Reducing equivalents were extracted from in vitro photosynthesis and used to drive cell-free and enzyme-free biochemical reduction reactions in this research. To investigate photosynthetic electron flow, an algal extract dense in chloroplasts was made from the microalga Scenedesmus sp. A6. The algal extract was subjugated to a variety of environmental parameters and exogenous quinones in order to optimize electron extraction. To monitor electron extraction and donation to metabolites, a novel assay was created that monitored the chemiluminescence (CL) produced by superoxide radicals formed during the process. In particular, these formed when a reduced exogenous quinone oxidized spontaneously. Our studies found that calcium chloride improved the reduction of low redox potential mediators along with prolonged exposure to red light. Other salts and environmental conditions examined had diverse effects on the quinones based on structure, redox potential, and site of electron extraction. We next applied our assay for monitoring the reduction of different metabolites. The CL recorded for different metabolites was compared to the Gibbs free energy of reduction and a highly correlated relationship was found. The assay was then applied to the reduction of metabolites via the oxidation of glucose in an alkaline environment. To exhibit the diverse application of the CL assay, urine of healthy individuals, patients with chronic kidney disease (CKD), and patients with bladder cancer (BCa) were characterized through their interactions with different quinones. The CL output was compared to that of SurineTM and urea followed by ANOVA analysis. Statistically significant differences were found for all quinones with 1,2-napthoquinone-4-sulfonate (NQS) producing significant differences between all groups examined. Monitoring algal phenotypes for biofuels or photosynthetic output requires arduous protocols and advanced instrumentation. Both of these energy producing options were explored along with rapid, high-throughput protocols for measuring reduction reactions. To monitor the phenotypes and health of our microalgae, Raman microscopy was applied to algal cultures of Scenedesmus sp. A6 grown under stress. Statistically unique phenotypes were found based on environmental factors during cell growth. ANOVA analysis determined the effect of stressors that caused significant change to algal phenotypes related to photosynthesis and lipids. / Doctor of Philosophy / Photosynthesis is the process by which plants and algae harness sunlight to convert CO2 to plant mass. Photosynthesis is performed in the chloroplast which can excite electrons and use them to generate energy. Detecting how much energy a chloroplast can produce and what chemicals effect the chloroplast requires complex procedures with complicated instruments. In this thesis the chloroplast from the microalgae Scenedesmus sp. A6 were isolated to evaluate how they are affected by different chemicals in the environment using a new, rapid and robust assay. Then, a group of chemicals called quinones were used to steal electrons (plant energy), and this process was optimized in this research. The purpose of stealing this plant energy from photosynthesis was so it could be re-directed into synthesizing valuable chemicals that are normally produced from fossil fuels. A new sensor was also developed in this research that would "light-up" the environment whenever this plant energy (electron) stealing process was successful allowing us to measure the efficiency of this energy transfer. Once a quinone stole an electron, it would spontaneously give up the electron to oxygen, creating an unstable molecule that could then react with the chemical luminol, forming a strong luminescence (light) signal. We found that calcium chloride greatly enhanced a quinone's ability to harvest electrons from the chloroplast. We also reported unique effects caused by salt, magnesium, phosphate, a mild detergent, and changing the amount of light the chloroplast would receive. This information was then used to transfer electrons from the chloroplast to make new valuable chemicals. We found that electrons could be donated to multiple chemicals using a quinone, chloroplasts, and light. We were also able to take electrons from glucose with our quinones when glucose was in an environment with a high pH. Electrons from glucose could also be donated to chemicals of interest using quinones. In addition, Quinones were used once more to find differences in the urine composition of healthy individuals and those with chronic kidney disease or bladder cancer. The urine from healthy individuals produced a unique luminescence signal when interacting with the quinones. Thus, quinones could be used for rapidly detecting changes in a patient's kidney and bladder function. We also developed a new method for detecting changes in the health of an algal culture. Algal cultures are used for producing biofuel, food, and pharmaceuticals, therefore it is imperative to track the growth of a culture to avoid contamination and algal death. Scenedesmus sp. A6 was exposed to chemicals harmful to algal health to see how these chemicals caused the algae to grow differently. Raman spectroscopy was used to collect data on algae grown under different conditions. The Raman spectra obtained then underwent statistical analysis to determine the chemicals that had the greatest impact on algal function. Methyl viologen, nickel sulfate, salt, and light exposure had the greatest impact on the algae.

Computed tomography for flame chemiluminescence emissions allows for 3D spatially resolved flame measurements to be acquired using a series of discrete viewing angle camera images. To determine fuel/air ratios, the ratio of excited radical species (OH*/CH*) emissions using chemiluminescence can be employed. Following the process of high-resolution tomography reconstructions in this work allowed for flame tomography coupled with chemiluminescence emissions to be used for spatially resolved phase averaged equivalence ratio measurements. This is important as variations in local equivalence ratios can have a profound effect on flame behavior including but not limited to thermoacoustic instability, NOx and CO formation, and flame stabilization. Local equivalence ratios are determined from a OH*/CH* ratio of tomographically reconstructed intensity fields and relating them to equivalence ratio. The correlation of OH*/CH* to equivalence ratio is derived from an axisymmetric, commercially available flat flame burner (Holthuis and Associates Burner). To relate intensity field imaging (camera coordinate system) during the tomographic reconstruction to the real-world coordinate system of the burner a calibration procedure was performed and then validated. A calibration plate with 39 non-coplanar points was used in this procedure. It was then validated by comparing the Abel inverted flame images of the axisymmetric Holthuis and Associates burner with the tomographic reconstructed images. Results show a successful tomographic reconstruction of thermoacoustic self-excited cycle concluding equivalence ratio fluctuations coinciding with the 1st dominate frequency of the pressure fluctuations and influenced by a 2nd harmonic frequency. / Master of Science / In recent years tomographic reconstruction of flames have gained significant focus in understanding different flame phenomenon. One specific flame phenomenon is known as a thermoacoustic instability. Using highspeed cameras for chemiluminescence imaging of specific species can help define heat release rate, air/fuel ratio/equivalence ratio spatially. Coupling of pressure measurements to imaging methods can be used to determine the flames response to acoustic perturbations in the flow field. Every optics system has inherently different light transmission characteristics and therefore, needs to be calibrated/correlated using a known flame source. The work done in this paper used a Holthuis and Associates flat flame as the known flame source in conjunction with an optics system to correlate OH*/CH* ratio to equivalence ratio. This is possible due to the perfectly premixed nature the flat flame provides. The correlation curve for the optics system is then applied to the tomographically reconstructed chemiluminescence intensities during a self-excited thermo-acoustic instability. In addition, a flat flame burner was used to validate the tomography approach and calibration procedure. In conclusion the objective of this work develops and validates a method for use in tomographic reconstruction of spatially varying equivalence ratios.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Iron is an essential micronutrient for all phytoplankton and is found in seawater at picomolar-level concentrations. For the first time in South Africa, a technique that utilizes a flow injection analyser (FIA) coupled with a chemiluminescence reaction has been developed for the analysis of Fe in seawater samples. The developed method is an improvement on similar available methods and uses commercially available resin (IDA) as opposed to the one that requires synthesis in the laboratory. Furthermore, the method requires reduced reagent concentrations thereby providing better results in a cost-efficient and easy manner. The improvements resulted in better precision while eliminating the loss of resin through bleeding, a common problem when using 8-HQ resin as per prior methods. Method validation was performed using internationally calibrated reference material provided by GEOTRACES and the values obtained were within the error limits of certified range. An inter-laboratory calibration was also conducted as part of the verification of the system. Surface samples from the SANAE 51 cruise were analysed for dFe and TdFe. Subsequently, the method was implemented on the SANAE 53 voyage on board the SA Agulhas II, to assess trace metal sampling protocol for any contamination issues, as well as for the analyses of collected samples. Current results suggest some contamination during collection stages, but this is still to be verified by complementary data on macronutrients and chlorophyll. The method was successfully developed and implemented in a land based clean laboratory, as well as on board a vessel. / AFRIKAANSE OPSOMMING: Yster is „n noodsaaklike mikrovoedsel vir fitoplankton en word in seewater in pikomolêre konsentrasies gevind. Hierdie lae konsentrasies en potensiële besoedeling gedurende monsteropname vanaf „n skip se platform maak akkurate Fe-analise moeilik. Vir die eerste keer in Suid-Afrika is „n tegniek ontwikkel wat gebruik maak van „n vloei- inspuitinganaliseerder (VIA), met „n geassosieerde chemiluminessensiereaksie, om die analise van Fe in seewatermonsters uit te voer. In teenstelling met soortgelyke bestaande metodes wat labratorium-gesintetiseerde hars vereis, is die nuut-ontwikkelde metode „n verbetering wat gebruik maak van „n kommersieel-beskikbare hars (IDA). Verder vereis die metode verminderde reagenskonsentrasies wat sodoende beter resultate lewer op 'n koste-effektiewe en eenvoudiger wyse. Die verbeteringe het gelei tot verhoogde akkuraatheid en uitskakeling van die verlies van hars deur dreinering – „n algemene probleem wat ondervind is met die gebruik van 8-HQ hars in vorige metodes. Geldigheidsbepaling van die metode is met internasionaal-gekalibreerde verwysingsmateriaal, verskaf deur GEOTRACES, uitgevoer. Die waardes wat verkry is, was binne die foutgrense van die gesertifiseerde skaal. „n Interlaboratorium-kalibrasie is ook uitgevoer as deel van die verifikasie van die stelsel. Daarna is die metode geïmplementeer gedurende die SANAE 53 reis op die SA Agulhas II, om die spoormetaal-monsternemingprotokol vir enige besoedelingskwessies te evalueer, asook vir die ontleding van versamelde monsters. Huidige resultate dui op „n mate van besoedeling tydens die versamelingstadiums, maar dit moet nog geverifieer word deur aanvullende data van die totale oplosbare Fe, makrovoedingstowwe en chlorofil.

La structuration et la fonctionnalisation de réseaux de fibres optiques ont été utilisées afin de réaliser différents capteurs électrochimiques et luminescents. Ce type de support permet de concevoir des capteurs capables de détecter à distance dans un milieu confiné, difficilement accessible ou dangereux. Deux capteurs pour la détection du mercure cationique (Hg2+) sont décrits dans cette thèse. Le premier utilise un dérivé de la rhodamine, qui est lié de façon covalente à la surface en verre du réseau, par silanisation. Le signal fluorescent de cette sonde est augmenté en présence de mercure ce qui permet de le quantifier. Le second capteur combine l’électrochimie sur ces réseaux de fibres optiques, grâce à une fine couche conductrice déposée à sa surface. Le réseau est ensuite modifié avec un complexe de Ruthénium(II), qui peut être électropolymérisé par l’intermédiaire de la fonction cyclopentadithiophène (CPDT). La détection du mercure est alors réalisée par électrochimiluminescence (ECL), qui est collectée à distance, grâce aux propriétés électro-modulables du film polymère. Les réseaux de fibres optiques nanostructurés ont également été utilisés pour la réalisation d’une sonde SECM, présentant un réseau dense de nanoélectrodes collectives. La réalisation d’un tel outil utilise le positionnement basé sur les forces de cisaillement et peut être utilisé pour la structuration de surfaces conductrices ou isolantes par SECM. / The structuration and the functionalisation of optical fiber bundles have been used to design different optical and electrochemical sensors. The use of these tools allows the realisation of sensors for remote detection in a confined environment, which may be dangerous or not easily accessible. Two sensors for the detection of inorganic mercury (Hg2+) are described in this thesis. The first sensor uses a rhodamine derivative, which is covalently functionalized on the surface of the glass optical fibers by silanisation. The fluorescent signal of this probe increases in presence of mercury, which allows its quantification. The second sensor uses the combination of electrochemistry on the optical fiber bundle, thanks to a thin conductive layer deposited on its surface. The bundle is then modified by electropolymerisation of a Ruthenium (II) complex through the cyclopentadithiophene (CPDT) moiety. The detection of mercury is then realised by elecrogenerated chemiluminescence (ECL), which is collected through the optical fiber bundle, thanks to the optical properties of the polymer film. The nanostructured array of optical fibers has also been used to create a new SECM probe composed of a dense nanoelectrode array. Such a tool is obtained through Shearforce positioning and can be used for the structuration of conductive or insulating surfaces by SECM.

In this presentation it is demonstrated that the unique magnetic properties of superparamagnetic cobalt-spinel ferrite nanoparticles can be employed in several novel applications. A method to selectively capture and remove pathogens from infected organisms to improve longevity is presented. Evidence is provided to show that automated methods using modified forms of hemofiltration or peritoneal dialysis could be used to eliminate the particle/pathogen or particle/infected cell conjugates from the organism postoperatively. It is shown that disparately functionalized nanoparticles can be used in concert as drug carrier and release mechanisms. Lastly, we provide preliminary evidence to support the use of magnetic nanoparticles for controlling reaction kinetics.

Doctor of Philosophy / Department of Chemistry / Jun Li / A lot of materials exhibit novel properties when scaled down to nanoscale. Here we explore nanoelectrode arrays (NEAs) and nanoparticles in the application of high performance biosensors. We have developed an electrochemical (EC) method for measuring the activity of proteases using vertically aligned carbon nanofiber (VACNF) NEAs. VACNFs were grown on conductive substrates and encapsulated in SiO₂ matrix. After polishing and plasma etching, controlled VACNF tips are exposed to form an embedded NEA. Tetrapeptides specific to cancer-mediated proteases are covalently attached to the exposed tip, with a ferrocene (Fc) moiety linked at the distal end. The redox signal of Fc can be measured with AC voltammetry (ACV) at ~1 kHz frequency, showing distinct properties from macro-electrodes due to VACNF's unique interior structure. The enhanced ACV properties enable the kinetic measurements of proteolytic cleavage of the surface-attached tetrapeptides by proteases. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis. This EC method was further applied in whole lysate of human breast tissue and breast cells. The detected protease activity was found increased in cancer cells, with the metastatic cancer cell lysate showing the highest cathepsin B activity. The results indicated the potential of this technique as a portable multiplex electronic device for cancer diagnosis and treatment monitoring through rapid profiling of the activity of specific cancer-relevant proteases. In another exploratory study, we modified nanoparticles with luminol and viral nucleic acid to develop chemiluminescence (CL) biosensors for blood-borne pathogens. Luminol-labeled 10-nm-diameter gold nanoparticles (GNPs) served as a nanocarrier for enhancing CL signal. The CL signal can be observed over 8 orders of magnitude variations in GNP concentration. Using the same number of particles, luminol-labeled 30-nm-diameter latex beads showed ~3 orders of magnitude higher CL compared to 10-nm-diameter GNPs. Hybridization of target H1N1 nucleic acid on the latex beads and probe nucleic acid on the glass or optical fiber surface has been achieved. This assay will be incorporated into a simple hand-held device for routine assays in hospitals and clinics, or for large-scale screening of human populations as diagnostic tools to identify specific viral strains.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

Organic nitrates, such as nitroglycerine (NTG), are widely used for their potent vasodilator capacity in the management of coronary artery disease and heart failure. Unfortunately, their beneficial effect is rapidly lost due to the development of nitrate tolerance, which is translated by an impaired vasorelaxation to NTG and an increased oxidative stress production. Although the mechanisms of the development of nitrate tolerance are still not fully elucidated, much interest has been focused in treating nitrate-receiving patients together with other drugs in order to overcome the development of nitrate tolerance. The Nitric Oxide generating enzyme, eNOS, and the superoxide anion generating enzyme, NAD(P)H oxidase, have been suggested to play a role in the development of nitrate tolerance. The aim of this study was to analyse the underlying mechanism by which ramipril, an ACE inhibitor and rosuvastatin, a new molecule of the statin class, are able to protect against the development of nitrate tolerance in the aortas isolated from rats, wild-type (wt) and eNOS-/- mice. These results show that ramipril as well as rosuvastatin are able to protect against the development of nitrate tolerance in the wt and eNOS-/- mice aortas suggesting that eNOS is not necessary for their protective effect. The aortas from nitrate tolerant rats and mice showed a significant increase in the NAD(P)H oxidase activation compared to the aortas from the control and from the co-treated ramipril+NTG or rosuvastatin+NTG animals. In line with these findings were the results obtained by RT-PCR analysis: the mRNA expression of the different subunits of the NAD(P)H oxidase, such as gp91phox, p22phox, were significantly decreased after rosuvastatin or ramipril treatment in wt and eNOS-/- mice aortas. Apocynin, the NAD(P)H oxidase inhibitor was also able to inhibit the development of nitrate tolerance in the rat and mouse aortas. In conclusion, these results suggest that rosuvastatin and ramipril are able to protect against the development of nitrate tolerance by counteracting the nitrate-induced oxidative stress. The mechanism of protection involves a direct interaction with the NAD(P)H oxidase pathway and seems to be completely independent of the eNOS pathway.

<p>This thesis covers sampling and analytical procedures for isocyanates (R-NCO) and amines (R-NH₂), two kinds of chemicals frequently used in association with the polymeric material polyurethane (PUR). Exposure to isocyanates may result in respiratory disorders and dermal sensitisation, and they are one of the main causes of occupational asthma. Several of the aromatic diamines associated with PUR production are classified as suspected carcinogens. Hence, the presence of these chemicals in different exposure situations must be monitored. </p><p>In the context of determining isocyanates in air, the methodologies included derivatisation with the reagent di-<i>n</i>-butylamine (DBA) upon collection and subsequent determination using liquid chromatography (LC) and mass spectrometric detection (MS). A user-friendly solvent-free sampler for collection of airborne isocyanates was developed as an alternative to a more cumbersome impinger-filter sampling technique. The combination of the DBA reagent together with MS detection techniques revealed several new exposure situations for isocyanates, such as isocyanic acid during thermal degradation of PUR and urea-based resins. Further, a method for characterising isocyanates in technical products used in the production of PUR was developed. This enabled determination of isocyanates in air for which pure analytical standards are missing. Tandem MS (MS/MS) determination of isocyanates in air below 10^-6 of the threshold limit values was achieved.</p><p>As for the determination of amines, the analytical methods included derivatisation into pentafluoropropionic amide or ethyl carbamate ester derivatives and subsequent MS analysis. Several amines in biological fluids, as markers of exposure for either the amines themselves or the corresponding isocyanates, were determined by LC-MS/MS at amol level. In aqueous extraction solutions of flexible PUR foam products, toluene diamine and related compounds were found. </p><p>In conclusion, this thesis demonstrates the usefulness of well characterised analytical procedures and techniques for determination of hazardous compounds. Without reliable and robust methodologies there is a risk that exposure levels will be underestimated or, even worse, that relevant compounds will be completely missed.</p>

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