Innovations in Centrifugal Microfluidics for Pathogen Detection in Water
- Author(s): Gowda, Hamsa Nandini
- Advisor(s): Madou, Marc J
- et al.
Waterborne diseases cause millions of deaths worldwide, especially in developing communities. The detection of waterborne pathogens is a critical step for the selection of water treatment processes that will lead to the prevention of disease transmission. Unfortunately, analyzing water samples is a lengthy and laborious process that requires 1) collecting and transporting large volumes of samples (>1 L) to a centralized lab, 2) sample preparation for downstream analysis, 3) using appropriate detection methods to identify pathogens and 4) estimating the proportion of viable pathogens that pose a risk to public health. This lengthy “sample-to-answer” process significantly delays the risk mitigation actions, and subsequently exposes those using the water for daily necessities to pathogen infection. This dissertation aims to streamline the water sample analysis timeline by incorporating the major laboratory steps onto centrifugal microfluidic platforms for implementation at the point-of-sample collection, mitigating many of the aforementioned limitations. We first investigate pathogen detection methods by describing the integration of a droplet digital loop mediated isothermal DNA amplification assay onto a centrifugal microfluidic disc that coupled with a portable analysis instrument, can detect E. faecalis, a common waterborne pathogen. We highlight the fluidic and functional integration of the major steps of the assay onto the disc as well as the rapid, quantitative, hands-free aspects of the system. Next, to process samples in our detection system, we tackle upstream sample preparation by focusing on adopting simple and accessible water sample concentration mechanisms onto a centrifugal microfluidic platform. We integrate super absorbent polymer beads with the disc system to concentrate E.coli, another common waterborne pathogen, and observe how the concentration is affected by various relevant parameters. We conclude by discussing strategies for downstream assessment of pathogen viability and relevant infectivity in water samples. We describe the method and initial feasibility of a RNA-based, enzyme-mediated signal amplification strategy to detect E.coli and highlight its integration into modified culture-based assays for rapid, viable pathogen detection and risk assessment. Together, this work highlights the critical challenges in water analysis and innovations in centrifugal microfluidics for pathogen detection at the point-of-sample collection.