Effect of Gold Nanoparticles on the Structure and Electron‐Transfer Characteristics of Glucose Oxidase Redox Polyelectrolyte‐Surfactant Complexes

Efficient electrical communication between redox proteins and electrodes is a critical issue in the operation and development of amperometric biosensors. The present study explores the advantages of a nanostructured redox‐active polyelectrolyte–surfactant complex containing [Os(bpy)₂Clpy]²⁺ (bpy=2,2′‐bipyridine, py= pyridine) as the redox centers and gold nanoparticles (AuNPs) as nanodomains for boosting the electron‐transfer propagation throughout the assembled film in the presence of glucose oxidase (GOx). Film structure was characterized by grazing‐incidence small‐angle X‐ray scattering (GISAXS) and atomic force microscopy (AFM), GOx incorporation was followed by surface plasmon resonance (SPR) and quartz‐crystal microbalance with dissipation (QCM‐D), whereas Raman spectroelectrochemistry and electrochemical studies confirmed the ability of the entrapped gold nanoparticles to enhance the electron‐transfer processes between the enzyme and the electrode surface. Our results show that nanocomposite films exhibit five‐fold increase in current response to glucose compared with analogous supramolecular AuNP‐free films. The introduction of colloidal gold promotes drastic mesostructural changes in the film, which in turn leads to a rigid, amorphous interfacial architecture where nanoparticles, redox centers, and GOx remain in close proximity, thus improving the electron‐transfer process.     

Flavin Derivatives with Tailored Redox Properties: Synthesis,Characterization, and Electrochemical Behavior

This study establishes structure–property relationships for four synthetic flavin molecules as bioinspired redox mediators in electro‐ and photocatalysis applications. The studied flavin compounds were disubstituted with polar substituents at the N1 and N3 positions (alloxazine) or at the N3 and N10 positions (isoalloxazines). The electrochemical behavior of one such synthetic flavin analogue was examined in detail in aqueous solutions of varying pH in the range from 1 to 10. Cyclic voltammetry, used in conjunction with hydrodynamic (rotating disk electrode) voltammetry, showed quasi‐reversible behavior consistent with freely diffusing molecules and an overall global 2e^?, 2H⁺ proton‐coupled electron transfer scheme. UV/Vis spectroelectrochemical data was also employed to study the pH‐dependent electrochemical behavior of this derivative. Substituent effects on the redox behavior were compared and contrasted for all the four compounds, and visualized within a scatter plot framework to afford comparison with prior knowledge on mostly natural flavins in aqueous media. Finally, a preliminary assessment of one of the synthetic flavins was performed of its electrocatalytic activity toward dioxygen reduction as a prelude to further (quantitative) studies of both freely diffusing and tethered molecules on various electrode surfaces.     

Direct electrochemistry and superficial characterization of DNA-cytochrome c-MUA films on chemically modified gold surface

Cytochrome c (Cyt. c) was immobilized on the 11-mercaptohendecanoic acid (MUA)-modified gold electrode. The electrode was stable and sensitive to Cyt. c. Later, DNA was also immobilized on the two-layer modified electrode. Cyclic voltammetry studies show that Cyt. c can interact with dsDNA and ssDNA. The binding site sizes were determined to be 15 base pairs per Cyt. c molecule with dsDNA and 30 nucleotides binding 1 Cyt. c molecule with ssDNA. The modified electrodes were characterized by quartz crystal microbalance (QCM), impedance spectroscopy and atomic force microscope (AFM). The modified electrode can be used for determining DNA.     

Electrochemical Modification of Gold Electrodes with Azobenzene Derivatives by Diazonium Reduction

An electrochemical study of Au electrodes electrografted with azobenzene (AB), Fast Garnet GBC (GBC) and Fast Black K (FBK) diazonium compounds is presented. Electrochemical quartz crystal microbalance, ellipsometry and atomic force microscopy investigations reveal the formation of multilayer films. The elemental composition of the aryl layers is examined by X‐ray photoelectron spectroscopy. The electrochemical measurements reveal a quasi‐reversible voltammogram of the Fe(CN)₆^3?/4? redox couple on bare Au and a sigmoidal shape for the GBC‐ and FBK‐modified Au electrodes, thus demonstrating that electron transfer is blocked due to the surface modification. The electrografted AB layer results in strongest inhibition of the Fe(CN)₆^3?/4? response compared with other aryl layers. The same tendencies are observed for oxygen reduction; however, the blocking effect is not as strong as in the Fe(CN)₆^3?/4? redox system. The electrochemical impedance spectroscopy measurements allowed the calculation of low charge‐transfer rates to the Fe(CN)₆^3? probe for the GBC‐ and FBK‐modified Au electrodes in relation to bare Au. From these measurements it can be concluded that the FBK film is less compact or presents more pinholes than the electrografted GBC layer.     

Oxygen-reducing electrodes based on layer-by-layer assemblies of cytochrome c and laccasse

Electroactive multilayer assemblies combining the redox protein cytochrome c and the enzyme laccase were fabricated by the layer-by-layer adsorption technique on gold electrodes and were shown to be capable of direct oxygen reduction. Laccase from trametes versicolor was electrostatically immobilized on multilayer films consisting of cytochrome c and the polyelectrolyte polyanilinesulfonic acid. The layer formation was monitored by quartz crystal microbalance. The electrochemical behavior of the electrodes was investigated by cyclic voltammetry. The resulting assembly exhibited a catalytic oxygen reduction current. This indicates a multi-step electron transport chain from the electrode via the cytochrome c layers towards laccase, and finally, to molecular oxygen. The catalytic efficiency of the electrodes was examined in the pH range from 4.5 to 7.0, showing highest enzymatic oxygen reduction at pH 4.5. Furthermore, the catalytic current was found to correlate linearly with the oxygen content of the solution. This suggests that the overall current is limited by the catalytic reduction of oxygen by the laccase rather than by the preceding electron transfer steps.     

Preparation and Redox Properties of N,N,N‐1,3,5‐Trialkylated Flavin Derivatives and Their Activity as Redox Catalysts

Eight different flavin derivatives have been synthesized and the electronic effects of substituents in various positions on the flavin redox chemistry were investigated. The redox potentials of the flavins, determined by cyclic voltammetry, correlated with their efficiency as catalysts in the H₂O₂ oxidation of methyl p‐tolyl sulfide. Introduction of electron‐withdrawing groups increased the stability of the reduced catalyst precursor.     

Preparation, characterization, and electrocatalytic properties of hybrid coatings of hexacyanometalate-doped-cationic films

Electrochemically active hybrid coatings based on cationic films, didodecyldimethylammonium bromide (DDAB), and poly(diallyldimethylammonium chloride) (PDDAC) are prepared on electrode surface by cycling the film-covered electrode repetitively in a pH 6.5 solution containing Fe(CN)₆ ³⁻ and Ru(CN)₆ ⁴⁻ anions. Modified electrodes exhibited stable and reversible voltammetric responses corresponding to characteristics of Fe(CN)₆ ^3−/4− and Ru(CN)₆ ^4−/3− redox couples. The cyclic voltammetric features of hybrid coatings resemble that of electron transfer process of surface-confined redox couple. Electrochemical quartz crystal microbalance results show that more amounts of electroactive anionic complexes partitioned into DDAB coating than those doped into PDDAC coating from the same doping solution. Peak potentials of hybrid film-bound redox couples showed a negative shift compared to those at bare electrode and this shift was more pronounced in the case of DDAB. Finally, the advantages of hybrid coatings in electrocatalysis are demonstrated with sulfur oxoanions.     

DYNAMICS OF EXCITED FLAVOPROTEINS—PICOSECOND LASER PHOTOLYSIS STUDIES

Abstract— Molecular mechanism of fluorescence quenching of flavins in flavodoxin from Desulfovibrio vulgaris , strain Miyazaki, and riboflavin binding protein from egg white has been investigated by means of picosecond laser photolysis technique. In the case of flavodoxin, a transient absorption band characteristic of the non-fluorescent exciplex formed by electron transfer from indole to excited flavins in model systems has been observed around 600 nm at the delay time of 33 ps from exciting ps pulse pulse width, 25 ps). In the case of riboflavin binding protein, the transient absorption spectra were different from those of flavin-indole exciplex and rather similar to the spectra of the model system of flavin-phenol. These results suggest that tryptophan residue exists near the isoalloxazine nucleus in flavodoxin, and in riboflavin binding protein, tyrosine residue exists near the flavin. Direct measurements of the ultrafast process of the electron transfer in flavoproteins as developed here could provide useful information for elucidating protein dynamics, associated with redox reaction, in the picosecond time region.     

Blocking properties of gold electrodes modified with 4-nitrophenyl and 4-decylphenyl groups

The electrochemical properties of Au electrodes grafted with 4-nitrophenyl and 4-decylphenyl groups have been studied. The electrografting of gold electrode surface with aryl groups was carried out by electroreduction of the corresponding diazonium salts in acetonitrile. The nitrophenyl film growth on gold was examined by atomic force microscopy, electrochemical quartz crystal microbalance and X-ray photoelectron spectroscopy. These measurements showed that a multilayer film of nitrophenyl groups was formed. Cyclic voltammetry was used to study the blocking properties of aryl-modified gold electrodes towards the Fe(CN)₆^3−/4− redox system. The reduction of oxygen was strongly suppressed on these electrodes as evidenced by the rotating disc electrode results.     

Chiral NADH model systems functionalized with Zn(II)-cyclen as flavin binding site

A series of chiral peptides has been prepared, bearing a 1,4-dihydronicotine amide and a zinc cyclen moiety. The metal complex reversibly binds flavins in aqueous solution, while the dihydronicotine amide serves as a NADH model transferring a hydride to the flavin within the assembly. The reaction rate of the redox reaction was monitored and determined by UV spectroscopy. The reaction rates of the substituted compounds were slower if compared to the non-substituted parent compound 1-H, but still show a 30-100 fold rate enhancement compared to the compound missing a flavin binding site. It was anticipated to probe the cryptic stereoselectivity of the hydride transfer from dihydropyridine to flavin. Spectroscopic data indicate that the introduction of deuterium labels upon reduction of the pyridinium salts to 1,4-dihydropyridine in D₂O proceeds diastereoselectively, but identical isotope effects on the rate of flavin reduction as with a non-chiral NADH model revealed that the hydride transfer within the assembly proceeds not stereoselective. A more rigid chiral NADH model compound must be prepared to achieve this goal.     

Flavin-sensitized Photo-oxidation of Lysozyme and Serum Albumin

The excited state processes of riboflavin, flavin mononucleotide and flavin adenine dinucleotide in argon-saturated aqueous solution were studied in the presence of lysozyme or bovine serum albumin (BSA). UV–Vis absorption and fluorescence spectroscopy indicates that the noncovalent flavin-protein binding is relatively weak. Quenching of the flavin triplet state by BSA, observed by time-resolved photolysis, is less efficient than by lysozyme. Light-induced oxidation of the two proteins and reduction of the three flavins were studied. The quantum yields of the former and latter in the absence of oxygen are up to 0.1 and 0.04, respectively. The effects of pH and sensitizer and protein concentrations were examined in greater detail. The proposed reaction is electron transfer from the tryptophan moiety to the flavin triplet rather than excited singlet state.     

The preparation of a novel polydopamine-graft-poly(2-methyl-2-oxazoline) protein-resistant coating and its applications in protein separation

A novel polydopamine-graft-poly(2-methyl-2-oxazoline)(PDA-g-PMOXA) coating was prepared by immobilizing poly(2-methyl-2-oxazoline)(PMOXA) onto material surfaces through polydopamine (PDA) anchored coating for the first time.And then,the chemical composition,hydrophilicity,and protein-resistant properties of the PDA-g-PMOXA coating were studied using X-ray photoelectron spectroscopy(XPS),contact angel(CA) test,surface plasmon resonance(SPR),and quartz crystal microbalance with dissipation(QCM-D) measurement.Finally,the coating was applied to the capillary inner surface for protein separation by capillary electrophoresis(CE).     

Fabrication and Characterization of DNA/QPVP‐Os Redox‐Active Multilayer Film

Calf thymus DNA was immobilized on functionalized glassy carbon, gold and quartz substrates, respectively, by the layer‐by‐layer (LBL) assembly method with a polycation QPVP‐Os, a quaternized poly(4‐vinylpyridine) partially complexed with osmium bis(2,2′‐bipyridine) as counterions. UV‐visible absorption and surface plasmon resonance spectroscopy (SPR) showed that the resulting film was uniform with the average thickness 3.4 nm for one bilayer. Cyclic voltammetry (CV) showed that the total surface coverage of the polycations increases as each QPVP‐Os/DNA bilayer added to the electrode surface, but the surface formal potential of Os‐centered redox reaction shifts negatively, which is mainly attributed to the intercalation of redox‐active complex to DNA chain. The electron transfer kinetics of electroactive QPVP‐Os in the multilayer film was investigated by electrochemical impedance experiment for the first time. The permeability of Fe(CN) in the solution into the multilayer film depends on the number of bilayers in the film. It is worth noting that when the multilayer film is up to 4 bilayers, the CV curves of the multilayer films display the typical characteristic of a microelectrode array. The nanoporous structure of the multilayer film was further confirmed by the surface morphology analysis using atomic force microscopy (AFM).     

A site-differentiated [4Fe–4S] cluster controls electron transfer reactivity of Clostridium acetobutylicum [FeFe]-hydrogenase I

One of the many functions of reduction–oxidation (redox) cofactors is to mediate electron transfer in biological enzymes catalyzing redox-based chemical transformation reactions. There are numerous examples of enzymes that utilize redox cofactors to form electron transfer relays to connect catalytic sites to external electron donors and acceptors. The compositions of relays are diverse and tune transfer thermodynamics and kinetics towards the chemical reactivity of the enzyme. Diversity in relay design is exemplified among different members of hydrogenases, enzymes which catalyze reversible H₂ activation, which also couple to diverse types of donor and acceptor molecules. The [FeFe]-hydrogenase I from Clostridium acetobutylicum (CaI) is a member of a large family of structurally related enzymes where interfacial electron transfer is mediated by a terminal, non-canonical, His-coordinated, [4Fe–4S] cluster. The function of His coordination was examined by comparing the biophysical properties and reactivity to a Cys substituted variant of CaI. This demonstrated that His coordination strongly affected the distal [4Fe–4S] cluster spin state, spin pairing, and spatial orientations of molecular orbitals, with a minor effect on reduction potential. The deviations in these properties by substituting His for Cys in CaI, correlated with pronounced changes in electron transfer and reactivity with the native electron donor–acceptor ferredoxin. The results demonstrate that differential coordination of the surface localized [4Fe–4S]His cluster in CaI is utilized to control intermolecular and intramolecular electron transfer where His coordination creates a physical and electronic environment that enables facile electron exchange between electron carrier molecules and the iron–sulfur cluster relay for coupling to reversible H₂ activation at the catalytic site.

Histidine coordination of the distal [4Fe–4S] cluster in [FeFe]-hydrogenase was demonstrated to tune the cluster spin-states, spin-pairing and surrounding molecular orbitals to enable more facile electron transfer compared to cysteine coordination.     

Multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole DNA biosensor for label-free detection of genetically modified organisms by QCM and EIS

In this paper, we describe DNA electrochemical detection for genetically modified organism (GMO) based on multi-wall carbon nanotubes (MWCNTs)-doped polypyrrole (PPy). DNA hybridization is studied by quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). An increase in DNA complementary target concentration results in a decrease in the faradic charge transfer resistance (R_ct) and signifying “signal-on” behavior of MWCNTs-PPy-DNA system. QCM and EIS data indicated that the electroanalytical MWCNTs-PPy films were highly sensitive (as low as 4 pM of target can be detected with QCM technique). In principle, this system can be suitable not only for DNA but also for protein biosensor construction.     

Reversible,electrochemical interconversion of NADH and NAD+ by the catalytic (Ilambda) subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I)

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the mitochondrial electron transport chain and catalyzes the oxidation of beta-NADH by ubiquinone, coupled to transmembrane proton translocation. It contains a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur (FeS) clusters, and at least one ubiquinone binding site. Little is known about the mechanism of coupled electron-proton transfer in complex I. This communication demonstrates how the catalytic fragment of complex I, subcomplex Ilambda, can be adsorbed onto a pyrolytic graphite edge electrode to catalyze the interconversion of NADH and NAD+, with the electrode as the electron acceptor or donor. NADH oxidation and NAD+ reduction are completely reversible and occur without the application of an overpotential. The potential of zero current denotes the potential of the NAD+/NADH redox couple, and the dependence of ENAD+ on pH, and on the NADH:NAD+ ratio, is in accordance with the Nernst equation. The catalytic potential of the enzyme, Ecat, is close to one of the two reduction potentials of the active site FMN and to the potential of a nearby [2Fe - 2S] cluster; therefore, either one or both of these redox couples is suggested to be important in controlling NADH oxidation by complex I.     

Calculating chemically accurate redox potentials for engineered flavoproteins from classical molecular dynamics free energy simulations

The tricyclic isoalloxazine nucleus of the redox cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) acts as an electron sink in life-sustaining biological electron transfer (eT). The functional diversity of flavin-containing proteins (flavoproteins) transcends that of free flavins. A large body of experimental evidence attributes natural control of flavoprotein-mediated eT to tuning of the thermodynamic driving force by the protein environment. Understanding and engineering such modulation by the protein environment of the flavin redox potential (DeltaE(o)) is valuable in biotechnology and device design. In this study we employed classical molecular dynamics free energy simulations (MDFES), within a thermodynamic integration (TI) formalism, to calculate the change in FMN first reduction potential (DeltaDeltaE(o)(ox/sq)) imparted by 6 flavoprotein active site mutations. The combined performance of the AMBER ff03 (protein) and GAFF (cofactor) force fields was benchmarked against experimental data for mutations close to the isoalloxazine re- and si-faces that perturb the wild-type DeltaE(o)(ox/sq) value in Anabaena flavodoxin. The classical alchemical approach used in this study overestimates the magnitude of DeltaE(o) values, in common with other studies. Nevertheless, chemically accurate DeltaDeltaE(o) values--calculated to within 1 kcal mol(-1) of the experimental value--were obtained for five of the six mutations studied. We have shown that this approach is practical for quantitative in silico screening of the effect of mutations on the first reduction potential where experimental values and structural data are available for the wild-type flavoprotein. This approach promises to be useful as an integral part of future interdisciplinary strategies to engineer desired thermodynamic properties in flavoproteins of biotechnological interest.     

Combining metal-microbe and microbe-microbe dual direct electron transfer on Fe(0)-cathode of bio-electrochemical system to enhance anaerobic digestion of cellulose wastewater

Considering that cathode of microbial electrochemical system (MES) is a good electrons source for methane production via direct/indirect electron transfer to electroactive microorganisms, and that Fe(0) is also a confirmed electron donor for some electroactive microorganisms through metal-microbe direct electron transfer (DET), Fe(0)-cathode was equipped into an MES digester to enhance cathodic methane production. The results of this study indicated that the potential DET participator, Clostridium possibly obtained electrons directly from Fe(0)-cathode via metal-microbe electrons transfer, then transferred electrons directly to the definite DET participators, Methanosarcina/Methanothrix via microbe-microbe electrons transfer for CH₄ production. In addition, Methanobacterium is another specially enriched methanogen on Fe(0)-cathode, which might obtain electrons directly from Fe(0)-cathode to produce CH₄ via metal/electrode-microbe DET. The increment of conductivity of cathodic sludge in Fe(0)-cathode MES digester (R1) further confirmed the enrichment of electroactive microorganisms participating in DET process. As a consequence, a higher CH₄ production (1205–1508 mL/d) and chemical oxygen demand (COD) removal (79.0%-93.8%) were achieved in R1 compared with graphite-cathode MES digester (R2, 720–1090 mL/d and 63.6%-85.6%) and the conventional anaerobic digester (R3, 384–428 mL/d and 35.2%-41.0%). In addition, energy efficiency calculated indicated that the output energy of CH₄ production was 8.16 folds of electricity input in Fe(0)-cathode MES digester.     

Electrochemical Quartz Crystal Microbalance Study of FeS Particles Attached to Au Surface

Electrochemical quartz crystal microbalance (EQCM) measurements were employed for studying of the redox processes of FeS microparticles immobilized on Au electrode surface in contact with aqueous solutions of NaCl, NaCl‐NaHCO₃, and NaCl‐Na₂S. The objective was to shed light on the complexity of the iron sulfide electrochemistry. For the sake of comparison electrochemical behavior of FeS suspension was also investigated on the paraffin‐impregnated graphite (PIGE) and hanging mercury drop (HMDE) electrodes. In order to understand the complex nature of the redox transformations of FeS, Mohr‐salt was dissolved in NaCl and NaCl‐NaHCO₃ solutions, respectively, and the deposition‐dissolution processes occurring in the course of the reduction of Fe(II) and reoxidation of Fe(0) were also monitored.     

Effects of slow substrate binding and release in redox enzymes: theory and application to periplasmic nitrate reductase

For redox enzymes, the technique called protein film voltammetry makes it possible to determine the entire profile of activity against driving force by having the enzyme exchanging directly electrons with the rotating-disc electrode onto which it is adsorbed. Both the potential location of the catalytic response and its detailed shape report on the sequence of catalytic events, electron transfers and chemical steps, but the models that have been used so far to decipher this signal lack generality. For example, it was often proposed that substrate binding to multiple redox states of the active site may explain that turnover is greater in a certain window of electrode potential, but no fully analytical treatment has been given. Here, we derive (i) the general current equation for the case of reversible substrate binding to any redox states of a two-electron active site (as exemplified by flavins and Mo cofactors), (ii) the quantitative conditions for an extremum in activity to occur, and (iii) the expressions from which the substrate-concentration dependence of the catalytic potential can be interpreted to learn about the kinetics of substrate binding and how this affects the reduction potential of the active site. Not only does slow substrate binding and release make the catalytic wave shape highly complex, but we also show that it can have important consequences which will escape detection in traditional experiments: the position of the wave (this is the driving force that is required to elicit catalysis) departs from the reduction potential of the active site even at the lowest substrate concentration, and this deviation may be large if substrate binding is irreversible. This occurs in the reductive half-cycle of periplasmic nitrate reductase where irreversibility lowers the driving force required to reduce the active site under turnover conditions and favors intramolecular electron transfer from the proximal [4Fe4S]+ cluster to the active site Mo(V).