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Larry O’Connell

Optical bacteriophage susceptibility test by Surface Plasmon Resonance

Published on 31 May 2022
Thesis presented May 31, 2022

In this work, a novel bacteriophage susceptibility testing modality was proposed, based on SPR monitoring of interactions between immobilized phage and challenge strains of bacteria.
SPR exhibits high sensitivity within the penetration depth of an evanescent field (a few hundred nanometers) which necessitates immobilization of probes at a metal-analyte interface. The cultivation of phages involves the lysis of host bacteria and the generation of large amounts of debris which must be separated from the phages before immobilization chemistry can be performed on them, an exercise that proved to be far from trivial.
Several methods were explored for purification of phage gh-1, including ultrafiltration, density gradient ultracentrifugation, PEG-precipitation, and tangential flow filtration. The comparison was made based on figures of merit such as a high level of infectious titer, low aggregation of phages, and consistent removal of contaminants from the phage lysate. While no one method satisfied all criteria, a combination of these methods was found to yield highly pure and monodisperse phage suspensions that retained their infectivity.
A review of the phage immobilization literature informed the selection of a set of strategies that were explored for the immobilization of phages on gold substrates. After comparing the results of different immobilization methods, the tried-and-tested thiolated self-assembled monolayer was shown to produce a dense, homogenous layer of infective bacteriophages on gold substrates.
This immobilization chemistry was then employed for the micro-arraying of multiple phages on the surface of a sensor exploiting SPR for parallel, multiplexed monitoring of phage-host interactions. This SPR sensor was shown to reveal specific interactions between phage 44AHJD and its host S. aureus; and between phage gh-1 and its host P. putida. During the course of experimentation, it was noticed that phage titer unexpectedly dropped while in storage, occasionally leading to failures in phage culture or functionalization of substrates. A large-scale, multi-week experiment was carried out to investigate the root cause of this phenomenon. Complementary plaque-counting measurements and nanoparticle tracking analysis of phage suspensions revealed new insights into multi-modal loss of infectious titer due to adsorption, inactivation, and aggregation — which was found to vary as a function of container material.
In the interest of improving the sensitivity of plasmonic sensing, a novel SPR sensor was conceived, which featured an embedded interdigitated electrode array (IDE) to permit electrokinetic mass transport of analyte to the sensing region. To produce IDE arrays on SPRi prisms, a novel rapid prototyping method was developed based on laser ablation with a commercial printed circuit board prototyper. The resulting electrokinetic SPR (EK-SPR) sensor was demonstrated for the detection of S. aureus by leveraging the affinity of an immobilized array of antimicrobial peptides.

phage therapy, bacteriophage susceptibility testing, surface plasmon resonance imaging, purification, immobilization, microfluidics, bioactive surfaces, nanoparticle tracking analysis, phage microarray, laser ablation, rapid prototyping, dielectrophoresis, electro-osmosis