Thesis

Surface enhanced spatially offset raman spectroscopy (SESORS) imaging of bacterial biofilms

Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2019
Thesis identifier
  • T15700
Person Identifier (Local)
  • 201458133
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • Bacterial biofilm formation is crucial to establishing chronic infections including respiratory infection, orthopaedic infection and medical device infection et.al. Many antibiotics are unable to eradicate dense biofilms since extracellular polymeric substances (EPS) make up the matrix of the biofilm which retard the diffusion penetration of antibiotics. Current methods of bacteria detection rely upon laboratory-based techniques that are time-consuming and costly and require specialist trained users. Hence, there is an urgent need for in-situ methodologies to detect and prevent the formation of bacterial biofilms. Raman spectroscopy (RS) is based on the inelastic scattering of photons following monochromatic laser excitation. This powerful technique has the advantages of being non-destructive, non-invasive and label-free. However, the main disadvantage is that spontaneous Raman spectroscopy has low signal levels and long acquisition time. To address these issues, surface-enhanced Raman scattering (SERS) has been used to enhance the Raman signal up to 1013 - 1015 orders of magnitudes and can increase acquisition speeds as well as improving the accuracy of detection. Therefore, the focus of this research is to use specially designed bionanosensors (lectin and DNA aptamer) with resonant nanotag chalcogenpyrylium dyes and low-pH sensing probes PhagoGreen as optical imaging tools showing spectral change in response to the interaction with defined target molecules via enhanced SERRS signals to detect biofilm. This research focuses on developing new biomolecular sensing Raman-active nanotags as highly sensitive surface enhanced Raman probes. The specific nanosnesor was designed such that they will detect bacterial biofilms in vitro. This approach involves using galactophilic lectin PA-IL functionalised silver nanoparticles as a molecular recognition agent to detect the carbohydrates on the surface of bacteria using SERS. This research demonstrated this lectin biosensor is not only capable of detecting bacteria but also providing a rapid, sensitive discrimination between Gram-negative and Gram-positive bacteria, offering opportunities for future SERS biosensing in biomedical applications. None of current biofilm models can mimic the complexity of the 3D microenvironment and host defence mechanisms. In this study, clinically relevant bacterial species including Escherichia coli (E.coli), methicillinsensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. 3D biofilm constructs produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing. The advantage of using Raman rather than fluorescence as the optical imaging technique is the molecular specificity of the optical response, however more importantly in this case, is the combination of surface enhanced spectroscopy and spatially offset Raman (SESORS) which allows detection of Raman signals at depth. Herein, we have developed a novel approach for the detection of bacterial biofilms at depth using a 3D bioprinted biofilm model combined with gold nanoparticles functionalised with resonant Raman reporters and bacteria specific DNA aptamers. Detection was carried out using surface enhanced spatially offset resonant Raman spectroscopy (SESORRS) allowing detection of the bacterial biofilms to be achieved at penetration depths up to 2.1 cm through tissue for single bacteria and 1.5 cm for multiple bacteria. This work uses a low-pH sensing fluorescent probe, PhagoGreen, as a Raman reporter attached to a silver nanoparticle, to detect phagosome acidification in Gram-negative bacteria strain Escherichia coli activated macrophages by surface enhanced Raman spectroscopy (SERS). The SERS intensity of PhagoGreen conjugates at peak 759 cm-1 was shown to be highly responsive at a lower pH range (pH5-pH3).
Advisor / supervisor
  • Vendrell, Marc
  • Graham, Duncan (Professor of Chemistry)
  • Faulds, Karen
Resource Type
Note
  • This thesis was previously held under moratorium until 19th October 2022.
DOI
Date Created
  • 2019
Former identifier
  • 9912922291902996
Embargo Note
  • Access to the digital copy of this thesis is restricted to Strathclyde users until 19th October 2025.

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