Investigating the use of novel fluorescent nanosensors to measure chemical changes in bacterial surroundings

Perkins, Mark Edwin (2021) Investigating the use of novel fluorescent nanosensors to measure chemical changes in bacterial surroundings. PhD thesis, University of Nottingham.

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The formation and persistence of microbial biofilms play an important role in infection and the biofouling of the environment. In order to eradicate these complex structures, further characterisation of the microniches that form within biofilms is vital. This study aimed to investigate whether fluorescent nanosensors could map pH and oxygen gradients in microbial biofilms.

Chapter One outlined the interaction of both neutral and cationic pH-sensitive, polyacrylamide nanosensors with the opportunistic pathogen Pseudomonas aeruginosa. When added to both planktonic cultures and biofilms, cationic pH-sensitive nanosensors co-localised with P. aeruginosa where they were likely interacting with extracellular components coating the bacterial cells. In a P. aeruginosa biofilm, this co localisation led to thicker biofilm formation. Conversely, neutral pH-sensitive nanosensors became dispersed within a planktonic culture; whilst in a biofilm the neutral nanosensors formed distinct aggregation between the microcolonies.

Chapter Two showed the optimisation of oxygen-sensitive polyacrylamide nanosensor use with P. aeruginosa. During planktonic growth, P. aeruginosa produced the auto-fluorescent virulence factor, pyoverdine, which matched the fluorescence spectra used to detect platinum (II) porphyrin, the oxygen-sensitive fluorophore used to functionalise polyacrylamide nanoparticles. By using PAO1 NΔpvdD, the oxygen-sensitive nanosensors were capable of measuring real-time oxygen consumption in planktonic culture. However, incorporation into a P. aeruginosa biofilm required further optimisation to prevent microcolony disruption.

Finally, Chapter Three used Streptococcus mutans, a predominant acid-producing oral bacteria, to determine whether the pH-sensitive nanosensors could detect pH changes induced by glucose treatment. Confocal laser scanning microscopy revealed that the addition of 1% w/v glucose to an established S. mutans biofilm, embedded with pH-sensitive nanosensors, resulted in a gradual reduction in the fluorescence intensity ratio during a 30 min period. This reduction in the fluorescence intensity ratio indicated a reduction in pH of the biofilm over time as the glucose was fermented.

These findings will help to improve technologies used to detect, measure, and map both pH and oxygen gradients in microbial biofilms in order to develop potential methods of biofilm treatment that either bypass or utilise these gradients.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Aylott, Jonathan
Hardie, Kim
Keywords: biofilms, fluorescent nanosensors, Pseudomonas aeruginosa
Subjects: Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Q Science > QR Microbiology > QR100 Microbial ecology
Faculties/Schools: UK Campuses > Faculty of Science > School of Pharmacy
Item ID: 65516
Depositing User: Perkins, Mark
Date Deposited: 04 Aug 2021 04:42
Last Modified: 04 Aug 2023 04:30

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