Artificial Redox Mediators Research Articles

Amperometric glucose biosensor based on gold nanorods and chitosan comodified Au electrode

Au nanorods were prepared by a seeding growth approach and used in fabricating the nanorod-enhancing glucose biosensor. The high affinity of chitosan for Au nanorods associated with its amino groups resulted in the formation of a layer of Au nanorods on the surface of Au electrode. It served as an intermediator to retain high efficient and stable immobilization of the enzyme. The performance of biosensors was investigated by cyclic voltammetry (CV), in the presence of artificial redox mediator, ferrocenecarboxaldehyde. The biosensor had a fast response to glucose, and the response time was less than 10 s. The results indicated that the gold nanorods could enhance the current response to glucose. The detection limits of glucose can reach 10 mM, and the Michaelis-Menten constant K app m is 13.62 mM.

Study on a novel non-dissolved redox mediator catalyzing biological denitrification (RMBDN) technology

There are little literatures about the accelerating effect of redox mediators on the denitrification processes. In this paper, a novel non-dissolved redox mediator catalyzing biological denitrification (RMBDN) technology was first explored, and the accelerating effect of redox mediator on the denitrification processes was conducted with immobilized anthraquinone. Anthraquinone as a redox mediator was able to increase the denitrification rate, and was immobilized by entrapment in calcium alginate (CA). The results suggested that the artificial redox mediator anthraquinone was found to be capable of raising about 2-fold denitrification rate, and the stabilized oxidation–reduction potential (ORP) values with anthraquinone immobilization beads were lower around 20 mV than the control without anthraquinone immobilization beads. The results of repeated-batch operations shown that anthraquinone immobilization beads appeared to exhibit good reusability. The study explored a great improvement of the redox mediator application and the new bio-treatment concept for the denitrification processes.

Amperometric glucose biosensor based on NiFe2O4 nanoparticles and chitosan

Abstract A novel glucose biosensor was fabricated by integrating glucose oxidase (GOx) with chitosan/NiFe2O4 nanoparticles (CHIT/NiFe2O4NPs) on a glassy carbon electrode (GCE). The properties of CHIT/NiFe2O4NPs/GOx were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), UV–vis spectroscopy, Fourier transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS). The GCE modified with the CHIT/NiFe2O4NPs/GOx showed excellent electrocatalytical response to the oxidation of glucose when ferrocene carboxylic acid was used as an artificial redox mediator, which was studied by cyclic voltammetry (CV). Different parameters including GOx concentration, working potential and pH of supporting electrolyte that governed the analytical performance of the biosensor, have been studied in detail and optimized. The biosensor was applied to detect glucose with a linear range of 1 × 10−4–2.0 × 10−2 mol L−1. The biosensor exhibited excellent performance for glucose at applied potential of 0.6 V with a fast response time (

Biocatalytic cascade oxidation using laccase for pyranose 2-oxidase regeneration

The interactions between two oxidoreductases coupled by an artificial redox mediator have been described quantitatively to increase both stability and productivity. In this cascade oxidation, pyranose 2-oxidase oxidizes several aldoses at the C-2 position to 2-ketoaldoses. A redox mediator is used as electron acceptor for pyranose 2-oxidase because it shows more favourable kinetics in comparison to oxygen. The reduced redox mediator is in turn re-oxidized by laccase, which uses oxygen as the terminal electron acceptor, reducing it fully to water. However, pyranose 2-oxidase is capable of using oxygen as an electron acceptor in a competing side reaction, leading to the formation of hydrogen peroxide, which is detrimental for both enzymes and seriously limits the operational stability of both enzymes. The experimental results showed full conversion of the aldose to the 2-ketoaldose and a good agreement with the simulations of the process.

Application of biocathode in microbial fuel cells: cell performance and microbial community

Instead of the utilization of artificial redox mediators or other catalysts, a biocathode has been applied in a two-chamber microbial fuel cell in this study, and the cell performance and microbial community were analyzed. After a 2-month startup, the microorganisms of each compartment in microbial fuel cell were well developed, and the output of microbial fuel cell increased and became stable gradually, in terms of electricity generation. At 20 ml/min flow rate of the cathodic influent, the maximum power density reached 19.53 W/m3, while the corresponding current and cell voltage were 15.36 mA and 223 mV at an external resistor of 14.9 Omega, respectively. With the development of microorganisms in both compartments, the internal resistance decreased from initial 40.2 to 14.0 Omega, too. Microbial community analysis demonstrated that five major groups of the clones were categorized among those 26 clone types derived from the cathode microorganisms. Betaproteobacteria was the most abundant division with 50.0% (37 of 74) of the sequenced clones in the cathode compartment, followed by 21.6% (16 of 74) Bacteroidetes, 9.5% (7 of 74) Alphaproteobacteria, 8.1% (6 of 74) Chlorobi, 4.1% (3 of 74) Deltaproteobacteria, 4.1% (3 of 74) Actinobacteria, and 2.6% (2 of 74) Gammaproteobacteria.

Bacteria and yeasts as catalysts in microbial fuel cells: electron transfer from micro-organisms to electrodes for green electricity

This article reviews the use of micro-organisms as catalysts at the electrodes of microbial fuel cells (MFCs). The principle of MFCs and their intended use for water treatment and clean electricity production is discussed. We address the different microbial structure and metabolic pathways found in prokaryote (bacteria) and eukaryote (yeasts) that allow the understanding of why electron transfer is possible between a microbe and an electrode. The different mechanisms of microbe–electrode electron transfer are discussed: direct electron transfer or through natural nanowires (pili), mediated electron transfer by natural or artificial redox mediator and finally direct redox transformation of excreted metabolites at the electrodes. This is followed by a review of the different bacteria that have been found and studied in MFCs mainly in the anodic compartment but also more recently in the cathodic side of the fuel cells. A perspective on the possible advantages and challenges of the use of yeasts in MFCs is provided, as this aspect has not been thoroughly studied so far. The fourth section of the review focuses on how to improve the performance and sustainability of MFCs through the functionalisation of the electrode surface, for instance with the covalent grafting of redox mediators and/or enzymes.

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: Direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E0′) of −0.420V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (ks) of GOx immobilized on NiO film glassy carbon electrode are 9.45×10−13molcm−2 and 25.2±0.5s−1, indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis–Menten constant 2.7mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24μM and wide concentration range of 30μM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.

Multilayered construction of glucose oxidase and silica nanoparticles on Au electrodes based on layer-by-layer covalent attachment

A feasible approach to construct multilayer films of aminated silica nanoparticles/glucose oxidase (ASNPs/GOx) on the Au electrode surface using a glutaraldehyde as a covalent attachment cross-linker is described. The covalent attachment processes were followed and confirmed by electrochemical impedance spectroscopy (EIS), which demonstrated that the ASNPs/GOx multilayer films are formed in a progressive and uniform manner. The gold electrodes modified with the ASNPs/GOx multilayer films were studied by cyclic voltammetry (CV) and showed excellent electrocatalytical response to the oxidation of glucose when ferrocenemethanol was used as an artificial redox mediator. From the analysis of voltammetric signals, the coverage of active enzyme on the electrode was estimated, which showed a linear relationship with the number of ASNPs/GOx bilayers. This suggests that the analytical performance such as sensitivity, detection limit is tunable by controlling the number of attached bilayers. The linear response range of the biosensor constructed with four bilayers of ASNPs/GOx to the concentration of glucose can extend at least to 8 m m and reached 95% of the steady-state current in less than 4 s with the sensitivity of 5.11 μA/m m cm 2 and the detection limit of 9 μ m. In addition, the sensor exhibited good stability and long-term life.

Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor

A feasible method to fabricate glucose biosensor was developed by covalent attachment of glucose oxidase (GOx) to a gold nanoparticle monolayer modified Au electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of ferrocyanide followed and confirmed the assemble process of biosensor, and indicated that the gold nanoparticles in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. CV performed in the presence of excess glucose and artificial redox mediator, ferrocenemethanol, allowed to quantify the surface concentration of electrically wired enzyme ( Γ E 0) on the basis of kinetic models reported in literature. The Γ E 0 on proposed electrode was high to 4.1×10 −12 mol·cm −2, which was more than four times of that on electrode direct immobilization of enzyme by cystamine without intermediate layer of gold nanoparticles and 2.4 times of a saturated monolayer of GOx on electrode surface. The analytical performance of this biosensor was investigated by amperometry. The sensor provided a linear response to glucose over the concentration range of 2.0×10 −5–5.7×10 −3 M with a sensitivity of 8.8 μA·mM −1·cm −2 and a detection limit of 8.2 μM. The apparent Michaelis–Menten constant ( K m app) for the sensor was found to be 4.3 mM. In addition, the sensor has good reproducibility, and can remain stable over 30 days.

Multilayered construction of glucose oxidase on gold electrodes based on layer-by-layer covalent attachment

A feasible approach to construct multilayered enzyme film on the gold electrode surface for use as biosensing interface is described. The film was fabricated by alternate layer-by-layer deposition of periodate-oxidized glucose oxidase (GOx) and poly(allylamine) (PAA). The covalent attachment process was followed and confirmed by electrochemical impedance spectroscopy (EIS). X-ray diffraction (XRD) experiments revealed that the film was homogeneous and formed in an ordered manner with a thickness of 2.6 ± 0.1 nm per bilayer. The gold electrodes modified with the GOx/PAA multilayers showed excellent electrocatalytical response to the oxidation of glucose when ferrocenemethanol was used as an artificial redox mediator, which was studied by cyclic voltammetry (CV). From the analysis of voltammetric signals, the coverage of active enzyme on the electrode surface was estimated, which had a linear relationship with the number of GOx/PAA bilayers. This suggests that the analytical performance such as sensitivity, detection limit, and so on, is tunable by controlling the number of attached bilayers. The six GOx/PAA bilayer electrode exhibited a sensitivity of 15.1 μA mM −1 cm −2 with a detection limit of 3.8 × 10 −6 M. In addition, the sensor exhibited good reproducibility and stability.

Construction of layer-by-layer self-assemblies of glucose oxidase and cationic polyelectrolyte onto glassy carbon electrodes and electrochemical study of the redox-mediated enzymatic activity

Build-up of enzyme–polyelectrolyte multilayer onto glassy carbon (GC) surfaces by electrostatic self-assembling method has been investigated. In order to functionalize GC surface by a starting negatively charged layer, two approaches have been carried out: (i) covalent linkage of phenyl acetic acid through electroreduction of 4-phenylacetic diazonium salt (GC A surface), and (ii) formation of a glucose oxidase (GOD) monolayer through an affinity reaction between a GOD conjugated antibody and a previously adsorbed antigen monolayer (GC B surface). GC A and GC B surfaces have been modified by a precursor film (PF) composed of one layer of poly(styrenesulfonate) (PSS) sandwiched between two layers of poly(dimethyldiallylammonium) (PDDA), which improves the further assembling of enzymes. GOD, used as a model enzyme, has been self-assembled with PDDA onto these GC/PF surfaces. Enzymatic activity of immobilized GOD has been electrochemically assessed layer-by-layer during the build-up of (GOD–PDDA) n multilayers. Cyclic voltammetry (CV) performed in the presence of excess glucose and using ferrocene derivatives as artificial redox mediators allowed to quantify the amount of electrically wired enzyme on the basis of kinetic models reported in literature. By using three mediators bearing different electrical charges we conclude that electrostatic interactions between the redox mediator and enzyme microenvironment play a key role in determining the rate of enzyme active site regeneration.

In vitro hydrogen photoproduction using Pyrococcus furiosus sulfhydrogenase and TiO 2

The [NiFe] sulfhydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus has been tested as a biological catalyst in a light-driven in vitro hydrogen evolution system, using a solar simulator apparatus as a source of light. The enzyme is active when coupled to photo-induced titanium dioxide (TiO 2) dispersed as submicronic powder in a buffer solution and in the presence of the artificial redox mediator methylviologen (MV), carrying electrons from the TiO 2 particle to the enzyme. To optimize the system towards practical applications, a simplified version which works in the absence of MV was set up and its performance was assayed with different reductants, as a function of time and over a wide range of temperatures. The comparison of MV-mediated and not-mediated conditions in terms of rate and amount of H 2 photoproduced, highlights a lower efficiency, but an improved overall stability of the system when a direct electron transfer from the TiO 2 to the enzyme takes place. As a consequence, the reaction lifetime is productively extended counteracting the effect of the low H 2 evolution rate with regard to the total amount of H 2 photoproduced. In particular, at 60°C the reaction lifetime is prolonged to 8 h, thus resulting in significantly higher amount of H 2 evolved. The flexibility of the P. furiosus sulfhydrogenase/TiO 2 in vitro system with respect to the redox mediator MV and the evidence that the enzyme performance can be maintained over a wide range of temperatures (60–80°C) are promising advances in the identification of the optimal conditions for the development of an economically feasible hydrogen production process exploiting solar light as a source of energy and waste compounds as electron donors.

Outward photocurrent component in chloroplasts of Peperomia metallica and its assignment to the `closed thylakoid' recording configuration

The photoinduced electrical events at energy-conserving chloroplast membranes can be studied in whole plastids using suction electrodes. In chloroplasts of Peperomia metallica the kinetic profile of photocurrent contains a minor outward component that occurs prior to and differs in polarity from the main component. The origin of this outward current was analyzed using single-turnover flashes in combination with prolonged light exposures and differential physicochemical treatments of tip-located (internal) and the exposed parts of a chloroplast. The outward current signal was higher after 10- to 20-s preillumination and gradually reduced in darkness. The relative amplitude of the outward peak current was enhanced when photosystem II (PS II) was excited by flashes given in the presence of far-red background light ( λ=712 nm). The outward current was small or absent under conditions promoting activity of photosystem I (cyclic electron transport supported by artificial redox mediators in the presence of diuron) and was particularly high in the presence of PS II electron acceptors (e.g., p-phenylenediamine). This indicates the predominant association of the outward current with activity of PS II. The external application of diuron strongly inhibited the inward current, giving rise to a temporal increase in the outward current. On the contrary, when diuron was added into the suction pipette, the outward current was inhibited soon after sealing. The data suggest that the outward current originated in the tip-located portions of the thylakoid membrane that have orientation opposite to the exposed part of `whole thylakoid'. These tip-located membrane portions are least accessible for inhibitors added into the outer medium and are highly sensitive to inhibitors (diuron), ionophores (gramicidin D), and detergents (Triton X-100) added into the pipette. Differential involvement of two photosystems in generation of the outward current may be caused by uneven structural distribution of photosystems I and II between appressed (granal) and nonappressed (stromal) thylakoids and by different recording configurations for these thylakoids.

Localization of the Enzyme System Involved in Anaerobic Reduction of Azo Dyes by Sphingomonas sp. Strain BN6 and Effect of Artificial Redox Mediators on the Rate of Azo Dye Reduction

The effect of different artificial redox mediators on the anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 or activated sludge was investigated. Reduction rates were greatly enhanced in the presence of sulfonated anthraquinones. For strain BN6, the presence of both cytoplasmic and membrane-bound azo reductase activities was shown.

Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Electrochemical data obtained with TMPD+‐sensitive electrodes indicate that ammonium‐uncoupled chloroplasts retain TMPD (N,N,N′,N′‐tetramethyl‐p‐phenylenediamine) mainly in the reduced form during illumination, whereas uncoupled DCMU‐treated chloroplasts accumulate TMPD in the oxidized form (TMPD+). This observation indicates that the reduced plastoquinol is the preferred electron donor for photosystem I (PSI) and TMPD can only compete efficiently when plastoquinone reduction is blocked. After adding DCMU the formation of a transmembrane gradient for TMPD+ is reflected by a slow‐down of the electrogenic electron transport and by the emerging of the overshoot of the membrane current in the light‐off response. A light‐dependent increase in photoelectric current generated by chloroplasts in the presence of NH4Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.

Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO3-→H2S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c3 from D. desulfuricans. The specific activity of mSiR was 103 nmol H2 min-1 mg-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO3-→H2S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.

The (2R)-hydroxycarboxylate-viologen-oxidoreductase from Proteus vulgaris is a molybdenum-containing iron-sulphur protein.

An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2-oxocarboxylates at the expense of reduced artificial redox mediators to (2R)-hydroxycarboxylates has been purified to a specific activity of up to 1800 mumol.min-1.mg-1 for the reduction of phenylpyruvate. The membrane-bound non-pyridine nucleotide-dependent enzyme appears in the form of various oligomers of the 80-kDa monomer. The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on 99Mo-labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis. Permanganate oxidation delivers 0.7 mol pterin-6-carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron-sulphur clusters.

On the redox equilibrium between H 2 and hydrogenase

Redox titrations of the nickel ion in active hydrogenase from Methanobacterium thermoautotrophicum and Chromatium vinosum were performed in the absence of artificial redox mediators, by variation of the H2-partial pressure. These experiments revealed a redox behaviour of the nickel ion which differed remarkably from previous redox titrations in the presence of redox mediators. Notably the EPR signal of the species earlier characterized as monovalent nickel with bound hydrogen, behaved as an n = 2 redox component upon reduction under varying H2-partial pressures. The EPR signal was not a transient one and persisted upon removal of hydrogen. Possible redox processes to explain these observations are discussed. A similar behaviour of nickel was also observed in enzyme as present in intact cells of M. thermoautotrophicum. These results suggest that nickel hydrogenases possess a second site for reaction with H2.

The stimulation of photophosphorylation and ATPase by artificial redox mediators in chromatophores ofRhodopseudomonas capsulata at different redox potentials

(1) Inhibition of cyclic phosphorylation in chromatophores of Rhodopseudomonas capsulata by antimycin A can be fully reversed by artificial redox mediators, provided the ambient redox potential is maintained around 200 mV. The redox mediator need not be a hydrogen carrier in its reduced form, N-methyl-phenazonium methosulfate and N,N,N',N'-tetramethyl-p-phenylenediamine being equally effective. However, the mediator needs to be lipophilic. Endogenous cyclic phosphorylation is fastest around 130 mV. A shift to 200 mV can also be observed if high concentrations of artificial redox mediator are present in the absence of antimycin. (2) ATPase activity of Rhodopseudomonas capsulata, in the light as well as in the dark, activated or not activated by inorganic phosphate, can also be stimulated by N-methylphenazonium methosulfate. This stimulation is highest at redox potentials between 60 to 80 mV and is sensitive to antimycin A. In this case N,N,N',N-tetramethyl-p-phenylenediamine is much less effective.