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.

Trialkoxyheptazine-Based Glyconanoparticles for Fluorescence in Aqueous Solutions and on Surfaces via Controlled Binding in Space.

The fluorescent organic 2,5,8-tris((adamantan-1-yl)-methoxy)-heptazine (HTZ-Ad) was solubilized in water by inclusion of adamantane groups into free ß-cyclodextrins or a cyclodextrin shell of glyconanoparticles. These glyconanoparticles with average diameters between 40 and 60 nm result from the self-assembly of polystyrene-block-ß-cyclodextrin copolymers. Under UV irradiation at 365 nm, the modified nanoparticles exhibit fluorescence emission in aqueous media as well as in their adsorbed state. This constitutes the first spectroscopic characterization of a trialkoxyheptazine in aqueous medium. The specific binding of the glyconanoparticles to a surface was achieved via host-guest interactions with an electrochemically generated poly(pyrrole-adamantane) film. An interdigitated microelectrode modified with poly(pyrrole-adamantane) film and glyconanoparticles was incubated in HTZ-Ad, resulting in a substrate with spatially controlled fluorescence. The same modified electrode was incubated with an aqueous suspension of glyconanoparticles previously functionalized by HTZ-Ad, resulting in a fluorescent 3D assembly.

Organic ß-cyclodextrin Nanoparticle: An Efficient Building Block Between Functionalized Poly(pyrrole) Electrodes and Enzymes.

Glyconanoparticles (GNPs) made by self-assembly of carbohydrate-based polystyrene-block-ß-cyclodextrin copolymer are used as a building block for the design of nanostructured biomaterials of electrode. The firm immobilization of GNPs is carried out on electrochemically generated polymer, poly(pyrrole-adamantane), and copolymer, poly(pyrrole-adamantane)/poly(pyrrole-lactobionamide) via host-guest interactions between adamantane and ß-cyclodextrin. The ability of GNPs for the specific anchoring of biological macromolecules is investigated using glucose oxidase enzyme modified by adamantane groups as a protein model (GOx-Ad). The immobilization of GOx-Ad is carried out by incubation of an aqueous enzyme solution on a coating of GNPs adsorbed on a platinum electrode. The presence of immobilized GOx-Ad is evaluated in aqueous glucose solution by potentiostating the underlying platinum electrode at 0.7 V/SCE for the electro-oxidation of H2 O2 generated by the enzyme. The analytical performance of the bioelectrodes for the detection of glucose is compared to control electrodes prepared without GNPs or without electropolymerized films. The better permeability of copolymer compared to polymer and the possibility to elaborate two alternating layers of GNPs and GOx-Ad are clearly observed. The best amperometric response is recorded with a multilayered bioelectrode displaying a wide linear range linear range of the calibration curve: 68 µmol L-1 to 0.1 mol L-1 .

Functionalizable Glyconanoparticles for a Versatile Redox Platform.

A series of new glyconanoparticles (GNPs) was obtained by self-assembly by direct nanoprecipitation of a mixture of two carbohydrate amphiphilic copolymers consisting of polystyrene-block-ß-cyclodextrin and polystyrene-block-maltoheptaose with different mass ratios, respectively 0-100, 10-90, 50-50 and 0-100%. Characterizations for all these GNPs were achieved using dynamic light scattering, scanning and transmission electron microscopy techniques, highlighting their spherical morphology and their nanometric size (diameter range 20-40 nm). In addition, by using the inclusion properties of cyclodextrin, these glyconanoparticles were successfully post-functionalized using a water-soluble redox compound, such as anthraquinone sulfonate (AQS) and characterized by cyclic voltammetry. The resulting glyconanoparticles exhibit the classical electroactivity of free AQS in solution. The amount of AQS immobilized by host-guest interactions is proportional to the percentage of polystyrene-block-ß-cyclodextrin entering into the composition of GNPs. The modulation of the surface density of the ß-cyclodextrin at the shell of the GNPs may constitute an attractive way for the elaboration of different electroactive GNPs and even GNPs modified by biotinylated proteins.

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.

Photoelectrochemically-assisted biofuel cell constructed by redox complex and g-C3N4 coated MWCNT bioanode.

Herein, we report a membraneless glucose and air photoelectrochemical biofuel cell (PBFC) with a visible light assisted photobioanode. Flavin adenine dinucleotide dependent glucose dehydrogenase (FADGDH) was immobilized on the combined photobioanode for the visible light assisted glucose oxidation (GCE|MWCNT|g-C3N4|Ru-complex|FADGDH) with a quinone mediated electron transfer. Bilirubine oxidase (BOx) immobilized on MWCNT coated GCE (GCE|BOx) was used as the cathode with direct electron transfer (DET). An improvement of biocatalytic oxidation current was observed by 6.2% due in part to the light-driven electron-transfer. The large oxidation currents are probably owing to the good contacting of the immobilized enzymes with the electrode material and the utilization of light assisted process. Under the visible light, the photobioanode shows an anodic photocurrent of 1.95 µA cm2 at attractively low potentials viz. -0.4 vs Ag/AgCl. The lower-lying conduction band of g-C3N4 as compared to Ru-complexes decreases the rate of hole and electron recombination and enhances the charge transportation. The bioanode shows maximum current density for glucose oxidation up to 6.78 µA cm-2 at 0.2 V vs Ag/AgCl at pH:7. The performance of three promising Ru-complexes differing in chemical and redox properties were compared as electron mediators for FADGDH. Upon illumination, the PBFC delivered a maximum power density of 28.5 ± 0.10 µW cm-2 at a cell voltage of +0.4 V with an open circuit voltage of 0.64 V.

Highly Sensitive Bisphenol-A Electrochemical Aptasensor Based on Poly(Pyrrole-Nitrilotriacetic Acid)-Aptamer Film.

An electrochemical highly sensitive aptasensor was developed based on electropolymerized poly(pyrrole-nitrilotriacetic) acid film and a new aptamer functionalized by a pentahistidine peptide for the quantification of bisphenol A. A surface coverage of antibisphenol A aptamer of 1.84 × 10(-10) mol cm(-2) was estimated from the electrochemical signal of the [Ru(III)(NH3)6](3+) complex bound by electrostatic interactions onto the aptamer-modified electrode. The binding of bisphenol A onto the polymer film was successfully characterized by electrochemical methods as square wave voltammetry and electrochemical impedance spectroscopy measurements. The designed label-free impedimetric aptasensor displayed a wide linear range from 10(-11) to 10(-6) mol L(-1) with a sensitivity of 372 Ω per unit of log of concentration and an excellent specificity toward interfering agents such as 4,4'-dihydroxybiphenyl and bisphenol P.

First Occurrence of Tetrazines in Aqueous Solution: Electrochemistry and Fluorescence.

The photophysical and electrochemical properties of tetrazines substituted by linear 2,3-naphtalimide antennas and/or adamantane groups specifically dedicated to host-guest interactions with cyclodextrins are studied both in organic and aqueous media. In acetonitrile solvent, the reduction potential of tetrazine leading to the anion radical is shifted, depending on the electron-withdrawing power of the substituent of the tetrazines. Due to the hydrophobic character of these compounds, their solubilization in aqueous solution is achieved successively in presence of either ß-cyclodextrins or gold nanoparticules modified by ß-cyclodextrins. We demonstrate that the formation of the inclusion compound tetrazine-cyclodextrin allows the solubilization of the tetrazines in aqueous solution. The supramolecular assemblies obtained in water retain tetrazine's emission properties, yielding a yellow fluorescence.

Label-free photoelectrochemical detection of double-stranded HIV DNA by means of a metallointercalator-functionalized electrogenerated polymer.

The design of photoactive functionalized electrodes for the sensitive transduction of double-stranded DNA hybridization is reported. Multifunctional complex [Ru(bpy-pyrrole)2 (dppn)](2+) (bpy-pyrrole=4-methyl-4'-butylpyrrole-2,2'-bipyridine, dppn=benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) exhibiting photosensitive, DNA-intercalating, and electropolymerizable properties was synthesized and characterized. The pyrrole groups undergo oxidative electropolymerization on planar electrodes forming a metallopolymer layer on the electrode. Thanks to the photoelectrochemical and intercalating properties of the immobilized Ru(II) complex, the binding of a double-stranded HIV DNA target was photoelectrochemically detected on planar electrodes. Photocurrent generation through visible irradiation was correlated to the interaction between double-stranded DNA and the metallointercalator polymer. These interactions were well fitted by using a Langmuir isotherm, which allowed a dissociation constant of 2×10(6)  L mol(-1) to be estimated. The low detection limit of 1 fmol L(-1) and sensitivity of 0.01 units per decade demonstrate excellent suitability of these modified electrodes for detection of duplex DNA.

Biofunctionalization of multiwalled carbon nanotubes by electropolymerized poly(pyrrole-concanavalin A) films.

The synthesis and electropolymerization of a pyrrolic concanavalin A derivative (pyrrole-Con A) onto a multiwalled carbon nanotube (MWCNT) deposit is reported. Glucose oxidase was then immobilized onto the MWCNT-poly(pyrrole-Con A) coating by affinity carbohydrate interactions with the polymerized Con A protein. The resulting enzyme electrode was applied to the amperometric detection of glucose exhibiting a high sensitivity of 36 mA cm(-2) mol(-1) L and a maximum current density of 350 µA cm(-2) .

Permeability improvements of electropolymerized polypyrrole films using dissolvable nano-CaCO3 particle templates.

The electropolymerisation of N-substituted pyrroles on a dissolvable calcium carbonate nanoparticle template was investigated in order to improve the film permeabilities in aqueous solution. After deposition of CaCO3 nanoparticles on the electrode surface, poly(pyrrole-ammonium) or poly(pyrrole-NTA) (NTA: nitrilotriacetic acid) were electrogenerated around the template structures of the electrodes using potentiostatic methods. The dissolution of nanoparticles in acidic medium leads to the formation of nano-porous structures increasing, therefore, the polypyrrole permeability in aqueous solutions. Histidine-tagged glucose oxidase, chosen as an enzyme model, was immobilised on the modified polypyrrole-NTA via the NTA-Cu(2+)-histidine interactions to validate the proposed method. The described setup led to a twofold increase in the maximum current density from 5 to 10 µA cm(-2) after template dissolution.

Biofunctionalization of multiwalled carbon nanotubes by irradiation of electropolymerized poly(pyrrole-diazirine) films.

A photoactivatable poly(pyrrole-diazirine) film was synthesized and electropolymerized as a versatile tool for covalent binding of laccase and glucose oxidase on multiwalled carbon nanotube coatings and Pt, respectively. Irradiation of the functionalized nanotubes allowed photochemical grafting of laccase and its subsequent direct electrical wiring, as illustrated by the electrocatalytic reduction of oxygen. Moreover, covalent binding of glucose oxidase as model enzyme, achieved by UV activation of electropolymerized pyrrole-diazirine, allowed a glucose biosensor to be realized. This original method to graft biomolecules combines electrochemical and photochemical techniques. The simplicity of this new method allows it to be extended easily to other biological systems.

Label-free impedimetric thrombin sensor based on poly(pyrrole-nitrilotriacetic acid)-aptamer film.

A label-free and highly sensitive impedimetric aptasensor was developed based on electropolymerized film for the determination of thrombin. The first step is the electrogeneration of a poly(pyrrole-nitrilotriacetic acid) (poly(pyrrole-NTA)) film onto the surface of electrodes followed by complexation of Cu(2+) ions. Then, the histidine labeled thrombin aptamer was immobilized onto the electrode through coordination of the histidine groups on the NTA-Cu(2+) complex. The aptamer sensor was applied for the detection and quantification of thrombin via impedimetric detection without a labeling step. A linear quantification of thrombin was obtained in the range 4.7×10(-12)-5.0×10(-10) mol L(-1) with a sensitivity of 2838 Ω/log unit (R(2)=0.9984). The impedance modulus at 0.3 Hz as a function of thrombin concentration was used to elaborate a similar linear relationship from 4.7×10(-12) to 5×10(-10) mol L(-1). In addition, aptamer-poly(pyrrole-NTA) electrodes incubated for 40 min in aqueous solutions of bovine serum albumin (BSA), lysozyme and IgG (5×10(-7) mol L(-1)) did not exhibit non-specific adsorption of proteins. Moreover, it has been demonstrated that the selective sensor can be regenerated several times with a good reproducibility.

In situ synthesis of stable mixed ligand Fe2+ complexes on bipyridinyl functionalized electrodes and nanotube supports.

A simple method is presented to synthesize asymmetric mixed ligand iron(II) diimine complexes using bipyridinyl functionalized carbon nanotubes. The synthesis of these complexes was realized using subsequent dip coating processes. The in situ formed mixed ligand complexes were used in aqueous media to act as building blocks in biosensor devices.

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).

Influence of the presence of a gel in the water phase on the electrochemical transfer of ionic forms of beta-blockers across a large water 1,2-dichloroethane interface.

The transfer of ionic species of three beta-blockers (propranolol, sotalol and timolol) has been studied by cyclic voltammetry at a macroscopic water 1,2-dichloroethane (1,2-DCE) interface. The aqueous solution has been gellified in order to study the effect of the gel on the transport properties of the drugs. The gelling agent also stabilizes the interface overcoming mechanical instability. The standard potential and standard Gibbs energy of transfer across the interface, the partition coefficient and the diffusion coefficient of each drug were determined in the presence of a gelled interface. The diffusion coefficients were shifted relative to those obtained at normal water 1,2-DCE interfaces (free of gel).

Sulfide oxidation by hydrogen peroxide catalyzed by iron complexes: two metal centers are better than one.

Peroxoiron species have been proposed to be involved in catalytic cycles of iron-dependent oxygenases and in some cases as the active intermediates during oxygen-transfer reactions. The catalytic properties of a mononuclear iron complex, [Fe(II)(pb)(2)(CH(3)CN)(2)] (pb=(-)4,5-pinene-2,2'-bipyridine), have been compared to those of its related dinuclear analogue. Each system generates specific peroxo adducts, which are responsible for the oxidation of sulfides to sulfoxides. The dinuclear catalyst was found to be more reactive and (enantio)selective than its mononuclear counterpart, suggesting that a second metal site affords specific advantages for stereoselective catalysis. These results might help for the design of future enantioselective iron catalysts.