MICROSYSTEM TECHNOLOGIES FOR AIRBORNE VIRUS QUANTIFICATION

Abstract

Advancing our ability to measure and characterize viruses is essential for progress in clinical diagnostics, vaccine development, and understanding airborne transmission in infectious disease research. However, existing assays often involve long turnaround times, high costs, and labor-intensive procedures. This dissertation presents two complementary microfluidic platforms developed to address these challenges: a digital focus assay (dFA) for viable viral quantification and a droplet merging platform for the compositional analysis of bioaerosols. The dFA employs an array of independent nanoliter cell cultures, with cells in each microwell inoculated with virus and isolated by oil discretization to prevent cross-contamination. Following incubation, infected cells are detected by immunofluorescent staining, and automated image analysis generates a binary map of wells positive for viral antigen. Statistical analysis of these data yields infectious viral titers using significantly smaller sample and reagent volumes than conventional focus assays, while enhancing assay automation and endpoint time flexibility. Demonstrated with both model virus and clinical influenza A specimens, the platform provides an accurate, rapid, cost-effective, and convenient tool for viral load quantification with broad clinical, pharmaceutical, and research applications. The droplet merging platform addresses fundamental questions in aerovirology, such as the distribution of virus within aerosol droplets and the quanta of infection. Approaches for collecting aerosols as discrete aqueous droplets in oil are explored, along with model emulsions generated using a microfluidic droplet generator. Controlled merging of individual or small numbers of droplets with assay reagents is demonstrated, which provides a framework for compositional analysis at the single-particle level. Proof-of-concept assays are explored for viral protein detection, along with an assessment of potential limitations. Finally, integration of the droplet merging platform with the dFA is discussed as a strategy for quantifying viable viruses in aerosols, offering new tools to investigate key open questions in aerovirology.