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    Enhancing sensitivity in the analysis of small biomolecules by surface plasmon resonance : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2012) Dombroski, Gaile Suzanne
    Highly potent biological micro-pollutants in the aquatic environment can potentially have detrimental effects on marine and human health, but the development of highly sensitive test methods suitable for use in a field environment remains a challenge. Surface plasmon resonance (SPR) is an optical-electrical phenomenon, which can be applied to the monitoring of surface binding, allowing the measurement of bio-molecular interactions in real time, without the use of radioisotope or fluorescent labeling. The technique has wide utility in the application to biological sensing, including quantitative concentration measurements and the qualitative comparison of binding partners. The central focus of this study was to investigate quantitative techniques and improve sensitivity using various strategies, including the incorporation of linkers into one of the binding partners and exploiting the signal enhancement properties of secondary antibodies and gold nanoparticles. The use of functionalised terthiophene as an alternative scaffold for immobilising the binding partner was investigated. The effect of attaching the binding partner as a protein conjugate compared to its protein-free counterpart was explored. Presented here is the use of SPR to investigate an estrone-antibody binding system, which has potential application in the analysis of wastewaters. The binding of a number of estrone derivatives was evaluated, with a view to being able to ‘tune’ the binding system so that the sensitivity range fell within a range suitable for the application. The use of secondary antibodies and gold nanoparticles to enhance the sensitivity further was also examined in the estrone system. The findings were later applied to the development of a highly sensitive test method for the detection of the shellfish toxin, domoic acid. Finally, investigations into an alternative scaffold to which one binding partner was attached to form the recognition element on the biosensor surface were carried out with a view to creating a generic scaffold for SPR sensor surfaces.
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    Refinement of analytical technologies for detection of biomolecules of importance to the dairy sector : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Sciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2011) Sharma, Indu; Sharma, Indu
    There is a continuous push on the dairy industry to enhance livestock productivity to meet with the demands of ever increasing human population. This demand can be achieved by developing rapid and early diagnostic aids to help curtail various problems encountered in the livestock production. The first study of the present thesis was focussed on standardizing initial steps towards development of Surface Plasmon Resonance for progesterone and oestradiol 17- , both of which are critically implicated in animal reproduction. For progesterone, the binding response of two different length linkers, and the antigen-antibody binding response of two different source monoclonal antibodies (P1922 from Sigma vs SE-7720-1430 from Serotec) were evaluated. It was concluded that the long length linker had better binding response than the short length linker. The antibody obtained from Serotec (SE-7720-1430) had greater sensitivity but its binding response was inconsistent. On the other hand, the sensitivity of the monoclonal antibody from Sigma (P1922) was lower, although its binding response was consistent. For oestradiol 17- , antibody procured from Bio-trend (BT70-1020-06) was tested and its binding response was consistently low on all the test days. This study thus suggests that careful testing and selection of antibodies to achieve desired antigen-antibody binding response is a critical step towards development of SPR for progesterone and oestradiol 17- . The second study was undertaken to refine the currently existing fluorescent techniques to measure phytoporphyrin in the peripheral circulation of cows. Phytoporphyrin is implicated in facial eczema (FE), which is a photosensitization disease of high economical importance. This occurs due to disturbances in the chlorophyll metabolism as a result of liver damage and bile duct occlusions caused by fungal toxicity. The present study described new modified fluorescent methods to measure phytoporphyrin in the serum of cows. Further, the absorption and emission spectra of phytoporphyrin were compared with that of other chlorophyll metabolites and thus a currently existing anomaly in the chemical structure of phytoporphyrin was rectified.