Synthesis and evaluation of novel enzymatic substrates to aid the detection and identification of clinically important microorganisms

Truong, Thang Viet (2019) Synthesis and evaluation of novel enzymatic substrates to aid the detection and identification of clinically important microorganisms. Doctoral thesis, Northumbria University.

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Abstract

In clinical diagnostics, the speed and accuracy with which pathogenic microorganisms may be detected and identified are crucial, as any delay may have an impact on patient health. The vast variety of pathogenic microorganisms, along with their evolution in countering existing treatment and diagnostic methods presents an ever-changing challenge. As such, there is a constant need to develop newer and more effective diagnostic reagents for use in the clinic and industry.

The focus of this project was on the development and evaluation of novel fluorogenic substrates to be incorporated into culture media. The substrates prepared in this project exhibit weak/no fluorescence, but upon transformation by the targeted enzyme, the fluorophore is liberated and a fluorescent response is observed. As substrates demonstrating diagnostic utility will ultimately be considered for manufacture, keeping cost and complexity of synthesis to a minimum was a major goal.

First of all, we have successfully developed a new synthetic procedure that could reduce the cost and large scale applications of the 7-nitrocoumarin-3-carboxylic acid (I) since the effectiveness of this nitroreductase in automatic detection applications in the automated TEMPO® system for quality control. Besides, we also found 7-azidocoumarin-3 carboxylic acid (II) was not effective as detecting hydrogen sulfide gas in bacteria, but rather similar to enzymatic profile of the nitroreductase substrate.

Secondary, a library of aminopeptidase substrates were synthesised and evaluated that based on fluorophores 2 amino-7-acetyl-9,9-dimethylfluorene (III) and 2-amino-7 nitro-9,9-dimethylfluorene (IV) showing exciting activities. While aminopeptidase substrates based on acetyl (III) showed moderate enzymatic activity; L-alanine substrate (V) and β alanine substrate (VI) demonstrated a great chromogenic and fluorogenic responses to selective Gramnegative bacteria at low concentration. Especially, the β-alanyl substrate (VI) has selectively detected a variety of P. aeruginosa strains which is a clinically significant microorganism in cystic fibrosis patients. A simple and cost-effectively synthetic route of this substrate also been developed for large scale applications.

A series of aminopeptidase substrate based on dihydroxanthene core which emission expected in the near infrared region also showed promising results. In particular, L-alanine substrate (VII) and β-alanine substrate (VIII) possessed an intense chromogenic and fluorogenic responses and could be utilised in microbial detection in the future.

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