Direct electrochemical conversion of bilirubin oxidase at carbon nanotube-modified glassy carbon electrodes

We report on direct electron transfer reactions of bilirubin oxidase at multi-walled carbon nanotube (MWCNT) modified glassy carbon electrodes (GCE). The bioelectrocatalytic oxygen reduction was recorded using linear sweep voltammetry (LSV) with BOD in solution, adsorbed and covalently linked to the nanotubes. The MWCNT modification of GC electrodes strongly enhances the oxygen reduction compared to the signals at unmodified GCE. Under anaerobic conditions with a high protein concentration in solution a pair of redox peaks with a formal potential of 450 ± 15 mV vs Ag/AgCl, 1 M KCl (pH 7.4) was found with cyclic voltammetry. The redox conversion is indicated to be surface-controlled and pH-dependent (54.5 mV/pH). The quasi-reversible redox reaction might be attributed to the trinuclear T2/T3 cluster of BOD.     

Direct electron transfer reaction of glucose oxidase at bare silver electrodes and its application in analysis

The direct electron transfer reaction of glucose oxidase (GOx) at a bare silver electrode is verified. The electron transfer number n = 2, electron transfer coefficient α = 0.45 and rate constant of the electrochemical reaction K_s = 0.1 s⁻¹ are obtained. This communication presents a multimolecular adsorption model to explain the properties of the direct electron reaction between GOx and bare silver electrodes. The residual valence force may be an important factor to ensure a direct electron transfer reaction on the bare electrode. On the basis of the experimental fact that only biologically active GOx exhibits electrochemical activity in solution, a facile analytical method for analyzing the active GOx concentration is developed. The results determined correspond very well to that of a spectrometric method.     

Direct electron transfer in immobilized flavoenzyme chemically modified graphite electrodes

Direct electron transfer between covalently immobilized flavoenzymes and a cyanuric chloride-modified graphite electrode is observed via differential pulse voltammetry. L-Amino acid oxidase and xanthine oxidase display peaks arising from the reduction of flavin adenine dinucleotide. Peak current enhancements are observed for both covalently attached enzymes compared to their free and adsorbed state voltammograms. Studies concerning flavin removal and reconstitution indicate that xanthine oxidase contains multiple flavin chromophores which are nonequivalent.     

Direct electron transfer reactions between human ceruloplasmin and electrodes

In an effort to find conditions favouring bioelectrocatalytic reduction of oxygen by surface-immobilised human ceruloplasmin (Cp), direct electron transfer (DET) reactions between Cp and an extended range of surfaces were considered. Exploiting advances in surface nanotechnology, bare and carbon-nanotube-modified spectrographic graphite electrodes as well as bare, thiol- and gold-nanoparticle-modified gold electrodes were considered, and ellipsometry provided clues as to the amount and form of adsorbed Cp. DET was studied under different conditions by cyclic voltammetry and chronoamperometry. Two Faradaic processes with midpoint potentials of about 400 mV and 700 mV vs. NHE, corresponding to the redox transformation of copper sites of Cp, were clearly observed. In spite of the significant amount of Cp adsorbed on the electrode surfaces, as well as the quite fast DET reactions between the redox enzyme and electrodes, bioelectrocatalytic reduction of oxygen by immobilised Cp was never registered. The bioelectrocatalytic inertness of this complex multi-functional redox enzyme interacting with a variety of surfaces might be associated with a very complex mechanism of intramolecular electron transfer involving a kinetic trapping behaviour.     

Direct electron transfer of PQQ-glucose dehydrogenase at modified carbon nanotubes electrodes

In order to establish efficient enzyme-electrode-contacts for the pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) different immobilisation strategies are investigated. Multi-walled carbon nanotubes (MWCNT) on gold electrodes are modified by chemical treatment and by (poly)-aniline derivatives. The electropolymerisation of methoxy-m-anilinesulfonic acid and m-aminobenzoic acid on the MWCNTs allows the covalent coupling of the PQQ-GDH. Such a poly-[ASA-ABA]/MWCNT/Au electrode can achieve current densities of up to 500 μA/cm² at a potential of 100 mV vs. Ag/AgCl. Furthermore investigations with small amounts of free PQQ indicate direct electron transfer between enzyme and electrode.     

Direct electron transfer of glucose oxidase on the carbon nanotube electrode

The direct electrochemistry of redox enzymes (or proteins) has received more and more attention[1—9]. These studies developed an electrochemical basis for the investigation of enzyme structure, mechanisms of redox transformations of enzyme molecules and metabolic processes involving redox transformations. From these studies, one can also find potential appli-cations of enzymes in biotechnology. For example, if an enzyme immobilized on electrode surface is ca-pable of the direct electron tra…     

Direct electron transfer reactions of laccases from different origins on carbon electrodes

Electrochemical studies of laccases from basidiomycetes, i.e., Trametes hirsuta, Trametes ochracea, Coriolopsis fulvocinerea, Cerrena maxima, and Cerrena unicolor, have been performed. Direct (mediatorless) electrochemistry of laccases on graphite electrodes has been investigated with cyclic voltammetry, square wave voltammetry as well as potentiometry. For all mentioned high potential laccases direct electron transfer (DET) has been registered at spectrographic graphite and highly ordered pyrolytic graphite electrodes. The characteristics of DET reactions of the enzymes were analysed under aerobic and anaerobic conditions. It is shown that the T1 site of the laccase is the primary electron acceptor, both in solution (homogenous case) and at surface of the graphite electrode (heterogeneous case). A mechanism of ET for the process of the electro-reduction of oxygen at the laccase-modified graphite electrodes is proposed and the similarity of this heterogeneous process to the laccase catalysed oxygen reduction homogeneous reaction is concluded.     

Direct electron transfer for hemoglobin in biomembrane-like dimyristoyl phosphatidylcholine films on pyrolytic graphite electrodes

Stable thin films made from dimyristoyl phosphatidylcholine (DMPC) with incorporated hemoglobin (Hb) on pyrolytic graphite (PG) electrodes were characterized by electrochemical and other techniques. Cyclic voltammetry (CV) of Hb-DMPC films showed a pair of well-defined and nearly reversible peaks at about -0.27 V vs. saturated calomel electrode (SCE) at pH 5.5, characteristic of Hb heme Fe(III)/Fe(II) redox couple. The electron transfer between Hb and PG electrodes was greatly facilitated in DMPC films. Apparent heterogeneous rate constants (ks) were estimated by fitting square wave voltammograms of Hb-DMPC films to a model featuring thin layer behavior and dispersion of formal potentials for redox center. The formal potential of Hb heme Fe(III)/Fe(II) couple in DMPC films shifted linearly between pH 4.5 to 11 with a slope of -48 mV pH-1, suggesting that one proton is coupled to each electron transfer in the electrochemical reaction. Soret absorption band positions suggest that Hb retains a near native conformation in DMPC films at medium pH. Differential scanning calorimetry (DSC) showed the phase transition for DMPC and Hb-DMPC films, suggesting DMPC has an ordered multibilayer structure. Trichloroacetic acid (TCA) was catalytically reduced by Hb-DMPC films with significant decreases in the electrode potential required.     

Modification of electrodes with nanostructured films containing dirhodium-substituted polyoxometalates

Layer-by-layer (LBL) electrostatic assembly was used to prepare films comprising generation-4 polyamidoamine dendrimers (G4-PAMAM) and dirhodium polyoxometalates (Rh₂POMs) on glassy carbon (GC) electrodes. To employ the propensity of P₂Mo₁₈O₆₂ ^6– (PMo) to directly adsorb on glassy carbon, a new compound, Rh₂PMo, was synthesized and characterized. The behavior was compared to that of a previously reported Rh₂POM prepared from PW₁₂O₄₀ ^3– (PW). The LBL assembly of (G4-PAMAM, Rh₂PW) _n bilayers on GC pretreated by formation of a monolayer of 4-aminobenzoic acid was studied by doing a linear least-squares fit of peak current to n (n=5); the result gave an r ² of only 0.90. With Rh₂PMo, the analogous experiments with n=7 gave an r ²=0.995. Strong adsorption of Rh₂PMo onto GC allowed the assembly of Rh₂PMo_adsorbed|(G4-PAMAM, Rh₂PMo) _n on conventional GC and on a 10-µm carbon fiber. The latter was used to demonstrate the bifunctional electrocatalytic property of these films through reduction of nitrite and the oxidation of arsenite.Contribution to the 3rd Baltic Conference on Electrochemistry, Gdansk-Sobieszewo, Poland, 23–26 April 2003Dedicated to the memory of Harry B. Mark, Jr. (28 February 1934–3 March 2003)     

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer-modified nanoporous gold electrodes

Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (±0.4), 16.0 (±0.1) and 2.0 (±0.1) μM were obtained for CtCDH-modified lactose and glucose biosensors and GOx-modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells composed of GOx- and CtCDH-modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase as cathodes, in biofuel cells. A maximum power density of 41 μW/cm² was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O₂-saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).     

Adsorption characteristics of electrodes containing nanostructured carbon of different morphology

The effect of camphor adsorption on the differential capacitance of electrodes of nanostructured carbon of different morphology (single-walled carbon nanotubes, filiform carbon, and columnar structures) in aqueous electrolyte solutions and also on the electrochemical reactions in these systems is studied. It is shown that irrespective of the ac frequency, the differential capacitance of the nanopaper and columnar electrodes increases 3–5-fold throughout the studied potential range. This experimental fact is explained by the substantial increase in the electrode surface accessible for electrolyte, which is a manifestation of the Rehbinder effect in electrochemistry. The revealed different kinds of effects of camphor adsorption layers formed at the nanostructured carbon/electrolyte interface on the electron transfer processes are as follows: partial inhibition of both the electron injection and the K₃[Fe(CN)₆] reduction; complete suppression of the reduction of sodium nitrate and nitrite; the absence of effects on the OH radical reduction and solvated electron oxidation.     

Direct electron transfer between heme-containing enzymes and electrodes as basis for third generation biosensors

Direct electron transfer (DET) between redox enzymes and electrodes found the basis for third generation biosensors. Recent investigations in the authors’ laboratories on the bioelectrochemistry of heme-containing proteins and enzymes, primarily peroxidases, but also cellobiose dehydrogenase, will be reviewed.     

Interprotein electron transfer in a confined space: uncoupling protein dynamics from electron transfer by sol-gel encapsulation

In this paper, we describe the first observations of photoinitiated interprotein electron transfer (ET) within sol-gels. We have encapsulated three protein-protein complexes, specifically selected because they represent a full range of affinities, are sensitive to different types of dynamic processes, and thus are expected to respond differently to sol-gel encapsulation. The three systems are (i) the [Zn, Fe(3+)L] mixed-metal hemoglobin hybrids, where the alpha(1)-Zn and beta(2)-Fe subunits correspond to a "predocked" protein-protein complex with a crystallographically defined interface (Natan, M. J.; Baxter, W. W.; Kuila, D.; Gingrich, D. J.; Martin, G. S.; Hoffman, B. M. Adv. Chem. Ser. 1991, 228 (Electron-Transfer Inorg., Org., Biol. Syst.), 201-213), (ii) the Zn-cytochrome c peroxidase complex with cytochrome c, [ZnCcP, Fe(3+)Cc], having an intermediate affinity between its partners (Nocek, J. M.; Zhou, J. S.; De Forest, S.; Priyadarshy, S.; Beratan, D. N.; Onuchic, J. N.; Hoffman, B. M. Chem. Rev. 1996, 96, 2459-2489), and (iii) the [Zn-deuteromyoglobin, ferricytochrome b(5)] complex, [ZnDMb, Fe(3+)b(5)], which is loosely bound and highly dynamic (Liang, Z.-X.; Nocek, J.; Huang, K.; Hayes, R. T.; Kurnikov, I. V.; Beratan, D. N.; Hoffman, B. M. J. Am. Chem. Soc. 2002, 124, 6849-6859. Intersubunit ET within the hybrid does not involve second-order processes or subunit rearrangements, and thus is influenced only by perturbations of high-frequency motions coupled to ET. For the latter two complexes, sol-gel encapsulation eliminates second-order processes: protein partners encapsulated as a complex must stay together throughout a photoinitiated ET cycle, while proteins encapsulated alone cannot acquire a partner. It further modulates intracomplex motions of the two partners.     

Direct electron transfer of glucose oxidase promoted by carbon nanotubes is without value in certain mediator-free applications

We have investigated the direct electron transfer (DET) promoted by carbon nanotubes (CNTs) on an electrode containing immobilized glucose oxidase (GOx) with the aim to develop a third-generation glucose biosensor and a mediator-free glucose biofuel cell anode. GOx was immobilized via chitosan (CS) on a glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes (MWCNTs). Cyclic voltammetric revealed that the GOx on the surface of such an electrode is unable to simultaneously demonstrate DET with the electrode and to retain its catalytic activity towards glucose, although the MWCNTs alone can promote electron transfer between GOx and electrode. This is interpreted in terms of two types of GOx on the surface, the distribution and properties of which are quite different. The first type exhibits DET capability that results from the collaboration of MWCNTs and metal impurities, but is unable to catalyze the oxidation of glucose. The second type maintains its glucose-specific catalytic capability in the presence of a mediator, which can be enhanced by MWCNTs, but cannot undergo DET with the electrode. As a result, the MWCNTs are capable of promoting the electron transfer, but this is without value in some mediator-free applications such as in third-generation glucose biosensors and in mediator-free anodes for glucose biofuel cells.

Long distance electron transfer in cytochrome c oxidase immobilised on electrodes. A surface enhanced resonance Raman spectroscopic study

Cytochrome c oxidase was tethered to a functionalised Ag electrode via a histidine-tag on the C-terminus of subunit I or II and embedded in a phospholipid bilayer. The uniformly oriented membrane-bound proteins were studied by surface enhanced resonance Raman spectroscopy (SERRS) that reveals preservation of the native structures of the heme a and heme a(3) sites. On the basis of time-dependent SERRS measurements, the rate constant for the heterogeneous electron transfer to heme a was determined to be 0.002 s(-1) independent of the enzyme orientation and the overpotential. Taking into account that the electrode-to-heme a distance is larger than 50 A, these findings suggest an electron hopping mechanism in which the Cu(A) center is not involved. Electrochemical reduction is restricted to heme a whereas electron transfer from heme a to heme a(3), which in solution occurs on the nanosecond time scale, is drastically slowed down. It may be that the network of cooperativities that links intramolecular electron transfer and proton translocation is perturbed in the immobilised enzyme, possibly due to the effect of the interfacial electric field.     

Organic interlayers for oxygen reducing electrodes based on bilirubin oxidase and MWCNT modified gold

Electrochemical properties of bilirubin oxidase (BOD) and multi-walled carbon nanotubes (MWCNT) modified gold electrodes were characterised by linear sweep voltammetry. For enhancement of the direct electron transfer of BOD an interlayer of different aromatic compounds was introduced between the adsorbed or covalently bound enzyme and the MWCNTs. By usage of pyrroloquinoline quinone (PQQ) an increased catalytic oxygen current was observed. The reduction process starts at a potential of +500 mV vs. Ag/AgCl, 1 M KCl. The peak current density in unstirred solution could be determined with about 500 μA/cm². A reduction of the diffusion layer thickness by stirring enhances the current density up to 1600 μA/cm² (at +250 mV).     

Proton-coupled electron transfer of flavodoxin immobilized on nanostructured tin dioxide electrodes: thermodynamics versus kinetics control of protein redox function

In this paper, we report a spectroelectrochemical investigation of proton-coupled electron transfer in flavodoxin D. vulgaris Hildenborough (Fld). Poly-L-lysine is used to promote the binding of Fld to the nanocrystalline, mesoporous SnO(2) electrodes. Two reversible redox couples of the immobilized Fld are observed electrochemically and are assigned by spectroelectrochemistry to the quinone/semiquinone and semiquinone/hydroquinone couples of the protein's flavin mononucleotide (FMN) redox cofactor. Comparison with control data for free FMN indicates no contamination of the Fld data by dissociated FMN. The quinone/semiquinone and semiquinone/hydroquinone midpoint potentials (E(q/sq) and E(sq/hq)) at pH 7 were determined to be -340 and -585 mV vs Ag/AgCl, in good agreement with the literature. E(q/sq) exhibited a pH dependence of 51 mV/pH. The kinetics of these redox couples were studied using cyclic voltammetry, cyclic voltabsorptometry, and chronoabsorptometry. The semiquinone/quinone reoxidation is found to exhibit slow, potential-independent but pH-sensitive kinetics with a reoxidation rate constant varying from 1.56 s(-)(1) at pH 10 to 0.0074 s(-)(1) at pH 5. The slow kinetics are discussed in terms of a simple kinetics model and are assigned to the reoxidation process being rate limited by semiquinone deprotonation. It is proposed that this slow deprotonation step has the physiological benefit of preventing the undesirable loss of reducing equivalents which results from semiquinone oxidation to quinone.     

Direct delocalization for calculating electron transfer in fullerenes

A method is introduced for simple calculation of charge transfer between very large solvated organic dimers (fullerenes here) from isolated dimer calculations. The individual monomers in noncentrosymmetric dimers experience different chemical environments, so that the dimers do not necessarily represent bulk‐like molecules. Therefore, we apply a delocalizing bias directly to the Fock matrix of the dimer system, and verify that this is almost as accurate as self‐consistent solvation. As large molecules like fullerenes have a plethora of excited states, the initially excited state orbitals are thermally populated, so that the rate is obtained as a thermal average over Marcus thermal transfers. © 2013 Wiley Periodicals, Inc.