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Extensive use of Unmanned Underwater Vehicles (UUVs) and remote equipment in future Naval operations leads to an energy logistics challenge for their rechargeable batteries. A solution to this challenge is a distributed network of renewable power sources detached from mainland power grids. One potential solution is Benthic Microbial Fuel Cell (BMFC) devices. BMFCs must be optimized and upscaled to produce power at relevant scales. 3-D printing is a promising manufacturing method to achieve scalable BMFC devices. In this thesis, we devised, developed, and optimized a protocol for clearance of microfluidic channels using square cross-sections embedded within PolyJet 3-D-printed chips. The developed protocol demonstrated 75% volumetric clearance of the embedded channels. The protocol was then used to clear channels with T-shaped cross-sections, which were used to produce an experimental proof of principle for self-assembled wiring within the microchannels due to fluidic surface tension. These advancements have opened the door to embedded wiring for 3-D printed biofuel cells and other microfluidic technologies. When perfected and manufactured at scale, biofuel cells could provide a solution to the critical logistics problem of recharging UUVs and similar equipment employed in remote maritime environments, thereby making an exceptional contribution to the U.S. Navy, U.S. national security, and the related research fields., Office of Naval Research, Arlington, VA 22203, Lieutenant, United States Navy, Approved for public release. Distribution is unlimited.

© 2016 Elsevier B.V. All rights reserved. The authors would be like to thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper. Financial support of this work by Banco de Santander and Universidad Complutense de Madrid within the framework of Projects 910358 and 910305 UCM Research Group-GR3/14 is gratefully acknowledged., Chronopotentiometric and swelling experiments have been conducted to characterize the behavior of a Nafion membrane in NaCl and KCl aqueous solutions without and with glucose. A mixture solution with similar composition to the cerebrospinal fluid and blood plasma has also been studied. From the chronotentiograms, current-voltage curves have been obtained, and the values of the limiting current density, diffusion boundary layer thickness, difference between counter-ion transport number in membrane and free solution, and transition times have been determined for the investigated membrane systems. The obtained results indicate that the presence of glucose affects the ion transport through the membrane depending on the electrolyte and glucose concentrations. At low electrolyte concentration, experimental transition times are found to be smaller in presence of glucose, which has been related to an effective membrane area reduction in presence of glucose. The membrane system corresponding to the mixture solution shows a behavior similar to the single high concentration NaCl membrane system, indicating that the observed behavior is mainly associated to the Na^+ ions transport in higher proportion. In this case, the glucose presence does not affect significantly the investigated properties of the membrane, which is interesting for its utilization in a glucose fuel cell., Banco Santander Central Hispano (BSCH), Universidad Complutense de Madrid (UCM), Depto. de Física de la Tierra y Astrofísica, Fac. de Ciencias Físicas, TRUE, pub

Thanks to rapid technological innovation over the last decade, a wide range of wearable devices have emerged, playing an inspiring role in healthcare, diagnostics, and sports monitoring. This review summarizes recent progress in the development and application of electrochemical (EC) sweat sensors and self-powered systems for wearable applications. Presented here are the most common types of EC sweat sensors, namely potentiometric ion-selective electrodes, amperometric enzymatic and non-enzymatic sensors, as well as differential pulse voltammetric sensors. The sensing principle and novel functional nanomaterials that are used to enhance the performance of wearable sweat sensors and their applications are summarized. The recent advancement of battery-free wearable devices is presented, including self-powered biofuel cells (BFC), energy harvesting from novel materials and its strategy, are also discussed. The challenges in, and opportunities for, the development of sweat sensor-based wearable systems moving forward are reviewed, with the aim of shedding some light on future directions. IEEE

Thanks to rapid technological innovation over the last decade, a wide range of wearable devices have emerged, playing an inspiring role in healthcare, diagnostics, and sports monitoring. This review summarizes recent progress in the development and application of electrochemical (EC) sweat sensors and self-powered systems for wearable applications. Presented here are the most common types of EC sweat sensors, namely potentiometric ion-selective electrodes, amperometric enzymatic and non-enzymatic sensors, as well as differential pulse voltammetric sensors. The sensing principle and novel functional nanomaterials that are used to enhance the performance of wearable sweat sensors and their applications are summarized. The recent advancement of battery-free wearable devices is presented, including self-powered biofuel cells (BFC), energy harvesting from novel materials and its strategy, are also discussed. The challenges in, and opportunities for, the development of sweat sensor-based wearable systems moving forward are reviewed, with the aim of shedding some light on future directions. IEEE

Thanks to rapid technological innovation over the last decade, a wide range of wearable devices have emerged, playing an inspiring role in healthcare, diagnostics, and sports monitoring. This review summarizes recent progress in the development and application of electrochemical (EC) sweat sensors and self-powered systems for wearable applications. Presented here are the most common types of EC sweat sensors, namely potentiometric ion-selective electrodes, amperometric enzymatic and non-enzymatic sensors, as well as differential pulse voltammetric sensors. The sensing principle and novel functional nanomaterials that are used to enhance the performance of wearable sweat sensors and their applications are summarized. The recent advancement of battery-free wearable devices is presented, including self-powered biofuel cells (BFC), energy harvesting from novel materials and its strategy, are also discussed. The challenges in, and opportunities for, the development of sweat sensor-based wearable systems moving forward are reviewed, with the aim of shedding some light on future directions. IEEE

The amalgamation of flexible electronics in biological systems has shaped the way health and medicine are administered. The growing field of flexible electrochemical bioelectronics enables the in situ quantification of a variety of chemical constituents present in the human body and holds great promise for personalized health monitoring owing to its unique advantages such as inherent wearability, high sensitivity, high selectivity, and low cost. It represents a promising alternative to probe biomarkers in the human body in a simpler method compared to conventional instrumental analytical techniques. Various bioanalytical technologies are employed in flexible electrochemical bioelectronics, including ion-selective potentiometry, enzymatic amperometry, potential sweep voltammetry, field-effect transistors, affinity-based biosensing, as well as biofuel cells. Recent key innovations in flexible electrochemical bioelectronics from electrochemical sensing modalities, materials, systems, fabrication, to applications are summarized and highlighted. The challenges and opportunities in this field moving forward toward future preventive and personalized medicine devices are also discussed.

The amalgamation of flexible electronics in biological systems has shaped the way health and medicine are administered. The growing field of flexible electrochemical bioelectronics enables the in situ quantification of a variety of chemical constituents present in the human body and holds great promise for personalized health monitoring owing to its unique advantages such as inherent wearability, high sensitivity, high selectivity, and low cost. It represents a promising alternative to probe biomarkers in the human body in a simpler method compared to conventional instrumental analytical techniques. Various bioanalytical technologies are employed in flexible electrochemical bioelectronics, including ion-selective potentiometry, enzymatic amperometry, potential sweep voltammetry, field-effect transistors, affinity-based biosensing, as well as biofuel cells. Recent key innovations in flexible electrochemical bioelectronics from electrochemical sensing modalities, materials, systems, fabrication, to applications are summarized and highlighted. The challenges and opportunities in this field moving forward toward future preventive and personalized medicine devices are also discussed.

The amalgamation of flexible electronics in biological systems has shaped the way health and medicine are administered. The growing field of flexible electrochemical bioelectronics enables the in situ quantification of a variety of chemical constituents present in the human body and holds great promise for personalized health monitoring owing to its unique advantages such as inherent wearability, high sensitivity, high selectivity, and low cost. It represents a promising alternative to probe biomarkers in the human body in a simpler method compared to conventional instrumental analytical techniques. Various bioanalytical technologies are employed in flexible electrochemical bioelectronics, including ion-selective potentiometry, enzymatic amperometry, potential sweep voltammetry, field-effect transistors, affinity-based biosensing, as well as biofuel cells. Recent key innovations in flexible electrochemical bioelectronics from electrochemical sensing modalities, materials, systems, fabrication, to applications are summarized and highlighted. The challenges and opportunities in this field moving forward toward future preventive and personalized medicine devices are also discussed.

This chapter will review the currently available strategies for interfacing transition metal nanoparticles with enzymes and other more complex biological systems, as well as the applications of such biometal hybrids in the areas of catalysis, energy production, environmental remediation, and medicine. In the first part of this chapter, the focus will be on the many nanometal-enzyme hybrids that have been developed for applications in organic synthesis. Within the field of organic chemistry, nanometal-enzyme hybrids are often used as bifunctional catalysts to mediate different multistep transformations, as for example the dynamic kinetic resolution of alcohols and amines. The second part of this chapter will offer an overview of nanometal-enzyme hybrids that are used as bioelectrodes in biofuel cells. This area of research has grown significantly during the past decades, much because of the many potential future applications of such devices for medical purposes. Here, nanometal-enzyme hybrid based biofuel cells hold particular promise for biosensing applications, as well as for replacing battery-based solutions in actuator devices such as mechanical valves and pacemakers. In the final part of this chapter, the different strategies to use bacteria to synthesize metal nanoparticles will be reviewed. As will be shown by the many examples in this part, biologically synthesized and supported transition metal nanoparticles constitute interesting catalytic systems that could for example be used for energy production, pollutant degradation, and small molecule synthesis.

This chapter will review the currently available strategies for interfacing transition metal nanoparticles with enzymes and other more complex biological systems, as well as the applications of such biometal hybrids in the areas of catalysis, energy production, environmental remediation, and medicine. In the first part of this chapter, the focus will be on the many nanometal-enzyme hybrids that have been developed for applications in organic synthesis. Within the field of organic chemistry, nanometal-enzyme hybrids are often used as bifunctional catalysts to mediate different multistep transformations, as for example the dynamic kinetic resolution of alcohols and amines. The second part of this chapter will offer an overview of nanometal-enzyme hybrids that are used as bioelectrodes in biofuel cells. This area of research has grown significantly during the past decades, much because of the many potential future applications of such devices for medical purposes. Here, nanometal-enzyme hybrid based biofuel cells hold particular promise for biosensing applications, as well as for replacing battery-based solutions in actuator devices such as mechanical valves and pacemakers. In the final part of this chapter, the different strategies to use bacteria to synthesize metal nanoparticles will be reviewed. As will be shown by the many examples in this part, biologically synthesized and supported transition metal nanoparticles constitute interesting catalytic systems that could for example be used for energy production, pollutant degradation, and small molecule synthesis.

The amalgamation of flexible electronics in biological systems has shaped the way health and medicine are administered. The growing field of flexible electrochemical bioelectronics enables the in situ quantification of a variety of chemical constituents present in the human body and holds great promise for personalized health monitoring owing to its unique advantages such as inherent wearability, high sensitivity, high selectivity, and low cost. It represents a promising alternative to probe biomarkers in the human body in a simpler method compared to conventional instrumental analytical techniques. Various bioanalytical technologies are employed in flexible electrochemical bioelectronics, including ion-selective potentiometry, enzymatic amperometry, potential sweep voltammetry, field-effect transistors, affinity-based biosensing, as well as biofuel cells. Recent key innovations in flexible electrochemical bioelectronics from electrochemical sensing modalities, materials, systems, fabrication, to applications are summarized and highlighted. The challenges and opportunities in this field moving forward toward future preventive and personalized medicine devices are also discussed.

A point-of-care (POC) device to enable de-centralized diagnostics can effectively reduce the time to treatment, especially in case of infectious diseases. However, many of the POC solutions presented so far do not comply with the ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment free, and deliverable to users) guidelines that are needed to ensure their on-field deployment. Herein, we present the proof of concept of a self-powered platform that operates using the analysed fluid, mimicking a blood sample, for early stage detection of HIV-1 infection. The platform contains a smart interfacing circuit to operate an ultra-sensitive electrolyte-gated field-effect transistor (EGOFET) as a sensor and facilitates an easy and affordable readout mechanism. The sensor transduces the bio-recognition event taking place at the gate electrode functionalized with the antibody against the HIV-1 p24 capsid protein, while it is powered via paper-based biofuel cell (BFC) that extracts the energy from the analysed sample itself. The self-powered platform is demonstrated to achieve detection of HIV-1 p24 antigens in fM range, suitable for early diagnosis. From these developments, a cost-effective digital POC device able to detect the transition from “healthy” to “infected” state at single-molecule precision, with no dependency on external power sources while using minimal components and simpler approach, is foreseen.

Considerable efforts in research have been dedicated to the advancement of enzyme-based biofuel cells (BFCs) for various applications. BFCs can be used as energy-conversion devices, converting biofuels into electricity. Self-powered biosensors can also be engineered from BFCs. Such self-sustainable BFC-based biosensors and bioelectronics open opportunities for various biomedical applications, ranging from wearable, ingestible, to implantable applications. However, irrespective of purposes, bioelectronics mandates sustainable energy sources. The goal in the research is to expand the spectrum of new BFCs for biomedical technologies. Valuably, BFCs working as self-powered biosensors and energy harvesters simplify overall systems by minimalizing energy-consuming compartments, allowing the miniaturization of biodevices that traditional devices cannot enable. This dissertation describes an example of the first textile-based BFCs as stretchable self-powered sensors that can autonomously extract the electricity from perspiration and use this electricity as an analytical signal to indicate metabolite levels. This successful demonstration of wearable self-powered sensors with real-time wireless communication is expected to step further the progress of energy-harvesting systems and self-sustainable bioelectronics. Moreover, this dissertation will describe the fully edible ethanol BFC, based merely on biocompatible mushroom/plant extracts and food-based materials without any additional external mediators. These edible BFC energy harvesters represent attractive opportunities for modernizing biosensors and bioelectronics for the use in the digestive system. Furthermore, the grand challenges of BFCs including oxygen dependency will be discussed. For example, this dissertation includes an approach to address the oxygen limitations by developing a nanocomposite of oxygen-rich cathode material to provide the internal oxygen for the BFC cathodic reaction. Therefore, the effect of fluc

Considerable efforts in research have been dedicated to the advancement of enzyme-based biofuel cells (BFCs) for various applications. BFCs can be used as energy-conversion devices, converting biofuels into electricity. Self-powered biosensors can also be engineered from BFCs. Such self-sustainable BFC-based biosensors and bioelectronics open opportunities for various biomedical applications, ranging from wearable, ingestible, to implantable applications. However, irrespective of purposes, bioelectronics mandates sustainable energy sources. The goal in the research is to expand the spectrum of new BFCs for biomedical technologies. Valuably, BFCs working as self-powered biosensors and energy harvesters simplify overall systems by minimalizing energy-consuming compartments, allowing the miniaturization of biodevices that traditional devices cannot enable. This dissertation describes an example of the first textile-based BFCs as stretchable self-powered sensors that can autonomously extract the electricity from perspiration and use this electricity as an analytical signal to indicate metabolite levels. This successful demonstration of wearable self-powered sensors with real-time wireless communication is expected to step further the progress of energy-harvesting systems and self-sustainable bioelectronics. Moreover, this dissertation will describe the fully edible ethanol BFC, based merely on biocompatible mushroom/plant extracts and food-based materials without any additional external mediators. These edible BFC energy harvesters represent attractive opportunities for modernizing biosensors and bioelectronics for the use in the digestive system. Furthermore, the grand challenges of BFCs including oxygen dependency will be discussed. For example, this dissertation includes an approach to address the oxygen limitations by developing a nanocomposite of oxygen-rich cathode material to provide the internal oxygen for the BFC cathodic reaction. Therefore, the effect of fluc

This work addresses the development of a compact paper-based enzymatic microfluidic glucose/O2 fuel cell that can operate using a very limited sample volume (≈35 µl) and explores the energy generated by glucose at concentrations typically found in blood samples at physiological conditions (pH 7.4). Carbon paper electrodes combined with a paper sample absorption substrate all contained within a plastic microfluidic casing are used to construct the paper-based fuel cell. The anode catalysts consist of glucose dehydrogenase and [Os(4,4′-dimethoxy-2,2′-bipyridine)2(poly-vinylimidazole)10Cl]+ as mediator, while the cathode catalysts were bilirubin oxidase and [Os(2,2′-bipyridine)2(poly-vinylimidazole)10Cl]+ as mediator. The fuel cell delivered a linear power output response to glucose over the range of 2.5–30 mM, with power densities ranging from 20 to 90 µW cm−2. The quantification of the available electrical power as well as the energy density extracted from small synthetic samples allows planning potential uses of this energy to power different sensors and analysis devices in a wide variety of in-vitro applications.

Highly stretchable textile-based biofuel cells (BFCs), acting as effective self-powered sensors, have been fabricated using screen-printing of customized stress-enduring inks. Due to synergistic effects of nanomaterial-based engineered inks and the serpentine designs, these printable bioelectronic devices endure severe mechanical deformations, e.g., stretching, indentation, or torsional twisting. Glucose and lactate BFCs with the single enzyme and membrane-free configurations generated the maximum power density of 160 and 250 µW cm-2 with the open circuit voltages of 0.44 and 0.46 V, respectively. The textile-BFCs were able to withstand repeated severe mechanical deformations with minimal impact on its structural integrity, as was indicated from their stable power output after 100 cycles of 100% stretching. By providing power signals proportional to the sweat fuel concentration, these stretchable devices act as highly selective and stable self-powered textile sensors. Applicability to sock-based BFC and self-powered biosensor and mechanically compliant operations was demonstrated on human subjects. These stretchable skin-worn "scavenge-sense-display" devices are expected to contribute to the development of skin-worn energy harvesting systems, advanced non-invasive self-powered sensors and wearable electronics on a stretchable garment.

Highly stretchable textile-based biofuel cells (BFCs), acting as effective self-powered sensors, have been fabricated using screen-printing of customized stress-enduring inks. Due to synergistic effects of nanomaterial-based engineered inks and the serpentine designs, these printable bioelectronic devices endure severe mechanical deformations, e.g., stretching, indentation, or torsional twisting. Glucose and lactate BFCs with the single enzyme and membrane-free configurations generated the maximum power density of 160 and 250 µW cm-2 with the open circuit voltages of 0.44 and 0.46 V, respectively. The textile-BFCs were able to withstand repeated severe mechanical deformations with minimal impact on its structural integrity, as was indicated from their stable power output after 100 cycles of 100% stretching. By providing power signals proportional to the sweat fuel concentration, these stretchable devices act as highly selective and stable self-powered textile sensors. Applicability to sock-based BFC and self-powered biosensor and mechanically compliant operations was demonstrated on human subjects. These stretchable skin-worn "scavenge-sense-display" devices are expected to contribute to the development of skin-worn energy harvesting systems, advanced non-invasive self-powered sensors and wearable electronics on a stretchable garment.

In the face of todays rapidly growing energy demands accompanied by limited, non-renewable supplies, development of novel energy alternatives that are both renewable and inexpensive has become more important than ever. Development of 3D structures exploring the properties of nano-materials and biological molecules has been shown through the years as an effective path forward for the design of advanced bio-nano architectures for enzymatic fuel cells (EFCs). Despite advantages over conventional fuel cells, EFCs still suffer from several problems including low efficiency and stability. Overcoming these limitations in order to make them more viable for real world application is an ongoing challenge for researchers. Functionalized carbon nanotubes (CNTs) were covalently bonded to diazonium salt modified gold surfaces through carbodiimide chemistry creating a brush-type nanotube alignment. Having CNTs ordered in this nature developed a highly ordered structure with markedly high surface area that allowed for the attachment of protein/DNA assembly. The specificity of the enzyme immobilization was controlled by small protein structural motifs, called zinc fingers (ZnF) that bind to specific dsDNA sequences and may be genetically bound to small laccase (SLAC-3ZnF) or other redox enzymes. Utilizing scaffolds with gold nanoclusters (AuNC) for mediated electron transfer (MET), we test the capabilities for oxygen reduction reactions (ORR) by SLAC-3ZnF. Direct absorption on SWNT results in poor ORR while using DNA results in slowed reaction kinetics. With the addition of AuNC, ORR and electron transfer are improved. Analytical techniques such as x-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and enzymatic activity analysis, allowed characterization at each stage of development. Additionally, two methods for co-immobilization on consecutive tricarboxylic acid cycle (TCA) enzymes were investigated. Encapsulation using deacylated chitosan (chit) and tether

The multicopper oxidase Trametes hirsuta laccase (ThLc) served as a bioelectrocatalyst on nanostructured cathodes. Nanostructuring was provided by gold nanorods (AuNRs), which were characterized and covalently attached to electrodes made of low-density graphite. The nanostructured electrode was the scaffold for covalent and oriented attachment of ThLc. The bioelectrocatalytic currents measured for oxygen reductionwere as high as 0.5 mA/cm2 and 0.7 mA/cm2, which were recorded under direct and mediated electron transfer regimes, respectively. The experimental data were fitted to mathematical models showing that when the O2 is bioelectroreduced at high rotation speed of the electrode the heterogeneous electron transfer step is the rateliming stage. The electrochemical measurement hints a wider population of non-optimally wired laccases than previously reported for 5-8 nmsize Au nanoparticle-modified electrode, which could be due to a larger size of the AuNRs when compared to the laccases as well as their different crystal facets. © 2015 Elsevier B.V.

Miniaturized, self-sufficient bioelectronics powered by unconventional micropower may lead to a new generation of implantable, wireless, minimally invasive medical devices, such as pacemakers, defibrillators, drug-delivering pumps, sensor transmitters, and neurostimulators. Studies have shown that micro-enzymatic biofuel cells (EBFCs) are among the most intuitive candidates for in vivo micropower. In the fisrt part of this thesis, the prototype design of an EBFC chip, having 3D intedigitated microelectrode arrays was proposed to obtain an optimum design of 3D microelectrode arrays for carbon microelectromechanical systems (C-MEMS) based EBFCs. A detailed modeling solving partial differential equations (PDEs) by finite element techniques has been developed on the effect of 1) dimensions of microelectrodes, 2) spatial arrangement of 3D microelectrode arrays, 3) geometry of microelectrode on the EBFC performance based on COMSOL Multiphysics. In the second part of this thesis, in order to investigate the performance of an EBFC, behavior of an EBFC chip performance inside an artery has been studied. COMSOL Multiphysics software has also been applied to analyze mass transport for different orientations of an EBFC chip inside a blood artery. Two orientations: horizontal position (HP) and vertical position (VP) have been analyzed. The third part of this thesis has been focused on experimental work towards high performance EBFC. This work has integrated graphene/enzyme onto three-dimensional (3D) micropillar arrays in order to obtain efficient enzyme immobilization, enhanced enzyme loading and facilitate direct electron transfer. The developed 3D graphene/enzyme network based EBFC generated a maximum power density of 136.3 μWcm-2 at 0.59 V, which is almost 7 times of the maximum power density of the bare 3D carbon micropillar arrays based EBFC. To further improve the EBFC performance, reduced graphene oxide (rGO)/carbon nanotubes (CNTs) has been integrated onto 3D mciropilla

Enzymatic biofuel cells are a promising source of alternative energy for small device applications, but still face the challenge of achieving direct electron transfer with high enzyme concentrations in a simple system. In this dissertation, methods of constructing electrodes consisting of enzymes attached to nanoparticle-enhanced substrates that serve as high surface area templates are evaluated. In the first method described, glucose oxidase is covalently attached to gold nanoparticles that are assembled onto genetically engineered M13 bacteriophage. The resulting anodes achieve a high peak current per area and a significant improvement in enzyme surface coverage. In the second system, fructose dehydrogenase, a membrane-bound enzyme that has the natural ability to achieve direct electron transfer, is immobilized into a matrix consisting of binders and carbon nanotubes to extend the lifetime of the anode. For the cathode, bilirubin oxidase is immobilized in a carbon nanotube and sol-gel matrix to achieve direct electron transfer. Finally, a full fuel cell consisting of both an anode and cathode is constructed and evaluated with each system described.

The novel composites of gold nanoparticles and polypyrrole nanotubes (Au-x@PNT) were prepared and used as a platform for fabrication of bioelectrode interfaces. Changing the conditions of composite preparation caused variations in a gold architecture, electrical conductivity and a biocompatibility. These features could be easily adjusted by setting up a proper fabrication protocol. The Au-x@PNT-chitosan matrix was utilized for fabrication of bioelectrode interfaces with physisorbed oxidoreductases. Biocatalytic activity of such physisorbed fructose dehydrogenase (FDH), laccase (Lac) and bilirubin oxidase (BOD) was investigated with biocatalytic current density up to j = 2.45 mA cm(-2) obtained for a bioanode based on direct electron transfer of FDH. Performance of biocathodes with immobilized Lac and BOD showed current density up to 232 mu A cm(-2) in presence of a redox mediator.

The novel composites of gold nanoparticles and polypyrrole nanotubes (Au-x@PNT) were prepared and used as a platform for fabrication of bioelectrode interfaces. Changing the conditions of composite preparation caused variations in a gold architecture, electrical conductivity and a biocompatibility. These features could be easily adjusted by setting up a proper fabrication protocol. The Au-x@PNT-chitosan matrix was utilized for fabrication of bioelectrode interfaces with physisorbed oxidoreductases. Biocatalytic activity of such physisorbed fructose dehydrogenase (FDH), laccase (Lac) and bilirubin oxidase (BOD) was investigated with biocatalytic current density up to j = 2.45 mA cm(-2) obtained for a bioanode based on direct electron transfer of FDH. Performance of biocathodes with immobilized Lac and BOD showed current density up to 232 mu A cm(-2) in presence of a redox mediator.

Enzymatic biofuel cells are a promising source of alternative energy for small device applications, but still face the challenge of achieving direct electron transfer with high enzyme concentrations in a simple system. In this dissertation, methods of constructing electrodes consisting of enzymes attached to nanoparticle-enhanced substrates that serve as high surface area templates are evaluated. In the first method described, glucose oxidase is covalently attached to gold nanoparticles that are assembled onto genetically engineered M13 bacteriophage. The resulting anodes achieve a high peak current per area and a significant improvement in enzyme surface coverage. In the second system, fructose dehydrogenase, a membrane-bound enzyme that has the natural ability to achieve direct electron transfer, is immobilized into a matrix consisting of binders and carbon nanotubes to extend the lifetime of the anode. For the cathode, bilirubin oxidase is immobilized in a carbon nanotube and sol-gel matrix to achieve direct electron transfer. Finally, a full fuel cell consisting of both an anode and cathode is constructed and evaluated with each system described.

The novel composites of gold nanoparticles and polypyrrole nanotubes (Au-x@PNT) were prepared and used as a platform for fabrication of bioelectrode interfaces. Changing the conditions of composite preparation caused variations in a gold architecture, electrical conductivity and a biocompatibility. These features could be easily adjusted by setting up a proper fabrication protocol. The Au-x@PNT-chitosan matrix was utilized for fabrication of bioelectrode interfaces with physisorbed oxidoreductases. Biocatalytic activity of such physisorbed fructose dehydrogenase (FDH), laccase (Lac) and bilirubin oxidase (BOD) was investigated with biocatalytic current density up to j = 2.45 mA cm(-2) obtained for a bioanode based on direct electron transfer of FDH. Performance of biocathodes with immobilized Lac and BOD showed current density up to 232 mu A cm(-2) in presence of a redox mediator.

One of the major advantage of enzymes as catalyst is that many of them work in similar conditions of pH, temperature, etc. and thus can be combined in one-pot multi-step reactions pathways. The jointed action of a sequence of enzymes allows to build complex structures from simple elements; to make irreversible a reversible process or to shift the equilibrium reaction in a way that enantiomerically pure products can be obtained from racemic or prochiral substrates. In this chapter we want to highlight the developments of recent studies involving multi-enzyme cascade reactions for the synthesis of different classes of organic compounds.

This work aimed at determining the amount of energy that can be harvested by implementing microbial fuel cells (MFC) in horizontal subsurface constructed wetlands (HSSF CWs) during the treatment of real domestic wastewater. To this aim, MFC were implemented in a pilot plant based on two HSSF CW, one fed with primary settled wastewater (Settler line) and the other fed with the effluent of a hydrolytic upflow sludge blanket reactor (HUSB line). The eubacterial and archaeal community was profiled on wetland gravel, MFC electrodes and primary treated wastewater by means of 16S rRNA gene-based 454-pyrosequencing and qPCR of 16S rRNA and mcrA genes. Maximum current (219 mA/m(2)) and power (36 mW/m(2)) densities were obtained for the HUSB line. Power production pattern correlated well with water level fluctuations within the wetlands, whereas the type of primary treatment implemented had a significant impact on the diversity and relative abundance of eubacteria communities colonizing MFC. It is worth noticing the high predominance (13-16% of relative abundance) of one OM belonging to Geobacter on active MFC of the HUSB line that was absent for the settler line MFC. Hence, MFC show promise for power production in constructed wetlands receiving the effluent of a HUSB reactor. (C) 2015 Elsevier Ltd. All rights reserved., Postprint (published version)

peer-reviewed, Two adjacent electrode surfaces were modified in a sequential manner with self-assembled thiol layers from the same solution using conditions (aqueous buffer at neutral pH) suitable for applications with proteins. A faradaic response was obtained from the redox protein, cytochrome c, independently immobilised at each surface., ACCEPTED, peer-reviewed

Presented at the AFOSR Spring Review 2013, 4-8 March, Arlington, VA.

Low‐density graphite electrodes have been covalently modified with gold nanoparticles and used as scaffold for the oriented immobilization of Myrothecium verrucaria bilirubin oxidase. The developed nanobioelectrodes have been tested as a biocathode in a human‐serum surrogate buffer that mimics physiological conditions. The nanostructured bioelectrode offered a −140 µA cm−2 current density under serum‐mimic conditions at 0.2 V, and was operational during at least 6 days.

Direct electrochemistry of redox proteins provides exciting platforms for performance improvement of biosensorsand power enhancement of biofuel cells. Nanomaterials with tailored structures and unique properties are promisingbuilding blocks to promote direct electron transfer (DET) between redox-active cofactors of proteins and electrodes.This paper reviews the advances of nanostructured materials for DET of redox proteins in recent years, and isdivided into applications in biosensors and biofuel cells. Both of them focus on the performance improvement, togetherwith discussion on major challenges and opportunities for future research. The enhancement mechanism fordirect electrochemistry is also reviewed for fundamental insights.

The original document contains color images.

Three immobilization protocols were investigated with respect to direct electron transfer between hierarchical carbon microfibers/carbon nanotubes composite material on graphite rod electrodes and Trametes hirsuta laccase. Immobilization was done by covalent binding of laccase to aminophenyl-modified electrodes via amide-bond formation with carboxylic acid residues or imino-bond formation with aldehyde groups introduced by oxidation of sugar residues of the enzyme's glycosylation shell. Moreover, immobilization was achieved by adsorbing laccase to electrodes hydrophilized with pyrene-hexanoic acid. High current densities for biocatalytic oxygen reduction were obtained for all immobilization strategies. The formation of the imino bonds let to the binding of laccase in close to 100% direct electron transfer configuration and consequently to the highest oxygen reduction currents.

A new conceptual approach to improve the performance of a laccase-based cathode at neutral pH is presented. The working pH of Trametes hirsuta laccase, typically acidic, can be achieved by oxidation of biological compounds such as glucose catalyzed by a second enzyme immobilized in the vicinity of the laccase electrode

We have purchased a state-of-the-art MicroCal Auto-iTC200 Isothermal Titration Calorimetry (ITC) System. ITC is the gold standard for measuring biomolecular interactions. ITC simultaneously determines all binding parameters in a single experiment which is information that cannot be obtained from any other single method. The system will dramatically enhance the activities in several interdisciplinary projects at Columbia University that have been funded by, or will be submitted for funding to, the Department of Defense., Sponsored in part by the Construction Engineering Research Laboratory of the Engineering Research and Development Center, Army Corps of Engineers.

One of the key goals of enzymatic biofuel cells research has been the development of a fully enzymatic biofuel cell that operates under a continuous flow-through regime. Here, we present our work on achieving this task. Two NAD+-dependent dehydrogenase enzymes; malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were independently coupled with poly-methylene green (poly-MG) catalyst for biofuel cell anode fabrication. A fungal laccase that catalyzes oxygen reduction via direct electron transfer (DET) was used as an air-breathing cathode. This completes a fully enzymatic biofuel cell that operates in a flow-through mode of fuel supply polarized against an air-breathing bio-cathode. The combined, enzymatic, MDH-laccase biofuel cell operated with an open circuit voltage (OCV) of 0.584 V, whereas the ADH-laccase biofuel cell sustained an OCV of 0.618 V. Maximum volumetric power densities approaching 20 mW cm-3 are reported, and characterization criteria that will aid in future optimization are discussed., Published in Biosensors and Bioelectronics v27 p132-136, 2011.

In this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.

In this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.

In this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.

Properties of FMN-containing NADH:quinone oxidoreductases (WrbA) from the hyperthermophile Archaeoglohus fudgidus and the mesophile Escherichia coli were investigated to gain an understanding of the mechanisms by which proteins in extremophilic organisms adapt to high temperature. A model of the thermophilic enzyme was constructed based on the crystal structure of the mesophilic counterpart to guide experiments. An electrochemical cell was designed and constructed to probe the redox properties of FMN. Only subtle differences in the midpoint potential were recorded for the two enzymes consistent with congruent physiological functions. Contrary to redox potentials, the two proteins showed differences in FMN binding. Strong cooperativity in flavin binding for the mesophilic enzyme was dependent on the concentration of FMN whereas binding for the thermophilic enzyme was independent of ligand concentration. Comparison of enzyme activities between the two proteins with a variety of quinones suggested differences in the active site architecture accommodating electron acceptors. Properties of the mesophilic wild-type versus single amino acid substitution variants identified residues adjacent to FMN influencing the redox potential, oligomerization and enzyme activity. Both enzymes were suitable catalysts for biofuel cells and NADH-dependent amperometric NADH biosensors, although the thermophilic enzyme was found to be superior.

Multicopper oxidases linked to multiwall carbon nanotubes via the molecular tethering reagent, 1-pyrenebutanoic acid, succinimydl ester, diplayed high bioelectrocatalytic activity for oxygen reduction., Prepared in cooperation with University of New Mexico, Albuquerque, NM; and Air Force Research Laboratory (AFRL), Tyndall AFB, FL. Published in Chemical Communications, v46 n33 p6045-6047, 7 September 2010.

The design of the coordination shell of an Os-complex and its integration within an electrodeposition polymer enables fast electron transfer between an electrode and a polymer entrapped high-potential laccasefrom the basidiomycete Trametes hirsuta. The redox potential of the Os3+/2+-centre tethered to the polymer backbone (+720 mV vs. NHE) is perfectly matching the potential of the enzyme (+780 mV vs. NHE at pH 6.5). The laccase and the Os-complex modified anodic electrodeposition polymer were simultaneously precipitated on the surface of a glassy carbon electrode by means of a pH-shift to 2.5. The modified electrode was investigated with respect to biocatalytic oxygen reduction to water. The proposed modified electrode has potential applications as biofuel cell cathode.

The design of the coordination shell of an Os-complex and its integration within an electrodeposition polymer enables fast electron transfer between an electrode and a polymer entrapped high-potential laccasefrom the basidiomycete Trametes hirsuta. The redox potential of the Os3+/2+-centre tethered to the polymer backbone (+720 mV vs. NHE) is perfectly matching the potential of the enzyme (+780 mV vs. NHE at pH 6.5). The laccase and the Os-complex modified anodic electrodeposition polymer were simultaneously precipitated on the surface of a glassy carbon electrode by means of a pH-shift to 2.5. The modified electrode was investigated with respect to biocatalytic oxygen reduction to water. The proposed modified electrode has potential applications as biofuel cell cathode.

This review summarizes and discusses from a protein engineering point of view strategies to improve performances of biocatalysts in enzymatic biofuel cells. Emphasis will be given on biocatalysts employed in biofuel cells and protein engineering principles for achieving an efficient electrical communication between electrode and biocatalyst(s). Biocatalyst engineering by Rational Design and Directed Evolution offers opportunities to redesign and to improve biocatalysts for biofuel cell applications instead of accepting insufficient biocatalyst properties as unalterable limitations in the development of biofuel cells.

This review summarizes and discusses from a protein engineering point of view strategies to improve performances of biocatalysts in enzymatic biofuel cells. Emphasis will be given on biocatalysts employed in biofuel cells and protein engineering principles for achieving an efficient electrical communication between electrode and biocatalyst(s). Biocatalyst engineering by Rational Design and Directed Evolution offers opportunities to redesign and to improve biocatalysts for biofuel cell applications instead of accepting insufficient biocatalyst properties as unalterable limitations in the development of biofuel cells.

This review summarizes and discusses from a protein engineering point of view strategies to improve performances of biocatalysts in enzymatic biofuel cells. Emphasis will be given on biocatalysts employed in biofuel cells and protein engineering principles for achieving an efficient electrical communication between electrode and biocatalyst(s). Biocatalyst engineering by Rational Design and Directed Evolution offers opportunities to redesign and to improve biocatalysts for biofuel cell applications instead of accepting insufficient biocatalyst properties as unalterable limitations in the development of biofuel cells.