A Conductive Microcavity Created by Assembly of Carbon Nanotube Buckypapers for Developing Electrochemically Wired Enzyme Cascades.

We describe the creation of a conductive microcavity based on the assembly of two pieces of carbon nanotube buckypaper for the entrapment of two enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOx), as well as a redox mediator: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS). The hollow electrode, employing GOx, HRP, and the mediator, as an electrochemical enzyme cascade model, is utilized for glucose sensing at a potential of 50 mV vs. Ag/AgCl. This bienzyme electrode demonstrates the ability to oxidize glucose by GOx and subsequently convert H2O2 to water via the electrical wiring of HRP by ABTS. Different redox mediators (ABTS, potassium hexacyanoferrate (III), and hydroquinone) are tested for HRP wiring, with ABTS being the best candidate for the electroenzymatic reduction of H2O2. To demonstrate the possibility to optimize the enzyme cascade configuration, the enzyme ratio is studied with 1 mg HRP combined with variable amounts of GOx (1-4 mg) and 2 mg GOx combined with variable amounts of HRP (0.5-2 mg). The bienzyme electrode shows continuous operational stability for over a week and an excellent storage stability in phosphate buffer, with a decay of catalytic current by only 29% for 1 mM glucose after 100 days.

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.

Functionalizing Carbon Nanotubes with Bis(2,9-dialkyl-1,10-phenanthroline)copper(II) Complexes for the Oxygen Reduction Reaction.

A new ligand, namely, 2-(5-(pyren-1-yl)pentyl)-9-methyl-1,10-phenanthroline, as well as new bis(2,9-dialkyl-1,10-phenanthroline)copper(II) complexes were designed, which were immobilized on multiwalled carbon nanotube (MWCNT) electrodes. These complexes show a high tendency of autoreduction into their copper(I) form according to electrochemical and EPR experiments. These complexes exhibit strong interactions with MWCNT sidewalls either with or without anchor functions such as the pyrene moiety. The pyrene-modified derivative can be electropolymerized on glassy carbon and MWCNT electrodes to form a poly-[bis(2-(5-(pyren-1-yl)pentyl)-9-methyl-1,10-phenanthroline)copper(II)] metallopolymer film. Furthermore, these MWCNT-supported bis(2,9-dialkyl-1,10-phenanthroline)copper complexes demonstrate a low overpotential for a 4H+/4e- oxygen reduction reaction at pH 5 with an onset potential of 0.86 V versus RHE. Integration of these functionalized MWCNTs at gas-diffusion electrodes of H2/air fuel cells led to a high open-circuit voltage of 0.84 V and a maximum current density of 1.77 mW cm-2 using a Pt/C anode.

Hollow Bioelectrodes Based on Buckypaper Assembly. Application to the Electroenzymatic Reduction of O2.

A new concept of hollow electrode based on the assembly of two buckypapers creating a microcavity which contains a biocatalyst is described. To illustrate this innovative concept, hollow bioelectrodes containing 0.16-4 mg bilirubin oxidase in a microcavity were fabricated and applied to electroenzymatic reduction of O2 in aqueous solution. For hemin-modified buckypaper, the bioelectrode shows a direct electron transfer between multi-walled carbon nanotubes and bilirubin oxidase with an onset potential of 0.77 V vs. RHE. The hollow bioelectrodes showed good storage stability in solution with an electroenzymatic activity of 30 and 11% of its initial activity after 3 and 6 months, respectively. The co-entrapment of bilirubin oxidase and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the microcavity leads to a bioelectrode exhibiting mediated electron transfer. After 23 h of intermittent operation, 5.66 × 10-4 mol of O2 were electroreduced (turnover number of 19,245), the loss of catalytic current being only 54% after 7 days.

Microcapsule-based biosensor containing catechol for the reagent-free inhibitive detection of benzoic acid by tyrosinase.

A biosensor based on the release of the enzyme substrate from its structure was developed for the inhibitive detection of benzoic acid. A polyurethane support comprising two perforated microcapsules (800 µm in diameter) filled with methylene blue as a model compound and covered with a conductive deposit of multiwalled carbon nanotubes, continuously released this stored dye for 24 h. An increase in methylene blue concentration of 0.5-0.75 µmol L-1 h-1 and 1.5-2 µmol L-1 h-1, in the presence and absence of the multiwalled carbon nanotube coating, respectively, was demonstrated by UV-vis spectroscopy in a 2 mL UV cuvette. The same configuration with microcapsules filled with catechol was modified by a laponite clay coating containing tyrosinase enzyme. The resulting biosensor exhibits a constant cathodic current at -0.155 V vs AgCl/Ag, due to the reduction of the ortho-quinone produced enzymatically from the released catechol. The detection of benzoic acid was recorded from the decrease in cathodic current due to its inhibiting action on the tyrosinase activity. Reagentless biosensors based on different deposited quantity of tyrosinase (100, 200, 400 and 600 µg) were investigated for the detection of catechol and applied to the detection of benzoic acid as inhibitor. The best performance was obtained with the 400 µg-based configuration, namely a detection limit of 0.4 µmol L-1 and a sensitivity of 228 mA L mol-1. After the inhibition process, the biosensors recover 97-100% of their activity towards catechol, confirming a reversible inhibition by benzoic acid.

From Graphite to Laccase Biofunctionalized Few-Layer Graphene: A "One Pot" Approach Using a Chimeric Enzyme.

A chimeric enzyme based on the genetic fusion of a laccase with a hydrophobin domain was employed to functionalize few-layer graphene, previously exfoliated from graphite in the presence of the hydrophobin. The as-produced, biofunctionalized few-layer graphene was characterized by electrochemistry and Raman spectroscopy, and finally employed in the biosensing of phenols such as catechol and dopamine. This strategy paves the way for the functionalization of nanomaterials by hydrophobin domains of chimeric enzymes and their use in a variety of electrochemical applications.

Assembly and Stacking of Flow-through Enzymatic Bioelectrodes for High Power Glucose Fuel Cells.

Bioelectrocatalytic carbon nanotube based pellets comprising redox enzymes were directly integrated in a newly conceived flow-through fuel cell. Porous electrodes and a separating cellulose membrane were housed in a glucose/oxygen biofuel cell design with inlets and outlets allowing the flow of electrolyte through the entire fuel cell. Different flow setups were tested and the optimized single cell setup, exploiting only 5 mmol L-1 glucose, showed an open circuit voltage (OCV) of 0.663 V and provided 1.03 ± 0.05 mW at 0.34 V. Furthermore, different charge/discharge cycles at 500 Ω and 3 kΩ were applied to optimize long-term stability leading to 3.6 J (1 mW h) of produced electrical energy after 48 h. Under continuous discharge at 6 kΩ, about 0.7 mW h could be produced after a 24 h period. The biofuel cell design further allows a convenient assembly of several glucose biofuel cells in reduced volumes and their connection in parallel or in series. The configuration of two biofuel cells connected in series showed an OCV of 1.35 V and provided 1.82 ± 0.09 mW at 0.675 V, and when connected in parallel, showed an OCV of 0.669 V and provided 1.75 ± 0.09 mW at 0.381 V. The presented design is conceived to stack an unlimited amount of biofuel cells to reach the necessary voltage and power for portable electronic devices without the need for step-up converters or energy managing systems.

Carbon-Nanotube-Supported Bio-Inspired Nickel Catalyst and Its Integration in Hybrid Hydrogen/Air Fuel Cells.

A biomimetic nickel bis-diphosphine complex incorporating the amino acid arginine in the outer coordination sphere was immobilized on modified carbon nanotubes (CNTs) through electrostatic interactions. The functionalized redox nanomaterial exhibits reversible electrocatalytic activity for the H2 /2 H+ interconversion from pH 0 to 9, with catalytic preference for H2 oxidation at all pH values. The high activity of the complex over a wide pH range allows us to integrate this bio-inspired nanomaterial either in an enzymatic fuel cell together with a multicopper oxidase at the cathode, or in a proton exchange membrane fuel cell (PEMFC) using Pt/C at the cathode. The Ni-based PEMFC reaches 14 mW cm-2 , only six-times-less as compared to full-Pt conventional PEMFC. The Pt-free enzyme-based fuel cell delivers ≈2 mW cm-2 , a new efficiency record for a hydrogen biofuel cell with base metal catalysts.