School of Chemistry - Theses
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ItemModel chemistry relevant to the molybdenum copper active site of CO dehydrogenase(University of Melbourne, 2007)
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ItemToward the synthesis and analysis of selenium-containing glucocorticoid prodrugs(University of Melbourne, 2007)
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ItemNMR studies of amyloid ab-peptide in membranes(University of Melbourne, 2006)
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ItemBiomaterial porous networks of hydroxyapatite and titanium dioxide( 2014)A gap in a bone that exceeds 2.5 times the bone radius is termed a critical size bone defect and will not heal naturally. The defect needs to be filled with a synthetic bone substitute (a biomaterial scaffold). \(\textit{In situ}\) delivery of medicinal drugs may assist with treating a bone defect, but current drug delivery vehicles (DDVs) are neither able to controllably release drug molecules nor allow for targeted delivery. Porous networks of either hydroxyapatite (HA) or anatase titanium dioxide could be used as biomaterial scaffolds or DDVs. Using sol-gel chemistry and a templating technique, the preparation of such networks, with potential as materials for biomedical applications, forms the research objective. Polyurethane sponge (PU), polyethylenimine-modified polyurethane sponge, polyurethane-agarose gel composite sponge (AG) and natural sea sponge were used as templates for open-celled, porous HA networks. Two concentrations of sol-gel precursor solutions were employed; the higher concentration obscured the template structure in the final network. The choice of template, multiple sol-gel coating, and the rate of temperature increase when removing the template by calcination led to evolution of the HA fibre surface. PU-templated and AG-templated HA networks were contrasted against each other, with the agarose gel component influencing results. All final networks were HA, but other calcium species were present as well. As an initial alternative to the HA networks, titanium dioxide networks were templated on PU sponge, but these lacked both high surface area and mesoporosity. Next, a Type I collagen gel was employed as a template for anatase titanium dioxide networks composed of mesoporous fibres. A standard method for titanium dioxide network preparation is firstly described, where selective solvothermal treatment preceded calcination. This is followed by modified preparations exploring the morphological transition from the collagen to titanium dioxide network structures, and solvothermal fluids containing varying solvent ratios or ammonia. The collagen fibres were 50-100 nm thick, while the titanium dioxide fibres had walls up to 300 nm thick but retained the collagen structure. Compared to networks that only underwent calcination, solvothermal treatment altered the fibre morphology and enhanced the textural properties (surface area, mesopore diameter and total pore volume). Three titanium dioxide networks, previously templated on collagen gel, and spanning a large surface area range were studied for possible biomedical applications. Biomineralisation took place in a simulated body fluid. Apatite grew on each network indicating in vitro bioactivity, but surface area may affect sustained biomineralisation. Ibuprofen drug delivery was monitored by two methods, with a loading of 58.9 mg/g achieved on the highest surface area network. The drug release was modelled as a sustained diffusion mechanism. Ibuprofen could be stored in mesopores or adsorb to the titanium dioxide fibre surface. Overall, HA and titanium dioxide porous networks were fabricated by sol-gel chemistry and templating. In general, morphology and textural properties were influenced by the choice of template, precursor concentrations and processing conditions, including the rate of heating, calcination time and solvothermal treatment. The collagen-templated titanium dioxide networks have potential as materials for biomedical applications.
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ItemPhotoisomerisation action spectroscopy( 2014)A custom ion mobility mass spectrometer has been designed and built to investigate the photoisomerisation of molecular ions in the gas phase. The instrument differs from existing ion mobility mass spectrometers in that there is provision for optical access to the drift region to allow photo-excitation of the drifting ions as they drift. Photoisomerisation manifests as a change in the ions’ drift mobility, altering the time they take to drift through the instrument. By monitoring changes in the arrival time distribution as a function of laser wavelength it is possible to to collect photoisomerisation action spectra. The instrument has been used to probe the photoisomerisation of selected cationic carbocyanine dyes. Comparison of the dyes’ laser-off and laser-on arrival time distributions reveals that these dyes have a multitude of isomers, that can be interconverted using laser light. The peak absorptions of these dyes are shown to be blue-shifted relative to their absorption spectra in solution by 30-50nm. This work demonstrates that ion mobility can form the basis of a new spectroscopic technique for molecular ions that are not responsive to existing methods.
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ItemControlling the microbial deterioration of cultural materials: Investigations into potential free radical, nitric oxide-based treatments( 2014)The biodeterioration of cultural heritage objects can cause adverse changes to the physical and chemical properties of cultural materials. This can lead to a reduction in the integrity of materials and changes to the aesthetic qualities of objects that can have a negative impact on their long-term preservation and may ultimately result in the loss/damage/alteration of unique objects of cultural significance. The biodeterioration of cultural heritage caused by microorganisms is a consequence of the successful establishment and activities communities of microorganism contained within the protective environment of a biofilm. Treatment methods aimed at reducing biofilm formation and enhancing biofilm dispersal can potentially reduce bacterial activity associated with bio-decay. The free-radical molecule nitric oxide (NO•) has been shown to possess both anti-biofilm properties. The approach to and design of investigations that underpin this thesis aimed to assess the potential and suitability of NO• as an intervention material in the treatment of cultural heritage objects. These investigations into NO• based treatments have examined the effects of NO• on biofilm formation, biofilm dispersal and cell motility in test populations that have included a model microorganism Pseudomonas. aeruginosa PAO1 as well as microorganisms representative of those found in association with cultural materials (CMO). The NO• based treatments studied include the nitric oxide donor (Z)- 1-[N-(2-aminoethyl)-N-(2 ammonioethyl) amino]diazen-1-ium- 1,2- diolate (DETA/NO•) and potassium nitrate (KNO3) as a source of nitrate in the bacterial generation of bioactivated NO•. These investigations demonstrate that treatment effects are concentration dependent; are evident at sub-lethal concentrations and are influenced by the duration of a NO• based treatment. Fluorescence based imaging techniques, using confocal laser scanning microscopy (CLSM), have been used to confirm the presence of bioactivated NO• in cells, as well as the properties of biofilms to which NO• based treatments have been administered. The results indicate that NO• based treatments have a dose dependant, non-toxic, anti-biofilm effect on the test populations studied.
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ItemInvestigating the structure and dynamics of DNA with fluorescence and computational techniques( 2014)Nucleic acids, such as DNA, play an essential role in all known forms of life; however, despite their fundamental importance, there is still a significant lack of understanding surrounding their functional behaviour. This thesis explores the structure and dynamics of DNA by employing methods based on fluorescence and through the use of computational calculations. Time-resolved fluorescence experiments have been performed on dinucleotides containing 2-aminopurine (2AP) in various alcohol-water mixtures. 2AP, a fluorescent analogue of the nucleobase adenine, has been used extensively to investigate nucleic acids because of its ability to be incorporated into their structures with minimal perturbation and its high sensitivity to its local environment. Direct solvent effects on 2AP were established through measurements on the free fluorophore. Analysis of the complex fluorescence decays associated with the dinucleotides was challenging but has provided insight into their conformational dynamics. Solvent polarity was found to play a significant role in determining both photophysical and conformational properties in these systems. The complicated fluorescence decay of 2AP in nucleic acids highlights the need for accurate and unbiased analysis methods. Various time-resolved fluorescence analysis methods, including iterative reconvolution and the exponential series method, have been investigated with real and simulated data to obtain an overview of their benefits and limitations. The main outcome of the evaluation is that no single method is preferred in all situations and there is likely to be value in using a combination when there is ambiguity in the interpretation of the results. Regardless of the analysis technique used, the parameterised description of the observed fluorescence decay is meaningless if the underlying physical model is unrealistic. The advance of computational methods has provided a new means to rigorously test the viability of proposed models. Calculations have been performed at the M06-2X/6-31+G(d) level of theory to investigate the stability of 2AP-containing dinucleotides in conformations similar to those observed in the double-helical structure of DNA. The results help to explain the similarity of the time-resolved fluorescence behaviour of 2AP in dinucleotide and DNA systems but also bring to light subtle differences that could perhaps account for experimental discrepancies. The recent emergence of advanced optical microscopy techniques has offered the prospect of being able to directly visualise nucleic acid structure at the nanoscale but, unfortunately, limitations of existing labelling methods have hindered delivery of this potential. To address this issue, a novel strategy has been used to introduce reversible fluorescence photoswitching into DNA at high label density. Photophysical studies have implicated aggregation and energy-transfer as possible quenching mechanisms in this system, which could be detrimental to its future application. The reliability of fluorescence photoswitching was investigated at ensemble and single-molecule level and by performing optical lock-in detection imaging. These developments lay the foundations for improved and sequence-specific super-resolution microscopy of DNA, which could offer new insights into the 3D nanoscale structure of this remarkable biopolymer. In summary, the work presented in this thesis outlines important observations and developments that have been made in the study of the structure and dynamics of nucleic acids.
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ItemUltrasonic formation of stable food emulsions for the delivery of nutrients( 2014)Growing market trend for the consumption of healthy food has led to production of foods rich in bioactives. This thesis reports on the outcomes of an investigation into the formation of stable emulsions of a bioactive, flaxseed oil, in two complex liquid food matrices, namely, skim milk and carrot juice by employing low frequency high intensity ultrasound, particularly in the absence of external food additives, pre-emulsification procedures and external heat and/or pressure. Initially, the study focussed at analysing the process-induced changes to skim milk at sono-emulsification experimental conditions. Sonication of skim milk for up to 8 min (maximum emulsification time) caused minor modifications to about less than 20% of total whey proteins. The modifications to whey proteins is also an advantage since they can act as emulsifiers to stabilise ultrasonically generated flaxseed oil emulsion droplets. The minimal change noticed at short sonication time indicates the safety of the technique. The changes to whey proteins resulted in a decrease in the turbidity of milk but did not affect the viscosity of milk. The casein micelles showed stability against disruption even until 60 min of sonication, except for the very minor changes in their sizes. This work is published in Innovative Food Science and Emerging Technologies and presented in Chapter 3. The feasibility of making finer and stable 7% flaxseed oil-skim milk emulsions was explored at different power levels ranging from 88 to 176 W and processing time from 1 to 8 min. Highly stable emulsions were obtained at 3 min & 176 W and 5 min & 132 W in the absence of external food additives. The emulsion stability test reported a shelf life of 9 days at 4±2ºC against phase separation of emulsions. The efficiency of sono-emulsification was checked up to oil loading of 21%. The factors responsible for emulsion stability were identified to be partially denatured whey proteins and the optimum power-process time combinations employed in the process. The study also drew comparisons between conventional and sono-emulsification processes. High shear mixing and pressure homogenization processes were compared against ultrasound at equivalent energy densities. On comparison, it was observed that at similar energy densities, high shear process of 12.5 min at 70 W & 17500 rpm and pressure homogenization at 200 bar (5 pass) & 655 W did not produce finer and stable emulsions as observed with 5 min of ultrasound process at 176 W. This work is published in Food Hydrocolloids and presented in Chapter 4. After characterization of emulsions, the functionality studies of 7% flaxseed oil-skim milk emulsions were conducted primarily to understand the nature/characteristics of downstreamed products, viz., beverage emulsions (ready-to-drink) and yoghurts (lactic acid gel). Results showed that sonication process did not induce oxidation in emulsions and did not change their viscosity until 9 days of storage at 4±2ºC. Also, the acid gels obtained from milk emulsions showed improved functionalities such as higher gel strength, reduced syneresis, higher storage modulus and reduced gelation time against controls. The evidences obtained suggested that the newly formed fat globular surface (by sonication) stabilized by denatured whey proteins played a major role in functionality, mainly by interacting with other whey proteins and caseins during gelation and formation of network. All the observations helped at arriving at a complete mechanism of gelation of those sono-emulsions. This work is published in Ultrasonics Sonochemistry and presented in Chapter 5. Finally, the feasibility of sonication to disperse flaxseed oil in carrot juice was explored. Stable emulsions of 1% bioactives were obtained at 4 min & 176 W and the size of emulsions were 0.6 μm until 8 days of storage at 4±2ºC. The pectins and/or proteins of carrot juice were responsible for emulsion stability, either by electrostatic repulsion or steric stabilization. The emulsions showed high negative zetapotential values of above -30 mV until 9 days of storage at 4±2ºC. The results of oxidative stability of emulsions study suggested the need for inert atmospheric conditions while processing. It showed that carotenoids exhibited pro-oxidant behaviour mainly because of its concentration, presence of oxygen rich atmosphere and formation of few process-induced free radicals during sonication. The study also looked at delivering flaxseed oil in an aqueous mixture of milk and carrot juice system. The sonication of carrot juice under proposed emulsification conditions also led to many advantages in the juices, such as improvement in cloud stability, increased carotenoid content, reduction in pectin methyl esterase activity, absence of non-enzymatic browning, etc. This work is submitted in Food and Bioprocess Technology and presented in Chapter 6.
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ItemQuantum dots for biological applications( 2014)The use of Quantum Dots (QDs) as an alternative to fluorescent organic dyes for the labelling of biological molecules has attracted a significant amount of interest in recent years. Due to their unique spectral and physical properties, QDs promise to overcome some of the limitations of organic dyes, such as broad emission profiles and low photobleaching thresholds. In this thesis, a flexible platform for the use of QDs in biology is presented. CdSe/ZnS core-shell QDs are rendered water-soluble and biocompatible through a polymer encapsulation technique that ensures colloidal and optical stability, low non-specific binding and that allows the nanocrystal surface to be equipped with a tunable amount of various functional groups (e.g. azides). The conjugation to biomolecules is achieved through the use of bifunctional linkers able to react via strain-promoted click-chemistry with the azide groups present on the QDs. The elements of this conjugation strategy are extremely versatile and can be used in a range of systems. As a proof-of-concept, the protein transferrin is conjugated to QDs and followed during its internalisation path in cell. Next, QDs are conjugated to antibodies and a QD-based ELISA assay is developed. Finally, the concepts are applied to a novel approach for the delivery of photodynamic therapy.
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ItemDevelopment of highly sensitive and selective microfluidic paper-based analytical devices (µPADs) for environmental monitoring( 2014)The increase in world population and the level of industrialisation of many developed and developing countries have affected the quality of the world’s environment significantly since last century. High demand for raw materials and agricultural products has resulted in the release of increasing amounts of mining toxins, fertilisers and biocides. Electronic wastes contain large quantities of hazardous materials, including heavy metals, and their disposal presents huge environmental challenges to all developed and most developing countries. These problems highlight the necessity for low-cost, portable and accurate chemical analysers as indispensable monitoring and analysis tools for environmental protection activities. The objective of the work described in this thesis was to develop microfluidic paper-based analytical devices (µPADs) for the detection of reactive phosphate, ammonia, nitrate/nitrite and copper to meet the increasing need for rapid, sensitive and selective analysis of nutrients in drinking, natural, and industrial and domestic wastewaters. Interest in the determination of reactive phosphate in aquatic systems stems from the realisation in the late-1960s to mid-1970s that phosphorus was a critical nutrient that promoting eutrophication in aquatic systems. A simple µPAD was prepared and evaluated for the determination of reactive phosphate based on the phosphomolybdenum reaction. Hydrophilic reagent zones were defined by printing filter paper with a hydrophobic paper-sizing agent using an inkjet printer. Under optimal conditions, the µPAD is characterised by a working range of 0.1–10 mg L-1 P, and limits of detection (LOD) and quantitation (LOQ) of 0.05 and 0.16 mg L-1 P, respectively. There was no significant difference between the results obtained using the µPAD and reference methods. A simple and effective µPAD for the determination of ammonia in wastewaters was based on gas-diffusion separation combined with colourimetric detection. The sample was spotted onto the µPAD and ammonium ion present in the sample reacted with sodium hydroxide to form molecular ammonia, which diffused through a hydrophobic microporous membrane into an adjacent paper zone containing an acid-base indicator (3-nitrophenol or bromothymol blue). The resultant change in indicator colour was monitored using a desktop scanner and the Red, Green and Blue (RGB) signal was used for quantification. Under optimal conditions, the µPAD gave LOD of 0.8 and 1.8 mg L-1 N and repeatability, expressed as relative standard deviation (RSD) (n ≥ 10), of 3.1% and 3.7% at 20 mg L-1 N for 3-nitrophenol and bromothymol blue, respectively. The small dimensions, minimal reagent consumption, low cost and simplicity of application make the proposed µPAD an attractive option for on-site wastewater process and discharge monitoring. A low-cost disposable colourimetric µPAD was developed for the determination of nitrite and nitrate by the Griess reaction. First, nitrate is reduced to nitrite in the µPAD’s hydrophilic channel using immobilised zinc microparticles. Under optimal conditions, the LOD and LOQ for nitrite are 1.0 and 7.8 µM respectively, while the corresponding values for nitrate are 19 and 48 µM respectively. This µPAD is suitable for on-site measurement of environmental and drinking waters. A µPAD based on a polymer inclusion membrane (PIM) was developed for the selective measurement of Cu(II) in drinking and mine-tailing waters. The PIM consists of di(2-ethlyhexyl) phosphoric acid as the carrier, dioctyl phthalate as a plasticiser, poly(vinyl chloride) as the base polymer and 1-(2’-pyridylazo)-2-naphthol as the colourimetric reagent. Under optimal conditions, the device is characterised by LOD and LOQ of 0.06 and 0.21 mg L-1 Cu(II), respectively. This µPAD was stable for more than 5 months under ambient conditions.