Development of a series of modified acridinium esters for use in clinical diagnostics

Alamri, Mesfer 2022. Development of a series of modified acridinium esters for use in clinical diagnostics. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Acridinium esters (AE) have been traditionally used as chemiluminescent reporter molecules in clinical diagnostic assays due to their high quantum yields and detection limits in the attomole range. The aim was to produce a family of novel acridinium esters with modified chemiluminescent properties for use in clinical diagnostics. The hypothesis was that, by changing the substituents on the acridinium ring and the phenoxy ring of the acridinium ester molecule, a series of compounds with distinct chemiluminescent characteristics could be generated which would support dual analyte measurement. Two series of compounds were synthesized based on the addition of two methoxy (electron donating) or two bromo (electron withdrawing) groups to the 2 and 7 positions of the acridinium ring. Additionally, the effect of adding methyl or methoxy groups to the 2- and 6-positions of the phenoxy ring of the acridinium ester molecule was compared to the unsubstituted state. Five distinct acridinium esters were synthesized and their excitation and emission spectra determined. Two of the compounds were linked to a DNA oligonucleotide and incorporated into a Hybridisation Protection Assay (HPA), a direct and highly specific nucleic acid assay. The performance of these chemiluminescent labelled probes within the HPA (kinetics of light output and the rate of hydrolysis) was characterised. Major findings included a shift in the fluorometric spectra resulting from the substitution on the acridinium ring, resulting in the peak emission wavelengths of Nmethylacridone, 2,7-dibromo-10-methyl-9-acridone and 2,7-dimethoxy-10-methyl-9- acridone being observed at 430, 440 and 480 nm, respectively. Furthermore, the optimal pH for discrimination between hybridised and unhybridised probes for the dimethoxysubstituted acridinium ester 34cx was between 9.2 and 9.6 whilst the pH optimum of the routinely used unsubstituted compound 15 ranged from 7.5 to 8.5. Theoretically, the differential properties of dimethoxy-substituted AE DNA probes, the shift in emission wavelength and hydrolysis pH, may form the basis for a novel dual analyte HPA format to be developed.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	21 March 2023
Last Modified:	30 Mar 2023 11:31
URI:	https://orca.cardiff.ac.uk/id/eprint/157859

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