Electrochemical oxidation behavior of guanine and adenine on graphene–Nafion composite film modified glassy carbon electrode and the simultaneous determination

The electrochemical behavior of guanine and adenine on the graphene and Nafion composite film modified glassy carbon electrode was investigated by differential pulse voltammetry (DPV). The results indicated that the modified electrode exhibited an excellent electrocatalytic activity towards the oxidation of guanine and adenine, testified by the increased oxidation peak current and decreased oxidation potential. The experimental conditions were optimized. The separation of the two oxidation peaks was 0.364 V in 0.1 M pH 4.4 acetate buffer solution (ABS). Based on this, a novel electrochemical method was proposed to simultaneously determine guanine and adenine with the detection limit of 0.58 (guanine) and 0.75 (adenine) μM (S/N = 3). The proposed method was applied to determine guanine and adenine in milk powder, urine and herring sperm DNA samples with satisfactory results. The value of (G + C)/(A + T) in herring sperm DNA was calculated to be 0.8065. The fabricated electrode showed excellent reproducibility, stability and anti-interference.     

Amperometric electrode for determination of urea using electrodeposited rhodium and immobilized urease

An amperometric biosensor was developed for determination of urea using electrodeposited rhodium on a polymer membrane and immobilized urease. The urease catalyzes the hydrolysis of urea to NH₄⁺ and HCO₃⁻ ions and the liberated ammonia is catalytically and electrochemically oxidized by rhodium present in the rhodinized membrane on the Pt working electrode. Three types of rhodinized polymer membranes were prepared by varying the number of electrodeposition cycles: membrane 1 with 10 deposition cycles, membrane 2 with 40 cycles and membrane 3 with 60 cycles. The morphologies of the rhodinized membranes were investigated by scanning electron microscopy and the results showed that the deposition of rhodium was like flowers with cornices-like centers. The influence of the amount of electrodeposited rhodium over the electrode sensitivity to different concentrations of ammonia was examined initially based on the cyclic voltammetric curves using the three rhodium modified electrodes. The obtained results convincingly show that electrode with rhodinized membrane 1, which contain the lowest amount of electrodeposited rhodium is the most active and sensitive regarding ammonia. It was found that the anodic oxidation peak of ammonia to nitrogen occurs at 0.60 V. In order to study the performance of urease amperometric sensor for the determination of urea, experiments at constant potential (0.60 V) were performed. The current–time experiments were carried out with urease rhodinized membrane 1 (10 cycles). The amperometric response increased linearly up to 1.75 mM urea. The detection limit was 0.05 mM. The urea biosensor exhibited a high sensitivity of 1.85 μA mM⁻¹ cm⁻² with a response time 15 s. The Michaelis–Menten constant K_m for the urea biosensor was calculated to be 6.5 mM, indicating that the immobilized enzyme featured a high affinity to urea. The urea sensor showed a good reproducibility and stability. Both components rhodium and urease contribute to the decreasing of the production cost of biosensor by avoiding the use of a second enzyme.     

A nylon membrane based amperometric biosensor for polyphenol determination

A nylon membrane based amperometric biosensor employing banana fruit polyphenol oxidase (PPO) is presented for polyphenol detection. Nylon membrane was first activated and then coupled with chitosan. PPO was covalently attached to this membrane through glutaraldehyde coupling. The membrane bioconjugate was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) study and then mounted onto Au electrode using parafilm to construct a working electrode. Once assembled along with Ag/AgCl as reference and Pt as auxiliary electrode, the biosensor gave optimum response within 15 s at pH 7.5 and 30 °C, when polarized at +0.4 V. The response (in mA) was directly proportional to polyphenol concentration in the range 0.2–400 μM. The lower detection limit of the biosensor was 0.2 μM. The biosensor was employed for determination of polyphenols in tea, beverages and water samples. The enzyme electrode showed 25% decrease in initial activity after 150 reuses over 6 months, when stored at 4 °C.     

Debranching enzyme concentration effected on physicochemical properties and α-amylase hydrolysis rate of resistant starch type III from amylose rice starch

The effect of debranching enzyme concentration on physicochemical properties and α-amylase hydrolysis rate of resistant starch type III from high amylose rice starch were studied. The pullulanase enzyme (8, 10, 12, 14 and 16 U/g starch) was introduced to modify amylopectin molecules of 15% (w/w) gelatinized rice starches at 55 °C for 16 h. The debranched starches with different degrees of hydrolysis (0.14–5.27%), and having 66.60–98.82% β-amylolysis limit were then induced at 4 °C for 16 h, afterward a one cycle of freeze–thaw process (?10/30 °C) was applied. The results showed that a pullulanase hydrolysis improved the degree of syneresis (51.64–54.85% from 8 to 16 U/g starch). Resistant starch content increased sharply as the amount of the enzyme increased, reaching the highest (19.81%) for a 12 U/g starch and decreased to 13.16% by 16 U/g starch. α-Amylase hydrolysis rate showed that incompletely-debranched had a lower estimated glycemic index than completely debranched rice starches. Microstructure of the selected RS III samples using X-ray diffraction and scanning electron microscopy revealed a crystal pattern change from A- to V-type pattern and formed a coarse honeycomb-like and a filamentous network structure.     

Overoxidized polypyrrole/multi-walled carbon nanotubes composite modified electrode for in vivo liquid chromatography–electrochemical detection of dopamine

Overoxidized polypyrrole/multi-walled carbon nanotubes (OPPy/MWNTs) modified electrode has been developed for sensitively detecting dopamine (DA). OPPy films developed outside MWNTs might have a porous morphology. Thus, OPPy/MWNTs films developed by this method do not reject ascorbic acid (AA). However, OPPy/MWNTs modified electrode shows largely enhancing oxidative current responses of DA. When combined with liquid chromatography, it not only obtains a low detection limit of 7.5 × 10^?10 mol L^?1 for DA, but also improves the selectivity of DA detection. Mechanisms for the enhancement are also well discussed in this paper. With this approach, microdialysis has been employed for successful assessment of DA in rat striatum.     

Partial nitrification under limited dissolved oxygen conditions

Partial nitrification to nitrite is technically feasible and economically favourable, especially when wastewaters contained high ammonium concentrations or low C/N ratios. Partial nitrification can be obtained by selectively inhibiting nitrite-oxidizing bacteria (NOB) through appropriate regulation of the pH, temperature and dissolved oxygen (DO) concentrations. The effect of pH, DO levels and temperature on ammonia oxidation rate and nitrite accumulation was investigated in order to determine the optimal conditions for partial nitrification of synthetic wastewater with high ammonia concentration. The experiments performed at low DO levels to lower the total oxygen needed in the nitrification step, which means great saving in aeration. During the start-up stage pH and DO were set at 7.0–7.4 and 0.5 mg/l, respectively. The reactor was operated until complete partial nitrification was achieved. The effect of pH, DO on partial nitrification was studied, as pH was kept at 6.5, 7.5, 8.5, 9.5 and DO at 0.5±0.2, 1.5±0.2 and 2.5±0.2 mg/l, and temperature at 30 °C. The influence of temperature on k_a value was studied by keeping pH=7.5, DO=1.5 mg/l and temperature was controlled at 12, 20 and 30 °C, respectively. The results showed that partial nitrification to nitrite was steadily obtained and the optimal operational parameters were pH=7.5, DO=1.5 mg/l, T=30 °C based on ammonia oxidation rate and nitrite accumulation rate. The maximum k_a was achieved and to be 115.1×10⁻³ mg NH₄⁺–N (mg VSS h)⁻¹ under this condition.     

Construction of d-amino acid biosensor based on d-amino acid oxidase immobilized onto poly (indole-5-carboxylic acid)/zinc sulfide nanoparticles hybrid film

d-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto poly indole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodeposited on surface of an Au electrode. A highly sensitive d-amino acid biosensor was constructed using this enzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5 and 35 °C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor response (mA) and d-alanine concentration in the range 0.001–2.0 mM. The sensitivity of the biosensor was 58.85 μA cm^?2 mM^?1 with a detection limit of 0.001 mM (S/N = 3). The enzyme electrode was used 120 times over a period of 2 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor was evaluated and employed for measurement of d-amino acid level in fruits and vegetables.     

An amperometric H2O2 biosensor based on cytochrome c immobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H₂O₂ biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM. The sensor had a detection limit of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM^?1 cm^?2. The sensor gave accurate and satisfactory results, when employed for determination of H₂O₂ in different fruit juices.     

Immobilization of lysine oxidase on a gold–platinum nanoparticles modified Au electrode for detection of lysine

A commercial lysine oxidase (LyOx) from Trichoderma viride was immobilized covalently onto gold nanoparticles (AuNPs) and platinum nanoparticles (PtNPs) electrodeposited onto Au electrode using 3-aminopropyltriethoxy silane (3-APTES) and glutaraldehyde cross linking chemistry. A lysine biosensor was fabricated using LyOx/3-APTES/AuNPs-PtNPs/Au electrode as a working electrode, Ag/AgCl (3 M KCl) as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The cumulative effect of AuNPs and PtNPs showed excellent electrocatalytic activity at low applied potential for detection of H₂O₂, a product of LyOx reaction. The sensor showed its optimum response within 4 s, when polarized at 0.2 V vs. Ag/AgCl in 0.1 M phosphate buffer, pH 7.5 at 30 °C. The linear range and detection limit of the sensor were 1.0–600 μM and 1.0 μM (S/N = 3), respectively. Biosensor measured lysine level in sera, milk and amino acid tablet, which correlated well with those by standard HPLC method. The enzyme electrode lost 50% of its initial activity after 200 uses over a period of 4 months.     

Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin–fluorescein isothiocyanate conjugates

A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein–hapten binding in the skin, is presented. Mass spectra of BSA–FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 mM. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined, 28 possible and 2 non-binding sites for FITC.     

Electrochemical studies of biocatalytic anode of sulfonated graphene/ferritin/glucose oxidase layer-by-layer biocomposite films for mediated electron transfer

In this study, a bioanode was developed by using layer-by-layer (LBL) assembly of sulfonated graphene (SG)/ferritin (Frt)/glucose oxidase (GOx). The SG/Frt biocomposite was used as an electron transfer elevator and mediator, respectively. Glucose oxidase (GOx) from Aspergillus niger was applied as a glucose oxidation biocatalyst. The electrocatalytic oxidation of glucose using GOx modified electrode increases with an increase in the concentration of glucose in the range of 10–50 mM. The electrochemical measurements of the electrode was carried out by using cyclic voltammetry (CV) at different scan rates (20–100 mV s⁻¹) in 30 mM of glucose solution prepared in 0.3 M potassium ferrocyanide (K₄Fe(CN)₆) and linear sweep voltammetry (LSV). A saturation current density of 50 ± 2 mA cm⁻² at a scan rate of 100 mV s⁻¹ for the oxidation of 30 Mm glucose is achieved.     

Amylolytically-resistant tapioca starch modified by combined treatment of branching enzyme and maltogenic amylase

Tapioca starch was modified using branching enzyme (BE) isolated from Bacillus subtilis 168 and Bacillus stearothermophilus maltogenic amylase (BSMA), and their molecular fine structure and susceptibility to amylolytic enzymes were investigated. By BE treatment, the molecular weight decreased from 3.1 × 10⁸ to 1.7 × 10⁶, the number of shorter branch chains (DP 6–12) increased, the number of longer branch chains (DP >25) decreased, and amylose content decreased from 18.9% to 0.75%. This indicated that α–1,4 linkages of amylose and amylopectin were cleaved, and moiety of glycosyl residues were transferred to another amylose and amylopectin to produce branched glucan and BE-treated tapioca starch by forming α–1,6 branch linkages. The product was further modified with BSMA to produce highly-branched tapioca starch with 9.7% of extra branch points. When subject to digestion with human pancreatic α-amylase (HPA), porcine pancreatic α-amylase (PPA) and glucoamylase, highly-branched tapioca starch gave significantly lowered α-amylase susceptibility (7.5 times, 14.4 times and 3.9 times, respectively), compared to native tapioca starch.     

Dominance of yeast in activated sludge under acidic pH and high organic loading

Flow cytometry-fluorescent in situ hybridization (FC-FISH) was used to investigate the effect of controlled pH and/or varied organic loading on the content of yeast and bacterial cells in an activated sludge system (AS) individually operating in continuous and batch mode for treatment of high-strength industrial wastewater. Specifically, we attempted to develop a yeast-predominant activated sludge system (Y-AS). For the batch-mode AS, bacteria-dominated AS (B-AS) obtained at pH 6.5–7.5 induced higher chemical oxygen demand (COD) removal than Y-AS obtained at acidic pH (5.0–6.0 and 4.0–5.0). For the continuous-mode AS operating at COD loadings of 2.5–2.8 kg COD m⁻³ d⁻¹, it was difficult to achieve a Y-AS solely by controlling the pH level at 7.0 to 5.1 then to 4.1 because bacteria stably accounted for greater than 98% of the total cells, regardless of the pH levels. Therefore, the effects of varied COD loadings (2.1, 8.7 and 21.0 kg COD m⁻³ d⁻¹) on continuous-mode AS operation at acidic pH (4.5) was investigated. Both acidic pH and high COD loading levels were found to be prerequisites for yeast to dominate the sludge microbial community in the continuous-mode AS.     

Purification and biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

Electrochemical analysis of autodisplayed adrenodoxin (Adx) on the outer membrane of E. coli

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron–sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron–sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10 mM HEPES buffer, pH 7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at + 0.4 V (vs. Ag/AgCl) and a reduction peak at − 0.3 V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron–sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron–sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.     

Development of Novel Glucose oxidase Immobilization on Graphene/Gold nanoparticles/Poly Neutral red modified electrode

Glucose oxidase (GOx) was immobilized onto glassy carbon electrode (GCE) that modified by reduced graphene oxide-gold nanoparticles- poly neutral red (RGO/AuNPs/PNR) nanocomposite. The composite was analyzed by scanning electron microscope (SEM), energy dispersive x-ray (EDX) spectroscopy, atomic force microscopy (AFM), attenuated total reflectance (ATR), cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). SEM/EDX analysis showed the morphological of the nanocomposite. AFM results showed the morphology and structure of the RGO/AuNPs and RGO surfaces. The covalent bonding between glucose oxidase and composite was confirmed by ATR technique. The electrochemical experiments were done in 100 mM phosphate buffer at pH 7 and temperature of 25 °C with three electrodes including Ag/AgCl, platinum wire and the modified GCE as the reference electrode, the auxiliary electrode and working electrode respectively. The electrochemical results confirmed the activity and direct electron transfer of immobilized enzyme. The immobilized electroactive GOx concentration was estimated 3.06 × 10⁻¹¹ mol cm⁻². The results showed the immobilized enzyme had a good stability and maintained 90% of its performance after two weeks. The nanocomposite bioanode in an air-birthing biofuel cell and 100 mM glucose concentration showed 176 μWcm⁻² Power density. This strategy could be used for GOx-based biofuel cells.     

Laccase mediator system for activation of agarose gel: Application for immobilization of proteins

Cross-linked Sepharose beads were treated with laccase–TEMPO system for oxidation of the primary alcohol groups on the sugar moieties. Optimal activation conditions using Trametes versicolor laccase were at pH 5 and 22 °C, giving an aldehyde content of 55 μmol g⁻¹ Sepharose with 28 units g⁻¹ of laccase and 12.5 mM TEMPO. The activated Sepharose was used for immobilization of trypsin as model protein. Highest degree of immobilization was obtained at pH 10.5 but the activity yield was only 31% of that loaded on the gel. The yield of gel bound trypsin activity was increased to 76% (corresponding to about 43 U g⁻¹ Sepharose) when the immobilization was performed in the presence of trypsin inhibitor, benzamidine. The immobilization yields were comparable to that obtained on the matrix activated using sodium periodate (containing 72 μmol aldehyde per g Sepharose). Recycling and storage of the immobilized trypsin preparations showed high stability of the enzyme bound to laccase–TEMPO activated gel.     

Production of novel halo-alkali-thermo-stable xylanase by a newly isolated moderately halophilic and alkali-tolerant Gracilibacillus sp. TSCPVG

An aerobic xylanolytic Gracilibacillus sp. TSCPVG growing at moderate to extreme salinity (1–30%) and neutral to alkaline pH (6.5–10.5) was isolated from the salt fields near Sambhar district of Rajasthan, India. β-xylanase (18.44 U/ml) and β-xylosidase (1.01 U/ml) were produced in 60 h in the GSL-2 mineral base medium with additions of (in g/l) Birchwood xylan (7.5), yeast extract (10.0), tryptone (8.0), proline (2.0), thiamine (2.0), Tween-40 (2.0) and NaCl (35) at pH 7.5, 30 °C and 180 rpm. The β-xylanase was active within a broad salinity range (0–30% NaCl), pH (5.0–10.5) and temperature (50–70 °C). It exhibited maximal activity with 3.5% NaCl, pH 7.5 at 60 °C. It was extremely halotolerant retaining more than 80% of activity at 0 and 30% NaCl and alkali-tolerant retaining 76% of activity at pH 10.5. The acetone precipitated xylanase was highly stable (100%) at variable salinities of 0–30% NaCl, pH of 5.0–10.5 and temperatures of 0–60 °C for 48 h. HPLC analysis showed xylose, arabinose and xylooligosaccharides as hydrolysis products of xylan. This is the first report on hemi-cellulose degrading halo-alkali-thermotolerant enzyme from a moderately halophilic Gram-positive Gracilibacillus species.     

Electrochemical detection of xanthine in fish meat by xanthine oxidase immobilized on carboxylated multiwalled carbon nanotubes/polyaniline composite film

A commercial xanthine oxidase (XOD) was immobilized covalently onto carboxylated multiwalled carbon nanotubes (c-MWCNT) and polyaniline (PANI) composite film electrodeposited on the surface of a Pt electrode, using N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. A xanthine biosensor was fabricated using XOD/c-MWCNT/PANI/Pt electrode as a working electrode, Ag/AgCl (3 M KCl) as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrophotometry and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 4 s at pH 7.0 and 35 °C, when polarized at 0.4 V. The optimized xanthine biosensor showed linear response range of 0.6–58 μM, with a detection limit of 0.6 μM (S/N = 3), and a correlation coefficient of 0.98. The biosensor was applied to determine xanthine in fish meat. The biosensor lost 50% of its initial activity after its 200 uses over a period of 100 days.     

Ethanol/O2 biofuel cell using a biocathode consisting of laccase/ HOOC-MWCNTs/polydiallyldimethylammonium chloride

In the present report we focused on the substitution of metallic catalysts by biocatalysts to develop a high efficient biofuel cell. A bioanode and a biocathode were designed using ADH and laccase, respectively. Carboxylated multiwall carbon nanotubes (HOOC-MWCNTs) and polydiallyldimethylammonium chloride (PDDA) were used for immobilizing the enzymes on either polymethylene green (PMG) modified glassy carbon or graphite electrodes. In this way, an ethanol–oxygen biofuel cell was designed in which PDDA/ADH/PDDA/HOOC-MWCNTs/PMG/GC and PDDA/Lac/PDDA/HOOC-MWCNTs/PMG/Gr operated as bioanode and biocathode, respectively. In the optimized condition of O₂ saturated PBS (0.1 M, pH 7.5) containing 1 mM ethanol and 1 mM NAD⁺ the open-circuit voltage reached to a plateau at 504 mV based of which the power density of 3.98 mW cm⁻² was obtained.