Chlorhexidine digluconate exerts bactericidal activity vs Gram positive Staphylococci with bioelectrocatalytic compatibility: High level disinfection for implantable biofuel cells.

Implanted devices destined for contact with sterile body tissues, vasculature or fluids should be free of any microbial contamination that could lead to disease transmission. The disinfection and sterilisation of implantable biofuel cells is a challenging and largely overlooked subject due to the incompatibility of fragile biocatalytic components with classical treatments. Here we report the development of a convenient "soft" chemical treatment based on immersion of enzymatic bioelectrodes and biofuel cells in dilute aqueous chlorhexidine digluconate (CHx). We show that immersion treatment in a 0.5 % solution of CHx for 5 min is sufficient to remove 10-6 log colony forming units of Staphylococcus hominis after 26 h while shorter treatments are less effective. Treatments with 0.2 % CHx solutions were ineffective. Bioelectrocatalytic half-cell voltammetry revealed no loss in activity at the bioanode after the bactericidal treatment, while the cathode was less tolerant. A maximum power output loss of ca. 10 % for the glucose/O2 biofuel cell was observed following the 5 min CHx treatment, while the dialysis bag had a significant negative impact on the power output. Finally, we report a proof-of-concept in vivo operation for 4 days of a CHx-treated biofuel cell with a 3D printed holder and additional porous surgical tissue interface. Further assessments are necessary to rigorously validate sterilisation, biocompatibility and tissue response performance.

Nanostructured electrodes based on multiwalled carbon nanotube/glyconanoparticles for the specific immobilization of bilirubin oxidase: Application to the electrocatalytic O2 reduction.

Here we describe the design and the characterization of novel electrode materials consisting of multi-walled carbon nanotubes coated with glyconanoparticles (GNPs) functionalized with anthraquinone sulfonate. The resulting modified electrodes were characterized by scanning electron microscopy and cyclic voltammetry. Their electrochemical behavior reveals a stable pH-dependent redox signal characteristic of anthraquinone sulfonate. Immobilization of bilirubin oxidase on these three-dimensional electrodes leads to the electroenzymatic reduction of O2 to water with an onset potential of 0.5 V/SCE (saturated calomel electrode). A catalytic cathodic current of 174 µA (0.88 mA cm-2) at 0.1 V/SCE, demonstrates that glyconanoparticles modified by anthraquinone sulfonate were able to interact and orientate bilirubin oxidase by electrostatic interactions.

Monofunctional pyrenes at carbon nanotube electrodes for direct electron transfer H2O2 reduction with HRP and HRP-bacterial nanocellulose.

The non-covalent modification of carbon nanotube electrodes with pyrene derivatives is a versatile approach to enhance the electrical wiring of enzymes for biosensors and biofuel cells. We report here a comparative study of five pyrene derivatives adsorbed at multi-walled carbon nanotube electrodes to shed light on their ability to promote direct electron transfer with horseradish peroxidase (HRP) for H2O2 reduction. In all cases, pyrene-modified electrodes enhanced catalytic reduction compared to the unmodified electrodes. The pyrene N-hydroxysuccinimide (NHS) ester derivative provided access to the highest catalytic current of 1.4 mA cm-2 at 6 mmol L-1 H2O2, high onset potential of 0.61 V vs. Ag/AgCl, insensitivity to parasitic H2O2 oxidation, and a large linear dynamic range that benefits from insensitivity to HRP "suicide inactivation" at 4-6 mmol L-1 H2O2. Pyrene-aliphatic carboxylic acid groups offer better sensor sensitivity and higher catalytic currents at ≤ 1 mmol L-1 H2O2 concentrations. The butyric acid and NHS ester derivatives gave high analytical sensitivities of 5.63 A M-1 cm-2 and 2.96 A M-1 cm-2, respectively, over a wide range (0.25-4 mmol-1) compared to existing carbon-based HRP biosensor electrodes. A bacterial nanocellulose pyrene-NHS HRP bioelectrode was subsequently elaborated via "one-pot" and "layer-by-layer" strategies. The optimised bioelectrode exhibited slightly weaker voltage output, further enhanced catalytic currents, and a major enhancement in 1-week stability with 67% activity remaining compared to 39% at the equivalent electrode without nanocellulose, thus offering excellent prospects for biosensing and biofuel cell applications.

Redox-Active Glyconanoparticles as Electron Shuttles for Mediated Electron Transfer with Bilirubin Oxidase in Solution.

We demonstrate self-assembly, characterization and bioelectrocatalysis of redox-active cyclodextrin-coated nanoparticles. The nanoparticles with host-guest functionality are easy to assemble and permit entrapment of hydrophobic redox molecules in aqueous solution. Bis-pyrene-ABTS encapsulated nanoparticles were investigated electrochemically and spectroscopically. Their use as electron shuttles is demonstrated via an intraelectron transfer chain between neighboring redox units of clustered particles (Dh,DLS = 195 nm) and the mono- and trinuclear Cu sites of bilirubin oxidases. Enhanced current densities for mediated O2 reduction are observed with the redox nanoparticle system compared to equivalent bioelectrode cells with dissolved mediator. Improved catalytic stability over 2 days was also observed with the redox nanoparticles, highlighting a stabilizing effect of the polymeric architecture. Bioinspired nanoparticles as mediators for bioelectrocatalysis promises to be valuable for future biofuel cells and biosensors.

5,5-Dithiobis(2-nitrobenzoic acid) pyrene derivative-carbon nanotube electrodes for NADH electrooxidation and oriented immobilization of multicopper oxidases for the development of glucose/O2 biofuel cells.

We report the functionalization of multi-walled carbon nanotubes (MWCNTs) electrodes by a bifunctional nitroaromatic molecule accomplished via π-π interactions of a pyrene derivative. DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) has the particularity to possess both electroactivable nitro groups and negatively charged carboxylic groups. The integration of the DTNB-modified MWCNTs was evaluated for different bioelectrocatalytic systems. The immobilized DTNB-based electrodes showed electrocatalytic activity toward the oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) with low overpotential of -0.09V vs Ag/AgCl at neutral pH. Glucose dehydrogenase was successfully immobilized at the surface of DTNB-based electrodes and, in the presence of NAD+, the resulting bioelectrode achieved efficient glucose oxidation with high current densities of 2.03mAcm-2. On the other hand, the aromatic structure and the negatively charged nature of the DTNB provoked orientation of both laccase and bilirubin oxidase onto the electrode, which enhanced their ability to undergo a direct electron transfer for oxygen reduction. Due to the proper orientation, low overpotentials were obtained (ca. 0.6V vs Ag/AgCl) and high electrocatalytic currents of about 3.5mAcm-2 were recorded at neutral pH in O2 saturated conditions for bilirubin oxidase electrodes. The combination of these bioanodes and bilirubin oxidase biocathodes provided glucose/O2 enzymatic biofuel cells (EBFC) exhibiting an open-circuit potential of 0.640V, with an associated maximum current density of 2.10mAcm-2. Moreover, the fuel cell delivered a maximum power density of 0.50mWcm-2 at 0.36 V.

Hybrid enzymatic and organic electrocatalytic cascade for the complete oxidation of glycerol.

We demonstrate the complete electrochemical oxidation of the biofuel glycerol to CO2 using a hybrid enzymatic and small-molecule catalytic system. Combining an enzyme, oxalate oxidase, and an organic oxidation catalyst, 4-amino-TEMPO, we are able to electrochemically oxidize glycerol at a carbon electrode, while collecting up to as many as 16 electrons per molecule of fuel. Additionally, we investigate the anomalous electrocatalytic properties that allow 4-amino-TEMPO to be active under the acidic conditions that are required for oxalate oxidase to function.

Bilirubin oxidase bioelectrocatalytic cathodes: the impact of hydrogen peroxide.

Mediator-less, direct electro-catalytic reduction of oxygen to water by bilirubin oxidase (Myrothecium sp.) was obtained on anthracene-modified, multi-walled carbon nanotubes. H2O2 was found to significantly and irreversibly affect the electro-catalytic activity of bilirubin oxidase, whereas similar electrodes comprised of laccase (Trametes versicolor) were reversibly inhibited.

Hydrogen peroxide produced by glucose oxidase affects the performance of laccase cathodes in glucose/oxygen fuel cells: FAD-dependent glucose dehydrogenase as a replacement.

Hydrogen peroxide production by glucose oxidase (GOx) and its negative effect on laccase performance have been studied. Simultaneously, FAD-dependent glucose dehydrogenase (FAD-GDH), an O2-insensitive enzyme, has been evaluated as a substitute. Experiments focused on determining the effect of the side reaction of GOx between its natural electron acceptor O2 (consumed) and hydrogen peroxide (produced) in the electrolyte. Firstly, oxygen consumption was investigated by both GOx and FAD-GDH in the presence of substrate. Relatively high electrocatalytic currents were obtained with both enzymes. O2 consumption was observed with immobilized GOx only, whilst O2 concentration remained stable for the FAD-GDH. Dissolved oxygen depletion effects on laccase electrode performances were investigated with both an oxidizing and a reducing electrode immersed in a single compartment. In the presence of glucose, dramatic decreases in cathodic currents were recorded when laccase electrodes were combined with a GOx-based electrode only. Furthermore, it appeared that the major loss of performance of the cathode was due to the increase of H2O2 concentration in the bulk solution induced laccase inhibition. 24 h stability experiments suggest that the use of O2-insensitive FAD-GDH as to obviate in situ peroxide production by GOx is effective. Open-circuit potentials of 0.66 ± 0.03 V and power densities of 122.2 ± 5.8 µW cm(-2) were observed for FAD-GDH/laccase biofuel cells.

Membraneless glucose/O2 microfluidic enzymatic biofuel cell using pyrolyzed photoresist film electrodes.

Biofuel cells typically yield lower power and are more difficult to fabricate than conventional fuel cells using inorganic catalysts. This work presents a glucose/O2 microfluidic biofuel cell (MBFC) featuring pyrolyzed photoresist film (PPF) electrodes made on silicon wafers using a rapid thermal process, and subsequently encapsulated by rapid prototyping techniques into a double-Y-shaped microchannel made entirely of plastic. A ferrocenium-based polyethyleneimine polymer linked to glucose oxidase (GOx/Fc-C6-LPEI) was used in the anode, while the cathode contained a mixture of laccase, anthracene-modified multi-walled carbon nanotubes, and tetrabutylammonium bromide-modified Nafion (MWCNTs/laccase/TBAB-Nafion). The cell performance was studied under different flow-rates, obtaining a maximum open circuit voltage of 0.54 ± 0.04 V and a maximum current density of 290 ± 28 µA cm(-2) at room temperature under a flow rate of 70 µL min(-1) representing a maximum power density of 64 ± 5 µW cm(-2). Although there is room for improvement, this is the best performance reported to date for a bioelectrode-based microfluidic enzymatic biofuel cell, and its materials and fabrication are amenable to mass production.

A glucose biofuel cell implanted in rats.

Powering future generations of implanted medical devices will require cumbersome transcutaneous energy transfer or harvesting energy from the human body. No functional solution that harvests power from the body is currently available, despite attempts to use the Seebeck thermoelectric effect, vibrations or body movements. Glucose fuel cells appear more promising, since they produce electrical energy from glucose and dioxygen, two substrates present in physiological fluids. The most powerful ones, Glucose BioFuel Cells (GBFCs), are based on enzymes electrically wired by redox mediators. However, GBFCs cannot be implanted in animals, mainly because the enzymes they rely on either require low pH or are inhibited by chloride or urate anions, present in the Extra Cellular Fluid (ECF). Here we present the first functional implantable GBFC, working in the retroperitoneal space of freely moving rats. The breakthrough relies on the design of a new family of GBFCs, characterized by an innovative and simple mechanical confinement of various enzymes and redox mediators: enzymes are no longer covalently bound to the surface of the electron collectors, which enables use of a wide variety of enzymes and redox mediators, augments the quantity of active enzymes, and simplifies GBFC construction. Our most efficient GBFC was based on composite graphite discs containing glucose oxidase and ubiquinone at the anode, polyphenol oxidase (PPO) and quinone at the cathode. PPO reduces dioxygen into water, at pH 7 and in the presence of chloride ions and urates at physiological concentrations. This GBFC, with electrodes of 0.133 mL, produced a peak specific power of 24.4 microW mL(-1), which is better than pacemakers' requirements and paves the way for the development of a new generation of implantable artificial organs, covering a wide range of medical applications.

Impedimetric immunosensor based on a polypyrrole-antibiotic model film for the label-free picomolar detection of ciprofloxacin.

This paper describes the construction of an impedimetric immunosensor for the label-free detection of ciprofloxacin, an antibiotic belonging to synthetic fluoroquinolones. A poly(pyrrole-N-hydroxysuccinimide) film was electrogenerated onto electrodes and then used for the reagentless covalent binding of a fluoroquinolone model bearing an amino group. The resulting electrodes were utilized to immobilize a layer of anticiprofloxacin antibody onto the polymer surface by immunoreaction. In presence of ciprofloxacin, the antibody was displaced in solution inducing marked changes in the impedance of the sensor electrodes. These phenomena were detected and characterized by electrochemical impedance spectroscopy allowing the selective detection of extremely low ciprofloxacin concentration, namely, 1 x 10(-12) g mL(-1) or 3 pmol L(-1). Sensors exposed to ciprofloxacin showed a decrease in the sum of the interfacial resistances with the increase in ciprofloxacin concentration from 1 x 10(-12) to 1 x 10(-6) g mL(-1).