Electrospun fiber based colorimetric probes for aspartate aminotransferase and I7ß-estradiol
- Authors: Pule, Bellah Oreeditse
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54869 , vital:26623
- Description: Fabrication, characterization and application of electrospun polymer composite based colorimetric probes are presented in this thesis. The first part of the thesis involved the development of a protocol for in situ reduction of gold trication (Au³+) into metallic gold atoms with sodium borohydride. The prepared PS-Au NPs showed an SPR band at 542 nm. Furthermore the absorbance of the colloidal Au NPs in polystyrene exhibited a good linear correlation (r2 = 0.9934) to E2 concentration in the range 5 to 50 ppb. The lowest naked eye detection limit was found to be 0.5 ppb and could further be easily monitored by UV-vis spectrophotometer. Upon interaction with E2 Au NPs aggregated to give nanoparticle clusters, confirmed through TEM analysis. Different concentrations of Au NPs were found to have a significant effect on the conductivity of the PS-Au NPs solution. At low concentrations of Au NPs (0.002, 0.015 and 0.025% w/v) PS-Au NPs solution could be electrospun without clogging. The FE-SEM images showed a non-beaded morphology of PS-Au NPs composite fibers. Upon interaction of the colorimetric probe strips with various E2 concentrations it was observed that with increasing E2 concentrations (50 ng/ml to 1000 µg/ml) the colour of the probe changed gradually from white to shades of pink and eventually to shades of blue at higher E2 concentrations. The visible cut-off concentration was 100 ng/ml. The second component of the thesis focussed on the development of diazonium dye-nylon 6 colorimetric probe for aspartate aminotransferase. At optimal pH 7.4 the enzyme was stable, highly active and catalyzed a reaction that was susceptible to detailed kinetic analysis by continuous optical methods. The KM values for L-aspartate, a- ketoglutarate and oxaloacetate were 2.60, 0.59 and 0.066 mM, respectively. On the basis of these KM values the solid-state colorimetric probe was developed. A colour change occurred when an electrospun dye-N 6 probes were exposed to visibly detectable concentrations of oxaloacetate, an AST-catalyzed reaction product. While monitoring AST activity at 530 run, a linear relation was obtained between oxaloacetate concentrations ranging from 0.4 - 7.4 µg/ml. Naked eye detection limit of 2.4 µg/ml oxalaoacetate equivalence of 10 times the normal AST activity was attained. The colorimetric probe was in addition, tested against co-substrates aspartate, ketoglutarate and a variety of other compounds such as alanine, pryruvate, as well as glutamic, malaic and succinic acids known to interfere with AST activity. Each compound elicited a distinct and unambiguous colour change upon interaction with the colorimetric probe. Further X-ray powder diffraction (XRD), duNouy ring tensiometer, Brunauer- Emmett- Teller (BET) and energy dispersive X-ray spectroscopy (EDS/EDX) characterization confirmed composition and stability of the colorimetric probes. Colorimetric probes developed in this thesis are relatively cost effective, simple and "rugged" for measurement of analytes with visual detection without sample pretreatment in matrices, such as plasma and dairy effluents. The probes warrant further investigation as they have shown potential and offer a promising solid-state platform for both clinical diagnostics and environmental monitoring.
- Full Text:
- Date Issued: 2014
- Authors: Pule, Bellah Oreeditse
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54869 , vital:26623
- Description: Fabrication, characterization and application of electrospun polymer composite based colorimetric probes are presented in this thesis. The first part of the thesis involved the development of a protocol for in situ reduction of gold trication (Au³+) into metallic gold atoms with sodium borohydride. The prepared PS-Au NPs showed an SPR band at 542 nm. Furthermore the absorbance of the colloidal Au NPs in polystyrene exhibited a good linear correlation (r2 = 0.9934) to E2 concentration in the range 5 to 50 ppb. The lowest naked eye detection limit was found to be 0.5 ppb and could further be easily monitored by UV-vis spectrophotometer. Upon interaction with E2 Au NPs aggregated to give nanoparticle clusters, confirmed through TEM analysis. Different concentrations of Au NPs were found to have a significant effect on the conductivity of the PS-Au NPs solution. At low concentrations of Au NPs (0.002, 0.015 and 0.025% w/v) PS-Au NPs solution could be electrospun without clogging. The FE-SEM images showed a non-beaded morphology of PS-Au NPs composite fibers. Upon interaction of the colorimetric probe strips with various E2 concentrations it was observed that with increasing E2 concentrations (50 ng/ml to 1000 µg/ml) the colour of the probe changed gradually from white to shades of pink and eventually to shades of blue at higher E2 concentrations. The visible cut-off concentration was 100 ng/ml. The second component of the thesis focussed on the development of diazonium dye-nylon 6 colorimetric probe for aspartate aminotransferase. At optimal pH 7.4 the enzyme was stable, highly active and catalyzed a reaction that was susceptible to detailed kinetic analysis by continuous optical methods. The KM values for L-aspartate, a- ketoglutarate and oxaloacetate were 2.60, 0.59 and 0.066 mM, respectively. On the basis of these KM values the solid-state colorimetric probe was developed. A colour change occurred when an electrospun dye-N 6 probes were exposed to visibly detectable concentrations of oxaloacetate, an AST-catalyzed reaction product. While monitoring AST activity at 530 run, a linear relation was obtained between oxaloacetate concentrations ranging from 0.4 - 7.4 µg/ml. Naked eye detection limit of 2.4 µg/ml oxalaoacetate equivalence of 10 times the normal AST activity was attained. The colorimetric probe was in addition, tested against co-substrates aspartate, ketoglutarate and a variety of other compounds such as alanine, pryruvate, as well as glutamic, malaic and succinic acids known to interfere with AST activity. Each compound elicited a distinct and unambiguous colour change upon interaction with the colorimetric probe. Further X-ray powder diffraction (XRD), duNouy ring tensiometer, Brunauer- Emmett- Teller (BET) and energy dispersive X-ray spectroscopy (EDS/EDX) characterization confirmed composition and stability of the colorimetric probes. Colorimetric probes developed in this thesis are relatively cost effective, simple and "rugged" for measurement of analytes with visual detection without sample pretreatment in matrices, such as plasma and dairy effluents. The probes warrant further investigation as they have shown potential and offer a promising solid-state platform for both clinical diagnostics and environmental monitoring.
- Full Text:
- Date Issued: 2014
The development and implementation of a mental toughness training programme for cricketers
- Authors: McInerney, Michael
- Date: 2014
- Subjects: Sports -- Psychological aspects , Athletes -- Conduct of life , Mental discipline , Achievement motivation
- Language: English
- Type: Thesis , Masters , MA
- Identifier: vital:3234 , http://hdl.handle.net/10962/d1013155
- Description: The professional turn in sports has prompted the search for explanations beyond the physical when discussing quality of performance. Mental Toughness has been recognised as one of the most important psychological attributes in achieving performance excellence across multiple sport settings. Yet, despite this, little research has been conducted into mental toughness development. This research seeks to answer the question: What insights can the development and implementation of a mental toughness programme for a semi-professional South African cricket team provide regarding Mental Toughness development? Based on a theoretical framework of mental toughness in cricket, a programme was developed in collaboration with the Border Cricket Team and Coaches. Incorporating elements that focussed on education, environment, awareness and mental skills, the programme was implemented in both individual and group settings during the off- and pre-seasons. An analysis of the participants’ experiences identified the need for a theoretical understanding of mental toughness within teams, facilitating the design of interventions that integrate the team and the individual. Future programmes should include measures of mastery, and individually customised approaches to mental toughness enhancement that acknowledge the potential contextual and individual variation in mental toughness.
- Full Text:
- Date Issued: 2014
- Authors: McInerney, Michael
- Date: 2014
- Subjects: Sports -- Psychological aspects , Athletes -- Conduct of life , Mental discipline , Achievement motivation
- Language: English
- Type: Thesis , Masters , MA
- Identifier: vital:3234 , http://hdl.handle.net/10962/d1013155
- Description: The professional turn in sports has prompted the search for explanations beyond the physical when discussing quality of performance. Mental Toughness has been recognised as one of the most important psychological attributes in achieving performance excellence across multiple sport settings. Yet, despite this, little research has been conducted into mental toughness development. This research seeks to answer the question: What insights can the development and implementation of a mental toughness programme for a semi-professional South African cricket team provide regarding Mental Toughness development? Based on a theoretical framework of mental toughness in cricket, a programme was developed in collaboration with the Border Cricket Team and Coaches. Incorporating elements that focussed on education, environment, awareness and mental skills, the programme was implemented in both individual and group settings during the off- and pre-seasons. An analysis of the participants’ experiences identified the need for a theoretical understanding of mental toughness within teams, facilitating the design of interventions that integrate the team and the individual. Future programmes should include measures of mastery, and individually customised approaches to mental toughness enhancement that acknowledge the potential contextual and individual variation in mental toughness.
- Full Text:
- Date Issued: 2014
Towards the development of a larval feeding strategy for the white-margined sole (Dagetichthys marginatus)
- Authors: Thompson, Ernst Frederick
- Date: 2014
- Subjects: Flatfishes -- South Africa , Soleidae -- South Africa , Soleidae -- Larvae , Soleidae -- Larvae -- Feeding and feeds , Soleidae -- Larvae -- Physiology , Artemia
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5364 , http://hdl.handle.net/10962/d1012982
- Description: The major bottleneck during the domestication of the white-margined sole Dagetichthys marginatus in South Africa has been low larval survival. The cause of this is not clear but considering current literature on flatfish culture and more specifically soleid culture, nutritional deficiencies are hypothesized to be the main possible cause. Following the importance of nutrition, the first aim of the research was to use an ontogenetic developmental approach to develop a species specific larval feeding strategy. Ontogenetic development of D. marginatus showed that weaning will take place at much later ages than other soleids currently being farmed. This makes the partial replacement of Artemia with a suitable inert diet in co-feeding strategies very important to cut the cost associated of live food production. This leads on to the second aim, in which an 'in vitro' approach was used to model the digestibility of Artemia, which could ultimately contribute towards designing inert feeds with similar digestibility characteristics to Artemia in the future. Obtaining nutrients from food is closely linked to the functional status of the digestive tract, the support organs and the external morphological characteristics required for the ingestion of live or inert feeds. Considering both morphological and physiological ontogenetic development, it is clear that D. marginatus follow a similar pattern to other soleids. Larvae can successfully feed on Artemia as early as 3 days after hatching but exhibit a slow metamorphosis into the juvenile stage when compared to other soleids. The absence of any detectable acidic protease activity during the first 45 days of development and the importance of exogenous enzymes from Artemia all points to limited capacity to digest artificial diets. Prior to modelling the 'in vitro' digestion of Artemia, digestive enzyme activity at different pH's were modelled using functional forms from the normal distributive category of functions. , As there is no substantiated information for the general effects of pH on enzyme activity in the literature for finfish larvae, three species occurring in the same bio-geographical region of Dagetichthys marginatus, namely Sarpa salpa, Diplodus sargus capensis and Argyrosomus japonicus were used to investigate this effect. The fitted parameters, namely the optimal pH and sigma (the slope around the optimal pH) showed two interesting results. When using a negative log likelihood ratio test to test for differences between species for a particular enzyme, the optimal pH for alkaline proteases (7.67), lipase (8.03), amylase (7.69) and phosphatase (9.84) activity was the same for all three species. Furthermore, the study illustrated the potential to detect dietary shifts during ontogenetic development based on changes in enzyme activity around the optimal pH using the sigma parameter. Sarpa salpa showed increased amylase activity and a decrease in protease activity around the optimal pH with increased size, corresponding to a change in diet from zooplankton to algae. The 'in vitro' modelling approach taken in this study was based on known enzyme interactions and dynamics which makes the results very interpretable. from Artemia is achieved based on gut evacuation time and enzyme levels. This 'in vitro' study furthermore clearly indicates the importance of exogenous enzymes from Artemia, contributing as much as 54 percent to protein digestion and 64 – 72 percent to carbohydrate digestion. This was however, only an initial investigation, and further expansion of the model is required to achieve a complete understanding of Artemia digestion and ultimately partial replacement with artificial diets. A feeding strategy for D. marginatus should therefore follow those of other farmed soleids, although there will be a general delay in implementation due to slower development. Problems can thus be solved and improvements made by transferring technology from other soleids to D. marginatus.
- Full Text:
- Date Issued: 2014
- Authors: Thompson, Ernst Frederick
- Date: 2014
- Subjects: Flatfishes -- South Africa , Soleidae -- South Africa , Soleidae -- Larvae , Soleidae -- Larvae -- Feeding and feeds , Soleidae -- Larvae -- Physiology , Artemia
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5364 , http://hdl.handle.net/10962/d1012982
- Description: The major bottleneck during the domestication of the white-margined sole Dagetichthys marginatus in South Africa has been low larval survival. The cause of this is not clear but considering current literature on flatfish culture and more specifically soleid culture, nutritional deficiencies are hypothesized to be the main possible cause. Following the importance of nutrition, the first aim of the research was to use an ontogenetic developmental approach to develop a species specific larval feeding strategy. Ontogenetic development of D. marginatus showed that weaning will take place at much later ages than other soleids currently being farmed. This makes the partial replacement of Artemia with a suitable inert diet in co-feeding strategies very important to cut the cost associated of live food production. This leads on to the second aim, in which an 'in vitro' approach was used to model the digestibility of Artemia, which could ultimately contribute towards designing inert feeds with similar digestibility characteristics to Artemia in the future. Obtaining nutrients from food is closely linked to the functional status of the digestive tract, the support organs and the external morphological characteristics required for the ingestion of live or inert feeds. Considering both morphological and physiological ontogenetic development, it is clear that D. marginatus follow a similar pattern to other soleids. Larvae can successfully feed on Artemia as early as 3 days after hatching but exhibit a slow metamorphosis into the juvenile stage when compared to other soleids. The absence of any detectable acidic protease activity during the first 45 days of development and the importance of exogenous enzymes from Artemia all points to limited capacity to digest artificial diets. Prior to modelling the 'in vitro' digestion of Artemia, digestive enzyme activity at different pH's were modelled using functional forms from the normal distributive category of functions. , As there is no substantiated information for the general effects of pH on enzyme activity in the literature for finfish larvae, three species occurring in the same bio-geographical region of Dagetichthys marginatus, namely Sarpa salpa, Diplodus sargus capensis and Argyrosomus japonicus were used to investigate this effect. The fitted parameters, namely the optimal pH and sigma (the slope around the optimal pH) showed two interesting results. When using a negative log likelihood ratio test to test for differences between species for a particular enzyme, the optimal pH for alkaline proteases (7.67), lipase (8.03), amylase (7.69) and phosphatase (9.84) activity was the same for all three species. Furthermore, the study illustrated the potential to detect dietary shifts during ontogenetic development based on changes in enzyme activity around the optimal pH using the sigma parameter. Sarpa salpa showed increased amylase activity and a decrease in protease activity around the optimal pH with increased size, corresponding to a change in diet from zooplankton to algae. The 'in vitro' modelling approach taken in this study was based on known enzyme interactions and dynamics which makes the results very interpretable. from Artemia is achieved based on gut evacuation time and enzyme levels. This 'in vitro' study furthermore clearly indicates the importance of exogenous enzymes from Artemia, contributing as much as 54 percent to protein digestion and 64 – 72 percent to carbohydrate digestion. This was however, only an initial investigation, and further expansion of the model is required to achieve a complete understanding of Artemia digestion and ultimately partial replacement with artificial diets. A feeding strategy for D. marginatus should therefore follow those of other farmed soleids, although there will be a general delay in implementation due to slower development. Problems can thus be solved and improvements made by transferring technology from other soleids to D. marginatus.
- Full Text:
- Date Issued: 2014
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