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Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b3 and non-heme FeB, respectively. The determined sensitivity (-4.00 × 10-8 ± 1.84 × 10-9 A/μM and - 2.71 × 10-8 ± 1.44 × 10-9 A/μM for NO and O2, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O2) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O2, respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O2, while the O2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors., FG and LBM thank FCT/MCTES for the fellowship grants SFRH/BD/52502/2014 and SFRH/BPD/111404/2015, respectively, which are financed by national funds and co-financed by FSE. CMC acknowledges FCT-MCTES funding through project PTDC/BBB-BQB/0129/2014 (FCT/MCTES). This work was supported by the REQUIMTE, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2013 and UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265 and POCI-01-0145-FEDER-007728), and also by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES). Funding through REQUIMTE project entitled “NOR-based biosensor for nitric oxide detection in biological and environmental samples” is also acknowledged.

Direct electron transfer (DET) of enzymes on electrode surfaces is highly desirable both for fundamental mechanistic studies and to achieve membrane- and mediator-less bioenergy harvesting. In this report, we describe the preparation and comprehensive structural and electrochemical characterization of a three-dimensional (3D) graphene-based carbon electrode, onto which the two-domain redox enzyme Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) is immobilized. The electrode is prepared by an entirely novel method, which combines in a single step electrochemical reduction of graphene oxide (GO) and simultaneous electrodeposition of positively charged polyethylenimine (PEI), resulting in a well dispersed MtCDH surface. The resulting MtCDH bio-interface was characterized structurally in detail, optimized, and found to exhibit a DET maximum current density of 7.7 ± 0.9 μA cm−2 and a half-lifetime of 48 h for glucose oxidation, attributed to favorable MtCDH surface orientation. A dual, entirely DET-based enzymatic biofuel cell (EBFC) was constructed with a MtCDH bioanode and a Myrothecium verrucaria bilirubin oxidase (MvBOD) biocathode. The EBFC delivers a maximum power density (Pmax) of 7.6 ± 1.3 μW cm−2, an open-circuit voltage (OCV) of 0.60 V, and an operational lifetime over seven days, which exceeds most reported CDH based DET-type EBFCs. A biosupercapacitor/EBFC hybrid was also constructed and found to register maximum power densities 62 and 43 times higher than single glucose/air and lactose/air EBFCs, respectively. This hybrid also shows excellent operational stability with self-charging/discharging over at least 500 cycles.

Hydrogenases are attractive biocatalysts for utilization in electrochemical devices as potential replacement for Pt in hydrogen evolving electrodes. In this work, we investigate the immobilization of ferredoxin tagged FeFe-hydrogenase (Fd-HydA1) on black TiO2 nanotubes (bTNTs), with uniform nanotube opening diameters of 140 nm. By utilizing an immunogold labelling method, we show that the enzymes attach on the top surface of the bTNTs film rather than on the inner nanotube walls, reflecting the difficulty to insert enzymes into high aspect-ratio nanomaterials for O2-shielding. Nevertheless, cyclic voltammetry demonstrates direct electron transfer between Fd-HydA1 and bTNTs for the hydrogen evolution reaction (HER) in neutral media. This work provides new insight towards design of new nanostructured electrodes for enzyme immobilization.

International audience; We present the effect of thermal treatment of carbon felt by gas flow containing 1% of oxygen at high temperature on the direct electron transfer and electrocatalytic oxygen reduction currents by a bilirubin oxidase (BOD) film directly adsorbed in the presence of carbon nanotubes on to a porous carbon felt (PCF). The upgraded properties (surface area, pore volume and hydrophilicity) of the resulting porous carbon felt (PCF) in comparison to commercial carbon felt (raw CF), creates a suitable support for the entrapment of MWCNTs bearing negative charges at neutral pH and BOD enzymes, all the components being entrapped in chitosan layer reticulated with glutaraldehyde. Since functional MWCNTs are 2 usually used to facilitate DET, we introduce COOH@MWCNTs, bearing negative charges at neutral pH, in the enzyme layer to evaluate their impact on the electron transfer properties with BOD. The enzyme immobilization efficiency is examined by varying the amount of the components and the immobilization procedure. Linear sweep voltammetry (LSV) and chronoamperometry measurements are used to evaluate the electrochemical behavior of the enzymatic biocathodes. Based on the experimental results, we show that the optimized bioelectrode delivers a current density of 3.70 mA cm-2at 0.15 V vs Ag/AgCl and could retain above 55 % of its initial response after 4 months, proving its outstanding performance. This new bioelectrode allows for optimal DET-type bioelectrocatalytic activity toward O2 reduction and is a very promising candidate for the construction of 3-dimensional cathodes in (bio)-electrochemical devices needing high current output.

Efficient direct electron transfer (DET) between cellobiose dehydrogenase from Corynascus thermophilus (CtCDH) and a novel gold electrode platform, obtained by covalent linking of green AuNPs and AgNPs modified with a dithiol self-assembled monolayer, consisting of biphenyl-4,4'-dithiol (BPDT), was presented. The green AuNPs and AgNPs were synthesized using quercetin as reducing agent at room temperature. TEM experiments showed that the AuNPs and AgNPs were circular in shape with an average diameter of 5 and 8nm, respectively. Cyclic voltammetry of CtCDH immobilized onto the AuNPs/BPDT/AuE and the AgNPs/BPDT/AuE electrode platforms were carried out and compared with naked AuE, BPDT/AuE, AuNPs/AuE, and AgNPs/AuE. A pair of well-defined redox waves in neutral pH solution due to efficient DET of CtCDH was present with both MNPs/BPDT/AuE platforms. No DET communication was found with platforms without MNPs linked to BPDT. The apparent heterogeneous electron transfer rate constants (kS) of CtCDH were calculated to be 21.5±0.8s-1 and 10.3±0.7s-1, for the AuNPs/BPDT/AuE and the AgNPs/BPDT/AuE platforms, respectively. The modified electrodes were successively used to develop an eco-friendly biosensor for lactose detection. The CtCDH/AuNPs/BPDT/AuE based biosensor showed the best analytical performances with an excellent stability, a detection limit of 3µM, a linear range between 5 and 400µM and a sensitivity of 27.5±2.5µAcm-2mM-1. Such performances were favorably compared with other lactose biosensors reported in literature. The biosensor was successively tested to quantify lactose content in real milk and cream samples. No significant interference present in the sample matrices was observed.

Quantitative routine detection of fucose, which is a cancer marker, in urine is effective for the preliminary screening of cancer. Amperometric biosensing methods have the advantage of being simple, rapid, and precise for urinalysis. However, coexisting electroactive interferences such as ascorbic acid (AA), dopamine (DA), and uric acid (UA) prevent accurate measurements. In this work, an amperometric L-fucose biosensor unaffected by interferences was developed and utilizes direct electron transfer type bioelectrocatalysis of pyrroloquinoline quinone (PQQ)-dependent pyranose dehydrogenase from Coprinopsis cinerea (CcPDH). The isolated PQQ domain from CcPDH was immobilized on gold nanoparticle (AuNP)-modified electrodes, which obtained a catalytic current at a lower potential than the oxidation potential of the interfering compounds. Applying an operating potential of −0.1 V vs. Ag|AgCl (3 M NaCl) enabled the detection of L-fucose while completely eliminating the oxidation of AA, DA, and UA on the electrodes. The increase in the specific area of the electrodes by increasing the AuNP drop-casting time resulted in an improvement in the sensor performance. The biosensor exhibited a linear range for L-fucose detection between 0.1 mM and 1 mM (R2 = 0.9996), including a cut-off value, the sensitivity was 3.12 ± 0.05 μA mM−1 cm−2, and the detection limit was 13.6 μM at a signal-to-noise ratio of three. The biosensor can be used to quantify the concentration of L-fucose at physiological levels and does not require urine preprocessing, making it applicable to practical use for point-of-care testing with urine.

The bioelectrocatalytic properties of membrane-bound aldehyde dehydrogenase (AlDH) from Gluconobacter oxydans NBRC12528 were evaluated. AlDH exhibited direct electron transfer (DET)-type bioelectrocatalytic activity for acetaldehyde oxidation at several kinds of electrodes. The kinetic and thermodynamic parameters for bioelectrocatalytic acetaldehyde oxidation were estimated based on the partially random orientation model. Moreover, at the multi-walled carbon nanotube-modified electrode, the coordination of CN− to AlDH switched the direction of the DET-type bioelectrocatalysis to acetate reduction under acidic conditions. These phenomena were discussed from a thermodynamic viewpoint.

Glucose-oxidizing enzymes are widely used in electrochemical biosensors and biofuel cells; in most applications glucose oxidase, an enzyme with non-covalently bound FAD and low capability of direct electronic communications with electrodes, is used. Here, we show that another glucose-oxidizing enzyme with a covalently bound FAD center, hexose oxidase (HOX), adsorbed on graphite, exhibits a pronounced non-catalytic voltammetric response from its FAD, at −307 mV vs. Ag/AgCl, pH 7, characterized by the heterogeneous electron transfer (ET) rate constant of 29.2 ± 4.5 s−1. Direct bioelectrocatalytic oxidation of glucose by HOX proceeded, although, with a 350 mV overpotential relative to FAD signals, which may be connected with a limiting step in biocatalysis under conditions of the replacement of the natural redox partner, O2, by the electrode; mediated bioelectrocatalysis was consistent with the potentials of a soluble redox mediator used. The results allow development of HOX-based electrochemical biosensors for sugar monitoring and biofuel cells exploiting direct ET of HOX, and, not the least, fundamental studies of ET non-complicated by the loss of FAD from the protein matrix. Keywords: Hexose oxidase (HOX), Bioelectrocatalysis, Direct electron transfer, Glucose oxidation

This work reports the non-covalent functionalization (dispersion) of multi-walled carbon nanotubes (MWCNTs) with cytochrome c (Cyt c), the direct electron transfer (DET) after drop-coating deposition of MWCNTs-Cyt c dispersion on glassy carbon electrodes (GCE), and the analytical applications for the highly sensitive quantification of hydrogen peroxide. The dispersion and the resulting modified electrodes were studied by UV-visible spectroscopy, scanning electron microscopy, and electrochemical techniques. The drastic treatment for dispersing the MWCNTs (5.0 min sonication in water with ultrasonic tip) produces a partial denaturation that facilitates the interaction of Cyt c with the CNTs and makes possible an efficient electron transfer between the heme group and the electrode. A critical analysis of the influence of different experimental conditions on the efficiency of the dispersion and on the performance of GCE modified with MWCNTs-Cyt c dispersion is also reported. The analytical parameters obtained with GCE/MWCNTs-Cyt c for the amperometric quantification of hydrogen peroxide at -0.100 V were: sensitivity of (43 ± 1) mA M-1 cm-2, linear range between 1.0 × 10-6 and 1.6 × 10-4 M, detection limit of 1.5 × 10-7 M, reproducibility of 3.1% and repeatability of 3.4%. The biosensor was successfully used for the quantification of hydrogen peroxide in mouthwash and spiked milk samples. Fil: Eguilaz Rubio, Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Gutierrez, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Rivas, Gustavo Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina

An electrochemical d -fructose sensor was fabricated by immobilizing a variant of d -fructose dehydrogenase on a porous gold microelectrode. The enzyme-modified electrode bioelectrocatalytically oxidizes d -fructose via direct electron transfer. The catalytic current reaches a steady-state limiting value at 0.05 V vs. Ag|AgCl (sat. KCl). The temperature dependence of the sensor suggests that its response is limited by the diffusion of d -fructose. Therefore, the sensor requires no calibration. The sensitivity of the sensor, (2.0 ± 0.2) × 102 μA cm−2 mM−1, is reproducible. The upper limit of detection is ~2.0 mM and the kinetics of the bioelectrocatalytic reaction is determined the limitation. The fabricated sensors were applied in the determination of d -fructose in commercial beverages as well as honey, and compared favorably with a more commonly used photometric method.

Graphite rods were modified by substituted aryldiazonium salts allowing subsequent laccase immobilisation and direct electron transfer at the cathode. Two covalent enzyme immobilisation methods were performed with carboxy and amino substituted grafted groups, either via the formation of an amide bond or a Schiff base between the glycosidic groups of the enzyme and the amino groups on the electrode surface, respectively. Laccase adsorption efficiency was consistently compared to the covalent attachment method on the same carbon surface, showing that the latter method led to a higher immobilisation yield when the electrode surface was functionalised with carboxylic groups, as shown from both laccase activity measurement towards an organic reducing substrate, ABTS, and quantitative XPS analysis. Both analytical methods led to similar laccase surface coverage estimations. From activity measurements, when laccase was covalently immobilised on the electrode functionalised with carboxylic groups, the surface coverage was found to be 43 ± 2% whereas it was only 10 ± 3% when laccase was adsorbed. Biocatalysed dioxygen reduction current was also higher in the case of covalent immobilisation. For the first time, oxidised laccase performances were compared to unmodified laccase, showing significant improved efficiency when using oxidised laccase: the current obtained with oxidised laccase was 141 ± 37 μA cm− 2 compared to 28 ± 6 μA cm− 2 for unmodified laccase after covalent immobilisation of the enzyme on a graphite electrode functionalised with carboxylic groups.

International audience; For the first time, gold nanoparticle-based electrodes have been used as platforms for efficient immobilization of the [NiFe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. AuNPs were characterized by electronic microscopy, dynamic light scattering and UV-Vis spectroscopy. Two sizes around 20.0 ± 5.3 nm and 37.2 ± 4.3 nm nm were synthesized. After thiol-based functionalization, the AuNPs were proved to allow direct H2 oxidn. over a large range of temps. A high c.d. up to 1.85 ± 0.15 mA·cm- 2 was reached at the smallest AuNPs, which is 170 times higher than the one recorded at the bare gold electrode. The catalytic current was esp. studied as a function of the AuNP size and amt., and procedure for deposition. A synergetic effect between the AuNP porous deposit and the increase surface area was shown. Compared to previously used nanomaterials such as carbon nanofibers, the covalent grafting of the enzyme on the thiol-modified gold nanoparticles was shown to enhance the stability of the hydrogenase. This bioanode was finally coupled to a biocathode where BOD from Myrothecium verrucaria was immobilized on AuNP-based film. The performance of the so-mounted H2/O2 biofuel cell was evaluated, and a power d. of 0.25 mW·cm- 2 was recorded. [on SciFinder(R)]

Gold disk electrodes modified with gold nanoparticles have been used as a scaffold for the covalent immobilization of bilirubin oxidase. The nanostructured bioelectrodes were tested as mediator-less biosensors for oxygen in a buffer that mimics the content and the composition of human physiological fluids. Chronoamperometry measurements showed a detection limit towards oxygen of 6 ± 1 μM with a linear range of 6-300 μM, i.e. exceeding usual physiological ranges of oxygen in human tissues and fluids. The biosensor presented is the first ever-reported oxygen amperometric biosensor based on direct electron transfer of bilirubin oxidase., This work was funded by the FP7 project “3D-Nanobiodevice” (NMP4-SL-2009-229255). We thank Novozymes A/S for the preparation of Myrothecium verrucaria BOx. M.P. acknowledges the 2009 Ramon y Cajal program from the Spanish MINECO.

A novel third generation biosensor was developed based on one-shot adsorption of chemically-modified cytochrome c (cyt c) onto bare gold electrodes. In contrast to the classic approach which consists of attaching cyt c onto an active self-assembled monolayer (SAM) priory chemisorbed on gold, here short-chain thiol derivatives (mercaptopropionic acid, MPA) were chemically introduced on cyt c protein shell via its lysine residues enabling the very fast formation ( < 5 min) of an electroactive biological self-assembled monolayer (SAM) exhibiting a quasi-reversible electrochemical behavior and a fast direct electron transfer (ET). The heterogeneous ET rate constant was estimated to be k(s)= 1600 s(-1), confirming that short anchors facilitate the ET via an efficient orientation of the heme pocket. In comparison, no ET was observed in the case of native and long-anchor (mercaptoundecanoic acid, MUA) modified cyt c directly adsorbed on gold. However, in both cases the ET was efficiently restored upon in-bulk generation of gold nanoparticles which acted as electron shuttles. This observation emphasizes that the lack of electroactivity might be caused by either an inappropriate orientation of the protein (native cyt c) or a critical distance (MUA-cyt c). Finally, the sensitivity of the bare gold electrode directly modified with MPA-cyt c to hydrogen peroxide (H2O2) was evaluated by amperometry and the so-made amperometric biosensor was able to perform real-time and non-invasive detection of endogeneous H2O2 released by unicellular aquatic microorganisms, Chlamydomonas reinhardtii, upon cadmium exposure. (c) 2012 Elsevier B.V. All rights reserved.

We report a simple and one-pot procedure for the simultaneous immobilization of poly(N-[3-(trimethoxy sily)propyl[aniline (PTMSPA) and horseradish peroxidase (HRP) on gold nanorods (GNRs) to yield HRP/PTMSPA@GNRs. HRP/PTMSPA@GNR was well characterized by field emission scanning electron microscopy and UV-visible spectroscopy. High resolution image of HRP/PTMSPA@GNR attributes GNRs are welded/connected to one another through the surface coated PTMSPA. Direct electron transfer was achieved at HRP/PTMSPA@GNR with an electron transfer rate constant (k(s)) of 3.2 +/- 0.1 s(-1). Cyclic voltammograms of H2O2 at HRP/PTMSPA@GNR modified electrode exhibited a stable reduction peak at approximately -0.25 V. Amperometric response of HRP/PTMSPA@GNR modified electrode showed quick response (

A highly sensitive biosensor was fabricated by incorporating myoglobin (Mb) within graphene oxide (GO) sheets and Nafion composite films. The stable composite Mb–GO–Nafion films were characterized by electrochemistry, scanning electron microscopy, Fourier transform infrared spectroscopy and UV–vis spectroscopy. It was found that Mb in Mb–GO–Nafion films retained its secondary structure similar to its native states. Cyclic voltammetry of Mb–GO–Nafion films showed a pair of well defined, quasi-reversible peaks at about −0.312 V vs saturated calomel electrode (SCE) at pH 5.5, corresponding to direct electron transfer (DET) between Mb and the glassy carbon electrode. Electrochemical parameter of Mb in Mb–GO–Nafion film such as apparent heterogeneous electron transfer rate constant (ks) and formal potential (Eo′) were obtained. The dependence of Eo′ on solution pH indicated that the DET reaction of Mb was coupled with proton transfer. Mb in the films displayed good electrocatalytic activities towards various substrates such as hydrogen peroxide, nitrite and oxygen, indicating that the composite films have potential applications in fabricating novel biosensors without using mediators.

Titanate nanotubes (TiNT) surface modified with (3-aminopropyl)trimethoxysilane were employed as a support for covalent immobilization of horseradish peroxidase (HRP) by using 1,4-benzoquinone as a coupling agent. Composite film-electrodes consisting of HRP-modified TiNT embedded into the porous carbon powder/Nafion matrix were fabricated and their applicability in direct bioelectrocatalytic reduction of H 2 O 2 and H 2 O 2 biosensing were investigated. An efficient direct electron transfer between the immobilized HRP molecules and the electrode was observed in the presence of H 2 O 2 at potentials lower than 600 mV (vs. Hg/Hg 2 Cl 2 /3.5 M KCl). For the HRP–TiNT-modified electrodes polarized at 0 mV, a linear dependence of the bioelectrocatalytic current on the concentration of H 2 O 2 was observed up to the concentration of H 2 O 2 equal to 10 μM, with the sensitivity of (1.10 ± 0.01) AM −1 cm −2 and the detection limit of 35 nM.

A new generation of mediatorless H2/O2 biofuel cells was designed based on a hyperthermophilic O2-tolerant hydrogenase and a thermostable bilirubin oxidase both immobilized on carbon nanofibers. A power density up to 1.5 ± 0.2 mW·cm−2 at 60 °C was reached. This first demonstration of a H2/O2 biofuel cell able to deliver electricity over a wide range of temperatures, from 30 °C up to 80 °C, and over a large pH window, allows considering this device as an alternative power supply for small portable applications in various environments, including extreme ones. Keywords: Enzymatic H2/O2 biofuel cell, Hydrogenase, Bilirubin oxidase, Thermostable enzymes, Direct electron transfer, Carbon nanofiber

Geobacter sulfurreducens bacteria increased the open circuit potential of 304L stainless steel by around 320 mV in only a few hours after inoculation. This represents a significant increase in the corrosion risk. In contrast, the oxidation of acetate, which is catalysed by well-established biofilms, shifted the pitting potential towards positive values. In acetate-lacking media, pitting occurred with and without bacteria in the same range of potential values, but the presence of bacteria drastically increased the size of pits. AFM showed pits more than 10 times broader and deeper due to the presence of bacteria.In the absence of acetate, the masking effect due to acetate oxidation disappeared and the full corrosive effect of the biofilm was revealed.This also fully explains why pitting was predominantly observed close to surface areas where bacterial settlement was the densest. Keywords: Microbial corrosion, Geobacter sulfurreducens, Stainless steel, Electrochemically active biofilms, Direct electron transfer

It has recently been discovered that many microbial species have the capacity to connect their metabolism to solid electrodes, directly exchanging electrons with them through membrane-bound redox compounds, nevertheless such a direct electron transfer pathway has been evoked rarely in the domain of microbial corrosion. Here was evidenced for the first time that the bacterium Geobacter sulfurreducens is able to increase the free potential of 304 L stainless steel up to 443 mV in only a few hours, which represents a drastic increase in the corrosion risk. In contrast, when the bacterial cells form a locally well-established biofilm, pitting potentials were delayed towards positive values. The microscopy pictures confirmed an intimate correlation between the zones where pitting occurred and the local settlement of cells. Geobacter species must now be considered as key players in the mechanisms of corrosion. Keywords: Microbial corrosion, Geobacter sulfurreducens, Direct electron transfer, 304 L stainless steel

We synthesized a kind of gold nanoparticle protected by a synthetic lipid (didodecyldimethylammonium bromide, DDAB). With the help of these gold nanoparticles, hemoglobin can exhibit a direct electron transfer (DET) reaction. The formal potential locates at −169 mV vs. Ag/AgCl. Spectral data indicated the hemoglobin on the electrode was not denatured. The lipid-protected gold nanoparticles were very stable (for at least 8 months). Their average diameter is 6.42 nm. It is the first time to use monolayer-protected nanoparticles to realize the direct electrochemistry of protein.

Covalently linked layers of glucose oxidase, single-wall carbon nanotubes and poly-l-lysine on pyrolytic graphite resulted in a stable biofuel cell anode featuring direct electron transfer from the enzyme. Catalytic response observed upon addition of glucose was due to electrochemical oxidation of FADH2 under aerobic conditions. The electrode potential depended on glucose concentration. This system has essential attributes of an anode in a mediator-free biocatalytic fuel cell. Keywords: Glucose oxidase, Direct electron transfer, Biofuel cell, Anode stability, Carbon nanotubes

We report herein a simple device for rapid biosensing consisting of a single microfluidic channel made from poly(dimethylsiloxane) (PDMS) coupled to an injector, and incorporating a biocatalytic sensing electrode, reference and counter electrodes. The sensing electrode was a gold wire coated with 5 nm glutathione-decorated gold nanoparticles (AuNPs). Sensitive detection of H2O2 based on direct bioelectrocatalysis by horseradish peroxidase (HRP) was used for evaluation. HRP was covalently linked the glutathione–AuNPs. This electrode presented quasi-reversible cyclic voltammetry peaks at −0.01 V vs. Ag/AgCl at pH 6.5 for the HRP heme FeIII/FeII couple. Direct electrochemical activity of HRP was used to detect H2O2 at high sensitivity with a detection limit of 5 nM in an unmediated system. Keywords: Microfluidics, Biosensor, H2O2, Electrocatalysis, Direct electron transfer