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

Abstract

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.