Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.681045
Title: Enzyme-triggered catalytic signal amplification
Author: Goggins, Sean
ISNI:       0000 0004 5918 4246
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2015
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Amplification is essential in order to achieve low limits of detection (LOD) within analyte detection assays. Over recent years, chemists have developed a multitude of amplification methodologies for the detection of a variety of analytes with varying signal readouts. Typically, these methodologies are focussed upon one of four main amplification strategies; target, label, signal or receptor amplification. Herein, a thorough review of the current literature has been compiled highlighting both the advantages and disadvantages of each strategy. Despite the increasing number of amplification methodologies being described, there is still significant demand for improving analyte assay efficiency and sensitivity. In particular, rapid and sensitive protein detection that can be performed at the point-of-care (POC) setting is highly desirable for the effective treatment and control of infectious diseases. In collaboration with medical diagnostics company Atlas Genetics Ltd., a new electrochemical substrate for the ratiometric detection of alkaline phosphatase (ALP), a commonly-used enzyme label within enzyme-linked immunosorbent assays (ELISA), was developed capable of delivering an ALP LOD of 0.3 pM within 30 minutes. When applied to the detection of C-reactive protein (CRP), a model biomarker used to diagnose inflammation, the substrate was shown to be significantly more reproducible than a commonly-used commercially-available electrochemical ALP substrate. In order to improve the sensitivity of an ELISA, a novel signal amplification methodology was then developed. Based upon a double-catalyst signal amplification strategy, an organometallic transfer hydrogenation catalyst was utilised in combination with the enzyme label to afford a hybrid synthetic and biological amplification cascade. To achieve selective enzyme-triggered catalyst activation, an enzyme substrate termed a proligand was synthesised. In the presence of the enzyme, self-immolation of the proligand occurs to release a ligand, capable of binding to an iridium pre-catalyst to generate a ligand-accelerated active catalyst. Catalyst activity could be observed through ratiometric electrochemical analysis through the use of an electroactive aldehyde as the catalyst substrate. An ALP LOD of 7.6 pM after 33 minutes was obtained for this amplification methodology but when applied to the detection of CRP however, a significant background reaction, found to be caused by the iridium pre-catalyst, limited sensitivity. The versatility of the signal amplification methodology was nevertheless demonstrated through the enzyme-triggered catalytic reduction of a range of different aldehydes with alternative signal readouts, enabling signal transduction and amplification to be easily achieved within a single procedure.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.681045  DOI: Not available
Share: