Enzymatic electrochemical cells (EECs) are a candidate for providing "green" solutions to a plethora of low-power, long-lifetime applications. A prototype three-electrode bio-battery configuration of an EEC has been designed and fabricated for neutron imaging and electrochemical testing to characterize cell performance. The working electrode (WE) was catalyzed by a polymer ink-based biocatalyst with carbon felt (CF) serving as the supporting material. Results of both ex situ and in operando neutron imaging are presented as methods for relating fuel distribution, the distribution of the enzymes, and cell electrochemical performance. Neutron radiography (NR) was also performed on fuel solutions of varied concentrations to calibrate fuel solution thickness and allow for transient mapping of the fuel distribution. The calibration data proved useful in mapping the thickness of fuel solution during transient radiography. When refueled after electrochemical testing and neutron imaging, the cell surpassed its original performance, indicating that exposure to the neutron beam had not detrimentally affected enzyme activity. In operando mapping of the fuel solution suggests that increased wetting of the catalyst region increases cell performance. The relation of this performance increase to active region wetting is further supported by fuel distributions observed via the ex situ tomography. While useful in mapping aggregate solution wetting, the calibration data did not support reliable mapping of detailed glucose concentration in the WE. The results presented further demonstrate potential for the application of neutron imaging for the study of EECs, particularly with respect to mapping the distribution of aqueous fuel solutions.

Enzymatic electrochemical cells (EECs) convert sugars, alcohols, and other fuels into electrical energy through electrochemical reactions catalyzed by naturally occurring enzymes. These devices typically consist of porous carbon electrodes, which may be separated by a polymer membrane. The porous electrodes in EECs are typically saturated with aqueous solutions to support electrochemical activity. The anode contains fuel within the liquid solution, while EECs often incorporate an air-breathing cathode. This work focuses on the characterization of the active anode assembled in a prototype three electrode cell developed to support investigation of the effects of varying environmental conditions on EEC performance. The aluminum three electrode cell reactor was designed and fabricated for in operando neutron imaging. This reactor consisted of two compartments for housing the working and counter electrodes. These halves of the reactor were separated by a Teflon gasket and filter paper. A three electrode cell arrangement was completed by embedding a silver reference electrode in the filter paper. Utilizing neutron tomography the active regions of the three electrode cell were mapped in the absence of the aqueous fuel solution. The reactor was then opened and wetted with a solution of 0.1 M glucose and 0.01 M hydroquinone in an aqueous sodium phosphate buffer. Cyclic voltammetry (CV) was performed to ensure proper function of the cell. Following CV in operando imaging was performed to generate radiographic representations of the anodic components prior to, during, and after discharge. Simultaneous chronoamperometry was performed to provide a connection between the dynamics of the aqueous fuel and buffer solutions and cell performance. Following discharge, tomographic scans were performed to allow for the observation of changes in the active regions of the half-cell. Following the initial measurements the three electrode cell was opened and flushed with fuel solution. The CV, in operando imaging, and tomographic measurements were then repeated. Increased aqueous solution content was found to improve the overall response of the system, yielding higher current in CV and a more consistent discharge behavior during chronoamperometry. Solution content is quantified based on tomographic scans and the related effects on performance are addressed. In general, the studies presented demonstrate a means of controlling test cell geometry to permit the study of EECs using neutron imaging techniques.

For the first time, oxygen reduction reaction has been demonstrated on a system which integrates enzymatic and non-platinum based catalysts simultaneously. This achievement is of a great importance as it offers the possibility of exploring concomitantly two very different types of catalysts, combining the advantages of both in enhancing oxygen reduction reaction rate. The engineered catalytic hybrid material not only possesses lower overpotentials compared to the purely non-PGM catalyst, but also is capable of achieving higher current densities in comparison to purely enzymatic catalyst. The hybrid catalyst undergoes oxygen reduction with the desired 4 electron transfer process, leading to the formation of water as a final product. The achieved current density of 1.2 mA cm(-2) is believed to be the highest reported for bilirubin oxidase-based gas-diffusion cathode reported so far. (C) 2016 Elsevier Ltd. All rights reserved.

Non-invasive monitoring of biomarkers in biological fluids through the use of wearable sensors gained significant interest in the past several decades [1]. Research efforts have been focused on the development of chemical and biochemical sensors for a range of metabolites, including glucose, lactate, ethanol, trace metals and various ions, targeting several bodily fluids such as tears, saliva, and sweat [2]. Work presented here will discuss the development of a sensor system, capable of real-time monitoring of lactate levels in sweat. Such a device is desired in order to correlate increasing levels of lactate to fatigue levels of a person performing physically demanding activities [3]. Most existing systems employ traditional batteries as power sources for sensors. The system described here utilizes a light weight biofuel cell as the power source giving a complete bio-friendly sensor system. The system is comprised of three main elements: a biosensor, a biofuel cell, and electronics interface. The biosensor detects lactate levels in sweat via enzymatic reaction of Lactate Dehydrogenase (LDH). Sensor patch features a three electrode design assembled onto generic athletic tape and covered with medical gauze, providing both flexibility and sweat collection capability. LDH is immobilized at the working electrode via conductive carbon-based ink. The electrode surface is then further modified by vapor-deposited tetramethyl orthosilicate (TMOS) coating in order to provide greater stability to the sensor against temperature and pH fluctuations. Sensor calibration time period was relatively quick (5 minutes) and response was immediate with addition of lactate. Test results for the sensor patch showed an open circuit potential of ~0.06V vs. Ag/AgCl and an equilibrated current of approximately 30µA when held at 0.3V vs. Ag/AgCl. The patch also demonstrated a sensitivity of 0.2µA/mM lactate when tested in a range of 5-100mM lactate. A glucose-based biofuel cell provides the power to operate the biosensor. The anode, driving glucose oxidation, is comprised of immobilized Glucose Dehydrogenase (GDH) on a high surface area carbon felt (CF) electrode. Prior to enzyme immobilization, CF electrode surface was modified with electrochemically deposited polymethylene green (PMG) as well as chemically tethered Nicotinamide Adenine Dinucleotide (NAD). An oxygen-reducing cathode was employed as counter electrode. The biofuel cell generated an open circuit potential of 0.7V vs. Ag/AgCl, a total current of 81.0mA, and total power of 16.7mW. The patch sensor is coupled to the biofuel cell via external electrical components: an energy harvester and a micropotentiostat. The energy harvester component is connected to the biofuel cell. Its primary function is to continuously provide stable output voltage to the micropotentiostat. Thus it is constantly drawing power from the biofuel cell and up-converting the voltage from 0.7 to 3V. The patch sensor is connected to the micropotentiostat. A constant potential of 0.3V vs. Ag/AgCl is needed to operate the sensor, which is supplied by the micropotentiostat. Additionally, it is used to provide a read-out of an electrical signal, i.e. current, generated during sensor operation. System performance was demonstrated, employing artificial sweat. Results showed linear sensor response with increasing lactate content of the artificial sweat solution. [1] A. J. Bandodkar and J. Wang, "Non-Invasive Wearable Electrochemical sensors: A Review," Trends in Biotechnology, vol. 32, no. 7, pp. 363-371, 2014. [2] J. Kim, W. R. de Araujo, I. A. Samek, A. J. Bandodkar, W. Jia, B. Brunetti, T. R. Paixao and J. Wang, "Wearable temporary tattoo sensor for real-time trace metal monitoring in human sweat," Electrochemistry Communications, vol. 51, pp. 41-45, 2015. [3] S. Jadoon, S. Karim, M. R. Akram, A. K. Khan, M. A. Zia, A. R. Siddqi and G. Murtaza, "recent Developments in Sweat Analysis and Its Applications," International Journal of Analytical Chemistry, pp. 1-7, 2015. Figure 1

Neutron imaging provides a route for studying the effect of environmental conditions on enzymatic electrochemical cell (EEC) performance. Here, the application of 2D and 3D neutron imaging is demonstrated as a means to investigate effects of environmental conditions on EEC performance. Changes in aqueous solutions that are vital to the operation of EECs have been directly observed in real time using neutron radiography (NR). These changes include uptake of aqueous solutions in electrode materials and in situ observation of water content during operation. The EEC structure has also been observed using ex situ neutron tomography. Initially samples constructed to simulate EEC geometry were imaged, and aqueous buffer solution uptake behavior was observed in carbon paper cell components. In follow-on studies neutron tomography was used to map regions of enzyme catalyst inks, defining active regions within the EEC electrode. The higher neutron attenuation of polymeric ink components enables this observation. Finally, in situ imaging of a prototype EEC pouch cell was performed using a standard chronoamperometry arrangement while performing real time neutron radiography. Neutron radiographs acquired during operation show a clear variation in transmission behavior, primarily due to drying. Together these studies demonstrate the applicability and attendant challenges of neutron imaging for the study of EEC performance and reliability.

Lactic acid is a key biomarker of anaerobic respiration and descriptor of an individual’s health state. Currently, lactate monitoring is performed though continuous invasive blood sampling and sample processing which is inconvenient and unpractical when trying to obtain real time measurements of an active individual. Non-invasive sensing methods are needed for monitoring human performance in sports, military and health care fields. Lactate is found in various bodily fluids including sweat and a correlation between blood and sweat lactate concentration has been determined (1). Hence, monitoring sweat lactate levels is a good noninvasive alternative to blood sampling methods. Here we report the development, characterization and optimization of an amperometric lactate sensor based on a lactate dehydrogenase and carbon nanotube chitosan electrode coupling system previously developed by our group (3). Due to the high overpotential of NADH oxidation, the electrocatalyst, polymethylene green (PMG) was electrochemically deposited onto the surface of multi-walled carbon nanotube (CNT) material called Bucky paper for NAD⁺ regeneration. Lactate dehydrogenase was immobilized onto the Bucky paper-PMG electrode with a chitosan-based carbon nanotubes (CNT) mixture. The bioelectrodes were tested in a standard three-electrode polycarbonate cell hardware consisting of enzymatic working electrode, Ag/AgCl reference and platinum wire counter electrode and operated in a chronoamperometric regime. Sweat composition varies between individuals and different parts of the human body resulting in variations in pH, salt concentration (related to conductivity,) and lactate levels before and after exercise with rates of release decreasing over time. In order to address some of these issues, calibration curves at various buffer pHs and buffer concentrations were created (Fig 1). The variation of pH of solutions from 5 to 7 showed an increase in bioelectrode response. The slope of the current/lactate concentration linear dependence was strongly influenced by the solution pH with low pH leading to decreased sensitivity most likely as a result of influenced enzyme activity. A similar trend was observed when the concentration of the electrolyte was varied from 0.01 to 0.245 M. The slope of the calibration curve was strongly influenced by the solution conductivity with low values leading to decreased reproducibility and sensitivity. In all cases the electrode response was linear with lactate concentration allowing one to tailor the system based on individual needs. An artificial sweat was prepared, composed of NaCl, urea, pH 6.5, glucose, NAD⁺, and having a conductivity of 16.6 mS/cm. As it was expected the initial tests of the LDH-electrode with the artificial sweat showed lower sensitivity most likely due to low conductivity of the solution, the generated current form the lactate oxidation followed linear dependence from lactate concentration (data not shown). The strong dependence of the slope of the sensor calibration curve from the electrolyte conductivity and pH can be minimized by buffering the sweat, which in real conditions is performed by impregnation of the sensor sweat collector with a buffer salts. A prototype of a patch lactate sensor was developed composed of a LDH-working electrode, a Ag/AgCl reference electrode and carbon yarn counter electrode. The three electrodes were placed on a medical adhesive and covered with a bandage, preventing a contact between electrodes and the skin and at the same time adsorbing and collecting the sweat. The designed patch sensor displayed an increase in current density with increasing lactate concentration with high sensitivity. To avoid artificial introduction of NAD + in the sweat, a method for NAD + immobilization of the electrode surface will be implemented along with encapsulation of the enzyme into silica gel matrix, which improve the linearity of the response and prolong the life -time of the sensor. The proposed amperometric enzyme electrode coupling approach along with optimization experiments provides the opportunity to monitor noninvasively and in real-time sweat lactate concentrations. (1) Sakharov, D.A. et al., 2010. Relationship between lactate concentrations in active muscle sweat and whole blood. Bull. Exp. Biol. Med.. 150 (1) 83-5. (2) Nikolaus, N., and Strehlitz B., 2008. Review. Amperometric lactate biosensors and their application in (sports) medicine, for life quality and wellbeing. Microchim Acta. 160, 15-55. (3). Narvaez Villarrubia C.W. et al., 2011. Biofuel cell anodes integrating NAD⁺-dependent enzymes and multiwalled carbón nanotube papers. ACS Appl. Mater Interfaces. 2011, 3(7), p. 2402-9 (7) Figure 1

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). References: Y. Ulyanova., US Air Force SBIR Phase I Award, Contract # FA8650-12-M-5165, 2012. Y. Ulyanova., US Air Force SBIR Phase II Award, Contract # FA8650-13-C-5071, 2013-2015.

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.

Very promising area of enzymatic fuel cell (EFC) research is in regards to a family of enzymes known as multi-copper oxidases (MCOs), which are capable of performing oxygen reduction reaction (ORR). Recently, MCOs have been shown to compare favorably to other state of the art ORR catalysts due to their activity at moderate temperatures and neutral pH at which they operate1. However, despite continuing research and on-going advancements in biocatalysis, relatively low catalyst stability (e.g. decreasing enzyme activity) and low current density, compared with traditional fuel cells, are impeding widespread application of EFCs. Specifically, enzyme biocatalyst activity and subsequent cell potential and current density decrease over time due to the inherently short lifetime of the enzyme and/or low interfacial electron transfer rate between the electrode and the enzyme. Improving enzyme stability and optimizing the interfacial electron transfer at the electrode is a key area of EFC research. The interfacial electron-transfer of MCOs is dependent on the spacing between the enzyme (the T1-Cu center) and the electrode surface2. To decrease the gap between the T1-Cu and the electrode, the enzyme should be “oriented” with the T1-Cu facing the underlying electrode surface. In this study a hypothesis was developed for orienting the T1-Cu site of Bilirubin oxidase (BOx) towards the electrode surface. Substrates of BOx, such as bilirubin, can be artificially attached to the electrode and subsequently used as “orienting agents” in the development of enzymatic oxygen reducing cathodes (Fig. 1)3. Two approaches were investigated in order to further improve current generation through favorable orientation of BOx on the electrode: i) “scale up” the substrate molecule through utilization of polymers or ii) “scale down” the substrate structure by the utilization of functional analogues. Multi-wall carbon nanotubes (MWNTs) were modified subsequently by physical adsorption of tethering 1-pyrenebutanoic acid succinimidyl ester (PBSE) and orienting agent (bilirubin). The interactions of bilirubin and PBSE with the MWNT scaffold are provided by π – π stacking. At the same time the tethering agent is attached to the enzyme by enzyme`s amino groups. The interaction of bilirubin and the enzyme is ensured by the key-lock principle. Thus the nanotube matrix modified by the application of tethering and orienting agents provides enhanced electrode stability and improved electron transfer efficiency showing increased bio-cathode performance (Fig. 2). The BiRu-PBSE modified cathode showed increase in current density, compared to the PBSE, bilirubin, and unmodified BOx cathodes of 0.4, 3.2, and a 9.5 times, respectively. As a macromolecular analogue of bilirubin a short-chained chemically polymerized polypyrrole was also tested. Unfortunately, the polypyrrole-modified cathode did not induce favorable enzyme orientation, most likely due to: i) the hydrophobicity of polypyrrole; ii) the lack of any carboxyl/negatively charged groups in the polypyrrole molecule; iii) the inhibition of the attached polypyrrole to the electronic communication between BOx and the electrode. The “scale down” approach involved the use of two bilirubin functional analogues, pyrrole-2-carboxaldehyde and 2,5-dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde (DPy-Carb), for enzyme orientation along with PBSE as the tethering agent. The DPy-Carb-PBSE-BOx modified cathode had a significantly higher generated current density compared to both the PBSE-BOx and BiRu-PBSE-BOx scaffolds (Fig. 3). The orientation efficiency of the enzyme after the modification procedure with DPy-Carb and PBSE was determined to be 90%. Therefore, int can be concluded that the enhanced performance of the modified cathodes is most likely due to improved interfacial electron transport as a result of favorable T1-Cu site orientation towards the electrode surface. Favorable orientation of bilirubin oxidase on a MWNT matrix and subsequent increase in electrocatalytic activity was achieved through cathode modification with a cross-linker and the natural substrate bilirubin or its artificial analogues as orienting agents. The described herein approach can be successfully applied for other reducing enzymes from the family of MCOs used at the cathode in the development of enzymatic biofuel cells. References: 1. Osman, M., A. A. Shah, F. C. Walsh, Biosensors & Bioelectronics 2011, 26, 3087-3102. 2. Hong, G., D. M. Ivnitski, G. R. Johnson, P. Atanassov, R. Pachter J. Am. Chem. Soc. 2011, 133, 4802–4809. 3. Lopez, R., S. Babanova, Y. Ulyanova, S. Singhal, P. Atanossov ChemElectroChem 2013, DOI: 10.1002/celc.201300085.

According to a number of reports about 90% of “used” water or wastewater remains untreated, and is pumped back into the environment, causing global pollution. Proper treatment is needed prior to releasing this water back into the ecosystem or re-purposing it for alternate uses. To meet the above-described need, a microbial fuel cell (MFC) has been developed, that can simultaneously generate energy from wastewater while also removing contaminants and allowing for its reuse, thereby greatly offsetting the net energy costs of water treatment. A MFC is a device that directly converts chemical energy, derived through microbial metabolism, to electrical energy. Specifically for this system the organic contaminants are processed at the anode, while heavy metal (ex. hexavalent chromium) contamination is removed at the cathode. This configuration of the MFC presents the novelty in the system’s design as compared to existing MFC technologies. The method for bioremediation of organic and heavy metal contaminants in wastewater is different. Organic contaminants are oxidized during wastewater treatment to carbon dioxide, whereas heavy metals are typically reduced to a safe metallic form. Therefore, separate types of bacteria are employed for anode and cathode fabrication. The bioanode bacteria, Shewanella oneidensis strain MR-1 [ 1 ] , has been widely studied [ 2 ] for the use in MFC. Bacterial attachment is facilitated by entrapment in biopolymers with carbon nanomaterials (including high surface area porous carbons and graphene) followed by incubation to allow for biofilm growth. Previous work has shown that these composite electrodes form very stable bioelectrodes that can be used for months without degradation [ 3 ] . Spectroscopic activity assays (Figure 1) along with amperometric activity assays for oxidation of organic substrates are employed to study the effects of bacterial immobilization and catalytic performance at modified bioelectrodes.

The search for alternative power sources able to generate electricity out of a fuel rather than just storing electrical current, like the battery, have turn into the development of fuel cells. Some of these fuel cells mimic the way the human body extracts energy from sugars or other organic compounds and these are called enzymatic biofuel cells (EFC). EFCs utilize enzymes to convert the chemical energy into electrical current. An important part of their design is the development of cathodes that can carry out oxygen reduction reaction (ORR) and explore the benefits of the abundant, cheap and easily available oxygen. The enzymes capable of ORR belong to the family of multi-copper oxidases, very well known in the area of EFCs. One particular enzyme, bilirubin oxidase (BOx), from that family is gaining more and more attention in the last years due to its superior performance and resistivity to halide ions. The main advantage of the enzymatic cathodes for ORR is the low overpotential of the reaction and thus the high open circuit potential (OCP) of the electrodes. At the same time, the maximum current generated from those types of cathodes is significantly lower in comparison to the traditional inorganic electrodes, which start the ORR at high overpotentials, but in contrast producing higher currents in this low potential region. In order to increase the performance of BOx cathodes for ORR, we combined the advantages of enzymatically catalyzed oxygen reduction with the advantages of ORR catalyzed by inorganic catalyst, such as non-platinum metal group catalyst (NPGM)1. Several NPGM catalysts were incorporated with BOx in ink type composite cathodes and tested using rotating disk electrode (RDE) technique in electrolyte with pH 7.5. Those catalysts were synthesized by sacrificial support method, as it was previously demonstrated1. Based on the performed screening study, two NPGM catalysts (Fe-DANM and Fe-AAPyr) demonstrated higher compatibility with the enzyme and as a result, higher performance was achieved. Fe-DANM catalyst used diaminomaleonitrile as a precursor and Fe-AAPyr proceeds from aminoantipyrine. Due to the high hydrophobicity of the NPGM catalyst, the interaction between the enzyme and the inorganic catalyst is hindered. Therefore, to provide more hydrophilic environment and enhance the enzyme immobilization, carbon nanotubes were introduced in the NPGM-BOx composite along with 1-pyrenebutanoic acid succinimidyl ester (PBSE) as a tethering agent. The ink composition was optimized in terms of: (1) NPGM:CNTs ratio, (2) amount of enzyme, (3) linker and (4) time of immobilization, showing that 1:1 NPGM:CNTs ratio with 10 mg/ml BOx, 10 mM PBSE and 16-18 hours of immobilization is the optimum ink formula. The aim of these NPGM-CNTs-BOx composites was to increase the cathodes final output in the broad potential region, combining the pronounced advantages of the enzymatic catalysis at high potentials and the ones of the inorganic catalysis at low potentials. Figure 1 clearly demonstrates that the simultaneous utilization of bio- and inorganic catalysis leads to improvement of the generated current densities in the whole range of potentials tested. After the positive effect of the combined NPGM-CNTs-BOx catalyst was demonstrated, a step forward was taken to improve the NPGM-CNTs interactions. NPGM catalyst (Fe-AAPyr) was in situ synthesized on multi-walled carbon nanotubes creating “fused” NPGM-CNTs composite. This composite was used for the development of the catalytic layer (CL) of gas-diffusion “hybrid” cathode. The gas-diffusion layer (GDL) of this cathode consisted on teflonized carbon black, on which the CL was pressed. BOx was physically adsorbed on the NPGM-CNTs “fused” catalyst and the performance of the cathodes was studied by polarization measurements (Fig.2). The comparison of the output of two identical cathodes differing in the preparation of the NPGM-CNTs composition shows the benefits of the in situ preparation technique. The introduction of NPGM catalysts into biological electrochemical systems, such as an enzymatic electrode for ORR, showed a notable improvement of the cathode’s performance, proving the described herein principal. This opens the venue for new applications of enzymatic biofuel cell, from biosensors to energy production. [1] Brocato, S., A. Serov, P. Atanassov Electrochimica Acta 2013, 87, 361– 365 [2] Brocato, S., C. Lau, P. Atanassov Electrochimica Acta 2012, 61, 44– 49

The 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-phenyl1H- 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.

Alkanes are attractive fuels for fuel cells due to their high energy density, but their use has not transitioned to biofuel cells. This paper discusses the development of a novel enzyme cascade utilizing alkane monooxygenase (AMO) and alcohol oxidase (AOx) to perform mediated bioelectrocatalytic oxidation of hexane and octane. This was then applied for the bioelectrocatalysis of the jet fuel JP-8, which was tested directly in an enzymatic biofuel cell to evaluate performance. The enzymatic catalysts were shown to be sulfur tolerant and produced power densities up to 3 mW/cm2 from native JP-8 without desulfurization as opposed to traditional metal catalysts, which require fuel preprocessing.

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 mu A 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. (C) 2013 Elsevier B.V. All rights reserved.

This paper describes the development of a molecularly imprinted polymer (MIP) for theophylline that can be used for electrochemical sensing. Theophylline is a commonly used medication for the treatment of asthma. Due to its very narrow therapeutic index, it may have toxic and potentially fatal effects on the individual. Electrochemical detection of theophylline is difficult, because its molecular structure and standard reduction potential are very similar to that of caffeine. A new method for fabricating molecularly imprinted polymers is proposed utilizing methylene green. Poly(methylene green)(PMG), prepared by electropolymerization of an azine, methylene green, was imprinted for theophylline. PMG-based MIP-coated electrodes showed sensitivity towards the presence of the imprint molecule in solutions, as well as selectivity for the imprint over the interferent molecule caffeine. The PMG-based MIP-coated electrode described in this paper had an improved selectivity factor and reproducibility compared to other theophylline-imprinted MIP-coated electrodes in literature.