Rapid molecular signature analysis of pseudomonas aeruginosa by direct nanomolar detection of quorum sensing and biomarker molecules at a boron-doped diamond electrode

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Buzid, Alyah
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University College Cork
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The thesis investigates a direct and sensitive method for the detection of biomolecules as indicators of Pseudomonas aeruginosa infection. This is of significant clinical importance for patients with cystic fibrosis (CF) of which Ireland has one of the highest rates in the world per head of population. Cell-to-cell communication (quorum sensing, QS) in P. aeruginosa is an important pathway to triggering biofilms which create added complications for patients. Different conventional analytical methods for the analysis and detection of P. aeruginosa QS signaling molecules exist. An overview of capillary electrophoresis (CE) and the boron-doped diamond (BDD) electrode are given. In this thesis, new approaches to separation and sensing these signaling molecules are developed. A method for the simultaneous separation of signaling molecules PYO, HHQ, and PQS using bare CE coupled with ultraviolet (UV) detection is an example. An optimized method on a bare CE is further applied to the analysis of P. aeruginosa PAO1 cell-free culture. Another solid-phase extraction (SPE) method for the signaling molecules PYO, HHQ, and PQS using mixed mode cation exchange (MCX) SPE is developed. Monitoring the production of signaling molecules of PA14 bacterial cultures was performed using CE-UV after MCX SPE. Instant feedback and detection is important in a clinical setting which is a challenging objective. Investigations into the simultaneous electrochemical detection of these microbial metabolites PYO, HHQ, and PQS using the BDD electrode are investigated. The detection limits obtained using the BDD electrode were 50 nM for PYO, and 250 nM for both HHQ and PQS. The direct detection of the three signaling molecules in bacterial cultures and spiked CF sputum samples through in situ membrane disruption using a cationic surfactant are also presented with success. Significantly this direct detection method avoids the need for biomarker enrichment using liquid-liquid extraction or SPE. The electroanalysis behavior at glassy carbon (GC) and BDD electrodes is compared for the detection of another biomolecule of P. aeruginosa specifically the synthesized ruminal biomarker “barakacin”. At pH 2.0, the detection limit (S/N = 3) on the BDD electrode is 5 nM, 100-fold lower than that obtained on the GC electrode (500 nM). An optimized method using the BDD electrode was successfully extended to both spiked bovine fecal matter and human sputum samples of a CF patient. The detection sensitivity for barakacin was improved using Nafion or poly (diallyldimethylammonium) chloride (PDDA) with multiwalled carbon nanotubes (MWCNTs) to modify the BDD electrode through a layer-by-layer assembly technique. Also, the performance of the modified BDD electrode for the detection of barakacin was investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The modified BDD electrode offers remarkable sensitivity, providing a limit of detection of 250 pM compared to 5 nM on the bare BDD electrode. The results presented represent a significant advance in the separation and sensing of biomarkers in diseases where outcomes can be greatly improved with early clinical intervention and where turnaround in analysis times is critical.
Pseudomonas aeruginosa , Signaling molecules , Biomarker , Cystic fibrosis , Boron-doped diamond electrode
Buzid, A. 2017. Rapid molecular signature analysis of pseudomonas aeruginosa by direct nanomolar detection of quorum sensing and biomarker molecules at a boron-doped diamond electrode. PhD Thesis, University College Cork.