Tailoring surfaces and supports for enzyme electrodes with application to biopower device development

Ó Conghaile, Peter
The objective of this thesis was to investigate the integration of enzymes and redox mediators capable of transferring electrons between enzymes and electrodes. This was done with a view to developing a semi- or fully implantable, miniature, membrane-less enzymatic fuel cell (EFC) exploiting enzymatic oxidation of glucose coupled to the enzymatic reduction of dissolved dioxygen. Miniaturisation is possible if appropriate enzymes are selected as catalysts, instead of non-selective precious metal catalysts, by removal of ion-exchange membrane from assembled fuel cells. Chapter two reports a novel method for the preparation of mediated biocatalytic enzyme electrodes for glucose oxidation, by cross-linking films of glucose oxidase, polymer supports, and a range of osmium complexes bearing functional groups on graphite electrodes. The redox potentials of the osmium complexes are manipulated by preparation of complexes using either 2,2'-bipyridine, or 2,2'-bipyridine with substitution of electron withdrawing or electron donating groups in the 4 and 4' positions, as ligands. Complexes with lower redox potential, desired for mediation of enzymatic glucose oxidation in fuel cell anodes, are based on use of 4,4'-dimethyl-2,2'-bipyridine or 4,4'-dimethoxy-2,2'-bipyridine as a ligand instead of 2,2'-bipyridine. Glucose oxidation current densities of 30 and 70 [mu]A cm(-2) at 0.2 and 0.35 V vs. Ag/AgCl applied potential were obtained for enzyme electrodes prepared using [osmium(4,4'-dimethoxy-2,2'-bipyridine)2(4-aminomethylpyridine)Cl]+ complexes and [osmium(4,4'-dimethyl-2,2'-bipyridine)2(4-aminomethylpyridine)Cl]+ compared to 120 [mu]A cm(-2) at 0.45 V vs. Ag/AgCl for the enzyme electrode using [osmium(2,2'-bipyridine)2(4-aminomethylpyridine)Cl]+, under pseudo-physiological conditions in 5 mM glucose, however stability of signals proved insufficient for long-term operation. The availability of redox mediators, polymer supports and cross-linkers offers wide scope for investigation of anchoring and cross-linking methodology that may improve current generation and stability to provide enzyme electrodes capable for application to longer-term glucose biosensors and anodes in enzymatic fuel cells. Chapter three focuses on an investigation of enzyme electrodes for oxygen reduction at high potentials, for application to EFC cathodes, based on co-immobilisation of multi-copper oxidases, such as bilirubin oxidase or a Streptomyces coelicolor laccase (SLac), with osmium redox complexes possessing an amine-functional group, in the presence of multi-walled carbon nanotubes (MWCNTs) at graphite electrodes. Oxygen reduction current densities of 0.8 mA cm(-2) under pseudo-physiological conditions at 0 V vs. Ag/AgCl are obtained by co-immobilisation of SLac, polyallylamine, MWCNTs and an [osmium(2,2'-bipyridine)2(4-aminomethylpyridine)Cl]+ complex. Enzyme electrodes prepared by incorporation of added MWCNT as a support in oxygen saturated, 150 mM NaCl, 50 mM phosphate buffer solution at 37°C, demonstrate a 3-fold increase in oxygen reduction current densities over those prepared without MWCNT. EFCs were assembled by combining the SLac-based enzyme electrode as a cathode, with glucose-oxidising anodes, based on either a pyrroloquinoline quinone or FAD-dependent glucose dehydrogenases and selected osmium redox complexes. The EFC assembled based on the PQQ-dependent glucose dehydrogenase enzyme electrode as the anode provides a maximum power density of 66 [mu]W cm(-2) in 5 mM glucose, 150 mM NaCl, phosphate buffer solution at 37°C. On operation in human serum, although the EFC power dropped to 37 [mu]W cm(-2), it still represents the highest reported power density to date for an enzymatic fuel cell operating in serum. Chapter four reports studies on enzyme electrodes for glucose oxidation prepared using films of novel osmium complex-modified redox polymers as mediators for application to biosensors or biofuel cells. These novel redox polymers were developed by coupling osmium complexes containing amine functional groups to synthetic epoxy-functionalised polymers, providing the possibility of tuning both redox polymer potential, by variation in Os complex ligand, and redox polymer physicochemical properties, by variation in monomer selection and ratio. The capability of the redox polymers to function as mediators for glucose oxidation was tested by co-immobilisation onto graphite with glucose oxidase or an FAD-dependent glucose dehydrogenase using a range of crosslinkers and in the presence and absence of MWCNT. Glucose oxidation current densities as high as 560 [mu]A cm(-2) were obtained in 100 mM glucose, 150 mM NaCl, phosphate buffer solution at 0.45 V vs. Ag/AgCl. Films prepared by crosslinking polymer bound-[Os(4,4'-dimethoxy-2,2'-bipyridine)2(4-aminomethylpyridine)Cl]+, an FAD-dependent glucose dehydrogenase, and carbon nanotubes provided current densities of 215 [mu]A cm(-2) in 5mM glucose at the lower potential of 0.2 V vs. Ag/AgCl, showing some promise for application to glucose oxidising EFCs. Chapter five reports on use of a fragmented form of a deglycosylated pyranose dehydrogenase (fdgPDH), produced when the deglycosylated enzyme (dgPDH) loses a C-terminal fragment when stored in buffer solution at 4 °C, as a glucose-oxidising catalyst for EFC anodes. A comparison of the capability of three forms of PDH, the native glycosylated enzyme (gPDH), the dgPDH and the fdgPDH, to function as catalysts for glucose oxidation when co-immobilised with osmium redox polymers on graphite electrodes, using flow injection amperometry and cyclic voltammetry, is reported. Higher glucose oxidation current densities are observed for using the fdgPDH when osmium redox polymers with low redox potentials, [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly-vinylimidazole)10Cl]+ and [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly-vinylimidazole)10Cl]+, are selected for the comparison. Under pseudo-physiological conditions, glucose oxidation current densities of ~0.3 mA cm(-2) are obtained from films containing [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly-vinylimidazole)10Cl]+ and fdgPDH at 0 V vs Ag/AgCl in 5 mM glucose, 150 mM NaCl, phosphate buffer solution. Improved access of the substrate to the active site and improved communication between enzyme and mediator within the film, possibly due to higher local concentration of redox complex, are suggested as two main reasons for the improved current generation of enzyme electrodes prepared using the fdgPDH when compared with thos prepared using the gPDH and dgPDH. Operation of an assembled, membrane-less EFC in physiological solutions, human saliva and blood is demonstrated to provide power to an electronic device to enable wireless transmission of sensing data. The EFC is prepared using the fdgPDH co-immobilised on graphite with [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly-vinylimidazole)10Cl]+ and MWCNT as anode, coupled to an oxygen-reducing cathode based on adsoprtion of a bilirubin oxidase on gold nanoparticles. Maximum power densities of up to 325 [mu]W cm(-2) were obtained in 5 mM glucose, 150 mM NaCl, phosphate buffer solution. When tested in whole human blood a power density of 80 [mu]W cm(-2) was achieved, the highest power density reported to date for EFCs operating in human blood. Finally, chapter 6 summarises my research attempts to address some the problems associated with the integration of biocatalysts and mediators, and looking at a range of techniques to improve the current output and stability of these modified enzyme-based electrodes through the incorporation of nanoparticles and/or different immobilising techniques. Chapter 6 also provides some opinion on the direction such research may take in the future. My PhD also included the synthesis of a range of redox polymers, which were also distributed to a range of collaborating partners resulting in a number of publications which continues to grow. Although these results are not discussed in this thesis, an appendix is included providing a list of my co-authored publications along with oral and poster presentations and research visits made over the course of my PhD studies.
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