Microbial fuel cells for remediation of metal rich wastewater coupled with bioelectricity generation
- Authors: Mshoperi, Edith
- Date: 2020
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/124112 , vital:35540
- Description: Expected release date-April 2022
- Full Text: false
- Date Issued: 2020
Microbial fuel cells for remediation of metal rich wastewater coupled with bioelectricity generation
- Authors: Mshoperi, Edith
- Date: 2020
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/124112 , vital:35540
- Description: Expected release date-April 2022
- Full Text: false
- Date Issued: 2020
Towards development of a malaria diagnostic: Generation, screening and validation of novel aptamers recognising Plasmodium falciparum lactate dehydrogenase
- Authors: Frith, Kelly-Anne
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Oligonucleotides , Lactate dehydrogenase , Biochemical markers , Systematic evolution of ligands through exponential enrichment (SELEX)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/142247 , vital:38062
- Description: Malaria, caused by infection with the Plasmodium parasite, is one of the leading causes of death in under-developed countries. Early detection is crucial for the effective treatment of malaria, particularly in cases where infection is due to Plasmodium falciparum. There is, therefore, an enduring need for portable, sensitive, reliable, accurate, durable, self-validating and cost-effective techniques for the rapid detection of malaria. Moreover, there is a demand to distinguish between various infectious species causing malaria. Research in the area of malarial biomarkers has identified a unique, species-specific, epitope of P. falciparum lactate dehydrogenase (PfLDH), enhancing prospects for the development of diagnostics capable of identifying the species causing malarial infection. In recent years, improvements have been made towards the development of rapid diagnostic tests for detecting malarial biomarkers. Owing to their low cost, ease of labeling, and high thermal stability (relative to antibodies), the development and synthesis of aptamers that target the malarial lactate dehydrogenase represents one of the key innovations in the field of rapid diagnostics for malaria. This study explored the generation of aptamers that specifically target P. falciparum. Two sets of aptamers with diagnostically-supportive functions were generated independently, through parallel SELEX of recombinantly-expressed, full-length Plasmodium falciparum lactate dehydrogenase (rPfLDH), and an oligopeptide comprising the P. falciparum-specific epitope on lactate dehydrogenase (LDHp). The latter offers a promising solution for generating aptamers capable of binding with high specificity to P. falciparum. In this work, an rLDH class of aptamers was generated when SELEX was performed using the full-length rPfLDH protein as the target and the LDHp class of aptamers was generated when SELEX was performed using the oligopeptide LDHp as a target. Aptamers were successfully generated through the process of SELEX (systematic evolution of ligands through exponential enrichment) following the study and application of several optimisation steps, particularly during the amplification stage of SELEX. Optimisation steps included the study of improvements in PCR conditions; role of surfactants (Triton-X), modifying the PCR clean-up protocol; and agarose gel excision. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers both reported here and previously published, confirming their importance in recognition of the target. Novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Clades of consensus sequences were identified in both the rLDH and LDHp groups of aptamers, where sequences in the rLDH clade did not show preferential binding to rPfLDH while those in the LDHp clade (particularly LDHp 3 and 18) were able to recognise and bind only LDHp. Of the 19 sequences returned from the parallel SELEX procedures for rPfLDH (11 sequences) and LDHp (8 sequences), six rPfLDH and all eight LDHp sequences underwent preliminary screening and those with low responses eliminated. Of the eight LDHp-targeting aptamer sequences, five were preliminarily shown to bind to LDHp, whereas only two rPfLDH-targeting sequences were shown to bind to the target (rLDH 4 and 7). To this small selection of rPfLDH oligonucleotide sequences, two more (rLDH 1 and 15) were chosen for further study based on their sequences, secondary and predicted tertiary conformations. Sequences chosen for further study were therefore: rLDH 1, 4, 7 and 15 in the rLDH class, and LDHp 1, 3, 11, 14 and 18 in the LDHp class. Binding properties of the aptamers towards their targets were investigated using enzyme-linked oligonucleotide assays (ELONA), fluorophore-linked oligonucleotide assays (FLONA), electromobility shift assays (EMSA), surface plasmon resonance (SPR), and GelRed dissociation assays, while applications towards aptasensors were explored using electrochemical impedance spectroscopy (EIS) and fluorescent microscopy. Some inconsistencies were seen for specific aptamer to target binding interactions using specific techniques; however, generally, binding to the targets was observed across the techniques assessed. These varied responses demonstrate the need to screen and validate aptamers using a variety of techniques and platforms not necessarily specific for the proposed application. From the aptamer binding screening studies using ELONA, the most promising aptamers generated were identified as LDHp 11, rLDH 4, rLDH 7 and rLDH 15. Aptamer rLDH 4, which was generated against rPfLDH, exhibited preferential and specific binding to the lactate dehydrogenase from P. falciparum, over the recombinantly-expressed lactate dehydrogenase from Plasmodium vivax (rPvLDH), albeit with lowered responses compared to LDHp 11 in ELONA and EMSA studies. However, in kinetic ELONA studies rLDH 4 showed binding to both rPfLDH and rPvLDH. Aptamer rLDH 7 showed high affinity for rPfLDH and rPvLDH in kinetic studies using ELONA. However, screening studies with ELONA indicates that aptamer rLDH 7 may not be suitable for diagnostic tests in serum samples given its non-specific binding to human serum albumin (HSA). Aptamer rLDH 15 exhibited species specificity for rPfLDH in screening studies using ELONA but showed affinity towards rPvLDH (albeit lower relative to its affinity for rPfLDH) in kinetic studies using ELONA. LDHp 11, generated against the PfLDH peptide, showed a clear preference for rPfLDH when compared to rPvLDH and other control proteins, in both sets of ELONA studies conducted, as well as EMSA, thus possessing a strong ability to identify the presence of Plasmodium falciparum owing to its generation against the species-specific epitope. While LDHp 1 demonstrated binding to plasmodial LDH in a flow-through system (SPR), so reiterating ELONA responses, it did not perform well in the remaining methodologies. Aptamers rLDH 1 and 15 and LDHp 3, 14 and 18 exhibited a mixed set of results throughout the target protein screening analyses and were, thus, not considered for selective binding in P. falciparum parasite bodies. In studies aimed at exploring biosensor assemblies utilising the developed aptamers, both rLDH 4 and LDHp 11, along with rLDH 7, LDHp 1 and pL1, demonstrated in situ binding to the native PfLDH in fluorescent microscopy. LDHp 11 exhibited FITC-based fluorescence equivalent to the anti-rPfLDHp IgY antibody in confocal fluorescent microscopy indicating superior binding to the native PfLDH compared to the remaining aptamers. An examination of electrochemical impedance as a platform for a biosensor assembly did not, in these studies, exhibit the required sensitivity using physiologically relevant concentrations of analyte expected for pLDH following infection with Plasmodium spp. Malstat/LDH activity was explored for application in a colorimetric aptasensor. A decrease in both rPfLDH and rPvLDH activity was observed following incubation with the tested aptamers, but rLDH 1, rLDH 7 and LDHp 14 did not exhibit similar decreases in rPvLDH activity. Aptamers rLDH 1, 4 and 7 and LDHp 11 and 14 were, therefore, not selected as candidates for LDH capture in LDH activity-based diagnostic devices for P. falciparum. The decreases in pLDH activity in the presence of aptamers could hold promise as direct or antagonistic malaria therapeutic agents. Preliminary studies on the application of aptamers as malaria therapeutic agents, while of interest, should be viewed with due caution given the challenges of aptamers reaching the intracellular native plasmodial LDH hosted within the red blood cells. In conclusion, this work has shown the ability of the LDHp 11 aptamer, generated in these studies, to selectively bind rPfLDH over rPvLDH, and to bind to the native PfLDH in fluorescent microscopy, indicating that this aptamer holds promise as a biorecognition element in malaria biosensors and other diagnostic devices for the detection, and differentiation, of P. falciparum and P. vivax. The use of a species-specific epitope of P. falciparum as a target in aptamer generation paves the way for similar such studies aimed at generating aptamers with species selectivity for other Plasmodium species.
- Full Text:
- Date Issued: 2020
- Authors: Frith, Kelly-Anne
- Date: 2020
- Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Oligonucleotides , Lactate dehydrogenase , Biochemical markers , Systematic evolution of ligands through exponential enrichment (SELEX)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/142247 , vital:38062
- Description: Malaria, caused by infection with the Plasmodium parasite, is one of the leading causes of death in under-developed countries. Early detection is crucial for the effective treatment of malaria, particularly in cases where infection is due to Plasmodium falciparum. There is, therefore, an enduring need for portable, sensitive, reliable, accurate, durable, self-validating and cost-effective techniques for the rapid detection of malaria. Moreover, there is a demand to distinguish between various infectious species causing malaria. Research in the area of malarial biomarkers has identified a unique, species-specific, epitope of P. falciparum lactate dehydrogenase (PfLDH), enhancing prospects for the development of diagnostics capable of identifying the species causing malarial infection. In recent years, improvements have been made towards the development of rapid diagnostic tests for detecting malarial biomarkers. Owing to their low cost, ease of labeling, and high thermal stability (relative to antibodies), the development and synthesis of aptamers that target the malarial lactate dehydrogenase represents one of the key innovations in the field of rapid diagnostics for malaria. This study explored the generation of aptamers that specifically target P. falciparum. Two sets of aptamers with diagnostically-supportive functions were generated independently, through parallel SELEX of recombinantly-expressed, full-length Plasmodium falciparum lactate dehydrogenase (rPfLDH), and an oligopeptide comprising the P. falciparum-specific epitope on lactate dehydrogenase (LDHp). The latter offers a promising solution for generating aptamers capable of binding with high specificity to P. falciparum. In this work, an rLDH class of aptamers was generated when SELEX was performed using the full-length rPfLDH protein as the target and the LDHp class of aptamers was generated when SELEX was performed using the oligopeptide LDHp as a target. Aptamers were successfully generated through the process of SELEX (systematic evolution of ligands through exponential enrichment) following the study and application of several optimisation steps, particularly during the amplification stage of SELEX. Optimisation steps included the study of improvements in PCR conditions; role of surfactants (Triton-X), modifying the PCR clean-up protocol; and agarose gel excision. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers both reported here and previously published, confirming their importance in recognition of the target. Novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Clades of consensus sequences were identified in both the rLDH and LDHp groups of aptamers, where sequences in the rLDH clade did not show preferential binding to rPfLDH while those in the LDHp clade (particularly LDHp 3 and 18) were able to recognise and bind only LDHp. Of the 19 sequences returned from the parallel SELEX procedures for rPfLDH (11 sequences) and LDHp (8 sequences), six rPfLDH and all eight LDHp sequences underwent preliminary screening and those with low responses eliminated. Of the eight LDHp-targeting aptamer sequences, five were preliminarily shown to bind to LDHp, whereas only two rPfLDH-targeting sequences were shown to bind to the target (rLDH 4 and 7). To this small selection of rPfLDH oligonucleotide sequences, two more (rLDH 1 and 15) were chosen for further study based on their sequences, secondary and predicted tertiary conformations. Sequences chosen for further study were therefore: rLDH 1, 4, 7 and 15 in the rLDH class, and LDHp 1, 3, 11, 14 and 18 in the LDHp class. Binding properties of the aptamers towards their targets were investigated using enzyme-linked oligonucleotide assays (ELONA), fluorophore-linked oligonucleotide assays (FLONA), electromobility shift assays (EMSA), surface plasmon resonance (SPR), and GelRed dissociation assays, while applications towards aptasensors were explored using electrochemical impedance spectroscopy (EIS) and fluorescent microscopy. Some inconsistencies were seen for specific aptamer to target binding interactions using specific techniques; however, generally, binding to the targets was observed across the techniques assessed. These varied responses demonstrate the need to screen and validate aptamers using a variety of techniques and platforms not necessarily specific for the proposed application. From the aptamer binding screening studies using ELONA, the most promising aptamers generated were identified as LDHp 11, rLDH 4, rLDH 7 and rLDH 15. Aptamer rLDH 4, which was generated against rPfLDH, exhibited preferential and specific binding to the lactate dehydrogenase from P. falciparum, over the recombinantly-expressed lactate dehydrogenase from Plasmodium vivax (rPvLDH), albeit with lowered responses compared to LDHp 11 in ELONA and EMSA studies. However, in kinetic ELONA studies rLDH 4 showed binding to both rPfLDH and rPvLDH. Aptamer rLDH 7 showed high affinity for rPfLDH and rPvLDH in kinetic studies using ELONA. However, screening studies with ELONA indicates that aptamer rLDH 7 may not be suitable for diagnostic tests in serum samples given its non-specific binding to human serum albumin (HSA). Aptamer rLDH 15 exhibited species specificity for rPfLDH in screening studies using ELONA but showed affinity towards rPvLDH (albeit lower relative to its affinity for rPfLDH) in kinetic studies using ELONA. LDHp 11, generated against the PfLDH peptide, showed a clear preference for rPfLDH when compared to rPvLDH and other control proteins, in both sets of ELONA studies conducted, as well as EMSA, thus possessing a strong ability to identify the presence of Plasmodium falciparum owing to its generation against the species-specific epitope. While LDHp 1 demonstrated binding to plasmodial LDH in a flow-through system (SPR), so reiterating ELONA responses, it did not perform well in the remaining methodologies. Aptamers rLDH 1 and 15 and LDHp 3, 14 and 18 exhibited a mixed set of results throughout the target protein screening analyses and were, thus, not considered for selective binding in P. falciparum parasite bodies. In studies aimed at exploring biosensor assemblies utilising the developed aptamers, both rLDH 4 and LDHp 11, along with rLDH 7, LDHp 1 and pL1, demonstrated in situ binding to the native PfLDH in fluorescent microscopy. LDHp 11 exhibited FITC-based fluorescence equivalent to the anti-rPfLDHp IgY antibody in confocal fluorescent microscopy indicating superior binding to the native PfLDH compared to the remaining aptamers. An examination of electrochemical impedance as a platform for a biosensor assembly did not, in these studies, exhibit the required sensitivity using physiologically relevant concentrations of analyte expected for pLDH following infection with Plasmodium spp. Malstat/LDH activity was explored for application in a colorimetric aptasensor. A decrease in both rPfLDH and rPvLDH activity was observed following incubation with the tested aptamers, but rLDH 1, rLDH 7 and LDHp 14 did not exhibit similar decreases in rPvLDH activity. Aptamers rLDH 1, 4 and 7 and LDHp 11 and 14 were, therefore, not selected as candidates for LDH capture in LDH activity-based diagnostic devices for P. falciparum. The decreases in pLDH activity in the presence of aptamers could hold promise as direct or antagonistic malaria therapeutic agents. Preliminary studies on the application of aptamers as malaria therapeutic agents, while of interest, should be viewed with due caution given the challenges of aptamers reaching the intracellular native plasmodial LDH hosted within the red blood cells. In conclusion, this work has shown the ability of the LDHp 11 aptamer, generated in these studies, to selectively bind rPfLDH over rPvLDH, and to bind to the native PfLDH in fluorescent microscopy, indicating that this aptamer holds promise as a biorecognition element in malaria biosensors and other diagnostic devices for the detection, and differentiation, of P. falciparum and P. vivax. The use of a species-specific epitope of P. falciparum as a target in aptamer generation paves the way for similar such studies aimed at generating aptamers with species selectivity for other Plasmodium species.
- Full Text:
- Date Issued: 2020
Key considerations for novel aptamer generation and aptasensor platform design: a case study on human α-thrombin and histamine as sensor targets
- Authors: Ho, Lance St John
- Date: 2018
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/63534 , vital:28432
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
- Authors: Ho, Lance St John
- Date: 2018
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/63534 , vital:28432
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
The current utility of oligonucleotide aptamers in targeting the MUC1 mucin tumour marker
- Authors: Flanagan, Shane Patrick
- Date: 2018
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62969 , vital:28348
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
- Authors: Flanagan, Shane Patrick
- Date: 2018
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62969 , vital:28348
- Description: Expected release date-April 2020
- Full Text:
- Date Issued: 2018
Towards a Mobile Bioethanol Unit for point of source conversion of sugar sources to bioethanol: design and feasibility study for South Africa
- Authors: Cech, Alexandra Louise
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/59141 , vital:27439
- Description: Restricted access-thesis embargoed for 5 years
- Full Text:
- Date Issued: 2015
- Authors: Cech, Alexandra Louise
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/59141 , vital:27439
- Description: Restricted access-thesis embargoed for 5 years
- Full Text:
- Date Issued: 2015
Critical studies in carbon electrode materials with applications in the electroanalysis of the mycotoxin citrinin
- Authors: Niland, Michael John
- Date: 2013
- Subjects: Electrodes, Carbon , Mycotoxins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4555 , http://hdl.handle.net/10962/d1018256
- Description: Guided by increasing legislation, the analysis of food borne toxins, including mycotoxins, seeks to address market related demands for the development of analytical systems to monitor this threat to food security and human health. This Thesis is directed at the assessment of the application of electrochemistry for direct electroanalysis and characterisation of the mycotoxin citrinin (CIT) in aqueous media as well as fundamental investigations of the surface of polished and oxidised glassy carbon electrodes (GCE). This study provides the first known account of CIT detection through electrochemical methods. Although electrochemically active, CIT current responses (Ip) were highly irreproducible at polished GCE with a coefficient of variation (C.V.) of 20.16 %. As stability of Ip across multiple electrode preparations is a key requirement in electroanalysis, investigations were directed at attaining stability in CIT Ip. Achieving stability in CIT Ip was investigated via two approaches, including: accounting for Ip variability between electrode preparations as a result of variable GCE surface conditions as a post-data-acquisition analysis and secondly, removing Ip variability through modification of GCE. Accounting for variability in Ip was investigated through the application of double layer capacitance as an indicator of the activity of an electrode, and in so doing serving as a relative mediator of Ip responses between electrodes. Application of this procedure dropped CIT C.V. to a third of starting value across polished GCE (C.V. = 7.18 %), chemically oxidised GCE (Pi-GCE, C.V = 8.47 %) and functionalised multi-walled carbon nanotube modified GCE (fMWCNT, C.V. = 25.79 %) and was effective with analysis of structurally distinct molecules, 2,4-dimethylaniline (2,4-DMA) and 1,2,4-trihydroxybenzene (Triol). Furthermore, it afforded the ability to determine discreet solution overlapping data sets of Ip. Stabilising Ip through GCE surface modification was achieved by anodic electro-oxidation of GCE and allowed for direct electroanalysis of CIT and subsequent characterisation and analysis of CIT in complex media as it reduced C.V. of CIT Ip to 0.73 %. Fundamental investigations of the electrode surface condition are described such that the source of variability could be identified and the interactions of CIT with the electrode understood. Two surface oxidation techniques were applied in modification of GCE; anodic electro-oxidation (EOx GCE) and chemical oxidation using piranha solution (Pi-GCE), analysis of which has previously not been reported. Fundamental analyses to determine surface morphology and chemistry of Pi-GCE, EOx-GCE and polished GCE were conducted using high resolution scanning electron microscopy (HRSEM), scanning electrochemical microscopy (SECM), energy dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FTIR) and via electroanalytical methods. These studies showed that both oxidation procedures introduced a variety of oxide species at GCE surface, and further that the extent of those species was similar with total % O being 27.67 % and 33.47 % at Pi-GCE and EOx-GCE respectively. Although chemically similar, each surface was morphologically distinct. Electrochemical analyses at the surfaces revealed Pi-GCE to behave more similarly to polished GCE than EOx-GCE. As CIT responses were found to be stable at EOx-GCE (C.V. = 0.73 %) as opposed to Pi-GCE (C.V. = 22.87 %), stability of CIT Ip was likely to be as a result of a physical interaction with electrode morphology rather than interaction on a chemical basis. Morphological analyses revealed polished GCE and Pi-GCE to be highly morphologically irregular at the micro-scale. Although comparatively smooth, the surface morphology of EOx-GCE does not account for the stability of Ip. This study thus proposed a theory to describe the mechanism by which the limited conductivity and porosity of EOx-GCE allow for it to provide a relatively stable surface area within the oxide layer, adjacent to the electrode surface, and thus provided a stable platform for electroanalysis. Voltammetric characterization of CIT at EOx-GCE revealed that anodic oxidation in aqueous media involved an uneven number of electrons to protons via an ECE mechanism. This was illustrated to be nt = 2e- accompanied by the transfer of 1H⁺ per molecule oxidised. A proposed reaction scheme for the initial stages of CIT oxidation was suggested to involve both hydroxyl and carboxyl moieties of the CIT molecule. CIT oxidation was shown to arise as a result of a relatively complex mass transport regime which included both adsorptive and diffusive derived Ip₁. The LOD in buffered aqueous media was found to be 16 nM, a highly competitive result in relation to chromatographic techniques. Further application of EOx-GCE in complex media illustrated that CIT associates non-specifically with the components of food samples, primarily proteins. As a result of this, extraction of CIT from such media is mandatory. Liquid-liquid extraction illustrated a recovery in CIT Ip₁ and in so doing provided a means of accurately and sensitively detecting CIT from food samples with an LOD of 20 nM. These responses were corroborated by HPLC analyses on the same extractions and illustrate the applicability of electroanalysis as an analytical technique.
- Full Text:
- Date Issued: 2013
- Authors: Niland, Michael John
- Date: 2013
- Subjects: Electrodes, Carbon , Mycotoxins
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4555 , http://hdl.handle.net/10962/d1018256
- Description: Guided by increasing legislation, the analysis of food borne toxins, including mycotoxins, seeks to address market related demands for the development of analytical systems to monitor this threat to food security and human health. This Thesis is directed at the assessment of the application of electrochemistry for direct electroanalysis and characterisation of the mycotoxin citrinin (CIT) in aqueous media as well as fundamental investigations of the surface of polished and oxidised glassy carbon electrodes (GCE). This study provides the first known account of CIT detection through electrochemical methods. Although electrochemically active, CIT current responses (Ip) were highly irreproducible at polished GCE with a coefficient of variation (C.V.) of 20.16 %. As stability of Ip across multiple electrode preparations is a key requirement in electroanalysis, investigations were directed at attaining stability in CIT Ip. Achieving stability in CIT Ip was investigated via two approaches, including: accounting for Ip variability between electrode preparations as a result of variable GCE surface conditions as a post-data-acquisition analysis and secondly, removing Ip variability through modification of GCE. Accounting for variability in Ip was investigated through the application of double layer capacitance as an indicator of the activity of an electrode, and in so doing serving as a relative mediator of Ip responses between electrodes. Application of this procedure dropped CIT C.V. to a third of starting value across polished GCE (C.V. = 7.18 %), chemically oxidised GCE (Pi-GCE, C.V = 8.47 %) and functionalised multi-walled carbon nanotube modified GCE (fMWCNT, C.V. = 25.79 %) and was effective with analysis of structurally distinct molecules, 2,4-dimethylaniline (2,4-DMA) and 1,2,4-trihydroxybenzene (Triol). Furthermore, it afforded the ability to determine discreet solution overlapping data sets of Ip. Stabilising Ip through GCE surface modification was achieved by anodic electro-oxidation of GCE and allowed for direct electroanalysis of CIT and subsequent characterisation and analysis of CIT in complex media as it reduced C.V. of CIT Ip to 0.73 %. Fundamental investigations of the electrode surface condition are described such that the source of variability could be identified and the interactions of CIT with the electrode understood. Two surface oxidation techniques were applied in modification of GCE; anodic electro-oxidation (EOx GCE) and chemical oxidation using piranha solution (Pi-GCE), analysis of which has previously not been reported. Fundamental analyses to determine surface morphology and chemistry of Pi-GCE, EOx-GCE and polished GCE were conducted using high resolution scanning electron microscopy (HRSEM), scanning electrochemical microscopy (SECM), energy dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), fourier transform infrared spectroscopy (FTIR) and via electroanalytical methods. These studies showed that both oxidation procedures introduced a variety of oxide species at GCE surface, and further that the extent of those species was similar with total % O being 27.67 % and 33.47 % at Pi-GCE and EOx-GCE respectively. Although chemically similar, each surface was morphologically distinct. Electrochemical analyses at the surfaces revealed Pi-GCE to behave more similarly to polished GCE than EOx-GCE. As CIT responses were found to be stable at EOx-GCE (C.V. = 0.73 %) as opposed to Pi-GCE (C.V. = 22.87 %), stability of CIT Ip was likely to be as a result of a physical interaction with electrode morphology rather than interaction on a chemical basis. Morphological analyses revealed polished GCE and Pi-GCE to be highly morphologically irregular at the micro-scale. Although comparatively smooth, the surface morphology of EOx-GCE does not account for the stability of Ip. This study thus proposed a theory to describe the mechanism by which the limited conductivity and porosity of EOx-GCE allow for it to provide a relatively stable surface area within the oxide layer, adjacent to the electrode surface, and thus provided a stable platform for electroanalysis. Voltammetric characterization of CIT at EOx-GCE revealed that anodic oxidation in aqueous media involved an uneven number of electrons to protons via an ECE mechanism. This was illustrated to be nt = 2e- accompanied by the transfer of 1H⁺ per molecule oxidised. A proposed reaction scheme for the initial stages of CIT oxidation was suggested to involve both hydroxyl and carboxyl moieties of the CIT molecule. CIT oxidation was shown to arise as a result of a relatively complex mass transport regime which included both adsorptive and diffusive derived Ip₁. The LOD in buffered aqueous media was found to be 16 nM, a highly competitive result in relation to chromatographic techniques. Further application of EOx-GCE in complex media illustrated that CIT associates non-specifically with the components of food samples, primarily proteins. As a result of this, extraction of CIT from such media is mandatory. Liquid-liquid extraction illustrated a recovery in CIT Ip₁ and in so doing provided a means of accurately and sensitively detecting CIT from food samples with an LOD of 20 nM. These responses were corroborated by HPLC analyses on the same extractions and illustrate the applicability of electroanalysis as an analytical technique.
- Full Text:
- Date Issued: 2013
Fundamental investigations into the factors affecting the response of laccase-based electrochemical biosensors
- Authors: Fogel, Ronen
- Date: 2011
- Subjects: Laccase Phenols Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4073 , http://hdl.handle.net/10962/d1007166
- Description: 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.
- Full Text:
- Date Issued: 2011
- Authors: Fogel, Ronen
- Date: 2011
- Subjects: Laccase Phenols Biosensors
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4073 , http://hdl.handle.net/10962/d1007166
- Description: 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.
- Full Text:
- Date Issued: 2011
Nanomaterial modified electrodes : optimization of voltammetric sensors for pharmaceutical and industrial application
- Authors: Brimecombe, Rory Dennis
- Date: 2011
- Subjects: Voltammetry , Electrochemistry , Nanotubes , Nanostructured materials
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4101 , http://hdl.handle.net/10962/d1009721
- Description: Nanomaterials, in particular carbon nanotubes have been shown to exhibit favourable properties for the enhancement of electrochemical detection of target analytes in complex matrices. There is however scope for improvement in terms of the optimization thereof in electrochemical sensors surface modification. The aim of this thesis was to examine methods that would result in increased current response, lowered passivation and application of such modified surfaces with application to pharmaceutically and industrially relevant analytes. Current methods for enhancing the performance of carbon nanotubes include acid functionalization which not only increases the hydrophilicity of the nanotubes, and consequently their ability to provide stable (aqueous) suspensions, but also introduces electrochemically active sites. This particular approach is however not normalized in the literature. Over-exposure to acid treatment results in loss of structural integrity of the carbon nanotubes, and as such a fine balance exists between achieving these dual outcomes. Guided by high resolution scanning electron microscopy, atomic force microscopy, voltammetric and impedance studies, this thesis examined the role of the length of time of the acid functionalization process as well as the impact of activation of carbon nanotubes and fullerenes on electrochemical sensor performance. Based on desired charge transfer resistances, rate transfer coefficients and sensitivity towards redox probes the optimal length of acid functionalization for multiwalled carbon nanotubes was 9 hours and 4 hours for single-walled carbon nanotubes. Further improvements in the desired outcomes were achieved through electrochemical activation of the modified electrode surface by cycling in the presence of catechol, in a novel approach. By employing electrochemical impedance spectroscopy it was observed that catechol activation resulted in lowered charge transfer resistance, before and after activation, with functionalized multi-walled carbon nanotubes (9 hours) exhibiting the greatest decrease of 90 % and functionalized single-walled carbon nanotubes (4 hours), a 50 % decrease. Corresponding increases in the heterologous rate transfer coefficient showed a 770 % increase for functionalized multi-walled carbon nanotubes (9 hours), following catechol activation. Comparative observations for fullerenes following partial reduction in potassium hydroxide yielded a 30 % decrease in charge transfer resistance, with an increased heterologous rate transfer coefficient at a fullerene modified surface The performance of the nanomaterial modified electrodes was applied to the detection of wortmannin with applications in bioprocess control and in the pharmaceutical sector as well as to the detection and monitoring of the industrial dye Reactive red. Of particular relevance to these analytes was the assessment of the nanomaterial modified electrodes for enhanced stability, reproducibility, sensitivity and decreased passivation effects. In this study the first known account of wortmannin detection through electrochemical methods is reported. Voltammetric characterization of wortmannin revealed an irreversible cathodic process with a total number of 4 electrons and a diffusion coefficient of 1.19 x 10-7 cm².s⁻¹. At a functionalized multiwalled carbon nanotubes modified glassy carbon electrode a limit of detection of 0.128 nmol.cm⁻³ was obtained, and with limited surface passivation the detection scheme afforded pertinent analyses in biological media representing a substantial improvement over chromatographic detection methods. This study also provided the first account of the voltammetric detection of reactive red, competing favourably with traditional spectroscopic methods for monitoring biodegradation of this compound in real time.
- Full Text:
- Date Issued: 2011
- Authors: Brimecombe, Rory Dennis
- Date: 2011
- Subjects: Voltammetry , Electrochemistry , Nanotubes , Nanostructured materials
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4101 , http://hdl.handle.net/10962/d1009721
- Description: Nanomaterials, in particular carbon nanotubes have been shown to exhibit favourable properties for the enhancement of electrochemical detection of target analytes in complex matrices. There is however scope for improvement in terms of the optimization thereof in electrochemical sensors surface modification. The aim of this thesis was to examine methods that would result in increased current response, lowered passivation and application of such modified surfaces with application to pharmaceutically and industrially relevant analytes. Current methods for enhancing the performance of carbon nanotubes include acid functionalization which not only increases the hydrophilicity of the nanotubes, and consequently their ability to provide stable (aqueous) suspensions, but also introduces electrochemically active sites. This particular approach is however not normalized in the literature. Over-exposure to acid treatment results in loss of structural integrity of the carbon nanotubes, and as such a fine balance exists between achieving these dual outcomes. Guided by high resolution scanning electron microscopy, atomic force microscopy, voltammetric and impedance studies, this thesis examined the role of the length of time of the acid functionalization process as well as the impact of activation of carbon nanotubes and fullerenes on electrochemical sensor performance. Based on desired charge transfer resistances, rate transfer coefficients and sensitivity towards redox probes the optimal length of acid functionalization for multiwalled carbon nanotubes was 9 hours and 4 hours for single-walled carbon nanotubes. Further improvements in the desired outcomes were achieved through electrochemical activation of the modified electrode surface by cycling in the presence of catechol, in a novel approach. By employing electrochemical impedance spectroscopy it was observed that catechol activation resulted in lowered charge transfer resistance, before and after activation, with functionalized multi-walled carbon nanotubes (9 hours) exhibiting the greatest decrease of 90 % and functionalized single-walled carbon nanotubes (4 hours), a 50 % decrease. Corresponding increases in the heterologous rate transfer coefficient showed a 770 % increase for functionalized multi-walled carbon nanotubes (9 hours), following catechol activation. Comparative observations for fullerenes following partial reduction in potassium hydroxide yielded a 30 % decrease in charge transfer resistance, with an increased heterologous rate transfer coefficient at a fullerene modified surface The performance of the nanomaterial modified electrodes was applied to the detection of wortmannin with applications in bioprocess control and in the pharmaceutical sector as well as to the detection and monitoring of the industrial dye Reactive red. Of particular relevance to these analytes was the assessment of the nanomaterial modified electrodes for enhanced stability, reproducibility, sensitivity and decreased passivation effects. In this study the first known account of wortmannin detection through electrochemical methods is reported. Voltammetric characterization of wortmannin revealed an irreversible cathodic process with a total number of 4 electrons and a diffusion coefficient of 1.19 x 10-7 cm².s⁻¹. At a functionalized multiwalled carbon nanotubes modified glassy carbon electrode a limit of detection of 0.128 nmol.cm⁻³ was obtained, and with limited surface passivation the detection scheme afforded pertinent analyses in biological media representing a substantial improvement over chromatographic detection methods. This study also provided the first account of the voltammetric detection of reactive red, competing favourably with traditional spectroscopic methods for monitoring biodegradation of this compound in real time.
- Full Text:
- Date Issued: 2011
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