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.

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/cm 2 from native JP-8 without desulfurization as opposed to traditional metal catalysts, which require fuel preprocessing.

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 O 2 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/O 2 biofuel cell based on DET-type bioelectrocatalysis via the BOx cathode and the glucose dehydrogenase (GDH) anode demonstrated peak power densities of 1 mW/cm 2 at pH 7.0 with 100 mM glucose/10 mM NAD fuel. The maximum current density of 1.6 mA/cm 2 and the maximum power density of 0.4 mW/cm 2 were achieved at 300 mV with nonmodified BOx cathode, while 3.5 mA/cm 2 and 1.1 mW/cm 2 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.

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.

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