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Abstract

The rapid growth of the diagnostics market is creating an ever-increasing demand for more sensitive, faster and cheaper detection systems. Horseradish peroxidase (HRP) is a relatively common enzyme with wide applications in chemiluminescence immunoassay.

It has been the intention of this work to make a contribution to this field by developing metalloporphyrin mimics of horse radish peroxidase enzyme. This was thought to have a two-fold importance. First it would offer a cheaper and more robust alternative to HRP and second, the simpler structure would facilitate investigations of the mechanism of the enzyme action which in turn could be used for improving enzyme performance.

To develop an artificial replacement for HRP a wide range of substituted metallo- tetraphenylporphyrins were synthesised and their structure-activity relationships in catalysing the oxidation of luminol by hydrogen peroxide were probed. The chemiluminescence resulting from the oxidation of luminol offered a reliable and quantifiable means of comparing the activities.

At the end of the screening programme tetra(aminophenyl)porphyrin manganese111 chloride (Mn(TNH2PP)Cl was identified as the best all-round catalyst and reaction mechanisms were proposed which go some way towards explaining this choice.

The practicality of using Mn(TNH2PP)Cl as a replacement for HRP in chemiluminescence immunoassay was demonstrated when the dose response curve produced a lower detection limit of lxlO"12 moles/ml and the functionalised metalloporphyrin was successfully conjugated to human IgG.

As in the case of HRP and /¿-peroxidase the catalytic performance of Mn(TNH2PP)Cl was found to be improved with the use of enhancers. Thus up to 20-fold increases in signal levels were observed when 1-methylimidazole was used as enhancer.

Additionally it was also found that the free-base porphyrin and some metalloporphyrins are capable of chemiluminescence upon oxidation by hydrogen peroxide under alkaline conditions. The analysis of the chemiluminescence spectrum revealed the existence of S, and S2 emission bands. Although under intense excitation energies the unusual phenomenon of S2 fluorescence emission has been reported for few limited cases, this is the first reporting of S2 chemiluminescence emission and was produced under relatively mild oxidation conditions.

An attempt was also made to open up the HRP molecule by unfolding the polypeptide chains surrounding the central heme by means of ultrasonic irradiation. Although the activity of the enzyme was reasonably enhanced when it was exposed to moderate irradiations, the technique needs to be refined before it can have any investigative value.

It is thought that the sensitivity of the artificial enzyme developed in the course of this research can be greatly improved by further optimisation of the reaction conditions thereby making its use as an HRP replacement viable.

Publication Type:	Thesis (Doctoral)
Subjects:	Q Science > QD Chemistry
Departments:	School of Science & Technology School of Science & Technology > School of Science & Technology Doctoral Theses

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