Development of Analytical Devices and Sensors for the Capture and Detection of Single Bacteria Cells

With the introduction of new drug treatments for a number of diseases comes a rise in observed multidrug-resistance amongst these targeted pathogens. The threat presented by these species has led to an urgent and increased need for methods to detect harmful microbes earlier and at lower concentrations, for the benefit of sick patients. One such microbe is the bacterium Pseudomonas aeruginosa which is known for infecting the lungs of cystic fibrosis patients and wounds of burn victims, for example, and is commonly found in soil and water. The resistance mechanisms and virulence of P. aeruginosa stem primarily from its ability to form biofilms, as well as several secreted virulence factors that can cause damage to the host immune system. Much work has been done on bulk cultures and biofilms, however detecting low cell counts could make a difference in the prognosis of a patient. Additionally, it is crucial to develop techniques that allow researchers to study how cellular heterogeneity within populations may play a role in the propagation of multidrug resistance and cellular communication. Some analytical techniques such as confocal microscopies, fluorescence, and electroanalytical methods have been used to study cellular communities, but few have achieved the ultimate limit of detection, a single bacterium.

The work described in this thesis discusses steps made to analyze and understand single P. aeruginosa cell behaviors through wide-field fluorescence spectroelectrochemical and traditional electrochemical methods. The development of a selective point-of-care whole-cell sensor for clinically relevant bacterial pathogens will also be addressed. In summary, the work will first address a micropore electrode array (MEA) developed to analyze the spectroelectrochemical fluorescence behaviors of parallel arrays of vertically-oriented single P. aeruginosa cells. We have demonstrated that single cells within the MEA can exhibit one of three fluorescence behaviors in response to an external applied potential across the array, an identification that had previously not been demonstrated due to ensemble averaging of bulk measurements. The fabricated MEA can also be tuned to trap different cells in different orientations. These devices enable future microbiology researchers to study mutations and drug effects down to a single cell level while still allowing for multiple cells to be studied at once. A comprehensive discussion of potential sources for each of three behaviors will follow, with a specific focus on the effect of nutritional and oxidative stress on individual cells. These stress responses activate regulatory pathways within the cell and can overlap, as well as initiate virulence, survival tactics, and intercellular communication, all of which play a role in multidrug resistance. Finally, a whole-cell biosensor designed to capture clinically relevant pathogens through selective aptamer binding and detect them through differential pulse voltammetry will also be exhibited, using P. aeruginosa as a model analyte of interest with Escherichia coli as a competing pathogen. This aptasensor can initially detect the presence of P. aeruginosa down to concentrations of 4080 colony forming units (cfu) mL^-1 and can be fabricated in a matter of hours compared to traditional diagnostic methods of bacteria culturing over the course of 1-5 days, making it an ideal and rapid point-of-care device for clinical settings.

The work described in this thesis addresses limitations in both understanding methods of antibacterial resistance heterogeneities at a single cell level and clinical diagnostic practices during the onset of infection or disease. Both the MEA device and the whole-cell aptasensor represent prominent steps forward in the fundamental study and clinical detection of pathogenic bacteria.