomena, including cell excitation, electroporation, and electrofusion. To study these phenomena, we developed an experimental system that provides easy sample insertion and removal, protection from airborne particles, observability during the experiment, accurate discharge origin positioning, discharge delivery into the sample either through an electric arc with adjustable air gap width or through direct contact, and reliable electrical insulation where required. We tested the system by assessing irreversible electroporation of Escherichia coli bacteria (15 mm discharge arc, 100 A peak current, 0.1 μs zero-to-peak time, 0.2 μs peak-to-halving time), and gene electrotransfer into CHO cells (7 mm discharge arc, 14 A peak current, 0.5 μs zero-to-peak time, 1.0 μs peak-to-halving time). Exposures to natural lightning stroke can also be studied with this system, as due to radial current dissipation, the conditions achieved by a stroke at a particular distance from its entry are also achieved by an artificial discharge with electric current downscaled in magnitude, but similar in time course, correspondingly closer to its entry.
Keywords: Electrostatic discharge; Lightning; Electroporation; Gene electrotransfer; Exposure system;

The application of thionine–graphene nanocomposite in chiral sensing for Tryptophan enantiomers by Liju Guo; Qing Zhang; Yihan Huang; Qian Han; Yonghua Wang; Yingzi Fu (87-93).
The thionine–graphene (THi–GR, positively charged) nanocomposite was successfully synthesized as a good biocompatible matrix for ds-DNA which acted as a chiral selector to construct an electrochemical chiral biosensor for Tryptophan (Trp) enantiomers sensing with the assistance of Cu(II). The nano-bionic interface was constructed as follows: Firstly, the nanocomposite was dropped on the surface of glassy carbon electrode (GCE), and then ds-DNA (negatively charged) was immobilized onto the nanocomposite film via the opposite-charged adsorption techniques. This biofunctionalized nanocomposite was characterized with scanning electron microscopy (SEM), ultraviolet–visible (UV–vis) spectrometry and cyclic voltammetry (CV). The chiral biosensor was employed to study the recognition effect between ds-DNA and Trp enantiomers by CV. The results show that larger electrochemical response was obtained from l-Trp when Cu(II) was present, indicating this strategy could be employed to enantioselectively recognize Trp enantiomers. Under optimum conditions, the chiral biosensor exhibited a good linear response to Trp enantiomers in the range of the concentration of [Cu(II)(Trp)2] from 5.0 × 10− 4 to 2.5 mM with a low limit of detection of 0.17 μM (S/N = 3). The binding constant was calculated to be 2.97 × 103  M− 1 for [Cu(II)(l-Trp)2] and 2.50 × 102  M− 1 for [Cu(II)(d-Trp)2].Display Omitted
Keywords: Nano-bionic interface; Thionine–graphene nanocomposite; ds-DNA (Herring Sperm); Chiral biosensor;

The direct electrochemistry and electrocatalysis of heme proteins entrapped in carbon-coated nickel magnetic nanoparticle–chitosan–dimethylformamide (CNN–CS–DMF) composite films were investigated in the hydrophilic ionic liquid [bmim][BF4]. The surface morphologies of a representative set of films were characterised via scanning electron microscopy. The proteins immobilised in the composite films were shown to retain their native secondary structure using UV–vis spectroscopy. The electrochemical performance of the heme proteins–CNN–CS–DMF films was evaluated via cyclic voltammetry and chronoamperometry. A pair of stable and well-defined redox peaks was observed for the heme protein films at formal potentials of − 0.151 V (HRP), − 0.167 V (Hb), − 0.155 V (Mb) and − 0.193 V (Cyt c) in [bmim][BF4]. Moreover, several electrochemical parameters of the heme proteins were calculated by nonlinear regression analysis of the square-wave voltammetry. The addition of CNN significantly enhanced not only the electron transfer of the heme proteins but also their electrocatalytic activity toward the reduction of H2O2. Low apparent Michaelis–Menten constants were obtained for the heme protein–CNN–CS–DMF films, demonstrating that the biosensors have a high affinity for H2O2. In addition, the resulting electrodes displayed a low detection limit and improved sensitivity for detecting H2O2, which indicates that the biocomposite film can serve as a platform for constructing new non-aqueous biosensors for real detection.
Keywords: Heme proteins; Carbon-coated nickel magnetic nanoparticles; Room-temperature ionic liquid; Hydrogen peroxide; Electrocatalysis;

In the present work differential pulse voltammetry coupled with multivariate curve resolution-alternating least squares (MCR-ALS) was applied for simultaneous determination of betaxolol (Bet) and atenolol (Ate) in 0.20 M Britton–Robinson (B-R) buffer solution at the surface of a multi-walled carbon nanotube modified carbon paste electrode (MWCNT/CPE). Characterization of the modified electrode was carried out by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A strategy based on experimental design was followed. Operating conditions were improved with central composite rotatable design (CCRD) and response surface methodology (RSM), involving several chemical and instrumental parameters. Then second order data were built from variable pulse heights of DPV and after correction in potential shift analyzed by MCR-ALS. Analytical parameters such as linearity, repeatability, and stability were also investigated and a detection limit (DL) of 0.19 and 0.29 μM for Bet and Ate was achieved, respectively. The proposed method was successfully applied in simultaneous determining the two analytes in human plasma.
Keywords: Multivariate curve resolution-alternating least squares; Central composite rotatable design; Differential pulse voltammetry; β-Blockers; Multi-wall carbon nanotube modified carbon paste electrode;