Dónal Leech

Application of Colloidal Gold in Protein Immobilization, Electron Transfer, and Biosensing

Abstract Direct electron transfer between redox proteins and electrodes is of practical and theoretical interest and can be improved by electrode or protein modification. The direct contact of protein with electrode surfaces can lead to a significant change of the protein structure and/or function. Immobilized colloidal gold on electrode surfaces provides a microenvironment similar to that of the redox protein in native systems and gives the protein molecules more freedom in orientation, thus reducing the insulating property of the protein shell for the direct electron transfer and facilitating the electron transfer through the conducting tunnels of colloidal gold. This brief review focuses on the current state of the colloidal gold used for protein immobilization, electron transfer and biosensing, with emphasis on recent advances, challenges and trends.

Geobacter sulfurreducens biofilms developed under different growth conditions on glassy carbon electrodes: insights using cyclic voltammetry

Growth of biofilms of G. sulfurreducens on glassy carbon that yield a bioelectrocatalytic response to acetate oxidation is achieved using a fixed applied potential, with current density for acetate oxidation scaling with applied potential. In contrast biofilms grown under electron acceptor-limiting conditions display redox signals shifted to lower potentials and do not oxidise acetate.

Characterisation of an antibody coated microcantilever as a potential immuno-based biosensor

In this study, we investigated the activity, stability, lifetime and re-usability of monoclonal antibodies to myoglobin covalently immobilised onto microfabricated cantilever surfaces. These sensing surfaces are of interest to us in the development of novel cantilever-based immunosensors. For such sensors the antibody layer represents the sensing element while the microcantilever acts as a mechanical transducer. A procedure for producing re-usable biological coatings has been tested with different independent techniques. An Enzyme Linked Immunosorbent Assay (ELISA) was used to determine the presence of an active antibody coating, and to monitor the lifetime and stability of the immobilised antibody. Through this analysis, the activity of the immobilised antibody layer was found to be more stable with the introduction of sucrose, as a stabilising agent. Sucrose was applied to the immobilised antibody layer after each regeneration step. The immobilised antibody was found to have a stable active lifetime for up to 7 weeks. Fluorescence microscopy was used to give information on the distribution of the coating on the gold and silicon nitride sides of the cantilever. Atomic Force Microscopy was used to determine the presence of the biological coating on the cantilever and to obtain information on the surface morphology of the biological element of the sensor. The combined results provide valuable information on the development of an optimised sensing element and demonstrate a set of methods to use for future sensor-to-sensor characterisation. Preliminary experimental results showing the antibody activity against myoglobin, detected with a microcantilever based sensor prototype confirmed the motivations and potentialities of the proposed immunosensing technique.

Reagentless Mediated Laccase Electrode for the Detection of Enzyme Modulators

We have investigated aerobic mediation of electron transfer to a laccase enzyme by the solution redox couples [Os(bpy)(2)Cl(2)](+/0) and [Os(bpy)(2)(MeIm)Cl](2+/+), where bpy is 2,2-bipyridine and MeIm is N-methylimidazole. The factors that influence the homogeneous mediation reaction are investigated and discussed. Investigation of ionic strength, pH, and temperature effects on the kinetics of intermolecular electron transfer elucidates the governing factors in the mediator-enzyme reactions. Coimmobilization of both enzyme and an osmium redox mediator in a hydrogel on glassy carbon electrodes results in a biosensor for the reagentless addressing of enzyme activity, consuming only oxygen present in solution. Thus, these immobilized enzyme biosensors can be utilized for the detection of modulators of laccase activity, such as the inhibitor sodium azide. The enzyme inhibition biosensor can detect levels of azide as low as 2.5 × 10(-6) mol dm(-3) in solution.

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

Electron transfer mediator systems for bleaching of paper pulp

The participation of biological agents in pulp bleaching systems has received a lot of attention from research teams around the world, driven by the environmental benefits that biobleaching could bring. Nature showed us the ability of some of its agents, such as wood-decaying fungi, to delignify and bleach wood and wood pulp. What we need to do is to enhance the efficiency of such agents to make them cope with the fast pace of our modern pulp mills. To do so, a profound understanding of the biobleaching system is required. Our efforts to discover new efficient mediators for the laccase-mediator system (LMS) brought us to use several techniques to analyse the reactions involved in mediated enzymatic delignification. Mostly based on electrochemistry, these techniques are reviewed in this paper, along with key results. Cyclic voltammetry was used to characterize electron transfer rates between each element of the LMS. We found, along with other authors, that the mediator redox potential has a great influence on its efficiency. We used bulk electrolysis to simulate the oxidative action of laccase on mediators and model compounds of lignin. Such electrolysis techniques allowed us to study mediated lignin oxidation outside of normal laccase working conditions. Finally, an electrolysis-based method for mediated pulp delignification that we developed, based upon our research on biobleaching, is presented.

Three-dimensional microchanelled electrodes in flow-through configuration for bioanode formation and current generation

Three-dimensional microchannelled nanocomposite electrodes fabricated by ice-segregation induced self-assembly of chitosan-dispersed multiwall carbon nanotubes are shown to provide a scaffold for growth of electroactive bacteria for use as acetate-oxidizing bioanodes in bioelectrochemical systems. The hierarchical structure provides a conductive surface area available for G. sulfurreducens colonization, with a flow through configuration along the electrode providing a substrate for bacterial colonization and bio-electrochemical processes. This configuration, whilst resulting in sub-monolayer biofilm coverage over the three-dimensional surface, is capable of providing acetate oxidation current densities of up to 24.5 A m−2, equating to a volumetric current density of 19 kA m−3, in the flow-through configuration. Such bioanodes, when operated in non-optimized flow-through microbial fuel cell configuration, provide a maximum power density of 2.87 W m−2, which is equivalent to 2.0 kW m−3 volumetric power density.

Mediated electron transfer in glucose oxidising enzyme electrodes for application to biofuel cells: recent progress and perspectives

Glucose oxidising enzyme electrodes have long been studied for their application to biosensors and, more recently, anodes in biofuel cells. At a fundamental level, insight into enzyme electron transfer and oxidation current generation at enzyme electrodes can be gained by systematic studies on integration of surfaces, biocatalysts, and artificial substrates (mediators). In this perspective, we present an overview of methods to aid the development of glucose oxidising enzyme electrodes based on mediated electron transfer for application to continuous-use anodes in a biofuel cell. Focus is placed on the rational design of mediators, based on osmium redox complexes, and screening of the activity of such complexes as mediators for glucose oxidising enzymes. An overview of the performance of enzyme electrodes, focused predominantly on crosslinked films of redox polymers and glucose oxidase, for glucose oxidation, is presented and approaches to improve both current output and stability of such enzyme electrodes are discussed.

Characterization of Nanoporous Gold Electrodes for Bioelectrochemical Applications

The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterization of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described. Robust np-Au electrodes were prepared by sputtering a gold-silver alloy onto a glass support and subsequent dealloying of the silver component. Alloy layers were prepared with either a uniform or nonuniform distribution of silver and, post dealloying, showed clear differences in morphology on characterization with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, A(real). Values of A(real) up to 28 times that of the geometric electrode surface area, A(geo), were obtained. For diffusion-controlled reactions, overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on A(geo). The importance of measuring the surface area available for the immobilization was determined using the redox protein, cyt c. The area accessible to modification by a biological macromolecule, A(macro), such as cyt c was reduced by up to 40% compared to A(real), demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (MvBOD) as a biocathode were performed; current densities of 500 μA cm(-2) were obtained in unstirred solutions.

Electrochemical study of a metallothionein modified gold disk electrode and its action on Hg2+ cations

Abstract A novel protein monolayer modified electrode has been prepared by the self-assembly of metallothionein (MT) at a gold disk electrode. The properties of MT in Tris–HCl buffer and in the monolayer are studied by using cyclic voltammetry and differential pulse voltammetry with a gold disk electrode. In the negative sweep, the voltammogram of MT in buffer shows two small peaks and different electrochemical behaviour from that at a mercury electrode. Cd 2+ complexed to the thionein can easily be replaced by Hg 2+ ions, and Hg 2+ ions can firmly adsorb in the MT monolayer with a saturation coverage of (2.78±0.29)×10 −10 mol cm −2 . This behaviour has been used to preconcentrate trace Hg 2+ for its determination by cathodic stripping differential pulse voltammetry. The cathodic stripping peak current is proportional to Hg 2+ concentration in the range of 0.15–3 μM and the detection limit is ca. 0.08 μM (16 ppb) with a 2 min open circuit accumulation step. The relative standard deviation is 7.2% at 0.4 μM Hg 2+ concentration ( n =4). At higher concentration the adsorption of Hg 2+ exhibits a response similar to that expected for a Langmuir adsorption isotherm with the stability constant of (4.0±0.2)×10 5 M −1 .