Bilirubin Oxidase Cathode Research Articles

Sustainably Sourced Mesoporous Carbon Molecular Sieves as Immobilization Matrices for Enzymatic Biofuel Cell Applications

Ordered mesoporous carbon CMK-3 sieves with a hexagonal structure and uniform pore size have recently emerged as promising materials for applications as adsorbents and electrodes. In this study, using sucrose as the sustainable carbon source and SBA-15 as a template, CMK-3 sieves are synthesized to form bioelectrocatalytic immobilization matrices for enzymatic biofuel cell (EFC) electrodes. Their electrochemical performance, capacitive features, and the stability of enzyme immobilization are analyzed and compared to commercially available multi-walled carbon nanotubes (MWCNT) using cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The anodic reaction in the presence of glucose oxidase (GOx) and ferrocene methanol (FcMeOH) on the sustainably sourced CMK-3-based electrodes produces bioelectrocatalytic current responses at 0.5 V vs. saturated calomel electrode (SCE) that are twice as high as on the MWCNT-based electrodes under saturated glucose conditions. For the cathodic reaction, the MWCNT-based cathode performs marginally better than the CMK-3-based electrodes in the presence of bilirubin oxidase (BOD) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−). The CMK-3-based EFCs assembled from the GOx anode and BOD cathode results in a power output of 93 μW cm−2. In contrast, the output power of MWCNT-based EFCs is approximately 53 μW cm−2. The efficiency of CMK-3 as a support material for biofuel cell applications is effectively demonstrated.

Carbon nanodots modified-electrode for peroxide-free cholesterol biosensing and biofuel cell design

The determination of cholesterol is greatly important because high concentrations of this biomarker are associated to heart disease. Moreover, cholesterol can be used as a fuel in enzymatic fuel cells operating under physiological conditions. Here, we present a cholesterol biosensor and a peroxide-free biofuel cell based on the electrocatalytic oxidation of the NADH generated during the enzymatic reaction of cholesterol dehydrogenase (ChDH) as an alternative to the H2O2 biosensing strategies used with cholesterol oxidase-bioelectrodes. Azure A functionalized-carbon nanodots were used as NADH oxidation electrocatalysts and for ChDH covalent immobilization. The biosensor responded linearly to cholesterol concentrations up to 1.7 mM with good sensitivity (4.50 mA cm−2 M−1) and at a low potential. The ChDH bioelectrode was combined with an O2-reducing bilirubin oxidase cathode to produce electrical energy using cholesterol as fuel and O2 as oxidant. Furthermore, the resulting enzymatic fuel cell was tested in human serum naturally containing free cholesterol.

A Self-Powered Biosensor for the Detection of Glutathione.

Glutathione is an important biological molecule which can be an indicator of numerous diseases. A method for self-powered detection of glutathione levels in solution has been developed using an enzymatic biofuel cell. The device consists of a glucose oxidase anode and a bilirubin oxidase cathode. For the detection of glutathione, the inhibition of bilirubin oxidase leads to a measurable decrease in current and power output. The reported method has a detection limit of 0.043 mM and a linear range up to 1.7 mM. Being able to detect a range of concentrations can be useful in evaluating a patient’s health. This method has the potential to be implemented as a quick, low-cost alternative to previously reported methods.

Enhancement of Electrochemical Performance of Bilirubin Oxidase Modified Gas Diffusion Biocathode by Porphyrin Precursor

Recent studies have focused on tailoring the catalytic currents of multicopper oxidase (MCO) enzymes-based biocathodes to enhance oxygen reduction. Biocathodes modified with natural substrates specific for MCO enzymes demonstrated drastic improvement for oxygen reduction. Performance of 1-pyrenebutanoic acid, succinimidyl ester (PBSE), and 2,5-dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde (Di-Carb) oriented bilirubin oxidase (BOx) modified gas diffusion biocathode has been highly improved by incorporating hematin, a porphyrin precursor as electron transfer enhancement moiety. Hematin modified electrodes demonstrated direct electron transfer reaction of BOx exhibiting larger O2 reduction in current density in phosphate buffer solution (pH 7.0) without the need of a mediator. A remarkable improvement in the catalytic currents with 2.5-fold increase compared to non-hematin modified oriented BOx electrodes was achieved. Moreover, a mediatorless and compartmentless glucose/O2 biofuel cell based on DET-type bioelectrocatalysis via the BOx cathode and the glucose dehydrogenase (GDH) anode demonstrated peak power densities of 1 mW/cm2 at pH 7.0 with 100 mM glucose/10 mM NAD fuel. The maximum current density of 1.6 mA/cm2 and the maximum power density of 0.4 mW/cm2 were achieved at 300 mV with nonmodified BOx cathode, while 3.5 mA/cm2 and 1.1 mW/cm2 of current and power density were achieved with hematin modified cathode. The performance improved 2.4 times which attributes to the hematin acting as a natural precursor and activator for BOx activity enhancement.

Enhancement of Electrochemical Performance of Bilirubin Oxidase Modified Gas Diffusion Biocathode By Porphyrin Precursor

Recent studies have focused on tailoring the catalytic currents of multicopper oxidase (MCO) enzymes-based biocathodes to enhance oxygen reduction. Biocathodes modified with natural substrates specific for MCO enzymes demonstrated drastic improvement for oxygen reduction. Performance of 1- pyrenebutanoic acid, succinimidyl ester (PBSE) and 2,5- dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde (Di-Carb) oriented bilirubin oxidase (BOx) modified gas diffusion biocathode has been drastically improved by incorporating, a porphyrin precursor as electron transfer moiety. Porphyrin precursor modified electrodes demonstrated direct electron transfer reaction of BOx exhibiting larger O2 reduction current density in phosphate buffer solution (pH 7.0) without the need of a mediator. A remarkable improvement in the catalytic currents with 2.5 fold increase was achieved compared to non-modified oriented BOx electrodes. Moreover, a mediator-less and compartment-less Glucose/O2 biofuel cell based on direct electron transfer (DET) type bioelectrocatalysis via the BOx-cathode and the glucose dehydrogenase (GDH)-anode demonstrated peak power densities of 1mW/cm2 at pH 7.0 with 100mM glucose/10mM NAD fuel. The maximum current density of 1.6mA/cm2 and the maximum power density of 0.4mW/cm2 were achieved at 300mV with non modified BOx cathode, while 3.5mA/cm2 and 1.1mW/cm2 of current and power density was achieved with porphyrin precursor modified cathode. The performance improved 2.4 times which attributes to the porphyrin precursor acting as a natural substrate and activator for BOx activity enhancement.

Pyrenyl-carbon nanostructures for scalable enzyme electrocatalysis and biological fuel cells.

The objective of this article is to demonstrate the electrode geometric area-based scalability of pyrenyl-carbon nanostructure modification for enzyme electrocatalysis and fuel cell power output using hydrogenase anode and bilirubin oxidase cathode as the model system.

Rechargeable membraneless glucose biobattery: Towards solid-state cathodes for implantable enzymatic devices

Enzymatic biobatteries can be implanted in living organisms to exploit the chemical energy stored in physiological fluids. Generally, commonly-used electron donors (such as sugars) are ubiquitous in physiological environments, while electron acceptors such as oxygen are limited due to many factors including solubility, temperature, and pressure. The wide range of solid-state cathodes, however, may replace the need for oxygen breathing electrodes and serve in enzymatic biobatteries for implantable devices. Here, we have fabricated a glucose biobattery suitable for in vivo applications employing a glucose oxidase (GOx) anode coupled to a solid-state Prussian Blue (PB) thin-film cathode. PB is a non-toxic material and its electrochemistry enables fast regeneration if used in a secondary cell. This novel biobattery can effectively operate in a membraneless architecture as PB can reduce the peroxide produced by some oxidase enzymes. The resulting biobattery delivers a maximum power and current density of 44 μW cm−2 and 0.9 mA cm−2, respectively, which is ca. 37% and 180% higher than an equivalent enzymatic fuel cell equipped with a bilirubin oxidase cathode. Moreover, the biobattery demonstrated a stable performance over 20 cycles of charging and discharging periods with only ca. 3% loss of operating voltage.

A Stretchable, Wearable Lactate Sensor with a Textile-Based Biofuel Cell

Wearable sensors for monitoring health status, exercise, etc. have received much attention. Commercial wearable sensors such as watches, patches, etc., measure physical quantities such as heart rate, body temperature, etc. In addition to those measurements, lactate is a promising chemical indicator of intensity of exercise. Lactate is generated in the body during exercise, and it is widely used for monitoring the level of exercise. Traditionally, blood samples have been taken for measurement of lactate concentration of athletes, but it is difficult to apply to continuous monitoring. Recently, it has been shown that the lactate concentration in blood is correlated with that in other body fluids such as perspiration, saliva, and tear, which shows feasibility of non-invasive and continuous lactate sensing with them. Biofuel cells, which use enzyme catalysts to convert chemical energy of biological molecules into electricity, can be a promising tool as a self-powered lactate sensor. Biofuel cells have advantages such as lightness, safety of materials, mild and ambient working conditions, high specificity to lactate by choosing appropriate enzymes. In a pioneering work of the lactate sensor for perspiration with a biofuel cell, a tattoo-shaped lactate sensor on skin was developed.1 The generated electrical current was correlated with the concentration of lactate, which enabled quantification of lactate concentration in perspiration. In this study, we developed a self-powered lactate sensor in perspiration using a stretchable biofuel cell. Stretchability of the sensor helps it fit to the skin, which enables the efficient interface with the skin during exercise (Fig.1). First, we fabricated a stretchable porous electrode for a biofuel cell from a stretchable textile and carbon nanotubes (CNTs), where fiber of the textile was coated with CNTs.2 CNTs give high conductivity and porous structures to the electrode, which are essential for high performance of the biofuel cell. Scanning electron microscopy (SEM) observation indicated that CNTs between fiber were peeled off while those on fiber remained intact in the pre-stretching (the first stretching). After the initial drop in conductivity on the pre-stretching, conductivity of the electrode remained stable. Next, the anode and cathode were prepared. The lactate oxidase (LOx) anode was prepared by immobilizing LOx on the stretchable CNT electrode fabricated above. LOx oxidizes lactate to pyruvate accompanied by the release of electrons, and the electrons are carried to the electrode via tetrathiafulvalene (TTF), the electron transfer mediator. To fabricate a cathode, bilirubin oxidase (BOD) that reduces O2 in the air to H2O was immobilized on the CNT electrode. After that, we further deposited CNTs to make the surface hydrophobic to form the three-phase boundary between solution, air, and the electrode. This three-phase boundary is an important factor in current generation with O2 in the air. To keep the three-phase boundary upon stretching, we used poly(tetrafluoroethylene) (PTFE) as a binder of CNTs. We investigated the performance of the LOx anode and the BOD cathode. Cyclic voltammetry (CV) of the LOx anode with and without lactate indicated that the anode generated current from lactate, and the anode maintained the current generation over 3 hours. We evaluated the stretchability of the anode and cathode by applying 50% stretching over 30 cycles. The generated current was constant after 5 cycles of pre-stretching. This is due to firm immobilization of the CNTs to the textile, the enzymes to the CNTs, and stable binding between the CNTs by PTFE. The LOx anode and BOD cathode were combined to assemble a biofuel cell, and it was put on the skin after intense exercise with O2-permeable medical tape. A LED device was connected to the biofuel cell on the skin, and blinking of the LED indicated that the biofuel cell generated current from lactate in perspiration (Fig.2). Finally, quantification of lactate concentration in perspiration by the biofuel cell was carried out. The biofuel cell was applied to the skin during exercise, and the current was measured and converted to lactate concentration from a calibration curve. The result obtained by the biofuel cell was correlated well with that by a commercial lactate sensor. Our stretchable lactate sensor could possibly be used for continuous monitoring of lactate on the skin. [1] Jia et al., Anal. Chem., 85, 6553-6560 (2013). [2] Ogawa et al., Biosens. Bioelectron., 74, 947-952 (2015). Figure 1

(Science for Solving Society’s Problems Challenge Grant Winner) Self-Powered Supercapacitive Microbial Fuel Cell

A self-powered supercapacitive microbial fuel cell (MFC) is here reported. Microbial fuel cell (MFC) is an interesting bio-electrochemical technology that is used for wastewater treatment with simultaneous organic removal and electricity generation. Due to the much lower current/power production compared to hydrogen and methanol fuel cells, a smart design is desired to increase performance up to the levels required for real applications. The most used method to store/deliver energy from an MFC at high rate is coupling it with an external supercapacitor with capacitances in the order of Farads1. The main disadvantage of this approach is that high capacitance supercapacitors require long time to be recharged at the low MFC current regimes. This makes the MFC-Supercapacitor system working with long recharge stand-by time and low switch-on/off frequency. Here, a microbial fuel cell has been integrated with internal supercapacitor (SC)2. The MFC anode was the SC negative electrode. Its potential (≈-0.5 V vs Ag/AgCl) was related to the presence of bacteria that consumed the oxygen present into the solution and colonize the carbon brush electrode. The MFC cathode, an air-breathing cathode working at ≈+0.1 V vs Ag/AgCl, was the SC positive electrode. Single chamber MFC filled with 50% volume of 0.1M phosphate buffer saline (PBS) solution and 50% activated sludge was used during the experiments. Galvanostatic discharges were performed with current pulses up to 4 mA. An MFC with activated carbon (AC)-based cathode featured a maximum power of 2.98 Wm-2 (5.36 Wm-3) which was mainly affected by the cathode ohmic losses. In order to further enhance the output, two strategies were adopted. The first one was simply enhance the overall cell voltage by utilizing cathode catalysts with higher potential like non-platinum based catalyst (iron-aminoantipyrine (Fe-AAPyr))3 or enzymatic-based catalyst (bilirubin oxidase (BOx)4). The second option was to utilize an additional high capacitive electrode of low resistance, short-circuited with the cathode, coupled with the negative electrode (MFC-AdE). The AdE used was a carbon brush coated with AC. The first option brought to the increase of the voltage from 0.6V with AC to 0.68V with Fe-AAPyr and to 0.78V with BOx cathode. Figure 1 showed the discharge of the MFC using Fe-AAPyr cathode with and without the AdE. Consequently, maximum power (Pmax) achieved 6.53 Wm-2 (11.76 Wm-3) and 4 Wm-2 (7.2 Wm-3) using BOx and Fe-AAPyr, respectively. The use of AdE significantly reduced the cell ohmic drop and led to a much important improvement (Figure 1). Pulses discharges up to 45 mA were performed. The cell resistance decreased by one order of magnitude allowing higher current pulses and power generated. In fact, Pmax was 84.4 Wm-2 (152 Wm-3) with BOx cathode, 62.2 Wm-2 (112 Wm-3) with Fe-AAPyr and 26.7 Wm-2 (49 Wm-3) with AC cathode. The addition of the AdE is a smart and easy way to overcome the ohmic losses of the system. This current/power output is in the same range of MFCs coupled with external supercapacitors. The main advantage of the system here reported is the much shorter recharging time (in the order of seconds/minutes) of interest for applications that have to be powered with more frequent pulses, like sensors. Also, utilization of the MFC electrodes as the supercapacitor electrodes makes the system simpler and more compact. Acknowledments CS was funded by the Electrochemical Society and Bill & Melinda Gates Foundation under initiative: “Applying Electrochemistry to Complex Global Challenges”. FS acknowledges financial support by Università di Bologna (Researcher Mobility Program).

Self-powered supercapacitive microbial fuel cell: The ultimate way of boosting and harvesting power

In this work, for the first time, we demonstrate a supercapacitive microbial fuel cell which integrates the energy harvesting function of a microbial fuel cell (MFC) with the high-power operation of an internal supercapacitor. The pursued strategies are: (i) the increase of the cell voltage by the use of high potential cathodes like bilirubin oxidase (BOx) or iron–aminoantipyrine (Fe–AAPyr); (ii) the use of an additional capacitive electrode (additional electrode, AdE) which is short-circuited with the MFC cathode and coupled with the MFC anode (MFC-AdE). The high working potential of BOx cathode and the low impedances of the additional capacitive electrode and the MFC anode permitted to achieve up to 19mW (84.4Wm−2, 152Wm−3), the highest power value ever reported for MFCs. Exploiting the supercapacitive properties of the MFC electrodes allows the system to be simpler, cheaper and more efficient without additional electronics management added with respect to an MFC/external supercapacitor coupling. The use of the AdE makes it possible to decouple energy and power and to achieve recharge times in the order of few seconds making the system appealing for practical applications.

Bilirubin oxidase based enzymatic air-breathing cathode: Operation under pristine and contaminated conditions

The performance of bilirubin oxidase (BOx) based air breathing cathode was constantly monitored over 45days. The effect of electrolyte composition on the cathode oxygen reduction reaction (ORR) output was investigated. Particularly, deactivation of the electrocatalytic activity of the enzyme in phosphate buffer saline (PBS) solution and in activated sludge (AS) was evaluated. The greatest drop in current density was observed during the first 3days of constant operation with a decrease of ~60μAcm−2day−1. The rate of decrease slowed to ~10μAcm−2day−1 (day 3 to 9) and then to ~1.5μAcm−2day−1 thereafter (day 9 to 45). Despite the constant decrease in output, the BOx cathode generated residual current after 45days operations with an open circuit potential (OCP) of 475mV vs. Ag/AgCl. Enzyme deactivation was also studied in AS to simulate an environment close to the real waste operation with pollutants, solid particles and bacteria. The presence of low-molecular weight soluble contaminants was identified as the main reason for an immediate enzymatic deactivation within few hours of cathode operation. The presence of solid particles and bacteria does not affect the natural degradation of the enzyme.

Contact Lens Biofuel Cell Tested in Conditions Similar to Human Eyes

Researchers in recent years have begun developing “smart” contact lenses for biomedical applications or for wearable electronic displays. Most of these devices require an external power source, which can be cumbersome, especially to people with an active lifestyle. In this work, we describe a biofuel cell contact lens prototype, which could someday replace external power sources for contact lens devices. Our first-generation prototype consisted of buckypaper electrodes with a poly(methylene green)/NAD+/lactate dehydrogenase anode and a bilirubin oxidase cathode (Fig. 1). These electrodes were molded into a silicone elastomer contact lens, and the prototype was tested in synthetic tears, both in vitro and on a test stand that mimicked eye conditions. The open circuit voltage was 0.413 ± 0.06 V and the maximum current and power density were 61.3 ± 2.9 µA cm-2 and 8.01 ± 1.4 µW cm-2, respectively. A COMSOL model was created and subsequently used to determine better electrode size and arrangement. These model results guided the design and fabrication of a second-generation contact lens biofuel cell, which showed improved performance compared to the first-generation lens. Fig. 1. Contact lens biofuel cell prototype. Modified buckypaper electrodes were integrated into a silicone elastomer lens. Connecting leads can be seen extending to the right. Figure 1

Insights on Hybrid Glucose Biofuel Cells Based on Bilirubin Oxidase Cathode and Gold‐Based Anode Nanomaterials

AbstractWe report a straightforward design for a hybrid glucose biofuel cell (h‐GBFC) operating at pH 7.4 with 10 mM glucose at 37 °C. Homemade electrospun carbon nanofibers were used as electrode support. Clean and highly active gold‐based nanomaterials (3–6 nm) were synthesized for glucose electrooxidation. Enhanced catalytic activity toward glucose oxidation has been highlighted. Bilirubin oxidase enzyme was used to catalyze the oxygen reduction reaction. The constructed h‐GBFCs exhibit an unexpected and highly improved open circuit voltage of 0.92 V, which is the best value so far reported for such cells. The abiotic Au60Pt20Pd20/C anode induces high electrical performance with a maximum power density of 91 µW cm−2 at 0.365 V. This improvement over monometallic anode catalysts has been assigned to synergistic effects between gold, platinum, and palladium. Strategies developed herein will serve as guidelines for the development of new rational pathways to more powerful, stable, and promising GBFC designs.

Effect of enzymatic orientation through the use of syringaldazine molecules on multiple multi-copper oxidase enzymes.

The effect of proper enzyme orientation at the electrode surface was explored for two multi-copper oxygen reducing enzymes: Bilirubin Oxidase (BOx) and Laccase (Lac). Simultaneous utilization of "tethering" agent (1-pyrenebutanoic acid, succinimidyl ester; PBSE), for stable enzyme immobilization, and syringaldazine (Syr), for enzyme orientation, of both Lac and BOx led to a notable enhancement of the electrode performance. For Lac cathodes tested in solution it was established that PBSE-Lac and PBSE-Syr-Lac modified cathodes demonstrated approximately 6 and 9 times increase in current density, respectively, compared to physically adsorbed and randomly oriented Lac cathodes. Further testing in solution utilizing BOx showed an even higher increase in achievable current densities, thus BOx was chosen for additional testing in air-breathing mode. In subsequent air-breathing experiments the incorporation of PBSE and Syr with BOx resulted in current densities of 0.65 ± 0.1 mA cm(-2); 2.5 times higher when compared to an unmodified BOx cathode. A fully tethered/oriented BOx cathode was combined with a NAD-dependent Glucose Dehydrogenase anode for the fabrication of a complete enzymatic membraneless fuel cell. A maximum power of 1.03 ± 0.06 mW cm(-2) was recorded for the complete fuel cell. The observed significant enhancement in the performance of "oriented" cathodes was a result of proper enzyme orientation, leading to facilitated enzyme/electrode interface interactions.

Enhanced performance of the wired-bilirubin oxidase oxygen cathode with incorporation of carboxylated single-walled carbon nanotubes

Single-walled carbon nanotubes (SWNTs) were oxidatively treated to introduce carboxyl groups on the carbon nanotube (CNT) surfaces. The carboxylated CNTs (CNT-COOHs) were incorporated into a redox hydrogel film made from a copolymer of polyacrylamide (PAA) and poly (N-vinylimidazole) (PVI) complexed with [Os(4,4′-dichloro-2,2′-bipyridine)2Cl]+/2+ (PAA-PVI-[Os(dCl-bpy)2Cl]+/2+) wiring bilirubin oxidase (BOD) enzyme molecules. The constructed BOD cathode was employed for the electrocatalytic O2 reduction. The performance was compared with that of the BOD cathode without the incorporation of CNT-COOHs. At an electrode rotating with a 500rpm speed in an air-saturated 0.15M NaCl buffer at pH 7.4, an enhancement of the O2 reduction current density from 0.24mA/cm2 to 0.51mA/cm2 after the CNT incorporation was observed. The BOD cathode containing CNT-COOHs showed less decay against halide ion inhibitions. For the BOD cathode with no CNT-COOHs incorporated, the current decreased by 22% with 0.9M NaCl added, and 27% with 0.9M KBr added, but, with CNT-COOHs incorporated, only 6% and 5% current decays were measured, respectively. Finally, the constructed BOD cathode with CNT-COOHs incorporated showed better stability. When a potential of 0.1V vs. Ag/AgCl was applied to the cathode being rotated with a speed of 200rpm for 8000 s, almost no current decay was measured, while 7% current decay was measured for the BOD cathode with no CNT-COOHs incorporated.

Improved Interfacial Electron Transfer in Modified Bilirubin Oxidase Biocathodes

AbstractThe reducing inhibition of interfacial electron transfer and the resulting impact on the catalytic current of bilirubin oxidase (BOx) biocathodes is explored. Polymer‐coated multi‐wall carbon nanotubes (MWNTs) are modified with tethering and orientating agents to provide stable immobilization and efficient enzyme orientation. 1‐pyrenebutanoic acid, succinimidyl ester (PBSE) is used as a cross‐linker. A BOx natural substrate, bilirubin, and its artificial analogues are explored as orientating agents. It is established that bilirubin/PBSE‐modified BOx cathodes show approximately 0.4‐ and 3.2‐fold increases in the current density compared to cathodes modified separately with either PBSE or bilirubin, respectively. In subsequent experiments, the incorporation of PBSE and 2,5‐dimethyl‐1‐phenyl‐1H‐pyrrole‐3‐carbaldehyde, a functional analogue of bilirubin, into the MWNT matrix results in a further 2–2.5‐fold increase in the generated current density compared to the hybrid bilirubin/PBSE‐modified cathode, which is, therefore, 20 times higher than the unmodified BOx cathode. This significant enhancement in the performance of the cathode is attributed to the concomitant covalent attachment and proper orientation of BOx, which leads to improved enzyme/electrode interactions.

Design of experiments and principal component analysis as approaches for enhancing performance of gas-diffusional air-breathing bilirubin oxidase cathode

Two statistical methods, design of experiments (DOE) and principal component analysis (PCA) are employed to investigate and improve performance of air-breathing gas-diffusional enzymatic electrodes. DOE is utilized as a tool for systematic organization and evaluation of various factors affecting the performance of the composite system. Based on the results from the DOE, an improved cathode is constructed. The current density generated utilizing the improved cathode (755 ± 39 μA cm−2 at 0.3 V vs. Ag/AgCl) is 2–5 times higher than the highest current density previously achieved. Three major factors contributing to the cathode performance are identified: the amount of enzyme, the volume of phosphate buffer used to immobilize the enzyme, and the thickness of the gas-diffusion layer (GDL). PCA is applied as an independent confirmation tool to support conclusions made by DOE and to visualize the contribution of factors in individual cathode configurations.

Optimizing the power of enzyme-based membrane-less hydrogen fuel cells for hydrogen-rich H2–air mixtures

The unusual ability of O2-tolerant hydrogenases (H2ase) to produce electricity from a H2–air mixture (when used as the anodic electrocatalyst in a simple, membrane-less fuel cell) is investigated with the aim of establishing a strategy for raising volume power density, the measure of importance for miniature devices. Compacted mesoporous carbon electrodes provide a simple and inexpensive method for obtaining a large increase in productive enzyme loading, greatly increasing current densities and stability. Operated under a 78% H2–22% air mixture at 25 °C, typical current densities at a stationary H2ase anode and bilirubin oxidase cathode are 4.60 ± 0.32 mA cm−2 and 1.23 ± 0.12 mA cm−2, respectively. The power limitation due to low O2 concentration is addressed by re-proportioning the cathode/anode area ratio to balance the cathodic and anodic currents. At room temperature, the maximum power density of the fuel cell with an anode/cathode (A/C) ratio of 1 : 3 (1A/3C) is 1.67 ± 0.24 mW cm−2 (per anode area) or 0.42 ± 0.06 mW cm−2 (per total area). Good prospects for stability are demonstrated by the fact that 90% of the power is retained after continuously working for 24 h, and more than half of the power is retained after one week of non-stop operation. Using an even weaker O2 mixture (89% H2, 11% air) the 1A/3C cell gives over 0.8 mW cm−2 (anode) or 0.2 mW cm−2 (total electrode area). The results demonstrate the feasibility of membrane-less hydrogen–air fuel cells delivering volume power densities well in excess of 1 mW cm−3.

A paradoxical method for NAD+/NADH accumulation on an electrode surface using a hydrophobic ionic liquid

In this communication, we describe a novel and facile method for the immobilization of NAD+/NADH on an electrode surface using a hydrophobic ionic liquid, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C4mim][Tf2N]). By taking advantage of the insolubility of NAD+/NADH in hydrophobic ionic liquids, it is expected that NAD+/NADH can be retained on the electrode's surface. Alcohol dehydrogenase (ADH) and NAD+/NADH were immobilized with a gelatin hydrogel on an electrode that was modified with an electropolymerized ruthenium complex containing 5-amino-1,10-phenanthroline (pAPRu) as a mediator for NADH oxidation. The (ADH, NAD+)/pAPRu-immobilized electrode exhibited the electrocatalytic oxidation of ethanol in [C4mim][Tf2N]. The obtained catalytic current in [C4mim][Tf2N] was comparable to that in buffer solution containing NAD+. It was confirmed by UV–vis spectroscopy that NAD+ did not dissolve in the [C4mim][Tf2N] and was retained on the electrode's surface. Furthermore, we succeeded in constructing an ethanol/O2 biofuel cell comprised of an (ADH, NAD+)/pAPRu anode and a bilirubin oxidase cathode using [C4mim][Tf2N] as an electrolyte.

Development of paper based electrodes: From air-breathing to paintable enzymatic cathodes

This work presents the results from the development of bio-cathodes for the application on paper-based biofuel cells. Our main goal here is to demonstrate the possibility of using different designs of air-breathing bio-cathodes and ink-based bio-cathodes for this new type of paper based electrochemical cell. The electrochemical performance for the bio-electrocatalytic oxygen reduction reaction was studied by using open circuit voltage and amperometry measurements, as well as polarization curves to probe the four-electron reduction reaction of ambient oxygen catalyzed by bilirubin oxidase (BOx). The electrochemical measurements showed that all procedures allowed the direct electron transfer from the active site of the bilirubin oxidase to the electrode surface with a limiting current density of almost 500μAcm−2 for an air-breathing BOx cathode and 150μAcm−2 for an ink based BOx cathode. Under a load of 300mV a stable current density was obtained for 12h of continuous operation.