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
Perceptions on ante-mortem welfare, quantitation of pain and pregnancy biomarkers, muscular fibre architecture and quality of Dohne Merino offal
- Authors: Fayemi, Peter Olutope
- Date: 2013
- Subjects: Merino sheep , Slaughtering and slaughter-houses -- By-products , Biochemical markers , Meat -- Quality , Consumers' preferences , Cooking (Variety meats) , Livestock -- Transportation
- Language: English
- Type: Thesis , Doctoral , PhD (Animal Science)
- Identifier: vital:11824 , http://hdl.handle.net/10353/d1007573 , Merino sheep , Slaughtering and slaughter-houses -- By-products , Biochemical markers , Meat -- Quality , Consumers' preferences , Cooking (Variety meats) , Livestock -- Transportation
- Description: Sheep farming is practiced extensively in South Africa for its significant contributions to the livestock, wool and meat industries. The sheep farming sector in the country has approximately 13,800 farmers with commercial and communal sheep farmers making up 58 percent and 42 percent of the entire work force (Directorate of Agricultural Information Services, 2008). An estimate of 28.8 million sheep and flock size ranging between ≤ 50 and ≥ 1800 exist in various South African provinces. Although the national herd size is unevenly distributed provincially most of the herds are found in the Eastern Cape (30 percent) followed by the Northern Cape (25 percent), Free State (20 percent) and the Western Cape (11 percent) respectively (Agriculture, Forestry and Fisheries, 2011). Over twenty indigenous and locally developed sheep breeds are managed where about 69 percent of the land area is available for their grazing nation-wide (Campher et al., 1998; Palmer and Ainslie, 2006). Common among the indigenous breeds are the Afrikaner, Blackhead Persian, Blackhead Speckled Persian, Blinkhaar Ronderib, Damara, Karakul, Namaqua Afrikaner, Pedi, Redhead Persian, Redhead Speckled, Swazi and Zulu. The locally developed breeds include Dorper, Van Rooy and Merinos. The local breeds developed from Merinos consist of the Afrino, Dormer, Dohne Merino and South African mutton Merino (Hammond, 2000; Pranisha, 2004; Hinton, 2006; Sorma et al., 2012). All these sheep breeds are best suited for providing by-products such as wool, meat, hide, milk or a combination of products (Dave and Meadowcroft, 1996; Jensen, 2009). The indigenous and locally developed sheep were bred to meet the growing demand for its by-products (Peters et al., 2010). Expectedly, sheep farmers therefore, make use of the products from these sheep as a means of livelihood and sustenance of a viable local society (Cloete and Olivier, 2010).
- Full Text:
- Date Issued: 2013
- Authors: Fayemi, Peter Olutope
- Date: 2013
- Subjects: Merino sheep , Slaughtering and slaughter-houses -- By-products , Biochemical markers , Meat -- Quality , Consumers' preferences , Cooking (Variety meats) , Livestock -- Transportation
- Language: English
- Type: Thesis , Doctoral , PhD (Animal Science)
- Identifier: vital:11824 , http://hdl.handle.net/10353/d1007573 , Merino sheep , Slaughtering and slaughter-houses -- By-products , Biochemical markers , Meat -- Quality , Consumers' preferences , Cooking (Variety meats) , Livestock -- Transportation
- Description: Sheep farming is practiced extensively in South Africa for its significant contributions to the livestock, wool and meat industries. The sheep farming sector in the country has approximately 13,800 farmers with commercial and communal sheep farmers making up 58 percent and 42 percent of the entire work force (Directorate of Agricultural Information Services, 2008). An estimate of 28.8 million sheep and flock size ranging between ≤ 50 and ≥ 1800 exist in various South African provinces. Although the national herd size is unevenly distributed provincially most of the herds are found in the Eastern Cape (30 percent) followed by the Northern Cape (25 percent), Free State (20 percent) and the Western Cape (11 percent) respectively (Agriculture, Forestry and Fisheries, 2011). Over twenty indigenous and locally developed sheep breeds are managed where about 69 percent of the land area is available for their grazing nation-wide (Campher et al., 1998; Palmer and Ainslie, 2006). Common among the indigenous breeds are the Afrikaner, Blackhead Persian, Blackhead Speckled Persian, Blinkhaar Ronderib, Damara, Karakul, Namaqua Afrikaner, Pedi, Redhead Persian, Redhead Speckled, Swazi and Zulu. The locally developed breeds include Dorper, Van Rooy and Merinos. The local breeds developed from Merinos consist of the Afrino, Dormer, Dohne Merino and South African mutton Merino (Hammond, 2000; Pranisha, 2004; Hinton, 2006; Sorma et al., 2012). All these sheep breeds are best suited for providing by-products such as wool, meat, hide, milk or a combination of products (Dave and Meadowcroft, 1996; Jensen, 2009). The indigenous and locally developed sheep were bred to meet the growing demand for its by-products (Peters et al., 2010). Expectedly, sheep farmers therefore, make use of the products from these sheep as a means of livelihood and sustenance of a viable local society (Cloete and Olivier, 2010).
- Full Text:
- Date Issued: 2013
Neuronal nitric oxide synthase : a biomarker for Alzheimers disease : interaction of neuronal nitric oxide synthase with beta-amyloid peptides in the brain
- Authors: Padayachee, Eden Rebecca
- Date: 2011 , 2013-07-19
- Subjects: Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4086 , http://hdl.handle.net/10962/d1007677 , Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Description: High levels of the amino acid arginine and low levels of the product citrulline in the cerebrospinal fluid of Alzheimer's patients could mean that there is a decrease in the enzymes that metabolize this amino acid. One such enzyme is neuronal nitric oxide synthase (nNOS). In this study, neuronal nitric oxide synthase (nNOS), sourced from bovine brain was extracted and concentrated using two methods of precipitation: poly (ethylene glycol) 20 000 (PEG) and ammonium sulphate [(NH₄)₂S0₄). These two techniques gave no increase in yield nor fold purification and hence were abandoned in favour of ion exchange chromatography by DEAE-Sepharose. The enzyme was then successfully purified by anion-exchange and after dialysis produced a 38% yield and three fold purification and yielded the highest specific activity of 2.27 U/mg. Neuronal nitric oxide synthase (nNOS) was a heterodimeric protein with a total molecular mass of ± 225 kDa (95 and 130 kDa monomers). The temperature and pH optima of the enzyme were 40⁰C and 6.5, respectively. The kinetic parameters (KM and Vmax) of nNOS were 70 μM and 0.332 μmol.min⁻¹, respectively. Moreover neuronal nitric oxide synthase (nNOS) was relatively stable at 40⁰C (t½ = 3 h). It was also confirmed that β-amyloid peptides inhibited nNOS when bound to the enzyme and that nNOS behaved as a catalyst in fibril formation through association-dissociation between enzyme and β-amyloid peptide. It was further shown that Aβ₁₇₋₂₈ inhibited nNOS the most with a Ki of 1.92 μM and also had the highest Stern-Volmer value (Ksv) of 0.11 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. Congo red, turbidity, thioflavin-T assays and transmission electron microscopy were successfully used to detect and visualize the presence of fibrils by studying the process of fibrillogenesis. Computerized molecular modeling successfully studied protein dynamics and conformational changes of nNOS. These results correlated with resonance energy transfer (FRET) results which revealed the distance of tryptophan residues from the arginine bound at enzyme active site. Both the aforementioned techniques revealed that in the natural state of the enzyme with arginine bound at the active site, the tryptophan residues (TRP₆₂₅ and TRP₇₂₁) were positioned at the surface of the enzyme 28 Å away from the active site. When the amyloid peptide (Aβ₁₇₋₂₈) was bound to the active site, these same two amino acids moved 14 Å closer to the active site. A five residue hydrophobic fragment Aβ₁₇₋₂₁ [Leu₁₇ - Val₁₈ - Phe₁₉ - Phe₂₀ - Ala₁] within Aβ₁₇₋₂₈ was shown by computer modeling to be critical to the binding of the peptide to the active site of nNOS.
- Full Text:
- Date Issued: 2011
- Authors: Padayachee, Eden Rebecca
- Date: 2011 , 2013-07-19
- Subjects: Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4086 , http://hdl.handle.net/10962/d1007677 , Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Description: High levels of the amino acid arginine and low levels of the product citrulline in the cerebrospinal fluid of Alzheimer's patients could mean that there is a decrease in the enzymes that metabolize this amino acid. One such enzyme is neuronal nitric oxide synthase (nNOS). In this study, neuronal nitric oxide synthase (nNOS), sourced from bovine brain was extracted and concentrated using two methods of precipitation: poly (ethylene glycol) 20 000 (PEG) and ammonium sulphate [(NH₄)₂S0₄). These two techniques gave no increase in yield nor fold purification and hence were abandoned in favour of ion exchange chromatography by DEAE-Sepharose. The enzyme was then successfully purified by anion-exchange and after dialysis produced a 38% yield and three fold purification and yielded the highest specific activity of 2.27 U/mg. Neuronal nitric oxide synthase (nNOS) was a heterodimeric protein with a total molecular mass of ± 225 kDa (95 and 130 kDa monomers). The temperature and pH optima of the enzyme were 40⁰C and 6.5, respectively. The kinetic parameters (KM and Vmax) of nNOS were 70 μM and 0.332 μmol.min⁻¹, respectively. Moreover neuronal nitric oxide synthase (nNOS) was relatively stable at 40⁰C (t½ = 3 h). It was also confirmed that β-amyloid peptides inhibited nNOS when bound to the enzyme and that nNOS behaved as a catalyst in fibril formation through association-dissociation between enzyme and β-amyloid peptide. It was further shown that Aβ₁₇₋₂₈ inhibited nNOS the most with a Ki of 1.92 μM and also had the highest Stern-Volmer value (Ksv) of 0.11 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. Congo red, turbidity, thioflavin-T assays and transmission electron microscopy were successfully used to detect and visualize the presence of fibrils by studying the process of fibrillogenesis. Computerized molecular modeling successfully studied protein dynamics and conformational changes of nNOS. These results correlated with resonance energy transfer (FRET) results which revealed the distance of tryptophan residues from the arginine bound at enzyme active site. Both the aforementioned techniques revealed that in the natural state of the enzyme with arginine bound at the active site, the tryptophan residues (TRP₆₂₅ and TRP₇₂₁) were positioned at the surface of the enzyme 28 Å away from the active site. When the amyloid peptide (Aβ₁₇₋₂₈) was bound to the active site, these same two amino acids moved 14 Å closer to the active site. A five residue hydrophobic fragment Aβ₁₇₋₂₁ [Leu₁₇ - Val₁₈ - Phe₁₉ - Phe₂₀ - Ala₁] within Aβ₁₇₋₂₈ was shown by computer modeling to be critical to the binding of the peptide to the active site of nNOS.
- Full Text:
- Date Issued: 2011
Selective and sensitive electrochemical detection of the Human Epidermal Growth Receptor 2 breast cancer biomarker, using Co (II) phthalocyanine-nanoparticle based platforms
- Centane, Sixolile Sibongiseni
- Authors: Centane, Sixolile Sibongiseni
- Date: 2024-10-11
- Subjects: Electrochemical sensors , HER-2 protein , Breast Cancer , Biochemical markers , Phthalocyanines , Nanoparticles
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/466569 , vital:76753 , DOI https://doi.org/10.21504/10962/466570
- Description: Breast cancer is the world’s leading cause of cancer related deaths in women worldwide. The main reason lies in its late detection, mostly in the metastatic stage resulting in poor after-therapy prognosis, despite advances in methods of diagnosis and therapy. The reason for late-stage detection, is because breast cancer like any other cancers is asymptomatic in its early stages. Significant and characterizable features present in the later stages. Furthermore, conventional methods for breast cancer detection are more useful in the identification of the phenotypic features of cancer cells that arise at a later stage of the disease. Another issue with conventional methods where cancer diagnosis is concerned is that they tend to be specialist-dependent, time consuming and costly. Thus, easy, fast and inexpensive detection methods need to be developed urgently. Biomarker-based cancer diagnosis has emerged as one of the most promising strategies for early diagnosis, monitoring disease progression, and subsequent cancer treatment. This thesis focuses on the design and development of novel electrochemical biosensor platforms towards the low cost, efficient, sensitive and simple detection of early-stage breast cancer biomarker, human epidermal growth factor 2 (HER2). The electrochemical method is preferred because of its moderate cost, rapid response, ease of operation, readily quantifiable signal as well as high sensitivity and selectivity with lower detection limits. This thesis reports on two strategies towards signal amplification and sensitive detection of HER2, namely signal based amplification and target-based amplification. The former focuses on electrode or transducer modification techniques for improved signal to noise ratio. In which case; novel nanocomposites of phthalocyanines, graphene quantum dots, gold nanoparticles and cerium oxide nanoparticles are used for electrode modification for signal amplification and biorecognition element immobilization. The biorecognition elements of choice, are an aptamer and antibody known to be specific to the HER2 antigen for an enhanced sensor sensitivity and specificity. The second strategy focuses on increasing the number of detectable targets on the electrode surface towards enhanced sensitivity, precision and sensor accuracy. In which case; the performance of the aptamer and the antibody as recognition elements was explored. Furthermore, the effect of arrangement of these recognition elements on the electrode surface is investigated and reported upon. The strategies covered in this thesis are expected to result in novel biosensor platforms that can detect the HER2 biomarker with high precision, reproducibility, sensitivity and stability; towards low cost and effective early-stage breast cancer diagnostic tools. , Thesis (PhD) -- Faculty of Science, Chemistry, 2024
- Full Text:
- Date Issued: 2024-10-11
- Authors: Centane, Sixolile Sibongiseni
- Date: 2024-10-11
- Subjects: Electrochemical sensors , HER-2 protein , Breast Cancer , Biochemical markers , Phthalocyanines , Nanoparticles
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/466569 , vital:76753 , DOI https://doi.org/10.21504/10962/466570
- Description: Breast cancer is the world’s leading cause of cancer related deaths in women worldwide. The main reason lies in its late detection, mostly in the metastatic stage resulting in poor after-therapy prognosis, despite advances in methods of diagnosis and therapy. The reason for late-stage detection, is because breast cancer like any other cancers is asymptomatic in its early stages. Significant and characterizable features present in the later stages. Furthermore, conventional methods for breast cancer detection are more useful in the identification of the phenotypic features of cancer cells that arise at a later stage of the disease. Another issue with conventional methods where cancer diagnosis is concerned is that they tend to be specialist-dependent, time consuming and costly. Thus, easy, fast and inexpensive detection methods need to be developed urgently. Biomarker-based cancer diagnosis has emerged as one of the most promising strategies for early diagnosis, monitoring disease progression, and subsequent cancer treatment. This thesis focuses on the design and development of novel electrochemical biosensor platforms towards the low cost, efficient, sensitive and simple detection of early-stage breast cancer biomarker, human epidermal growth factor 2 (HER2). The electrochemical method is preferred because of its moderate cost, rapid response, ease of operation, readily quantifiable signal as well as high sensitivity and selectivity with lower detection limits. This thesis reports on two strategies towards signal amplification and sensitive detection of HER2, namely signal based amplification and target-based amplification. The former focuses on electrode or transducer modification techniques for improved signal to noise ratio. In which case; novel nanocomposites of phthalocyanines, graphene quantum dots, gold nanoparticles and cerium oxide nanoparticles are used for electrode modification for signal amplification and biorecognition element immobilization. The biorecognition elements of choice, are an aptamer and antibody known to be specific to the HER2 antigen for an enhanced sensor sensitivity and specificity. The second strategy focuses on increasing the number of detectable targets on the electrode surface towards enhanced sensitivity, precision and sensor accuracy. In which case; the performance of the aptamer and the antibody as recognition elements was explored. Furthermore, the effect of arrangement of these recognition elements on the electrode surface is investigated and reported upon. The strategies covered in this thesis are expected to result in novel biosensor platforms that can detect the HER2 biomarker with high precision, reproducibility, sensitivity and stability; towards low cost and effective early-stage breast cancer diagnostic tools. , Thesis (PhD) -- Faculty of Science, Chemistry, 2024
- Full Text:
- Date Issued: 2024-10-11
Design of Immunobiosensors for Detection of Tumor-Associated Anti-P53 Autoantibodies: Method Development
- Authors: Adeniyi, Omotayo Kayode
- Date: 2020
- Subjects: Lungs Cancer Early detection , Autoantibodies , Biosensors , Immunochemistry , Biochemical markers , p53 antioncogene
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/162988 , vital:41002 , 10.21504/10962/162988
- Description: Thesis (PhD)--Rhodes University, Science Faculty, Department of Chemistry, 2020. , Detection and profiling of circulating tumor-associated autoantibodies (TAAbs) are useful for screening and early-stage diagnosis of asymptomatic lung cancer. Immunobiosensor technologies aimed to accomplish the highly sensitive, rapid and low-cost detection of TAAbs can improve the early-stage detection of lung cancer. Immunobiosensors for the detection of anti-P53-tumour associated autoantibodies have been developed in this work. The design of sensing interfaces with immobilized P53 protein (P53ag) as a sensing element layer on a solid interface was investigated. Several methods of detecting anti-P53-antibodies (anti-P53ab) were investigated. These methods are label-free detection using electrochemical impedance spectroscopy (EIS) and two label techniques. The label-free electrochemical techniques utilize gold electrode pre-modified with a conducting layer of electrochemically grafted phenylethylamine for covalent immobilization of P53ag. The limit of anti-P53ab detection with the label-free EIS was 103.0 pg.ml-1. The labeled technique developed utilizes fluorescent, and peroxidase-like nanomaterial labeled antibody as a detection probe. For the fluorescence detection, fluorescent silica nanoparticles were synthesized by overloading FITC into the silica matrix and conjugated to detection antibody (anti-IgG). The detection of the anti-P53ab was based on the dissolution of the silica nanoparticles to release the loaded dye as a signal amplification strategy. The fluorescence detection was carried out on a microplate, and magnetic bead modified P53-antigen platforms and limit of detection (LoD) were 42.0 fg.ml-1 and 3.3 fg.ml-1 for anti-P53ab; respectively. Fe3O4@SiNP-APTES-Au@Pd hybrid nanoparticles were synthesized, and their peroxidase-like activity and colorimetric detection were evaluated. The Fe3O4@SiNP-APTES-Au@Pd exhibited comparable activity to HRP. The Fe3O4@SiNP-APTES-Au@Pd was conjugated to protein-G-anti-IgG for the detection of anti-P53ab on a microplate and cellulose paper platforms. The LoD was 20.0 fg.ml-1 and 63.0 fg.ml-1 for the microplate and cellulose paper platform; respectively. The potential application of the designed immunobiosensor was evaluated in simulated serum samples. The developed sensors showed higher detection sensitivity, stability and had a lower detection limit for anti-P53ab when compared with the ELISA based detection. The results have provided alternative and effective quantification approaches to ELISA and a promising future for multiplexed detection of tumor-associated autoantibodies. The developed methodologies in this thesis could be applied for the detection of other autoantibodies in other cancer types and auto-immune diseases.
- Full Text:
- Date Issued: 2020
- Authors: Adeniyi, Omotayo Kayode
- Date: 2020
- Subjects: Lungs Cancer Early detection , Autoantibodies , Biosensors , Immunochemistry , Biochemical markers , p53 antioncogene
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/162988 , vital:41002 , 10.21504/10962/162988
- Description: Thesis (PhD)--Rhodes University, Science Faculty, Department of Chemistry, 2020. , Detection and profiling of circulating tumor-associated autoantibodies (TAAbs) are useful for screening and early-stage diagnosis of asymptomatic lung cancer. Immunobiosensor technologies aimed to accomplish the highly sensitive, rapid and low-cost detection of TAAbs can improve the early-stage detection of lung cancer. Immunobiosensors for the detection of anti-P53-tumour associated autoantibodies have been developed in this work. The design of sensing interfaces with immobilized P53 protein (P53ag) as a sensing element layer on a solid interface was investigated. Several methods of detecting anti-P53-antibodies (anti-P53ab) were investigated. These methods are label-free detection using electrochemical impedance spectroscopy (EIS) and two label techniques. The label-free electrochemical techniques utilize gold electrode pre-modified with a conducting layer of electrochemically grafted phenylethylamine for covalent immobilization of P53ag. The limit of anti-P53ab detection with the label-free EIS was 103.0 pg.ml-1. The labeled technique developed utilizes fluorescent, and peroxidase-like nanomaterial labeled antibody as a detection probe. For the fluorescence detection, fluorescent silica nanoparticles were synthesized by overloading FITC into the silica matrix and conjugated to detection antibody (anti-IgG). The detection of the anti-P53ab was based on the dissolution of the silica nanoparticles to release the loaded dye as a signal amplification strategy. The fluorescence detection was carried out on a microplate, and magnetic bead modified P53-antigen platforms and limit of detection (LoD) were 42.0 fg.ml-1 and 3.3 fg.ml-1 for anti-P53ab; respectively. Fe3O4@SiNP-APTES-Au@Pd hybrid nanoparticles were synthesized, and their peroxidase-like activity and colorimetric detection were evaluated. The Fe3O4@SiNP-APTES-Au@Pd exhibited comparable activity to HRP. The Fe3O4@SiNP-APTES-Au@Pd was conjugated to protein-G-anti-IgG for the detection of anti-P53ab on a microplate and cellulose paper platforms. The LoD was 20.0 fg.ml-1 and 63.0 fg.ml-1 for the microplate and cellulose paper platform; respectively. The potential application of the designed immunobiosensor was evaluated in simulated serum samples. The developed sensors showed higher detection sensitivity, stability and had a lower detection limit for anti-P53ab when compared with the ELISA based detection. The results have provided alternative and effective quantification approaches to ELISA and a promising future for multiplexed detection of tumor-associated autoantibodies. The developed methodologies in this thesis could be applied for the detection of other autoantibodies in other cancer types and auto-immune diseases.
- Full Text:
- Date Issued: 2020
Prevalence, associated risk factors and diagnostic biomarkers of schistosomiasis among school going children in Nelson Mandela Bay Municipality
- Authors: Vere, Maryline
- Date: 2025-04
- Subjects: Schistosomiasis , Schistosomiasis in children , Biochemical markers
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/75066 , vital:79879
- Description: Schistosomiasis is a significant parasitic infection impacting children in socioeconomically disadvantaged regions. The study aimed to determine the prevalence and risk factors, including knowledge, attitudes, and practices (KAP), associated with schistosomiasis among school-going children aged 5 to 14 years in Nelson Mandela Bay (NMB), South Africa. Additionally, the research assessed the diagnostic efficacy of conventional and advanced methods to improve detection accuracy. Methods: A comprehensive cross-sectional study was conducted to determine the prevalence, risk factors, and diagnostic biomarkers associated with schistosomiasis among school-going children aged 5–14 years in Nelson Mandela Bay (NMB). The study was structured into three distinct phases: phase 1 included assessing risk factors and knowledge, attitudes, and practices (KAP), phase 2 evaluated the prevalence of schistosomiasis, and phase 3 profiling diagnostic urinary biomarkers. In Phase 1, structured, close-ended, interview-administered questionnaires were used to assess socio-demographic characteristics, environmental risk factors, and KAP related to schistosomiasis. Data were analysed to identify factors influencing KAP and their associations with demographic and behavioural variables. In Phase 2, urine and stool samples were collected to diagnose urogenital and intestinal schistosomiasis. Urine analysis involved the use of dipsticks to detect haematuria, followed by urine filtration to identify S. haematobium eggs. Haematuria-positive samples (2+ and 3+) underwent molecular diagnostics (cPCR) targeting the Dra1 repeat sequence for cell-free DNA (cfDNA) confirmation. Stool analysis focused on detecting S. mansoni infections using Kato-Katz and POC-CCA tests, with cPCR targeting the cox-1 gene performed on POC-CCA-positive samples to confirm S. mansoni. In Phase 3, urinalysis was conducted using Siemens Multistix 10SG dipsticks to measure urinary biomarkers such as proteins, leukocytes, bilirubin, ketones, and glucose. Correlations among these biomarkers were evaluated to explore their potential association with schistosomiasis. A total of 759 children (58% males, 42% females; mean age 11 ± 1.5 years) participated in the study. In Phase 1, the study revealed poor knowledge, attitudes, and practices (KAP) among participants, with only 11% demonstrating knowledge about schistosomiasis. Key risk factors included swimming (44%), living near water bodies (21.1%), using bush toilets (5.1%), lacking access to household tap water (4.3%), and fishing (0.3%). Knowledge scores were significantly influenced by gender (p = 0.015) and grade level (p = 0.045), with females scoring lower than males (β = -0.15; p = 0.018). Attitude (p = 0.023) and practice (p = 0.001) scores improved in higher grades, though children in grades 4–7 displayed fewer positive attitudes (β = 0.07; p = 0.038) and practices (β = 0.11; p = 0.001). Unawareness of disease transmission was associated with lower knowledge (β = -0.24; p = 0.091) and attitude scores (β = -0.22; p = 0.001). Using a neighbour’s toilet was linked to significantly lower knowledge (β = -0.55; p = 0.020). In Phase 2, haematuria was detected in 33.6% of urine samples, but traditional urine filtration identified only one egg-positive case (0.1%). Molecular diagnostics confirmed S. haematobium in 31.4% of haematuria-positive samples. For S. mansoni, no infections were detected using Kato-Katz, but POC-CCA indicated a prevalence of 3.2%, with 32.1% of these confirmed by cPCR. In Phase 3, haematuria (33.6%) was the most prevalent biomarker, followed by leukocytes (21.3%) and proteins (15%). Other biomarkers, including bilirubin and ketones, were detected at lower frequencies. A strong correlation was observed between haematuria and leukocytes. Conclusion: This multi-phase study highlights poor KAP and significant environmental risk factors for schistosomiasis transmission. Molecular diagnostics proved more sensitive than traditional methods in detecting schistosomiasis, emphasizing the underestimation of disease burden in lowtransmission settings. Urinalysis provided additional insights into biomarkers potentially linked to schistosomiasis, paving the way for targeted control measures and further research in endemic areas. This study highlights the limitations of traditional diagnostic techniques in lowtransmission settings, emphasizing the need for sensitive molecular methods like cPCR to detect low-intensity infections. The significant prevalence of urogenital schistosomiasis identified through advanced diagnostics underscores ongoing transmission risks in NMB especially among school-going children. The findings stress the necessity for targeted educational programs to improve KAP, public health interventions tailored to mitigate risk factors, preventive chemotherapy with praziquantel and water sanitation and hygiene practices interventions among the school-going children in the study area. Urine reagent strips show promise as cost-effective initial screening tools, yet further validation with comprehensive diagnostics is recommended to enhance early detection and use in monitoring control efforts. , Thesis (DPhil) -- Faculty of Health Sciences, School of Behavioural & Lifestyle Sciences, 2025
- Full Text:
- Date Issued: 2025-04
- Authors: Vere, Maryline
- Date: 2025-04
- Subjects: Schistosomiasis , Schistosomiasis in children , Biochemical markers
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/75066 , vital:79879
- Description: Schistosomiasis is a significant parasitic infection impacting children in socioeconomically disadvantaged regions. The study aimed to determine the prevalence and risk factors, including knowledge, attitudes, and practices (KAP), associated with schistosomiasis among school-going children aged 5 to 14 years in Nelson Mandela Bay (NMB), South Africa. Additionally, the research assessed the diagnostic efficacy of conventional and advanced methods to improve detection accuracy. Methods: A comprehensive cross-sectional study was conducted to determine the prevalence, risk factors, and diagnostic biomarkers associated with schistosomiasis among school-going children aged 5–14 years in Nelson Mandela Bay (NMB). The study was structured into three distinct phases: phase 1 included assessing risk factors and knowledge, attitudes, and practices (KAP), phase 2 evaluated the prevalence of schistosomiasis, and phase 3 profiling diagnostic urinary biomarkers. In Phase 1, structured, close-ended, interview-administered questionnaires were used to assess socio-demographic characteristics, environmental risk factors, and KAP related to schistosomiasis. Data were analysed to identify factors influencing KAP and their associations with demographic and behavioural variables. In Phase 2, urine and stool samples were collected to diagnose urogenital and intestinal schistosomiasis. Urine analysis involved the use of dipsticks to detect haematuria, followed by urine filtration to identify S. haematobium eggs. Haematuria-positive samples (2+ and 3+) underwent molecular diagnostics (cPCR) targeting the Dra1 repeat sequence for cell-free DNA (cfDNA) confirmation. Stool analysis focused on detecting S. mansoni infections using Kato-Katz and POC-CCA tests, with cPCR targeting the cox-1 gene performed on POC-CCA-positive samples to confirm S. mansoni. In Phase 3, urinalysis was conducted using Siemens Multistix 10SG dipsticks to measure urinary biomarkers such as proteins, leukocytes, bilirubin, ketones, and glucose. Correlations among these biomarkers were evaluated to explore their potential association with schistosomiasis. A total of 759 children (58% males, 42% females; mean age 11 ± 1.5 years) participated in the study. In Phase 1, the study revealed poor knowledge, attitudes, and practices (KAP) among participants, with only 11% demonstrating knowledge about schistosomiasis. Key risk factors included swimming (44%), living near water bodies (21.1%), using bush toilets (5.1%), lacking access to household tap water (4.3%), and fishing (0.3%). Knowledge scores were significantly influenced by gender (p = 0.015) and grade level (p = 0.045), with females scoring lower than males (β = -0.15; p = 0.018). Attitude (p = 0.023) and practice (p = 0.001) scores improved in higher grades, though children in grades 4–7 displayed fewer positive attitudes (β = 0.07; p = 0.038) and practices (β = 0.11; p = 0.001). Unawareness of disease transmission was associated with lower knowledge (β = -0.24; p = 0.091) and attitude scores (β = -0.22; p = 0.001). Using a neighbour’s toilet was linked to significantly lower knowledge (β = -0.55; p = 0.020). In Phase 2, haematuria was detected in 33.6% of urine samples, but traditional urine filtration identified only one egg-positive case (0.1%). Molecular diagnostics confirmed S. haematobium in 31.4% of haematuria-positive samples. For S. mansoni, no infections were detected using Kato-Katz, but POC-CCA indicated a prevalence of 3.2%, with 32.1% of these confirmed by cPCR. In Phase 3, haematuria (33.6%) was the most prevalent biomarker, followed by leukocytes (21.3%) and proteins (15%). Other biomarkers, including bilirubin and ketones, were detected at lower frequencies. A strong correlation was observed between haematuria and leukocytes. Conclusion: This multi-phase study highlights poor KAP and significant environmental risk factors for schistosomiasis transmission. Molecular diagnostics proved more sensitive than traditional methods in detecting schistosomiasis, emphasizing the underestimation of disease burden in lowtransmission settings. Urinalysis provided additional insights into biomarkers potentially linked to schistosomiasis, paving the way for targeted control measures and further research in endemic areas. This study highlights the limitations of traditional diagnostic techniques in lowtransmission settings, emphasizing the need for sensitive molecular methods like cPCR to detect low-intensity infections. The significant prevalence of urogenital schistosomiasis identified through advanced diagnostics underscores ongoing transmission risks in NMB especially among school-going children. The findings stress the necessity for targeted educational programs to improve KAP, public health interventions tailored to mitigate risk factors, preventive chemotherapy with praziquantel and water sanitation and hygiene practices interventions among the school-going children in the study area. Urine reagent strips show promise as cost-effective initial screening tools, yet further validation with comprehensive diagnostics is recommended to enhance early detection and use in monitoring control efforts. , Thesis (DPhil) -- Faculty of Health Sciences, School of Behavioural & Lifestyle Sciences, 2025
- Full Text:
- Date Issued: 2025-04
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