Bioelectrochemistry (v.98, #C)

Pyrrole was electro-polymerized on the surface of the glassy carbon electrode (GCE) coated with a thin film of carbon nanomaterials, including carbon nanotubes (CNTs), carbon nanoparticles (CNPs), nanodiamond-graphite (NDG) or graphite nanopowder (GNP). Morphology, thickness, stability and loading of the polypyrrole (PPY) film were significantly affected by the structure and morphology of the sub-layer of carbon nanomaterials. Electrochemical oxidation of atorvastatin (ATOR) was investigated. Under the optimized conditions, a significant increase in the peak current (compared to other modified electrodes and bare GCE) and a negative shift in the peak potential (compared to bare GCE) were observed on the surface of the CNTs/PPY modified electrode. The electrode was able to completely resolve the voltammetric response of ATOR from potentially interfering species, e.g. ascorbic acid (AA), uric acid (UA) and dopamine (DA) which are present in many biological systems. Two linear dynamic ranges of 0.005–0.1 μM and 0.1–1 μM with a detection limit of 1.5 nM and a sensitivity of 267.68 (± 1.26) μA/μM were obtained for ATOR from linear sweep voltammetry (LSV) measurements. The modified electrode with high sensitivity, stability and good reproducibility showed promising results for determination of the ATOR concentration in pharmaceutical and clinical preparations.
Keywords: Carbon nanomaterials; Carbon nanotubes; Polypyrrole; Atorvastatin; Carbon nanoparticles;

Crystallographic orientation and electrode nature are key factors for electric current generation by Geobacter sulfurreducens by Beatriz Maestro; Juan M. Ortiz; Germán Schrott; Juan P. Busalmen; Víctor Climent; Juan M. Feliu (11-19).
We have investigated the influence of electrode material and crystallographic structure on electron transfer and biofilm formation of Geobacter sulfurreducens. Single-crystal gold—Au(110), Au(111), Au(210)—and platinum—Pt(100), Pt(110), Pt(111), Pt(210)—electrodes were tested and compared to graphite rods. G. sulfurreducens electrochemically interacts with all these materials with different attachment kinetics and final current production, although redox species involved in the electron transfer to the anode are virtually the same in all cases. Initial bacterial colonization was fastest on graphite up to the monolayer level, whereas gold electrodes led to higher final current densities. Crystal geometry was shown to have an important influence, with Au(210) sustaining a current density of up to 1442 ± 101 μA cm− 2 at the steady state, over Au(111) with 961 ± 94 μA cm− 2 and Au(110) with 944 ± 89 μA cm− 2. On the other hand, the platinum electrodes displayed the lowest performances, including Pt(210). Our results indicate that both crystal geometry and electrode material are key parameters for the efficient interaction of bacteria with the substrate and should be considered for the design of novel materials and microbial devices to optimize energy production.Display Omitted
Keywords: Geobacter sulfurreducens; Single-crystal electrode; Biofilm; Electron transport; Cytochrome;

Sensitive bi-enzymatic biosensor based on polyphenoloxidases–gold nanoparticles–chitosan hybrid film–graphene doped carbon paste electrode for carbamates detection by Thiago M.B.F. Oliveira; M. Fátima Barroso; Simone Morais; Mariana Araújo; Cristina Freire; Pedro de Lima-Neto; Adriana N. Correia; Maria B.P.P. Oliveira; Cristina Delerue-Matos (20-29).
A bi-enzymatic biosensor (LACC–TYR–AuNPs–CS/GPE) for carbamates was prepared in a single step by electrodeposition of a hybrid film onto a graphene doped carbon paste electrode (GPE). Graphene and the gold nanoparticles (AuNPs) were morphologically characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering and laser Doppler velocimetry. The electrodeposited hybrid film was composed of laccase (LACC), tyrosinase (TYR) and AuNPs entrapped in a chitosan (CS) polymeric matrix. Experimental parameters, namely graphene redox state, AuNPs:CS ratio, enzymes concentration, pH and inhibition time were evaluated. LACC–TYR–AuNPs–CS/GPE exhibited an improved Michaelis–Menten kinetic constant (26.9 ± 0.5 M) when compared with LACC–AuNPs–CS/GPE (37.8 ± 0.2 M) and TYR–AuNPs–CS/GPE (52.3 ± 0.4 M). Using 4-aminophenol as substrate at pH 5.5, the device presented wide linear ranges, low detection limits (1.68 × 10− 9  ± 1.18 × 10− 10–2.15 × 10− 7  ± 3.41 × 10− 9  M), high accuracy, sensitivity (1.13 × 106  ± 8.11 × 104–2.19 × 108  ± 2.51 × 107  %inhibition M− 1), repeatability (1.2–5.8% RSD), reproducibility (3.2–6.5% RSD) and stability (ca. twenty days) to determine carbaryl, formetanate hydrochloride, propoxur and ziram in citrus fruits based on their inhibitory capacity on the polyphenoloxidases activity. Recoveries at two fortified levels ranged from 93.8 ± 0.3% (lemon) to 97.8 ± 0.3% (orange). Glucose, citric acid and ascorbic acid do not interfere significantly in the electroanalysis. The proposed electroanalytical procedure can be a promising tool for food safety control.
Keywords: Bi-enzymatic biosensor; Graphene modified electrode; Gold nanoparticles; Chitosan hybrid film; Carbamates;

The environmentally sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole (NBD) is generally utilized to monitor dynamic properties of membrane lipids and proteins. Here we studied the behavior of a homologous series of 4-n-alkylamino-substituted NBD derivatives (NBD-Cn; n = 4, 6, 8, 9, 10, 12) in planar lipid bilayers, liposomes and isolated mitochondria. NBD-C10 induced proton conductivity in planar lipid membranes, while NBD-C4 was ineffective. The NBD-Cn compounds readily provoked proton permeability of neutral liposomes being less effective in negatively charged liposomes. NBD-Cn increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Remarkably, the bell-shaped dependence of the uncoupling activity of NBD-Cn on the alkyl chain length was found in mitochondria in contrast to the monotonous dependence in liposomes. The effect of NBD-Cn on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by mitochondria swelling. Binding of NBD-Cn to mitochondria increased with n, as shown by fluorescence correlation spectroscopy. It was concluded that despite a pKa value of the amino group in NBD-Cn being about 10, i.e. far from the physiological pH range, the expected hindering of the uncoupling activity could be overcome by inserting the alkyl chain of a certain length.Display Omitted
Keywords: Mitochondria; Bilayer lipid membrane; Uncoupler; Protonophore; NBD; Membrane potential;

The two step nanotube formation on TiZr as scaffolds for cell growth by Sabina Grigorescu; Vasile Pruna; Irina Titorencu; Victor V. Jinga; Anca Mazare; Patrik Schmuki; Ioana Demetrescu (39-45).
Various TiO2 nanotubes on Ti50Zr alloy have been fabricated via a two step anodization method in glycol with 15 vol.% H2O and 0.2 M NH4F under anodization controlled voltages of 15, 30 and 45 V. A new sonication treatment in deionized water with three steps and total sonication time as 1 min was performed after the first anodization step in order to remove the oxide layer grown during 2 h. The second step of anodization was for 1 h and took place at the same conditions. The role of removed layer as a nano-prepatterned surface was evidenced in the formation of highly ordered nanotubular structures and morphological features were analyzed by SEM, AFM and surface wettability. The voltage-controlled anodization leads to various nanoarhitectures, with diameters in between 20 and 80 nm. As biological assay, cell culture tests with MG63 cell line originally derived from a human osteosarcoma were performed. A correlation between nanostructure morphological properties as a result of voltage-controlled anodization and cell response was established.Display Omitted
Keywords: Anodization; TiO2 nanotubes; TiZr alloy; Cell response;

Titania nanotube-modified screen printed carbon electrodes enhance the sensitivity in the electrochemical detection of proteins by Soumit S. Mandal; Vikas Navratna; Pratyush Sharma; B. Gopal; Aninda J. Bhattacharyya (46-52).
The use of titania nanotubes (TiO2-NT) as the working electrode provides a substantial improvement in the electrochemical detection of proteins. A biosensor designed using this strategy provided a robust method to detect protein samples at very low concentrations (Cprotein ca 1 ng/μl). Reproducible measurements on protein samples at this concentration (Ip,a of 80  +  1.2 μA) could be achieved using a sample volume of ca 30 μl. We demonstrate the feasibility of this strategy for the accurate detection of penicillin binding protein, PBP2a, a marker for methicillin resistant Staphylococcus aureus (MRSA). The selectivity and efficiency of this sensor were also validated using other diverse protein preparations such as a recombinant protein tyrosine phosphatase (PTP10D) and bovine serum albumin (BSA). This electrochemical method also presents a substantial improvement in the time taken (few minutes) when compared to conventional enzyme-linked immunosorbent assay (ELISA) protocols. It is envisaged that this sensor could substantially aid in the rapid diagnosis of bacterial infections in resource strapped environments.
Keywords: Electrochemical biosensor; Penicillin binding protein 2a; Methicillin resistant Staphylococcus aureus; Titania nanotubes;

Direct electrochemistry of cytochrome c (Cyt c) is achieved via Zr(IV) ion as an immobilization matrix to interface Cyt c on gold surface via thiol self-assembled monolayers. Steps of surface modification and electrocatalytic activity of the immobilized Cyt c are followed by voltammetry, impedance spectroscopy, chronoampetrometry, and attenuated total reflectance Fourier transform infrared (ATR–FTIR) spectroscopy. The results indicate that the native structure of Cyt c is conserved during the immobilization process. The immobilization method is rather simple, effective and free of immobilizing activators and reagents. Direct electron transfer rate constant and surface coverage of the immobilized Cyt c are found as 8.62(± 1.98) s− 1 and 1.15(± 0.38) × 10− 11  mol cm− 2, respectively. Bioactivity studies of the immobilized Cyt c toward oxidation of the ascorbic acid (AA) substrate show a linear response, from 10.0 μM to 1.30 mM AA, with a detection limit of 5.0(± 1.8) μM AA and mean relative standard deviations varied from 13.7% to 3.7% for n = 4 at each point. A value of 1.6(± 0.8) mM AA is found for the Michaelis–Menten constant of Au-MPA-Zr(IV)-Cyt c toward AA for the first time. The tightly immobilized Cyt c maintains its bioactivity for more than 32 days storage at 4 °C.
Keywords: Cytochrome c; Zr(IV) ion; Impedance spectroscopy; Direct electrochemistry; Ascorbic acid;

In this manuscript, the electrocatalytic reduction of hydrogen peroxides on Prussian blue (PB) modified nanoporous gold film (NPGF) electrode is described. The PB/NPGF is prepared by simple anodizing of a smooth gold film followed by PB film electrodeposition method. The morphology of the PB/NPGF electrode is characterized using scanning electron microscopy (SEM). The effect of solution pH and the scan rates on the voltammetric responses of hydrogen peroxide have also been examined. The amperometric determination of H2O2 shows two linear dynamic responses over the concentration range of 1 μM–10 μM and 10 μM–100 μM with a detection limit of 3.6 × 10− 7  M. Furthermore, this electrode demonstrated good stability, repeatability and selectivity remarkably.
Keywords: Nanoporous gold film; Prussian blue; Hydrogen peroxide; Amperometric sensor; Electrocatalysis;

A simple and sensitive electrochemical sensor based on poly (l-lysine) modified glassy carbon electrode (PLL/GCE) was developed to sensitively detect methotrexate (MTX) in the presence of sodium dodecyl benzene sulfonate (SDBS). Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy and cyclic voltammetry were carried out to characterize PLL film which exhibits excellent electrocatalytic activity toward the oxidation of MTX in 0.1 M phosphate buffer solution. In addition, the oxidation peak of MTX remained stable at PLL/GCE in the presence of SDBS and its current increased 8 times compared with that at bare GCE without SDBS. Experimental parameters were optimized with regard to pH, electro-polymerization segment, accumulation time and concentration of SDBS. Under optimum conditions, the square wave voltammograms exhibited that the oxidation peak current was linearly proportional to the concentration of MTX in the range of 5 nM – 0.2 μM with detection limit of 1.7 (± 0.06) nM (S/N = 3). Moreover, this method was applied to detect MTX in medicinal tablets with satisfying results.
Keywords: Poly (l-lysine); Methotrexate; Sodium dodecyl benzene sulfonate; Modified electrode; Square wave voltammetry;

Dielectric models of biological cells are generally based on spherical or ellipsoidal geometries, where the different adjoining dielectric media are arranged as distinct core and shells, representing the cytosol and the cell membrane. For ellipsoidal particles, this approach implies the assumption of confocal shells that, in turn, means a cell membrane of ill-defined thickness. A quantitative analysis of the influence of a non-uniform thickness of the cell membrane has been not considered so far.In the case of a toroidal particle, this problem can be conveniently addressed by considering the solution of the Laplace equation in two different coordinate systems, i.e., toroidal coordinates (confocal shells and hence non-uniform thickness of the shell membrane) and toroidal polar coordinate, (concentric shells and hence a uniform thickness of the shell membrane).In the present paper, we compare the dielectric spectra of a toroidal particle aqueous suspension obtained from the two above stated solutions of the Laplace equation and we furnish a first quantitative estimate of the differences arising from considering the presence of confocal or concentric shells.This approach offers a complete view of the influence of the membrane thickness on the whole dielectric spectrum of a biological particle suspension, at least as far as toroidal objects are concerned.
Keywords: Dielectric spectroscopy technique; Permittivity of cell membrane; Conductivity of cell membrane; Toroidal particles;

We designed and synthesized a novel organic–inorganic hybrid material polypyrrole–Co3O4 (Ppy–Co3O4), then mixed it with ionic liquid (IL) to form stable composite films for the immobilization of Hemoglobin (Hb) and Glucose Oxidase (GOD). The combination of Ppy and Co3O4 as well as IL created a platform with exceptional characteristics, and the content of Ppy had an effect on the direct electron transfer (DET) of Hb/GOD. Notably, when weight percentage of pyrrole monomer was 20%, the heterogenous electron transfer rate constant (k s) for Hb and GOD was estimated to be 1.71 s− 1 and 1.67 s− 1, respectively. In the meantime, electrochemical and spectroscopic measurements showed that Hb/GOD remained their bioactivity, and achieved fast electron transfer on the Ppy–Co3O4/IL composite film modified electrode. Furthermore, the Ppy–Co3O4/IL/Hb composite film modified electrode was used as a biosensor, and exhibited a long linear range and lower detection limit to H2O2. The apparent Michaelis–Menten constant (K m) was found to be 0.53 mM. The sensing design based on the Ppy–Co3O4 hybrid material was demonstrated to be effective and promising in developing protein and enzyme biosensors.Display Omitted
Keywords: Polypyrrole–Co3O4; Hybrid material; Protein/enzyme; Direct electron transfer;

Information processing through a bio-based redox capacitor: Signatures for redox-cycling by Yi Liu; Eunkyoung Kim; Ian M. White; William E. Bentley; Gregory F. Payne (94-102).
Redox-cycling compounds can significantly impact biological systems and can be responsible for activities that range from pathogen virulence and contaminant toxicities, to therapeutic drug mechanisms. Current methods to identify redox-cycling activities rely on the generation of reactive oxygen species (ROS), and employ enzymatic or chemical methods to detect ROS. Here, we couple the speed and sensitivity of electrochemistry with the molecular-electronic properties of a bio-based redox-capacitor to generate signatures of redox-cycling. The redox capacitor film is electrochemically-fabricated at the electrode surface and is composed of a polysaccharide hydrogel with grafted catechol moieties. This capacitor film is redox-active but non-conducting and can engage diffusible compounds in either oxidative or reductive redox-cycling. Using standard electrochemical mediators ferrocene dimethanol (Fc) and Ru(NH3)6Cl3 (Ru3 +) as model redox-cyclers, we observed signal amplifications and rectifications that serve as signatures of redox-cycling. Three bio-relevant compounds were then probed for these signatures: (i) ascorbate, a redox-active compound that does not redox-cycle; (ii) pyocyanin, a virulence factor well-known for its reductive redox-cycling; and (iii) acetaminophen, an analgesic that oxidatively redox-cycles but also undergoes conjugation reactions. These studies demonstrate that the redox-capacitor can enlist the capabilities of electrochemistry to generate rapid and sensitive signatures of biologically-relevant chemical activities (i.e., redox-cycling).
Keywords: Biofabrication; Chitosan; Electrodeposition; Redox-cycling; Signal processing;

Riboflavin (RF), the primary redox active component of flavin, is involved in many redox processes in biogeochemical systems. Despite of its wide distribution and important roles in environmental remediation, its redox behaviors and reaction mechanisms in hydrophobic sites remain unclear yet. In this study, spectroelectrochemical analysis and density functional theory (DFT) calculation were integrated to explore the redox behaviors of RF in dimethyl sulfoxide (DMSO), which was used to create a hydrophobic environment. Specifically, cyclic voltafluorometry (CVF) and derivative cyclic voltafluorometry (DCVF) were employed to track the RF concentration changing profiles. It was found that the reduction contained a series of proton-coupled electron transfers dependent of potential driving force. In addition to the electron transfer-chemical reaction-electron transfer process, a disproportionation (DISP1) process was also identified to be involved in the reduction. The redox potential and free energy of each step obtained from the DFT calculations further confirmed the mechanisms proposed based on the experimental results. The combination of experimental and theoretical approaches yields a deep insight into the characteristics of RF in environmental remediation and better understanding about the proton-coupled electron transfer mechanisms.Display Omitted
Keywords: Spectroelectrochemistry; Riboflavin; Proton coupled electron transfer; Redox chemistry; Disproportionation process;