Microfluidic PCR with plasmonic imaging for rapid multiplexed characterization of DNA from microbial pathogens
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Bloodstream infections (BSIs) are a critical concern in modern medicine due to their continued prevalence in modern hospitals, along with high costs and attributable mortality, particularly among those who are immunocompromised. The current gold standard for detection and characterization of causative pathogens involves cell culture, which can take 24-48 hours to complete, increasing time to adequate treatment and thus mortality. The rise of antimicrobial resistance in hospital acquired infections has reduced the effectiveness of broad spectrum antimicrobial treatments, resulting in a clear need for a rapid, sensitive technique for characterization of resistance markers in microbial pathogens without cell culture. Here we present the development of a microfluidic platform for polymerase chain reaction (PCR) mediated amplification of microbial gene targets in a continuous flow system for potential coupling with sample preparation systems to reduce time to diagnosis from days to within two hours. This culminated in a thermoelectric cooler mediated fluidic thermocycler with a recirculating assay region for real-time hybridization measurements to minimize assay time. We subsequently demonstrated development of a low-cost optical system for localized surface plasmon resonance imaging using a digital micromirror device and tuned nanoprism monolayers for DNA hybridization with a spectral resolution of 2nm. This LSPR imaging system was integrated in-flow into the microfluidic thermocycler, enabling detection of input E. coli DNA samples at a minimum concentration of 5fg/ [microliter]. We further demonstrated multiplex detection of target markers, indicating potential for assaying target panels for characterization of pathogens. Overall, the studies in this dissertation demonstrate a microfluidic PCR system with built-in sensitive LSPR detection of DNA hybridization. It should serve as a starting point for exploration of and expansion with fluidic sample preparation with a focus on rapid characterization of pathogens.