Fundamental investigations into the factors affecting the response of laccase-based electrochemical biosensors

Given their widespread effects and distribution in both natural and industrial environments, the monitoring of phenolic compounds is of considerable analytical interest. Electrochemical biosensor technologies, in particular those comprising laccase enzymes, afford many potential benefits to address this analytical need. However, several key factors affecting sensor response currently limit their applicability. This Thesis reports on the fabrication and optimisation of an electrochemical laccase-based biosensor towards the application of the monitoring of phenolic compounds. Selected factors considered to affect sensor response were investigated using the optimised biosensor. These included: electrochemical, biochemical and substrate-dependent factors, which were found to intersect in modulating biosensor response signals. Through the application of transducer-dependent and substrate-dependent parameters, the selective and simultaneous detection of a mixture of different phenolic analytes is successfully demonstrated. This Thesis also investigates the use of Quartz-Crystal Microbalance with Dissipation (QCM-D) technology, an analytical technique that measures physical parameters of thin-film structures, towards the successful monitoring of enzyme immobilisation strategies. These strategies are fundamental to the successful fabrication of biosensors, and the real-time monitoring of immobilised film formations is of considerable research interest. In the studies reported on in this Thesis, QCM-D technology was demonstrated to be an effective complementary technology in the prediction of film immobilisation techniques on the resultant biochemical kinetics of immobilised enzymes

Fundamental investigations into the factors affecting the response of laccase-based electrochemical biosensors

Given their widespread effects and distribution in both natural and industrial environments, the monitoring of phenolic compounds is of considerable analytical interest. Electrochemical biosensor technologies, in particular those comprising laccase enzymes, afford many potential benefits to address this analytical need. However, several key factors affecting sensor response currently limit their applicability. This Thesis reports on the fabrication and optimisation of an electrochemical laccase-based biosensor towards the application of the monitoring of phenolic compounds. Selected factors considered to affect sensor response were investigated using the optimised biosensor. These included: electrochemical, biochemical and substrate-dependent factors, which were found to intersect in modulating biosensor response signals. Through the application of transducer-dependent and substrate-dependent parameters, the selective and simultaneous detection of a mixture of different phenolic analytes is successfully demonstrated. This Thesis also investigates the use of Quartz-Crystal Microbalance with Dissipation (QCM-D) technology, an analytical technique that measures physical parameters of thin-film structures, towards the successful monitoring of enzyme immobilisation strategies. These strategies are fundamental to the successful fabrication of biosensors, and the real-time monitoring of immobilised film formations is of considerable research interest. In the studies reported on in this Thesis, QCM-D technology was demonstrated to be an effective complementary technology in the prediction of film immobilisation techniques on the resultant biochemical kinetics of immobilised enzymes

Application of carbon black and iron phthalocyanine composites in bioelectricity production at a brewery wastewater fed microbial fuel cell

Aerobic cathode microbial fuel cells (MFCs) have been widely researched to provide bioremediation of wastewaters, coupled to sustainable energy production. In order to effectively accomplish this aim, suitable catalysts and catalyst supports for oxygen reduction reaction (ORR) are required. While iron phthaloycanine (FePc), supported on multi-walled carbon nanotubes has previously been studied for this function, cost of industrial production may hinder this. Importantly, this study examines the use of several available grades of carbon black for their relative suitability to perform as supports for FePc in MFC formats. Voltammetric studies showed that the different grades of carbon black provided varying lowering of the ORR overpotential, between ∼160 and ∼270 mV relative to unmodified GCEs, and an optimum grade (N326) was selected for further study. Carbon black/FePC composite electrodes exhibited comparable lowering of the ORR overpotential (606 mV) to potentials previously reported to nanotube/FePc composites (620 mV), as well as lowered charge-transfer resistance compared to electrodes solely modified with FePc. When applied as cathode modifiers in dual chambered MFCs utilising Enterobacter cloacae, the combined use of carbon black and FePc provided greater power densities than either alone; composite electrodes obtaining ∼400% power density, compared to unmodified electrodes. Modification of the anode with carbon black further increased power density, generating power densities an order of magnitude larger than those obtained at unmodified electrodes. The ability of beer brewery waste water (BBWW) to generate power at these modified surfaces yielded permissible power densities (∼40% that of reinforced clostridial media). Differences observed, in particular under agitation, are attributed to variations in nutrient content and nutrient complexity, between the two fuel substrates

Critical assessment of the quartz crystal microbalance with dissipation as an analytical tool for biosensor development and fundamental studies

One of the challenges in electrochemical biosensor design is gaining a fundamental knowledge of the processes underlying immobilisation of the molecules onto the electrode surface. This is of particular importance in biocomposite sensors where concerns have arisen as to the nature of the interaction between the biological and synthetic molecules immobilised. We examined the use of the Quartz Crystal Microbalance with Dissipation (QCM-D) as a tool for fundamental analyses of a model sensor constructed by the immobilisation of cobalt(II) phthalocyanine (TCACoPc) and glucose oxidase (GOx) onto a gold-quartz electrode (electrode surface) for the enhanced detection of glucose. The model sensor was constructed in aqueous phase and covalently linked the gold surface to the TCACoPc, and the TCACoPc to the GOx, using the QCM-D. The aqueous metallophthalocyanine (MPc) formed a multi-layer over the surface of the electrode, which could be removed to leave a monolayer with a mass loading that compared favourably to the theoretical value expected. Analysis of frequency and dissipation plots indicated covalent attachment of glucose oxidase onto the metallophthalocyanine layer. The amount of GOx bound using the model system compared favourably to calculations derived from the maximal amperometric functioning of the electrochemical sensor (examined in previously-published literature, Mashazi, P.N., Ozoemena, K.I., Nyokong, T., 2006. Electrochim. Acta 52, 177–186), but not to theoretical values derived from dimensions of GOx as established by crystallography. The strength of the binding of the GOx film with the TCACoPc layer was tested by using 2% SDS as a denaturant/surfactant, and the GOx film was not found to be significantly affected by exposure to this. This paper thus showed that QCM-D can be used in order to model essential processes and interactions that dictate the functional parameters of a biosensor

Correlating electrochemical impedance spectroscopy and quartz crystal microbalance with dissipation signals for optimisation of aptamer-based biosensors

DNA aptamers provide new avenues towards the development of a wide range of biosensors with high specificity and controlled surface chemistry for signal optimisation. Electrochemical Impedance Spectroscopy (EIS) is an extremely promising technique towards the development of DNA-based biosensors since it can be used to accurately monitor changes in the charge density of the DNA layer. However, EIS optimisation for DNA aptamers is somewhat complex due to the different characteristics that induce a signal change: namely DNA density, change in charge density close to the electrode upon DNA conformational changes, size and charge of the analyte, screening of DNA charges upon analyte binding. The use of techniques such as Quartz Crystal Microbalance with Dissipation mode (QCM-D) can provide valuable information about conditions for maximum analyte binding as well as the hydration, folding and behaviour of the aptamer distribution on the electrode.We here report on the correlation of EIS and QCM-D signals for optimisation of aptamer-based biosensors using a DNA aptamer against Prostate Specific Antigen (PSA) as a case study. Given the unreliability of current prostate cancer diagnostic tools, it is critical to develop prostate cancer biosensors with high selectivity and sensitivity. Although PSA by itself is not an ideal prostate cancer biomarker, it is believed that any reliable diagnosis will involve a panel of biomarkers comprising PSA. The sensor setup comprises a gold surface modified with a mixed Self Assembled Monolayer (SAM) made of 6-mercapto-hexanol (MCH) and thiolated-DNA aptamer. QCM-D results provided the optimal MCH:aptamer ratios, pH and buffer solutions for the binding of PSA to the aptamers. The efficiencies of the two techniques under similar conditions were compared and correlated in order to develop a reliable, label-free and cost effective biosensor for prostate cancer as well as to serve as support for further EIS aptamer-based sensors

International experience and FDI location choices of Chinese firms: The moderating effects of home country government support and host country institutions

We examine the extent to which Chinese government support of foreign direct investment (FDI) projects and host country institutional environments interact with prior entry experience by Chinese firms, and how this interrelationship affects FDI undertaken by Chinese firms. We hypothesize that home country government support and well-established host country institutions enhance organizational capabilities to take risks in FDI. As such, they reduce the need to accumulate experiential knowledge and capabilities relating to entering host countries based on prior entry experience in a particular country when undertaking follow-up investment projects. Using a unique, hand-collected panel data set of Chinese publicly listed firms during 2002–2009, we find that home government support and well-developed host country institutions reduce the importance of prior entry experience and significantly increase the likelihood of FDI entry into a host country. Further, from our subsample analyses we identify differences between entering developed and developing host countries in terms of the impact of home country government support and quality of host country institutions. Our findings help explain the puzzle concerning why emerging economy firms have rapidly internationalized in a short period of time and do not follow the pattern predicted by classical IB theories

Developing Biosensors in Developing Countries: South Africa as a Case Study

A mini-review of the reported biosensor research occurring in South Africa evidences a strong emphasis on electrochemical sensor research, guided by the opportunities this transduction platform holds for low-cost and robust sensing of numerous targets. Many of the reported publications centre on fundamental research into the signal transduction method, using model biorecognition elements, in line with international trends. Other research in this field is spread across several areas including: the application of nanotechnology; the identification and validation of biomarkers; development and testing of biorecognition agents (antibodies and aptamers) and design of electro-catalysts, most notably metallophthalocyanine. Biosensor targets commonly featured were pesticides and metals. Areas of regional import to sub-Saharan Africa, such as HIV/AIDs and tuberculosis diagnosis, are also apparent in a review of the available literature. Irrespective of the targets, the challenge to the effective deployment of such sensors remains shaped by social and economic realities such that the requirements thereof are for low-cost and universally easy to operate devices for field settings. While it is difficult to disentangle the intertwined roles of national policy, grant funding availability and, certainly, of global trends in shaping areas of emphasis in research, most notable is the strong role that nanotechnology, and to a certain extent biotechnology, plays in research regarding biosensor construction. Stronger emphasis on collaboration between scientists in theoretical modelling, nanomaterials application and or relevant stakeholders in the specific field (e.g., food or health monitoring) and researchers in biosensor design may help evolve focused research efforts towards development and deployment of low-cost biosensors

Developing Biosensors in Developing Countries: South Africa as a Case Study

A mini-review of the reported biosensor research occurring in South Africa evidences a strong emphasis on electrochemical sensor research, guided by the opportunities this transduction platform holds for low-cost and robust sensing of numerous targets. Many of the reported publications centre on fundamental research into the signal transduction method, using model biorecognition elements, in line with international trends. Other research in this field is spread across several areas including: the application of nanotechnology; the identification and validation of biomarkers; development and testing of biorecognition agents (antibodies and aptamers) and design of electro-catalysts, most notably metallophthalocyanine. Biosensor targets commonly featured were pesticides and metals. Areas  of regional import to sub-Saharan Africa, such as HIV/AIDs and tuberculosis diagnosis, are also apparent in a review of the available literature. Irrespective of the targets, the challenge to the effective deployment of such sensors remains shaped by social and economic realities such that the requirements thereof are for low-cost and universally easy to operate devices for field settings. While it is difficult to disentangle the intertwined roles of national policy, grant funding availability and, certainly, of global trends in shaping areas of emphasis in research, most notable is the strong role that nanotechnology, and to a certain extent biotechnology, plays in research regarding biosensor construction. Stronger emphasis on collaboration between scientists in theoretical modelling, nanomaterials application and or relevant stakeholders in the specific field (e.g., food or health monitoring) and researchers in biosensor design may help evolve focused research efforts towards development and deployment of low-cost biosensors