Maciej Sosna

Monolayer anthracene and anthraquinone modified electrodes as platforms for Trametes hirsuta laccase immobilisation

Surface modification techniques are essential to the construction of enzyme based elements of biofuel cells and biosensors. In this article we report on the preparation and characterisation of modified carbon electrodes which were used as supports for the immobilisation of laccase from Trametes hirsuta. The electrodes were electrochemically modified with diamine or diazonium linkers followed by attachment of either anthracene or anthraquinone head groups using solid phase chemical methodology. These well defined surfaces were found to effectively bind laccase and to provide direct electrical contact to the enzyme active site, as evidenced by XPS, EIS and voltammetry, respectively. The influence of the type of linker and head group on enzyme binding and bioelectrocatalytic activity are evaluated.

Mass transport controlled oxygen reduction at anthraquinone modified 3D-CNT electrodes with immobilized Trametes hirsuta laccase

Carbon nanotubes covalently modified with anthraquinone were used as an electrode for the immobilization of Trametes hirsuta laccase. The adsorbed laccase is capable of oxygen reduction at a mass transport controlled rate (up to 3.5 mA cm(-2)) in the absence of a soluble mediator. The storage and operational stability of the electrode are excellent.

Influence of macroporous gold support and its functionalization on lactate oxidase-based biosensors response.

A general bioanalytical platform for biosensor applications was developed based on three-dimensional ordered macroporous (3DOM) gold film modified electrodes using lactate oxidase (LOx) as a case study, within the framework of developing approaches of broad applicability. The electrode was electrochemically fabricated with an inverted opal template, making the surface area of the 3DOM gold electrode up to 18 times higher than that of bare flat gold electrodes. These new electrochemical transducers were characterized by using Field Emission Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM) and the X-ray diffraction (XRD). The biosensor was developed by immobilization of lactate oxidase (LOx), on a 3DOM gold electrode modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate (DTSP). The resulting lactate oxidase biosensor was characterized by electrochemical impedance spectroscopy (EIS). The 3DOM gold electrode not only provides a good biocompatible microenvironment but also promotes the increase of conductivity and stability. Thus, the developed lactate oxidase bioanalytical platforms showed higher mediated bioelectrocatalytic activity compared to others previously described based on polycrystalline gold transducers. The response to varying lactate concentrations has been obtained in the presence of hydroxymethylferrocene as redox mediator in solution. Under these conditions, the bioanalytical platform response for DTSP covalently bound enzyme was improved with respect to that obtained in absence of DTSP.

Direct electrochemistry and Os-polymer-mediated bioelectrocatalysis of NADH oxidation by Escherichia coli flavohemoglobin at graphite electrodes

Escherichia coli flavohemoglobin (HMP), which contains one heme and one FAD as prosthetic groups and is capable of reducing O₂ by its heme at the expense of NADH oxidized at its FAD site, was electrochemically studied at graphite (Gr) electrodes. Two signals were observed in voltammograms of HMP adsorbed on Gr, at -477 and -171 mV vs. Ag|AgCl, at pH 7.4, correlating with electrochemical responses from the FAD and heme domains, respectively. The electron transfer rate constant for ET reaction between FAD of HMP and the electrode was estimated to be 83 s⁻¹. Direct bioelectrocatalytic oxidation of NADH by HMP was not observed, presumably due to impeded substrate access to HMP orientated on Gr through the FAD-domain and/or partial denaturation of HMP. Bioelectrocatalysis was achieved when HMP was wired to Gr by the Os redox polymers, with the onset of NADH oxidation at the formal potential of the particular Os complex (+140 mV or -195 mV). Apparent Michaelis constants K(M)(app) and j(max) were determined, showing bioelectrocatalytic efficiency of NADH oxidation by HMP exceeding the one earlier shown with diaphorase, which makes HMP very attractive as a component of bioanalytical and bioenergetic devices.

High-Throughput Synthesis and Electrochemical Screening of a Library of Modified Electrodes for NADH Oxidation

We report the combinatorial preparation and high-throughput screening of a library of modified electrodes designed to catalyze the oxidation of NADH. Sixty glassy carbon electrodes were covalently modified with ruthenium(II) or zinc(II) complexes bearing the redox active 1,10-phenanthroline-5,6-dione (phendione) ligand by electrochemical functionalization using one of four different linkers, followed by attachment of one of five different phendione metal complexes using combinatorial solid-phase synthesis methodology. This gave a library with three replicates of each of 20 different electrode modifications. This library was electrochemically screened in high-throughput (HTP) mode using cyclic voltammetry. The members of the library were evaluated with regard to the surface coverage, midpeak potential, and voltammetric peak separation for the phendione ligand, and their catalytic activity toward NADH oxidation. The surface coverage was found to depend on the length and flexibility of the linker and the geometry of the metal complex. The choices of linker and metal complex were also found to have significant impact on the kinetics of the reaction between the 1,10-phenanthroline-5,6-dione ligand and NADH. The rate constants for the reaction were obtained by analyzing the catalytic currents as a function of NADH concentration and scan rate, and the influence of the surface molecular architecture on the kinetics was evaluated.

An analysis of the kinetics of oxidation of ascorbate at poly(aniline)-poly(styrene sulfonate) modified microelectrodes

A detailed kinetic study is provided for the oxidation of ascorbate at poly(aniline)-poly(styrene sulfonate) coated microelectrodes. Flat films with a low degree of polymer spillover and a thickness much lower than the microelectrode radius were produced by controlled potentiodynamic electrodeposition. The currents for ascorbate oxidation are found to be independent of the polymer thickness, indicating that the reaction occurs at the outer surface of the polymer film. At low ascorbate concentrations, below around 40 mM, the currents are found to be mass transport limited. At higher ascorbate concentrations the currents became kinetically limited. The experimental data for measurements at a range of potentials are fitted to a consistent kinetic model and the results summarized in a case diagram. The results obtained for the poly(aniline)-(polystyrene sulfonate) coated microelectrode are compared to those for a poly(aniline)-poly(vinyl sulfonate) coated microelectrode and to the results of an earlier study of the reaction at poly(aniline)-poly(vinyl sulfonate) coated rotating disc electrodes. For poly(aniline)-poly(styrene sulfonate) the oxidation of ascorbate is found to proceed by one electron reaction whereas for poly(aniline)-poly(vinyl sulfonate) the reaction is found to be a two electron oxidation.

Design of Maleimide‐Functionalised Electrodes for Covalent Attachment of Proteins through Free Surface Cysteine Groups

Mixed two-component monolayers on glassy carbon are prepared by electrochemical oxidation of N-(2-aminoethyl)acetamide and mono-N-Boc-hexamethylenediamine in mixed solution. Subsequent N-deprotection, amide coupling and solid-phase synthetic steps lead to electrode-surface functionalisation with maleimide, with controlled partial coverage of this cysteine-binding group at appropriate dilution for covalent immobilisation of a model redox-active protein, cytochrome c, with high coverage (≈7.5 pmol cm(-2) ).

A His‐Tagged Melanocarpus albomyces Laccase and its Electrochemistry upon Immobilisation on NTA‐Modified Electrodes and in Conducting Polymer Films

The article describes the construction, immobilisation and electrochemistry of histidine tagged laccase from Melanocarpus albomyces. A facile method of functionalisation of glassy carbon electrodes with nitrilotriacetic acid (NTA) using diazonium grafting and solid state chemistry is described. NTA-modified electrodes are shown to bind laccase which reduces oxygen at neutral pH in the presence of soluble redox mediator. Laccase-modified electrodes are also prepared by enzyme immobilisation within poly(aniline)/poly(vinylsulfonate) films. The polymer is found to efficiently retain the enzyme as well as provide direct electrical contact between the electrode and the enzyme active centre. Cyclic voltammetry reveals the direct electron transfer to the enzyme is dependent on the redox state of the polymer film.

Scanning electrochemical microscopy: using the potentiometric mode of SECM to study the mixed potential arising from two independent redox processes

This study demonstrates how the potentiometric mode of the scanning electrochemical microscope (SECM) can be used to sensitively probe and alter the mixed potential due to two independent redox processes provided that the transport of one of the species involved is controlled by diffusion. This is illustrated with the discharge of hydrogen from nanostructured Pd hydride films deposited on the SECM tip. In deareated buffered solutions the open circuit potential of the PdH in equilibrium between its β and α phases (OCP(β→α)) does not depend on the tip-substrate distance while in aerated conditions it is found to be controlled by hindered diffusion of oxygen. Chronopotentiometric and amperometric measurements at several tip-substrate distances reveal how the flux of oxygen toward the Pd hydride film determines its potential. Linear sweep voltammetry shows that the polarization resistance increases when the tip approaches an inert substrate. The SECM methodology also demonstrates how dissolved oxygen affects the rate of hydrogen extraction from the Pd lattice. Over a wide potential window, the highly reactive nanostructure promotes the reduction of oxygen which rapidly discharges hydrogen from the PdH. The flux of oxygen toward the tip can be adjusted via hindered diffusion. Approaching the substrate decreases the flux of oxygen, lengthens the hydrogen discharge, and shifts OCP(β→α) negatively. The results are consistent with a mixed potential due to the rate of oxygen reduction balancing that of the hydride oxidation. The methodology is generic and applicable to other mixed potential processes in corrosion or catalysis.

Development and first results of a new fast response microelectrode DO-sensor

A new dissolved oxygen sensor for marine application was developed and first tests in the field were carried out. The sensor uses a micro-electrode as the sensing element that has an inherently fast response and an increased resistance to bio-fouling due to the properties of the electrode material, platinum, and the employed operation regime. Introduction of a cleaning cycle, where the potential applied to the electrode is altered, allows the elimination of material building up at the electrode surface as well as the reconditioning of the electrode-surface, thus minimizing drift and the effect of bio-fouling. Microdisc electrodes ranging between 10 and 50 /spl mu/m diameter have been tested. The sensor has been adapted to operate on a CTD system or on towed instrument platforms. Results of test cruises will be presented.