Doctor of Philosophy

dissertationEnzymatic biofuel cells use enzymes to catalyze electrochemical reactions, directly converting chemical energy to electricity. In this research, three enzymatic biofuel cell devices were created and a focus was placed on their electrode structure in order to improve current density, power density, and/or biocompatibility. The first device, a flow-through glucose biofuel cell, was fabricated from laser-cut poly(methyl methacrylate) and utilized a porous anode to increase current density through improved mass transfer. The maximum current and power density of 705 μA cm-2 and 146 μW cm-2 were among the highest for a flowing biofuel cell in the literature. The second device was a contact lens lactate biofuel cell fabricated in two iterations: one using buckypaper electrodes and the other with carbon paste electrodes, both electrode types being molded into a contact lens. These were the first reported examples of a biofuel cell on a contact lens. The first prototype suffered from poor stability as well as biocompatibility issues, but the second prototype was more stable and amenable to possibly being worn on the eye. The current and power density of the second prototype were, respectively, 22 ± 4 μA cm-2 and 2.4 ± 0.9 μW cm-2 at 0.18 ± 0.06 V. As the device was limited by its cathode, simulations were created to investigate two important factors: carbon nanotube (CNT) connectivity to the electrode and enzyme loading on the CNT surface. It was found that ca. 20% of the CNTs were connected to the electrode; furthermore, only 1-2% of the enzyme was wired to the electrode through the CNT network and roughly 20% of the CNT surfaces were in communication with enzyme. The ferrocene redox polymer/lactate oxidase enzyme-mediator anode system used on the second contact lens biofuel cell prototype performed very well, so it was also used in the third device-a self-powered lactate sensor. Coupled with a bilirubin oxidase cathode, the sensor had a detection range between 0-5 mM lactate, a sensitivity of 45 μA cm-2 mM-1, and a current and power density of 657 ± 17 μA cm-2, 122 ± 5 μW cm-2, respectively

Biosensors & enzymatic fuel cells based on direct electron transfer of dehydrogenases: characterization and applications

Il lavoro svolto durante i tre anni di dottorato è stato indirizzato verso lo sviluppo di nuovi metodi di sintesi ed elettrosintesi di nanomateriali metallici o carboniosi per il miglioramento del trasferimento elettronico diretto tra l’enzima e l’elettrodo. Questo miglioramento si traduce in un notevole incremento della sensibilità, stabilità e selettività dei biosensori sviluppati nonché della potenza generata da una pila enzimatica a biocombustibile, (Biofuel Cell). La prima parte della tesi riguarda lo studio e l’ottimizzazione del trasferimento elettronico diretto della cellobiosio deidrogenasi (CDH), un enzima appartenente alle flavoemeossidoreduttasi, costituito da due subunità dotate rispettivamente di cofattore FAD (subunità I) e heme b (subunità II). In questa parte abbiamo sintetizzato nanoparticelle di oro e di argento con un nuovo metodo “green”, che impiega come agente riducente la quercetina, un noto flavonoide presente in numerosi alimenti e bevande (es. tè, capperi, mirtilli, etc.). La reazione è stata condotta a temperatura ambiente e a pressione atmosferica senza ulteriore purificazione in quanto la quercetina è nota avere un comportamento stabilizzante delle sospensioni colloidali. Le suddette nanoparticelle sono state impiegate nella costruzione di biosensori per la determinazione del lattosio e di una pila a biocombustibile glucosio/ossigeno. Successivamente, abbiamo sviluppato un nuovo metodo per l’elettrodeposizione di nanoparticelle di oro in modo da ottenere una superficie nanostrutturata ordinata che ha portato allo sviluppo di un biosensore per la determinazione del glucosio nella saliva. La seconda parte della tesi riguarda lo studio del meccanismo del trasferimento elettronico diretto della fruttosio deidrogenasi (FDH), con particolare attenzione rivolta all’influenza dei cationi monovalenti e bivalenti, all’influenza della forma delle nanoparticelle sulla catalisi enzimatica, all’individuazione dei siti “heme” coinvolti nel trasferimento elettronico diretto attraverso l’accesso ad una porzione idrofobica dell’enzima, ed infine allo sviluppo di un biosensore per la determinazione del fruttosio realizzato immobilizzando la FDH su elettrodi di oro altamente poroso.The aim of this thesis is the study and the enhancement of the direct electron transfer of two different dehydrogenases, by means of a proper nanostructuration of the electrodes, for biosensors and enzymatic fuel cells (EFCs) development. Cellobiose dehydrogenase (CDH) is an extracellular enzyme belonging to the oxidoreductase group. CDH contains two subunits: (a) subunit I is the dehydrogenase domain (DHCDH), similar to the domain of other oxidoreductases, which belongs to the glucose-methanol-choline (GMC) oxidoreductase superfamily with a flavin adenine dinucleotide (FAD) co-factor covalently bound to the enzyme structure; (b) subunit II is the cytochrome domain (CYTCDH), which contains a heme b and acts as a built-in mediator by shuttling the electrons to a modified electrode. Both subunits are connected through a flexible linker responsible of the modulation of the internal electron transfer (IET) rate by varying the experimental conditions, such as changes of pH and divalent cations the concentration. Fructose dehydrogenase (FDH) is a membrane-bound flavocytochrome oxidoreductase which also belongs to the hemoflavoproteins family. FDH is a heterotrimeric membrane-bound enzyme complex with a molecular mass of 146.4 kDa, consisting of three subunits: (a) subunit I (DHFDH) is the catalytic domain with a covalently bound flavin adenine dinucleotide (FAD) cofactor, where D-(-)-fructose is involved in a 2H+/2e- oxidation to 5-dehydro-D-(-)-fructose; (b) subunit II (CYTFDH) acts as a built-in electron acceptor with three heme c moieties covalently bound to the enzyme scaffold and two of them involved in the electron transfer pathway; (c) subunit III is not involved in the electron transfer but plays a key role for the enzyme complex stability. The central target of the present thesis is the possibility to improve the electron transfer through the electrode nanostructuration, which can be realized by exploiting new nanomaterials as well as new nanostructuration methods (e.g. “green” synthesized metal nanoparticles, electrodeposition etc.). In the thesis much attention has been paid also to the understanding of the electron transfer pathway of FDH, which would be of fundamental interest in the near future for the development of highly sensitive biosensors and efficient EFCs. The biosensors realized and optimized in this thesis are prototypes of devices that, hopefully, will be commercially available on the market in the next future

C-MEMS Based Micro Enzymatic Biofuel Cells

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 mciropillar arrays to further increase EBFC performance in the fourth part of this thesisThe developed rGO/CNTs based EBFC generated twice the maximum power density of rGO based EBFC. Through a comparison of experimental and theoretical results, the cell performance efficiency is noted to be 67%

Development of 3D printed enzymatic biofuel cells for powering implantable biomedical devices

The drive toward device miniaturisation in the field of enzyme-based bioelectronics established a need for multi-dimensional geometrically structured and highly effective microelectrodes, which are difficult to implement and manufacture in devices such as biofuel cells and sensors. Additive manufacturing coupled with electroless metal plating enables the production of three-dimensional (3D) conductive microarchitectures with high surface area for potential applications in such devices. However, interfacial delamination between the metal layer and the polymer structure is a major reliability concern, which results in device performance degradation and eventually device failure. This thesis demonstrates a method to produce a highly conductive and robust metal layer on a 3D printed polymer microstructure with strong adhesion by introducing an interfacial adhesion layer. Prior to 3D printing, multifunctional acrylate monomers with alkoxysilane (-Si-(OCH3)3) were synthesised via the Thiol-Michael addition reaction between pentaerythritol tetraacrylate (PETA) and 3-mercaptopropyltrimethoxysilane (MPTMS) with a 1:1 stoichiometric ratio. Alkoxysilane functionality remains intact during photopolymerisation in a projection micro-stereolithography (PµSLA) system and is utilised for the sol-gel reaction with MPTMS post-functionalisation of the 3D printed microstructure to build an interfacial adhesion layer. This functionalisation leads to the implementation of abundant thiol functional groups on the surface of the 3D printed microstructure, which can act as a strong binding site for gold during electroless plating to improve interfacial adhesion. The 3D conductive microelectrode prepared by this technique exhibited excellent conductivity of 2.2×107 S/m (53% of bulk gold) with strong adhesion between a gold layer and a polymer structure even after harsh sonication and adhesion tape test, which offers potential to build a robust 3D conductive microarchitecture for applications such as biosensors and biofuel cells. As a proof-of-concept, the microelectrode with gold-coated complex lattice geometry was employed as an enzymatic glucose anode, which showed a significant increase in the current output compared to the one in the simple cube form. As the first approach, glucose oxidase was used as an enzyme. To find the optimal protocol for the enzyme immobilisation, the enzyme was first immobilised on agarose to achieve the enzyme’s highest activity and stability. Then, this immobilisation protocol was applied to immobilise the enzyme on the gold electrode surface. Preliminary studies on the preparation of 3D gold diamond lattice microelectrode modified with cysteamine and glucose oxidase as a bioanode for single cell enzymatic biofuel cell (EFC) application were performed, which demonstrated high current density of 0.38 μA cm–2 at 0.35 V in glucose solutions. This method for fabrication of 3D conductive microelectrodes offers potential for several biological applications. Instead of using a thiol, the surface of the 3D-printed part can be functionalised with different other functional groups to create an appropriate surface for biomolecules and cell adhesion. Furthermore, the surface of thiol functionalised printed parts can be perfect for additional metal coatings, opening the door to the creation of highly efficient and customised implantable energy harvesters and biosensors