Enzymatic Anode Research Articles

3D printed cathodes for implantable abiotic biofuel cells

3D printing has recently triggered huge attention in several fields such as construction, artificial tissue engineering, food fabrication, wearable electronics, and electrochemical energy storage.This work investigates the fabrication of a 3D-printed abiotic cathode for implantable glucose/oxygen biofuel cells. The ink formulation was optimized to get printable ink with high electro-catalytic activity. Electrode macro porosity was screened in order to identify the better compromise between electrode density and electrochemical performance. A maximum current density of 260 μA/cm2 was obtained with cylindrical electrodes with linear mesh infill and a volumic infill rate of 40%.A complete biofuel cell was assembled using a 3D-printed abiotic cathode and an enzymatic anode in the form of a compressed pellet showing maximum power and current densities of 80 μW/cm2 and 320 μA/cm2, respectively. Moreover, the hybrid biofuel cell was implanted in the intraabdominal region of a rat for three months and after cell explantation, the abiotic cathode displayed a 50% decrease in the current density while the enzymatic anode did not display any residual activity.The 3D printed electrode displayed a 2–3.6 fold increase in current density when compared to homolog 2D electrodes.

Immobilization of Glucose Oxidase on Glutathione Capped CdTe Quantum Dots for Bioenergy Generation

An efficient immobilization of Glucose oxidase (GOx) on an appropriate substrate is one of the main challenges of developing fuel cells that allow energy to be obtained from renewable substrates such as carbohydrates in physiological environments. The research importance of biofuel cells relies on their experimental robustness and high compatibility with biological organisms such as tissues or the bloodstream with the aim of obtaining electrical energy even from living systems. In this work, we report the use of 5,10,15,20 tetrakis (1-methyl-4-pyridinium) porphyrin and glutathione capped CdTe Quantum dots (GSH-CdTeQD) as a support matrix for the immobilization of GOx on carbon surfaces. Fluorescent GSH-CdTeQD particles were synthesized and their characterization by UV-Vis spectrophotometry showed a particle size between 5–7 nm, which was confirmed by DLS and TEM measurements. Graphite and Toray paper electrodes were modified by a drop coating of porphyrin, GSH-CdTeQD and GOx, and their electrochemical activity toward glucose oxidation was evaluated by cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Additionally, GOx modified electrode activity was explored by scanning electrochemical microscopy, finding that near to 70% of the surface was covered with active enzyme. The modified electrodes showed a glucose sensitivity of 0.58 ± 0.01 μA/mM and an apparent Michaelis constant of 7.8 mM. The addition of BSA blocking protein maintained the current response of common interferent molecules such as ascorbic acid (AA) with less than a 5% of interference percentage. Finally, the complex electrodes were employed as anodes in a microfluidic biofuel cell (μBFC) in order to evaluate the performance in energy production. The enzymatic anodes used in the μBFC allowed us to obtain a current density of 7.53 mAcm−2 at the maximum power density of 2.30 mWcm−2; an open circuit potential of 0.57 V was observed in the biofuel cell. The results obtained suggest that the support matrix porphyrin and GSH-CdTeQD is appropriate to immobilize GOx while preserving the enzyme’s catalytic activity. The reported electrode arrangement is a viable option for bioenergy production and/or glucose quantification.

A hybrid hydrogel separated biofuel cell with a novel enzymatic anode and glucose tolerant cathode

Enzymatic biofuel cells offer a new avenue of eco-friendly energy generation which is highly essential to establish a sustainable energy infrastructure. Herein, a hybrid fuel cell using an immobilised enzyme-based anode, a hydrogel-based separator and a glucose tolerant catalyst based cathode has been fabricated and demonstrated. Multiwalled carbon nanotube-pyrene carboxylic acid (MWCNT–PCA) nanocomposite based anode has been used for the electrostatic immobilisation of glucose oxidase enzyme. Agar-polyvinyl alcohol (PVA) hydrogel is used as the separator which supplies the fuel cell with 500 mM glucose solution. Reduced graphene oxide-ceria (rGC) acts as the glucose tolerant non-enzymatic cathode catalyst. A sandwiched construction is used to fabricate the device with which an open circuit potential of 140 mV has been recorded with a peak power density of 6.25 μW/cm2 at 60 μA/cm2.

A “Press and Go” Thin Biofuel Cell Patch for Power Generation

Biofuel cells are devices that generate portable electrical power, typically from glucose, alcohols or related biofuels, by using enzymatic anodes and cathodes. We report on the performance of membrane- and mediator-free enzymatic glucose biofuel cells (BFCs) fabricated in a sandwiched, patch geometry with carbon nanotube nanopaper bio-electrodes prepared using a compression technique to immobilize the enzymes. A gel electrolyte comprised of agar mixed with glucose is sandwiched between the two bio-electrodes to form a patch. Power densities up to 111.90 μW/cm2 at a current density of 334.50 μA/cm2 at a voltage of 0.335 V were obtained for a typical single cell BFC of this design. The sandwich BFC also showed good stability with a half-life of about 1.5 days under continuous operation. The relatively high power density for this new design is attributed to the compression-induced immobilization of the enzymes by the nanotubes as indicated by scanning electron microscope images. This compression-induced immobilization of enzymes provides improved direct electron transfer at the carbon nanotube electrodes.

High-power hybrid biofuel cells using layer-by-layer assembled glucose oxidase-coated metallic cotton fibers

Electrical communication between an enzyme and an electrode is one of the most important factors in determining the performance of biofuel cells. Here, we introduce a glucose oxidase-coated metallic cotton fiber-based hybrid biofuel cell with efficient electrical communication between the anodic enzyme and the conductive support. Gold nanoparticles are layer-by-layer assembled with small organic linkers onto cotton fibers to form metallic cotton fibers with extremely high conductivity (>2.1×104 S cm−1), and are used as an enzyme-free cathode as well as a conductive support for the enzymatic anode. For preparation of the anode, the glucose oxidase is sequentially layer-by-layer-assembled with the same linkers onto the metallic cotton fibers. The resulting biofuel cells exhibit a remarkable power density of 3.7 mW cm−2, significantly outperforming conventional biofuel cells. Our strategy to promote charge transfer through electrodes can provide an important tool to improve the performance of biofuel cells.

Hybrid Non-Enzymatic and Enzymatic Cascade Bioanode for Glycerol/O2 Biofuel Cell Applications

Enzymatic biofuel cell technology has demonstrated the possibility of producing electrical power from chemical energy using biological catalysts for powering portable and implanted electronic devices. However, this technology has been limited by insufficient power and shell life compared to existing commercial, non-biological fuel cells. During the last 20 years, significant advancements have been made in improving the overall performance of the biofuel cells with incorporation of hybrid nanostructures including carbon and metal based nanomaterials and combination thereof with enzymes. Typically, biofuel cell cathodes utilize a single enzyme system, while a cascade of reactions for deeper and more complete oxidation of fuel is frequently necessary at the anodes. Here, we present the feasibility of using glycerol for the development of alcohol based biofuel cell. For the first time, the biofuel cells were extended via the assembly of hybrid enzymatic anode and bilirubin oxidase (BOx) biocathode. The hybrid anode was integrated with non-enzymatic oxidation of glycerol by inorganic nanoparticles and enzymatic nanocomposite conversion of final byproduct to achieve a cascade of efficient energy conversion from glycerol. The constructed mediator-less and membrane-less biofuel cell demonstrated maximum open circuit potential (OCV) greater than 600mV, power output of 0.7mW/cm2 and maximum current density of 3.0mA/cm2 with 0.1M glycerol at neutral pH. The fuel cell demonstrated remarkable discharge capacity of 600mAh/g under 0.25mA constant current load. The enzymatic/non enzymatic hybrid strategy can be further extended to develop other alcohol based biofuel cells.

Immobilized glucose oxidase biofuel cell anode by MWCNTs, ferrocene, and polyethylenimine: Electrochemical performance

AbstractFerrocene‐functionalized polyethylenimine and multiwalled carbon nanotubes were attached covalently by glutaraldehyde onto a carbon cloth to develop an immobilized enzyme (glucose oxidase) electrode for biofuel cell applications. Developed enzymatic anode was characterized by electrochemical methods to determine electrochemical performance. Anodic open‐circuit potential was measured as within 0–20 mV range. Cyclic voltammetry showed anodic peak for glucose oxidation around 400–600 mV (vs. sat. Ag/AgCl) varying with scan rate. An enzyme fuel cell with 2.5 mg/cm2 glucose oxidase‐loaded bioanode and 0.70 mg/cm2 Pt‐loaded cathode attached to Nafion™ 115 membrane has provided around 2.5 mA/cm2 current density at short‐circuit conditions. Enzymatic kinetic parameters of prepared anode were determined by electrochemical methods that surprisingly indicated less KM (i.e., better substrate affinity) than that of determined by conventional enzymatic methods. Enzymatic stability determined by electrochemical methods moreover indicated longer enzyme half‐life.

(Invited) Construction of an Enzymatic Biofuel Cell Which Produces Electric Energy and a Platform Chemical Simultaneously

Introduction Enzymes can catalyze selective and environmentally friendly production of platform chemicals from biomass. When a platform chemical is produced through one or more oxidation reactions, the electrons can obtain from the substrate. The electrons are usually just dumped to electron accepters. If a biofuel cell anode is used as an electron acceptor for the process, we can obtain the electric energy. Organic acids have especially attracted attention among the bio-derived chemical raw materials. The US Department of Energy (DOE) selected 12 chemicals from more than 300 biomass-derived chemicals on the basis of cooperative research with industry and academia, to develop the production methods preferentially [1]. More than half of the 12 chemicals are organic acids. The organic acids obtained by the oxidation of monosaccharides and oligosaccharides are referred to as sugar acids. The aldose oxidation at its aldehyde group forming into the carboxyl group produces aldonic acid while corresponding oxidation at its terminal hydroxyl group gives another mono-carboxylic acid, uronic acid. Aldaric acid is a di-carboxylic acid produced by the oxidation of both groups. Because various application studies of aldaric acids have been conducted, the development of their production methods are eagerly expected. Hence, we set a goal of producing aldaric acids and electric energy simultaneously from biomass with enzymatic biofuel cells. We struggle with the production of aldaric acids from cellulose and pectin so far. It is desirable to use a flow-cell so that the fuel and product can be provided and removed efficiently. To construct a flow-cell type biofuel cell, enzymes and electron mediators should be fixed on electrodes so that they do not flow out. Here, the construction of a flow-cell type enzymatic biofuel cell for the D-glucose oxidation are mainly reported (Fig. 1). Methods -Examination of poly(methylene green) as an electron mediator for enzymatic anode- Methylene green was polymerized on glassy carbon (GC) electrodes by cyclic potential sweeping method as previously reported [2]. Onto the poly(methylene green) (PMG) modified electrode, FAD-dependent glucose dehydrogenase (FAD-GDH) was cast. The electrode was evaluated by cyclic voltammetry (CV) measurement in 100 mM glucose / 90 mM potassium phosphate buffer solution. -Flow-cell type biofuel cell construction- A carbon fiber (CF) electrode was modified by PMG and immersed in FAD-GDH solution to use as the anode. Another CF electrode was immersed in bilirubin oxidase solution to use as an O2-reducing cathode. A flow-cell type biofuel cell consists of the anode and the cathode was constructed as Fig. 1. Evaluation of the cell was conducted by linear sweep voltammometry measurement with a flow of 100 mM glucose / 100 mM potassium phosphate buffer solution at a rate of 11 μL/min. Results and discussion Voltammograms of cyclic potential sweeping in methylene green solution by GC electrodes had the same features as previously reported one. This result shows that a film of PMG was formed on the surface of the electrode. When the PMG modified electrode was washed and examined in potassium phosphate buffer, the cyclic voltammogram showed a couple of peak currents around the redox potential previously reported [2]. This also confirmed adsorption of PMG on the electrode. The CV measurement of the FAD-GDH/PMG modified GC electrode exhibited a catalytic current in glucose solution. This demonstrated electron transfer mediation by PMG from FAD-GDH to the GC electrode. To maximize the catalytic current, the number of potential sweeping for PMG synthesis was optimized. The maximum catalytic current by the FAD-GDH/PMG/GC electrode was obtained when the PMG was synthesized by 20-time potential sweeping. Because cyclic voltammograms of the FAD-GDH/PMG/GC electrode displayed catalytic current in glucose solution, a flow-cell type biofuel cell was constructed. Evaluation of the flow-cell type biofuel cell demonstrated that the open-circuit voltage was 549 mV, maximum current density was 143 μA cm-2, and maximum power density was 23.6 μW cm-2. The current density and power density were lower than what was expected in advance taking the CV measurements of the anode and the cathode respectively. It can be considered a cause that less bioelectrocatalysis reactions proceeded due to outflow of enzymes on electrodes. Further studies to improve this issue are under way.

Simultaneous Production of Electricity and Galactaric Acid from Pectin with an Enzymatic Biofuel Cell

Introduction Enzymes, which are reproducible catalysts, can catalyze selective and environmentally friendly production of platform chemicals from biomass, in simple systems. When the platform chemical-production is through one or more oxidation reactions, the electrons obtained from the substrate are usually just dumped to electron accepters. However, when a biofuel cell anode is used as an electron acceptor, enzymatic, oxidative conversions on the anode can be combined with O2 reduction on the cathode to generate electricity. Hence, the simultaneous production of platform chemicals and electricity is possible through an oxidative conversion with enzymatic biofuel cells. D-Galacturonic acid is included in most land plants as the main component of pectin. Because it is a cheap raw material that can also be extracted in large quantities from food process residues, such as fruit peels and pulp of sugar beets and chicory, the conversion of pectin to fuels and chemicals has been studied. meso-Galactaric acid is a target compound of a pectin biorefinery, and is expected to be both a chelating agent and a precursor for polymers with various applications, a cross-linking agent, and other platform chemicals. Uronate dehydrogenase, uronate oxidase, and glucose oxidase are known to oxidize D-galacturonic acid to produce meso-galactaric acid, so far. However, these enzymes are not suitable for an electrode catalyst. In vitro use of uronate dehydrogenase requires an expensive NAD addition as its cofactor. The oxidases donate electrons to O2 to result in reducing the electric current. Glucose dehydrogenase (GDH) which binds pyrroloquinoline quinone (PQQ) tightly, but not covalently, as its cofactor has superior characteristics as an electrode catalyst. PQQ-GDH contains its cofactor and GDH catalysis does not use O2. To date, there have been no reports on uronic acid oxidation, including D-galactuornic acid, catalyzed by GDHs. However, we expected PQQ-GDH to catalyze the oxidation of D-galacturonic acid into meso-galactaric acid because PQQ-GDH is known to have the broad selectivity for substrates including D-galactose. Hence, we observed the oxidation reaction of D-galacturonic acid catalyzed by PQQ-GDH mainly through NMR spectrometry and constructed a PQQ-GDH bioanode to produce meso-galactaric acid. Experimental NMR measurements. Methylene green (MG: ca. 15 mmol), D-galacturonic acid Na salt (ca. 15 mmol), and 0.13 mg PQQ-GDH were added to 530 μL of deuterated phosphate buffer (pD 7.1). NMR measurements of the mixture were conducted 1 h and 2 days after the mixing at 500 MHz, holding the probe temperature constant at 20 °C. Electrode preparation and electrochemical measurements. A conventional three-electrode cell was used, in which a Pt wire served as the counter electrode and Ag/AgCl (3 M NaCl) served as the reference electrode. PolyMG (PMG) film was formed on the PFC electrode by 5 sets of continuous cyclic sweeps in 0.5 mM MG / 0.1 M phosphate buffer (pH 7.0). Then, 6 μL of 50 μg μL-1 PQQ-GDH / 0.1 M phosphate buffer (pH 7.0) was dropped onto the PMG-modified electrode. The cyclic voltammetry (CV) measurements of the PQQ-GDH-modified electrode were conducted in 0.1 M D-galacturonic acid Na salt / 0.1 M phosphate buffer (pH 7.0). Results & discussion The NMR spectra obtained from the samples with PQQ-GDH showed the production of D-galactaro-1,4-lactone and meso-galactaric acid 1 h and 2 day after reaction started, respectively. The spectra of control samples without PQQ-GDH showed no signal from products. These results clearly showed PQQ-GDH-catalyzed D-galacturonic acid oxidation. The delayed appearance of meso-galactaric acid in the spectra that followed the emergence of D-galactaro-1,4-lactone reflected the non-enzymatic equilibrium among these chemical species as previously reported. To construct an enzymatic anode that produces meso-galactaric acid by using an electrode as the electron acceptor, a PQQ-GDH-modified electrode was fabricated. A redox couple with a half-wave potential (E 1/2) of approximately -0.10 V was observed when CV measurements of the PQQ-GDH-modified electrodes were conducted in the control solution without D-galacturonic acid (Figure 1: broken line). In the D-galacturonic acid Na salt solution, an increase in the anodic current density was observed, which started from around E 1/2 (Figure 1: solid line). In contrast, no catalytic current was observed in the cyclic voltammogram of the PMG-modified electrodes without PQQ-GDH in the D-galacturonic acid Na salt solution. The voltammograms of the PMG-modified electrodes in the D-galacturonic acid Na salt solution and in the control solution without D-galacturonic acid were almost the same. These results demonstrated continuous D-galacturonic acid oxidation, which was catalyzed by PQQ-GDH, and the subsequent oxidation of PQQ-GDH by the electrode. We will also report a biofuel cell with this PQQ-GDH-modified anode, which will produce meso-galactaric acid and electricity simultaneously. Figure 1

Towards timely Alzheimer diagnosis: A self-powered amperometric biosensor for the neurotransmitter acetylcholine

Serious brain disorders, such as the Alzheimer's Disease (AD), are associated with a marked drop in the levels of important neurotransmitters, such as acetylcholine (ACh). Real time monitoring of such biomarkers can therefore play a critical role in enhancing AD therapies by allowing timely diagnosis, verifications of treatment effectiveness, and developments of new medicines. In this study, we present the first acetylcholine/oxygen hybrid enzymatic fuel cell for the self-powered on site detection of ACh in plasma, which is based on the combination of an enzymatic anode with a Pt cathode. Firstly, an effective acetylcholinesterase immobilized electrode was developed and its electrochemical performance evaluated. Highly porous gold was used as the electrode material, and the enzyme was immobilized via a one step rapid and simple procedure that does not require the use of harsh chemicals or any electrode/enzyme pre-treatments. The resulting enzymatic electrode was subsequently used as the anode of a miniature flow-through membrane-less fuel cell and showed excellent response to varying concentrations of ACh. The peak power generated by the fuel cell was 4nW at a voltage of 260mV and with a current density of 9μAcm−2. The limit of detection of the fuel cell sensor was 10μM, with an average response time as short as 3min. These exciting results open new horizons for point-of-care Alzheimer diagnosis and provide an attractive potential alternative to established methods that require laborious and time-consuming sample treatments and expensive instruments.

Transparent and Capacitive Bioanode Based on Specifically Engineered Glucose Oxidase

AbstractHere we detail an optimized transparent capacitive glucose oxidizing bioanode, capable of supplying current densities of 10 µA cm−2 at applied potentials of 0.1 V–0.2 V versus saturated calomel electrode, when continuously performing in a simple phosphate buffer, pH 7.4 and artificial human tears, both with a glucose concentration of 0.05 mM only. When operating in pulse mode, the bioanode was able to deliver current densities as high as 21 µA cm−2 at the beginning of the pulse with 571 µC cm−2 total charges stored. The biogenic part of the enzymatic device was a recombinant glucose oxidase mutant from Penicillium amagasakiense with high catalytic efficiency towards glucose, up to 14.5⋅104 M−1 s−1. The non‐biogenic part of the anodic system was based on a poly(3,4‐ethylenedioxythiophene)‐graphene nanocomposite, as a highly capacitive component with a capacitance density in the 1 mF cm−2 range, multi‐walled carbon nanotubes, as an additional nanostructuring element, and a conductive organic complex, as an electron shuttle between the redox enzyme and the electrode surface. The bioanode could potentially serve as a prototype of a double‐function enzymatic anode for hybrid electric power biodevices, energizing smart contact lenses.

Combined Experimental and Computational Approach for Rational Design of Bio-Nano Interfaces

Various approaches have been developed in the last 5 years regarding targeted enzyme orientation on the electrode surface but no explanation of enzyme orientation on electron transfer properties have been provided. The same can be said about the utilization of mediators for enhancement of the interfacial electron transfer. Facing this fundamental challenge we started using computational approaches along with experimental efforts to gain knowledge of the underlying processes. In our computational and experimental benchmark studies soluble PQQ-dependent glucose dehydrogenase (PQQ-sGDH) have been explored since this enzyme is known as capable of direct electron transfer (DET). Various techniques for PQQ-sGDH immobilization and/or enhanced electron transfer have been developed over the years and a key question regarding the “proper” orientation of PQQ-sGDH has been raised. Should the substrate-binding pocket and thus PQQ be placed facing the electrode surface or it should be fully exposed to the electrolyte? To resolve this question and to enhance the electron transfer at the enzyme-electrode interface, quinones were explored in solution and immobilized on carbon support material. In both cases the current densities generated form the glucose oxidation through PQQ-sGDH were increased from 1.5 to 5 times depending on the properties of the explored quinones. In addition to the electrochemical characterization of the designed enzymatic anodes, molecular computational calculations, such as Density Functional Theory (DFT) and Molecular Docking Simulations, were used to gain insight into interactions between the quinones and PQQ-sGDH from one hand and the quinones and the support material, from another (Fig. 1). The role of the two types of benzoquinones, 1,2- and 1,4-benzoquinone, and ubiquinone in the operation and mechanism of the electron transfer in PQQ-sGDH anodes has been addressed1. It was established that the higher electrochemical performance of PQQ-sGDH anodes in the presence of free 1,2- and 1,4-benzoquinones is due to the smaller distance between these molecules and the enzyme cofactor (PQQ). It was proposed that when 1,4-benzoquinone is adsorbed, it plays the role of a mediator as a result of its high electron transfer rate ability and smaller PQQ-quinone distance. At the same time when 1,2-benzoquinone is adsorbed on the electrode surface, the enzyme will transfer the electrons directly to the support and the modifier will mostly play the role of an orienting agent. Similar conclusion can be made for the PQQ-sGDH anode modified with physically adsorbed ubiquinone for which purely orientation role was proposed. 1.Babanova, S., Matanovic, I. & Atanassov, P. Quinone-modified surfaces for enhanced enzyme-electrode interactions in PQQ-dependent glucose dehydrogenase anodes. Chem. Electro. Chem. 1, 2017–2028 (2014). Figure 1

Engineering of pyranose dehydrogenase for application to enzymatic anodes in biofuel cells.

In the search for improved glucose oxidising enzymes for biofuel cells, a number of Agaricus meleagris (Am) pyranose dehydrogenase mutants (mPDHs) exhibiting different degrees of glycosylation were produced using site-directed mutagenesis and electrochemically characterised. The response of electrodes modified with different mPDHs is compared in a mediated electron transfer mode, where the electrodes are modified with each of the mutants covalently attached to redox polymers based on polyvinylimidazole-bound osmium complexes using a cross-linking agent. Coating of each of the enzymes onto the graphite electrode surface is also used to screen for their capacity for direct electron transfer. The double mutant PDH exhibits the highest response to glucose at physiological pH in both direct and mediated electron transfer modes, producing a Jmax of ≈800 μA cm(-2) at room temperature and when "wired" to the Os-polymer having the highest formal potential. From the results obtained the double mPDH is proposed as the most suitable candidate for application to bioanode fabrication.

Paper-Based, Multi-Fueled Enzymatic Fuel Cell with Passive Microfluidic Flow

Enzymatic fuel cell (EFC) technology offers several advantages over the conventional electrochemical power sources: higher energy density, low-cost, environmentally-friendly and renewable biocatalysts, room temperature and pH neutral operating environment, and fuel flexibility via a variety of renewable fuels (e.g. sugars and alcohols). The development of a flexible, paper-based system can significantly increase the utility of the device.The developed system was based on microfluidic passive evaporative pump action provided by the paper and ensured continuous delivery of fuel to the electrodes. The multi-enzymatic anode, consisting of several oxidative enzymes, was capable of simultaneously or separately converting glucose and/or ethanol fuels to electrical energy. Coupled with an air-breathing enzymatic cathode the complete cell produced 800 µW /cm2 at 0.3V.Employing our previously developed carbon nanotube (CNT) based ink we developed a series of enzymatic anodes: with immobilized glucose dehydrogenase (GDH); with immobilized alcohol dehydrogenase (ADH); and with co-immobilized GDH and ADH enzymes. Additionally, in order to investigate multi-oxidation of a single fuel (ethanol) anodes were developed with co-immobilized ADH and aldehyde dehydrogenase (AlDH). Half-cell experiments were performed on the various enzymatic anodes to determine limits of performance as well as stability and compatibility with non-complementary fuels. Results are depicted in Figure 1.Bilirubin oxidase (BOx) air-breathing cathode was also developed, characterized and optimized for best performance. The composite cathode consisted of three layers: Toray paper (TP) current collector layer; Teflon-treated carbon black (XC-35) gas diffusional layer, and high conductivity CNT paper catalytic layer. All three layers were fused together in a hydraulic press at 500 psi. BOx was tethered to the surface of the catalytic layer through the use of bi-functional chemical linker (1-Pyrenebutanoic acid, succinimidyl ester, PBSE), which forms peptide bonds with the enzyme through the succinimidyl moiety and π-π stacks on CNTs through the pyrene moiety. Such approach resulted in increased stability of the enzyme at the electrode surface when compared to physical adsorption deposition method.Once the individual components of the fuel cell were developed and optimized, the complete EFC was constructed and tested. The GDH-based and ADH/AlDH-based systems were analyzed in respective fuels. The experimental power curve for the GDH EFC is illustrated in Figure 2. Finally, in order to demonstrate actual application, three paper-based EFCs were connecter in series and immersed in Gatorade® (fuel). This system was then employed to power a digital clock continuously for several days (Figure 3).

Electron Transfer Reactions and Stability of Bio-Anodes with PQQ-Dependent Dehydrogenases

The performance of enzymatic electrodes for biofuel cells or bio-sensors is subject among other factors to the rate of interfacial electron transfer between the enzyme and the surface of the electrode as well as decrease in enzyme activity due to enzyme immobilization and operation. Therefore, one approach towards improving generated current, stability, and consistent reproducibility of enzymatic electrodes is to optimize the enzyme-electrode interactions. Towards this goal it has been demonstrated previously by our group that covalent attachment of multi-copper oxidase enzymes to carbonaceous electrodes through a bifunctional cross-linker, 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBSE), resulted in improved interfacial electron transfer and subsequent higher generated current relative to physisorbed enzymes on bare multi-walled carbon nanotubes (MWNT)[ 1 ].In the current work, a similar technique is applied for the immobilization of Pyrroloquinoline quinone (PQQ) dependent dehydrogenases using three cross-linkers (PBSE, N-(1-Pyrenyl) maleimide (PYMAL) and 1-Pyrenecarboxylic acid (PCA)) selected based on their similar structures to that of PBSE. PQQ-dependent dehydrogenases are of interest to the application of biofuel cells and sensors due to their high catalytic activity, insensitivity to oxygen and broader substrate specificity[ 2 ] relative to NAD+/FAD+ dependent proteins. Electrochemical characterization using linear voltammetry and potentiostatic polarization of anodes developed using soluble Pyrroloquinoline quinone-dependent Glucose dehydrogenase (sGDH-PQQ) indicate that incorporation of a cross-linker onto the anode surface results in a significant increase in generated current (Fig. 1). This result impliesthat the length of the linker “tail” determines the distance between the attached enzyme and the electrode; shorter tail – shorter hoping distance for the electrons, resulting in higher electrontransfer efficiency.Further chronoamperometry studies indicate an improvement in electrode stability and resistance to enzyme activity degradation when compared to physisorbed sGDH-PQQ on MWNT. Expanding on the close interaction of the enzyme to the electrode substrate provided by the cross-linker, this work further characterizes enzyme activity of sGDH-PQQ as well as various quinohaemoproteins in situ immobilized on MWNT-paper to determine the most effective cross-linker for improving interfacial electron transport and preserving enzyme activity when immobilized. Subsequent to the selection and incorporation of a cross-linker, heme and heme analogues are covalently attached to the surfaces of gold and MWNT electrodes via techniques including electrochemical grafting of diazonium salts possessing amide functional groups which can be used to covalently attach hemes and their analogues to the electrode (Fig. 2). The heme is a natural electron “mediator” that carries out the internal electron transfer within quinohaemoproteins. Therefore, incorporating hemes or other selected porphyrins possessing appropriate redox potential in close proximity to the enzyme could improve the electron transfer from the enzyme to the electrode. Enzymatic anodes containing immobilized hemes or heme analogues in combination with cross-linking agents are characterized for generated current, enzyme activity, and stability using various electrochemical techniques. [1] S. Brocato, C. Lau, P. Atanassov, Electrochimica Acta 2012, 61, 44-49.[2] C. Anthony, Antioxidants & Redox Signaling 2001, 3, 757-774.

Enhanced Enzyme-Electrode Interface Interactions in Bioanodes Based on PQQ-Dependent Enzymes

Enzymatic fuel cells (EFC) are one of the oldest “untraditional” fuel cells that have been developed and they are still an avenue of significant part of the research activities. The main application of EFC is the design of biosensors due to the specific and very high turn over rate of organic substrates by enzymes. The biggest hindrance in EFC operation is the interface interaction between the enzyme and the electrodes surface. Different approaches have been used to overcome it: i) use of mediators to facilitate and enhance the electron transfer rate; ii) utilization of carbon materials having a diameter comparable to the enzyme size; iii) covalent attachment of enzymes to the electrode surface to ensure the interactions between them and increase stability; iv) efficient enzyme orientation, etc. All of these approaches proved to be successful.Our earlier activities in the area of biofuel cells demonstrated the importance of the proper enzyme orientation towards efficient electron transfer from the enzyme to the electrode surface and thus increased biofuel cells performance. It was established that the subsequent utilization of tethering and orienting agents, providing stable enzyme immobilization and effective enzyme orientation, lead to significant enhancement of the generated current densities of bilirubin oxidase-based oxygen reducing cathode [1]. Therefore in this study the same approach was explored for the development of oriented enzyme based three-dimensional nanocomposites for anode application in enzymatic biofuel cells.For the design of the enzymatic anode pyrroloquinoline quinone (PQQ) dependent enzymes were explored as our team has been previously demonstrated [2,3]. PQQ-glucose dehydrogenases are ideal enzymes for anode development of biofuel cell especially for sensing applications, due to their oxygen insensitivity and high catalytic activity. However, the application of those enzymes is inherently limited because of the low efficiency of the electron transfer from the enzyme towards the anode. Therefore our efforts were associated with the study and enhancement of the enzyme/electrode interactions by the utilization of “orienting agent” that also plays the role of mediators.In anodic enzymes the substrate attaches to the substrate-binding pocket and the electrons gained from the substrate oxidation are most likely transferred to the electrode surface from a part of the enzyme molecule opposite to the position of the substrate-binding pocket (Fig. 1). This especially holds true when the enzyme has more than one cofactor, such as the quinohemoproteins, which can have one or more heme groups. Therefore the orientation of the oxidizing enzymes with their substrate-binding site facing the electrode, as it was explored for oxygen reducing enzymes, is not effective approach for oxidizing enzymes, used at the anode in biofuel cells.The critical hypothesis for orienting PQQ-dependent enzymes is to use their natural electron acceptor (ubiquinone) or its substitutes as orientation agent. The anode surface, composed of carbon nanotubes, was modified with the subsequent utilization of tethering and orienting agent. As orienting agents we explored ubiquinone, catechol, 1,4-benzoquinone and other quinones with specifically designed structure. The selection of the proper quinones was done on the base of Density Functional Theory (DFT) calculations. Using DFT the adsorption energy and theoretical redox potential of different quinones were determined (Fig. 2 and Table 1) and the proper ones were further explored in real electrochemical studies (Fig. 3). A 6-times increase in the anode performance was recorded in presence of catechol used as orienting agent. At the same time due to the electrochemical activity of catechol we assume that it acts as a mediator and thus facilitates the electron transfer mechanism.Due to the fundamental principle behind the approach demonstrated herein, it could be successfully applied for other oxidizing enzymes used at the anode in enzymatic biofuel cells and enhance the enzyme-electrode interface interactions in bioanodes.

Uncovering and Redesigning a Key Amino Acid of Glucose Oxidase for Improved Biotechnological Applications

AbstractHere we uncover a key amino acid at the entrance of the active site of glucose oxidase from Penicilium amagasakiense and we successfully redesign it by nonactive site mutations, leading to enzymatic anodes with 2.4 fold higher current densities. The increase in current density could be correlated with a better interaction between the negatively charged amino acid located in position 424 and the positively charged redox mediator. We also demonstrate that this increase in current was not specific to Osmium and that further introduction of negative charges around the active site of glucose oxidase lead to a decrease in current density, illustrating the uniqueness of this amino acid in position 424.

Designing Thin Films of Redox Hydrogel for Highly Efficient Enzymatic Anodes

A method to easily increase the catalytic efficiency of enzymatic electrodes based on redox hydrogel is described. Ketjen Black (KB) is used as carbon electrode material for the construction of enzymatic anodes made of osmium based redox hydrogel and glucose oxidase. Due to its porous structure, the use of KB permits to increase the catalytic efficiency of glucose oxidation by 2.9 compared to glassy carbon (GC) electrode where a thick layer is obtained for the same amount of electroactive material. In physiological conditions, the electrodes based on KB displays 20% more current than GC electrode and an onset potential for glucose oxidation 60 mV lower.

Direct hybrid glucose–oxygen enzymatic fuel cell based on tetrathiafulvalene–tetracyanoquinodimethane charge transfer complex as anodic mediator

TTF–TCNQ has been used for the first time as a mediator in a direct glucose fuel cell operating on gas-phase oxygen. It has been shown that TTF–TCNQ forms highly irregular porous structure, which emphasizes the importance of optimization of mass transport and kinetic resistance in the catalyst layer. Kinetics resistance can be optimized by variation of the mediator and/or enzyme loading, while mass transport resistance mainly by the variation of other structural parameters such as electrode thickness. The optimized anode reached limiting current densities of nearly 400 μA cm −2 in presence of 5 mM glucose under rotation. The enzymatic fuel cell exhibited unexpectedly high OCV values (up to 0.99 V), which were tentatively ascribed to different pH conditions at the anode and the cathode. OCV was influenced by glucose crossover and was decreasing with an increase of glucose concentration or flow rate. Although the performance of the fuel cell is limited by the enzymatic anode, the long-term stability of the fuel cell is mainly influenced by the Pt cathode, while the enzymatic anode has higher stability. The fuel cell delivered power densities up to 120 μW cm −2 in presence of 5 mM glucose, depending on the glucose flow rate.

Enzymatic Anodes for Hydrogen Fuel Cells based on Covalent Attachment of Ni‐Fe Hydrogenases and Direct Electron Transfer to SAM‐Modified Gold Electrodes

AbstractImmobilization of hydrogenases onto electrodes is of great interest for developing biofuel cells that use H2 as a fuel. In this way, hydrogenases replace Pt as electrocatalyst for oxidizing H2 in the anode. We have developed a method of covalent bonding of Desulfovibrio gigas Ni‐Fe hydrogenase and Desulfovibrio vulgaris Hildenborough Ni‐Fe‐Se hydrogenase to gold electrodes modified with a self assembled monolayer (SAM) of 4‐aminothiophenol for measuring high electrocatalytic currents of H2‐oxidation in the absence of redox mediators. Electrochemical measurements and atomic force microscopy characterization show that direct electron transfer between enzyme and the Au support is due to formation of an organized monolayer of hydrogenase over the SAM‐modified surface.