Kinetic study for implementation of a disposable “redox-flexible” glucose biosensor

A “redox-flexible” reagentless amperometric biosensor that can function either in oxidation or reduction and can cover large ranges of substrate concentrations has been developed for glucose measurements. Glucose oxidase (GOx) either uses O₂ as its natural electron acceptor and produces H₂O₂ or uses artificial electron acceptors like ferricinium derivatives. To broaden the range of working potentials, peroxidase enzyme (HRP) was added to GOx, offering the alternative to correlate the glucose concentration to the reduction current of the ferricinium that results from H₂O₂ oxidation of ferrocene. In such a configuration, the same ferrocene/ferricinium couple acts as a redox mediator for both enzymatic reactions involving GOx and HRP. Two ferrocenes were used in this study: Fc(CH₂OH)₂, substituted by electron-donor groups, and FcCOOH, substituted by an electron-withdrawing group. The rates of the reactions involved were determined, and the calibration curves in cathodic and anodic mode were drawn. The comparative study showed that for glucose measurement, the [FcCOOH]/[FcCOOH]⁺ couple is best suited to act as mediator in the construction of a “redox-flexible” glucose biosensor.     

Alcohol dehydrogenase amperometric biosensor based on a colloidal gold-carbon nanotubes composite electrode

A novel ethanol biosensor based on the bulk incorporation of alcohol dehydrogenase (ADH) into a colloidal gold (Au_coll)-multiwalled carbon nanotubes (MWCNTs) composite electrode using Teflon as binding material is reported. The composite Au_coll-MWCNTs-Teflon electrode exhibited significantly improved electrooxidation of NADH when compared with other carbon composite electrodes, including those based on carbon nanotubes. Amperometric measurements for NADH at +0.3 V showed significant differences in sensitivity between Au_coll-MWCNTs-Teflon and MWCNTs-Teflon composite electrodes. Incorporation of ADH into the bulk electrode material allowed the construction of a mediatorless ethanol biosensor. Both the enzyme loading and the NAD⁺ concentration in solution were optimized. The ADH-Au_coll-MWCNTs-Teflon biosensor allowed a limit of detection for ethanol of 4.7 μmol l⁻¹, which is remarkably better than those reported for other CNTs-based ADH biosensors. The apparent Michaelis-Menten constant was 4.95 mmol l⁻¹, which is much lower than that reported by immobilization of ADH onto a gold electrode. Both repeatability of the ethanol amperometric measurements, reproducibility with different biosensors, lifetime and storage ability can be, in general, advantageously compared with other ADH-CNTs biosensors. The biosensor was applied for the rapid determination of ethanol in commercial and certified beer samples.     

Room-temperature ionic liquids/multi-walled carbon nanotubes/chitosan composite electrode for electrochemical analysis of NADH

An electrochemical sensing platform was developed based on the integration of room-temperature ionic liquids (1-butyl-3-methylimidazolium tetrafluoroborate, BMIM·BF₄) and multi-walled carbon nanotubes (MWNTs) with polymeric matrix (chitosan, CHIT). The resulting composite were investigated and characterized by FTIR, TEM, SEM, EDS and electrochemical methods. The BMIM·BF₄/MWNTs/CHIT have good dispersibility in aqueous solution and can form a relative uniform film with unique structure. Electrochemical studies suggested that the BMIM·BF₄/MWNTs/CHIT composite system provided a synergistic augmentation on the voltammetric and amperometric behaviors of electrochemical oxidation of NADH, which indicated by the decrease of the peak potential of NADH oxidation and the improvement of amperometric response. Additionally, the BMIM·BF₄/MWNTs/CHIT/GC electrode shows good analytical performance such as low detection limit (0.06 μM), good regeneration and anti-fouling properties for determination of NADH. This nanomaterials-based composite may be used as electrochemical transducers and have potential application for designing a variety of NAD⁺-dependent electrochemical biosensors.     

Amperometric bienzyme glucose biosensor based on carbon nanotube modified electrode with electropolymerized poly(toluidine blue O) film

The amperometric bienzyme glucose biosensor utilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) immobilized in poly(toluidine blue O) (PTBO) film was constructed on multi-walled carbon nanotube (MWNT) modified glassy carbon electrode. The HRP layer could be used to analyze hydrogen peroxide with toluidine blue O (TBO) mediators, while the bienzyme system (HRP + GOx) could be utilized for glucose determination. Glucose underwent biocatalytic oxidation by GOx in the presence of oxygen to yield H₂O₂ which was further reduced by HRP at the MWNT-modified electrode with TBO mediators. In the absence of oxygen, glucose oxidation proceeded with electron transfer between GOx and the electrode mediated by TBO moieties without H₂O₂ production. The bienzyme electrode offered high sensitivity for amperometric determination of glucose at low potential, displaying Michaelis-Menten kinetics. The bienzyme glucose biosensor displayed linear response from 0.1 to 1.2 mM with a sensitivity of 113 mA M⁻¹ cm⁻² at an applied potential of −0.10 V in air-saturated electrolytes.     

Poly-phenothiazine derivative-modified glassy carbon electrode for NADH electrocatalytic oxidation

Electropolymerization of a new phenothiazine derivative (bis-phenothiazin-3-yl methane; BPhM) on glassy carbon (GC) electrode generates a conducting film of poly-BPhM, in stable contact with the electrode surface. The heterogeneous electron-transfer process corresponding to the modified electrode is characterized by a high rate constant (50.4 s⁻¹, pH 7). The GC/poly-BPhM electrode shows excellent electrocatalytic activity toward NADH oxidation. The rate constant for catalytic NADH oxidation, estimated from rotating disk electrode (RDE) measurements and extrapolated to zero concentration of NADH, was found to be 9.4 × 10⁴ M⁻¹ s⁻¹ (pH 7). The amperometric detection of NADH, at +200 mV vs. SCE, is described by the following electroanalytical parameters: a sensitivity of 1.82 mA M⁻¹, a detection limit of 2 μM and a linear domain up to 0.1 mM NADH.     

Fabrication of graphene/prussian blue composite nanosheets and their electrocatalytic reduction of H2O2

In this work, graphene/prussian blue (PB) composite nanosheets with good dispersibility in aqueous solutions have been synthesized by mixing ferric-(III) chloride and potassium ferricyanide in the presence of graphene under ambient conditions. Transmission electron microscopy (TEM) shows that the average size of the as-synthesized PB nanoparticles on the surface of graphene nanosheets is about 20 nm. Fourier-transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) patterns have been used to characterize the chemical composition of the obtained graphene/PB composite nanosheets. The graphene/PB composite nanosheets exhibit good electrocatalytic behavior to detection of H₂O₂ at an applied potential of −0.05 V. The sensor shows a good linear dependence on H₂O₂ concentration in the range of 0.02-0.2 mM with a sensitivity of 196.6 μA mM⁻¹ cm⁻². The detection limit is 1.9 μM at the signal-to-noise ratio of 3. Furthermore, the graphene/PB modified electrode exhibits freedom of interference from other co-existing electroactive species. This work provides a new kind of composite modified electrode for amperometric biosensors.     

Low potential detection of glucose at carbon nanotube modified glassy carbon electrode with electropolymerized poly(toluidine blue O) film

A mediator glucose biosensor has been constructed by immobilizing glucose oxidase at electropolymerized poly(toluidine blue O) film on carbon nanotube modified glass carbon electrode. The toluidine blue O moieties served as redox mediators for enzymatic glucose oxidation and as polymeric network to maintain the biosensor activity. Great enhancement in current response was observed for the glucose biosensor. The detection potential could be decreased to −0.1 V (versus Ag|AgCl), where common interferences such as ascorbic acid, uric acid and acetamidophenol were not oxidized to cause interferences. The amperometric glucose biosensor offered a sensitivity of 14.5 mA M⁻¹ cm⁻² for the linear range of 1-7 mM.     

Prussian Blue-modified microelectrodes for selective transduction in enzyme-based amperometric microbiosensors for in vivo neurochemical monitoring

Prussian Blue-modified carbon fiber microelectrodes (CFE/PBs) are proposed as an alternative to the more conventional metal transducers used for H₂O₂-detecting biosensors in brain extracellular fluid (ECF). The main advantages of this approach are the very small dimensions (∼10 μm diameter) and the low applied potentials needed (0.0 V versus SCE). Electrocatalytic and physiochemical properties of PB deposits were studied using cyclic voltammetric (CV), amperometric and spectroscopic methods (FTIR and VIS). Optimized CFE/PB microsensors displayed a H₂O₂ current density of 1.00 ± 0.04 A M⁻¹ cm⁻² with a detection limit of 10⁻⁸ M. Furthermore, to improve stability and selectivity properties, several polymeric films were investigated: Nafion, poly(o-phenylenediamine) (PoPD), and a hybrid configuration of these two polymers. Finally, the PoPD film was selected due to its excellent anti-interference properties. The use of this permselective film also enhanced the stability of PB against solubilization at high pH, albeit at the expense of slightly lower H₂O₂ sensitivity (0.48 ± 0.02 A M⁻¹ cm⁻²) and higher detection limit (∼10⁻⁷ M). However, the use of the PoPD film significantly enhanced the selectivity against the main endogenous brain interference species (ascorbic acid, uric acid, dopamine and 3,4-dihydroxyphenylacetic acid), expressed as the ratio of the sensitivity slopes (SH₂O₂/Sinterference), which was close to 600 for all interference molecules studied. A prototype of a CFE/PB-based glucose microbiosensor design is presented, together with preliminary studies of its characteristics in vitro and its functionality in brain ECF in vivo.     

Subaquatic oxidation of coal by water-dissolved oxygen

Subaquatic oxidation of two bituminous coals by water-dissolved oxygen was investigated using batch reactor equipped with membrane oxygen sensor. Effects of time, temperature and coal grain size were studied as basic parameters influencing the oxidation process. Obtained results showed the subaquatic coal oxidation can be considered as interaction of the first reaction order with respect to oxygen. From temperature dependence of oxidation rate, activation energies E = 72 ± 4 kJ mol⁻¹ and/or 50 ± 4 kJ mol⁻¹ were calculated. For the samples, oxygen consumption R_O2 was found to be in the range of 2 × 10⁻⁷ mol O₂ kg⁻¹ s⁻¹ to 6 × 10⁻⁷ mol O₂ kg⁻¹ s⁻¹, such values being quite comparable with R_O2 for aerial oxidation of bituminous coals.     

Comprehensive kinetic characterization of the oxidation and gasification of model and real diesel soot by nitrogen oxides and oxygen under engine exhaust conditions: Measurement, Langmuir-Hinshelwood, and Arrhenius parameters

The reaction kinetics of the oxidation and gasification of four types of model and real diesel soot (light and heavy duty vehicle engine soot, graphite spark discharge soot, hexabenzocoronene) by nitrogen oxides and oxygen have been characterized for a wide range of conditions relevant for modern diesel engine exhaust and continuously regenerating particle trapping or filter systems (0-20% O₂, 0-800 ppm NO₂, 0-250 ppm NO, 0-8% H₂O, 303-773 K, space velocities 1.3 × 10⁴-5 × 10⁵ h⁻¹). Soot oxidation and NO₂ adsorption experiments have been performed in a model catalytic system with temperature controlled flat bed reactors, novel aerosol particle deposition structures, and sensitive multicomponent gas analysis by FTIR spectroscopy. The experimental results have been analyzed and parameterized by means of a simple carbon mass-based pseudo-first-order rate equation, a shrinking core model, oxidant-specific rate coefficients, Langmuir-Hinshelwood formalisms (maximum rate coefficients and effective adsorption equilibrium constants), and Arrhenius equations (effective activation energies and pre-exponential factors), which allow to describe the rate of reaction as a function of carbon mass conversion, oxidant concentrations, and temperature. At temperatures up to 723 K the reaction was driven primarily by NO₂ and enhanced by O₂ and H₂O. Within the technically relevant concentration range the reaction rates were nearly independent of O₂ and H₂O variations, while the NO₂ concentration dependence followed a Langmuir-Hinshelwood mechanism (saturation above ∼200 ppm). Reaction stoichiometry (NO₂ consumption, CO and CO₂ formation) and rate coefficients indicate that the reactions of NO₂ and O₂ with soot proceed in parallel and are additive without significant non-linear interferences. The reactivity of the investigated diesel soot and model substances was positively correlated with their oxygen mass fraction and negatively correlated with their carbon mass fraction.     

Enhanced solid-state electrogenerated chemiluminescence of Au/CdS nanocomposite and its sensing to H2O2

Au nanoparticles (AuNPs) are good quenchers once they closely contact with luminophore. Here we reported a simple approach to obtain enhanced electrogenerated chemiluminescence (ECL) behavior based on Au/CdS nanocomposite films by adjusting the amount of AuNPs in the nanocomposite. The maximum enhancement factor of about 4 was obtained at an indium tin oxide (ITO) electrode in the presence of co-reactant H₂O₂. The mechanism of this enhancement was discussed in detail. The strong ECL emission from Au/CdS nanocomposites film was exploited to determine H₂O₂. The resulting ECL biosensors showed a linear response to the concentration of H₂O₂ ranging from 1.0 × 10⁻⁸ to 6.6 × 10⁻⁴ mol L⁻¹ with a detection limit of 5 nmol L⁻¹ (S/N = 3) and good stability and reproducibility.     

Synergistic effect of a catechin-immobilized poly(3,4-ethylenedioxythiophene)-modified electrode on electrocatalysis of NADH in the presence of ascorbic acid and uric acid

Catechin is a polyphenolic flavonoid that can be isolated from a variety of natural sources, including tea leaves, grape seeds, and the wood and bark of trees such as acacia and mahogany. In our experiments, catechin was immobilized on PEDOT/GC (poly(3,4-ethylenedioxythiophene)/glassy carbon)-modified electrodes and used as a mediator for NADH (nicotinamide adenine dinucleotide) oxidation. The effect of the PEDOT thickness on the surface coverage of the catechin molecules was studied using cyclic voltammetry. The electrochemical properties and the effect of pH on the redox properties of the immobilized catechin molecules were studied by cyclic voltammetry in phosphate solution. The electrocatalytic oxidation of NADH at different electrode surfaces such as the bare GC-, the PEDOT/GC-, the catechin/GC- and the catechin/PEDOT/GC-modified electrodes was explored in phosphate solution at pH 7. In the catechin/PEDOT/GC-modified electrode, the PEDOT film plays an important role in resolving the oxidation potentials of ascorbic acid and NADH into two peaks that occur at the same potential for the catechin/GC-modified electrode surface. The heterogeneous electron transfer rate constant for NADH oxidation at the catechin/PEDOT/GC-modified electrode was determined using the rotating disk electrode technique and found to be 9.88 × 10³ M⁻¹ s⁻¹. The amperometric determination of NADH at the catechin/PEDOT/GC electrode was tested. The sensitivity of the electrode was 19 nA/μM.     

Gold nanoparticles mediate the assembly of manganese dioxide nanoparticles for H2O2 amperometric sensing

Aggregates of gold nanoparticles (AuNPs) that mediate the assembly of manganese dioxide nanoparticles (nano-MnO₂) for hydrogen peroxide (H₂O₂) amperometric sensing have been developed. The aggregates were prepared by directly mixing citric-capped AuNPs and poly(allylamine hydrochloride) (PAH)-capped nano-MnO₂ using an electrostatic self-assembly strategy. The prepared sensor exhibited excellent electrochemical behaviors and a wide linear range from 7.80 × 10⁻⁷ to 8.36 × 10⁻⁴ M with a detection limit of 4.68 × 10⁻⁸ M (S/N = 3) because of the synergistic influence of excellent catalytic ability of MnO₂ and good electrical conductivity of AuNPs. In addition, its applicability to practical samples for measuring H₂O₂ in toothpastes has obtained a satisfactory result. Due to the ease of preparation and excellent properties of the sensor, indicating the MnO₂-AuNP material may be a potential H₂O₂ sensor.     

A comparison of the electrocatalytic activities of ordered mesoporous carbons treated with either HNO3 or NaOH

Ordered mesoporous carbon (OMC) was treated with HNO₃ or NaOH. The two treated OMCs have many oxygen-containing functional groups. Those treated with HNO₃ have more acidic surface groups than those treated with NaOH. Nicotinamide adenine dinucleotide (NADH) and H₂O₂ were selected as marker molecules for the comparison of the electrocatalytic property of the OMCs. A comparison between the cyclic voltammograms shows that the oxidation peak potential of NADH is 0.614 V at a bare glassy carbon electrode (GCE), 0.205 V at OMC/GCE, 0.223 V at NaOH-treated OMC/GCE, and 0.0 V at HNO₃-treated OMC/GCE (vs. Ag/AgCl). The results indicate that the HNO₃-treated OMC/GCE exhibits the highest electrocatalytic activity for NADH oxidation. Thus, acidic groups rather than other oxygen-containing functional groups, play a very important role in the catalytic activity of OMC.     

Preparation, characterization and electrocatalytic properties of an iodine|lignin-modified gold electrode

Hydrolytic lignin (HL) was adsorbed from an aqueous/organic solution on bare and iodine-modified gold electrode. Subsequent electrooxidation of the lignin adsorbate generated redox-active quinone-based groups in the biopolymer structure, exhibiting high reversibility during potential cycling and fast electron transfer kinetics. The presence of the chemisorbed iodine layer on the supporting gold electrode had a pronounced effect on the electrochemical properties of the final modified electrode in terms of double-layer capacitance (C_dl) and the observed surface coverage (Γ_obs). The high electrochemical activity in connection with low C_dl made it possible to apply the Au|I_(ads)|HL electrode as a fast-responding and sensitive electrochemical sensor for NADH. When tested in the amperometric mode at a constant potential of +0.4 V vs. Ag/AgCl, the modified electrode showed a linear current-concentration response over the range of 5-120 μM with a sensitivity of 2.39 nA μM⁻¹ cm⁻² and a detection limit of 1.0 μM (S/N = 3). Kinetic studies using the rotating disk electrode revealed that the mediated oxidation of NADH on the Au|I_(ads)|HL electrode was limited by the second order reaction of the analyte molecules with o-quinone moieties with a rate constant of ca. 4.7 × 10² M⁻¹ s⁻¹ (C_NADH → 0). The modified electrode showed high resistivity against fouling and retained ca. 65% activity after storage in phosphate buffer (pH 7.4) at room temperature for 1 week.     

The electrochemical redox processes in methacrylate-based polymer electrolytes II. - Study on microelectrodes

The electrochemical behaviour of ferrocene was studied in different gel polymer electrolytes based on methyl, ethyl and 2-ethoxyethyl methacrylate and compared to the liquid aprotic solution (propylene carbonate). Voltammetric and chronoamperometric measurements on microelectrodes were conducted in order to describe the qualitative as well as quantitative behaviour of ferrocene in different conditions. Heterogeneous electron-transfer rate constants and diffusion coefficients of ferrocene in polymer electrolytes were estimated to be 1.1-7.8 × 10⁻³ cm s⁻¹ and 4-13 × 10⁻⁸ cm² s⁻¹ depending on the electrolyte composition. The influence of the polymer polarity, ferrocene concentration and level of polymer cross-linkage on the kinetics of ferrocene oxidation and its transport was discussed. The electrolytes with poly(2-ethoxyethyl methacrylate) exhibit the highest ionic conductivity (2-4 × 10⁻⁴ S cm⁻¹) as well as diffusion coefficient of ferrocene (1.3 × 10⁻⁷ cm² s⁻¹) in their structure.     

Electrocatalytic activity of 2,3,5,6-tetrachloro-1,4-benzoquinone/multi-walled carbon nanotubes immobilized on edge plane pyrolytic graphite electrode for NADH oxidation

This work reports the electrocatalytic activity of 2,3,5,6-tetrachloro-1,4-benzoquinone (TCBQ)/multi-walled carbon nanotubes (MWCNT) immobilized on an edge plane pyrolytic graphite electrode for nicotinamide adenine dinucleotide (NADH) oxidation. Scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS) were used to confirms the presence of chloro after the nanotube modification with 2,3,5,6-tetrachloro-1,4-benzoquinone. The surface charge transfer constant, k_s, and the charge transfer coefficient for the modified electrode, α, were estimated as 98.5 (±0.6) s⁻¹ and 0.5, respectively. With this modified electrode the oxidation potential of the NADH was shifted about 300 mV toward a less positive value, presenting a peak current much higher than those measured on an unmodified edge plane pyrolytic graphite electrode (EPPG). Cyclic voltammetry and rotating disk electrode (RDE) experiments indicated that the NADH oxidation reaction involves 2 electrons and a heterogenous rate constant (k_obs) of 3.1 × 10⁵ mol⁻¹ l s⁻¹. The detection limit, repeatability, long-term stability, time of response and linear response range were also investigated.     

An enzyme-based microfluidic biofuel cell using vitamin K3-mediated glucose oxidation

Viamin K₃-modified poly-l-lysine (PLL-VK₃) was synthesized and used as the electron transfer mediator during catalytic oxidation of NADH by diaphorase (Dp) at the anode of biofuel cell. PLL-VK₃ and Dp were co-immobilized on an electrode and then coated with NAD⁺-dependent glucose dehydrogenase (GDH). The resulting enzymatic bilayer (abbreviated PLL-VK₃/Dp/GDH) catalyzed glucose oxidation. Addition of carbon black (Ketjenblack, KB) into the bilayer enlarged the effective surface area of the electrode and consequentially increased the catalytic activity. An oxidation current of ca. 2 mA cm⁻² was observed when the electrochemical cell contained a stirred 30 mM glucose, 1.0 mM NAD⁺, pH 7.0 phosphate-buffered electrolyte solution. The performance of glucose/O₂ biofuel cells, constructed as fluidic chips with controllable fuel flow and containing a KB/PLL-VK₃/Dp/GDH-coated anode and an Ag/AgCl or a polydimethylsiloxane-coated Pt cathode, were evaluated. The open circuit voltage of the cell with the PDMS-coated Pt cathode was 0.55 V and its maximum power density was 32 μW cm⁻² at 0.29 V when a pH 7.0-buffered fuel containing 5.0 mM glucose and 1.0 mM NAD⁺ was introduced into the cell at a flow rate of 1.0 mL min⁻¹. The cell's output increased as the flow rate increased. During 18 h of continuous operation of the cell with a load of 100 kΩ, the output current density declined by ca. 50%, probably due to swelling of the enzyme bilayer.     

Meldola blue immobilized on a new SiO2/TiO2/graphite composite for electrocatalytic oxidation of NADH

Meldola blue immobilized on a new SiO₂/TiO₂/graphite composite was applied in the electrocatalytic oxidation of NADH. The materials were prepared by the sol-gel processing method and characterized by several techniques including scanning electronic microscopy coupled to energy dispersive spectroscopy (SEM-EDS), X-ray photoelectron spectroscopy (XPS) and high-resolution transmission electronic microscopy (HRTEM). Si and Ti mapping profiles on the surface showed a homogeneous distribution of the components. Ti2p binding energy peaks indicate that the formation of Si-O-Ti linkage is presumably the responsible for the high rigidity of the matrices. The good electrical conductivity presented by the composites (5 and 11 S cm⁻¹) can be related to a homogeneous distribution of graphite particles observed by TEM. After the materials characterization, a SiO₂/TiO₂/graphite electrode was prepared and some chemical modifications were performed on its surface to promote the adsorption of meldola blue. The resulting system presented electrocatalytic properties toward the oxidation of NADH, decreasing the oxidation potential to −120 mV. The proposed sensor showed a wide linear response range from 0.018 to 7.29 mmol l⁻¹ and limit of detection of 0.008 mmol l⁻¹. SiO₂/TiO₂/graphite has shown to be a promising material to be used as a suitable support in the construction of new electrochemical sensors.     

Cofactor Regeneration of NAD+ from NADH: Novel Water‐Forming NADH Oxidases

Dehydrogenases with their superb enantioselectivity can be employed advantageously to prepare enantiomerically pure alcohols, hydroxy acids, and amino acids. For economic syntheses, however, the co‐substrate of dehydrogenases, the NAD(P)(H) cofactor, has to be regenerated. Whereas the problem of regenerating NADH from NAD⁺ can be considered solved, the inverse problem of regenerating NAD⁺ from NADH still awaits a definitive and practical solution. A possible solution is the oxidation of NADH to NAD⁺ with concomitant reduction of oxygen catalyzed by NADH oxidase (E.C. 1.6.‐.‐) which can reduce O₂ either to undesirable H₂O₂ or to innocuous H₂O. We have found and cloned two novel genes from Borrelia burgdorferi and Lactobacillus sanfranciscensis with hitherto only machine‐annotated NADH oxidase function. We have overexpressed the corresponding proteins and could prove the annotated function to be correct. As demonstrated with a more sensitive assay than employed previously, the two novel NADH oxidases reduce O₂ to H₂O.