HETEROFUNCTIONAL PARTICLES AS SINGLE CELL SENSORS FOR THE LIVE-CELL ANALYSIS AND QUANTIFICATION OF SECRETED PROTEINS

Identification of individual cells that are actively secreting a specific protein is a challenging proposition due the difficulty to assign secreted cellular products to the specific cell. Two challenges to individual cell secretion analysis and separation include the rapid diffusion and mixing of secreted products and the inability to relate the low levels of protein secretion to individual cells at scale. This thesis focuses on the design and testing of heterofunctional particles to target specific cells and collect and detect secreted protein that overcomes these limitations to enable a quantitative single-cell secretion analysis platform. The particles offer a non-destructive, simple protocol to bind to specific targeted cells and detect targeted secreted products through fluorescent labeling compatible with fluorescent activated cell sorting (FACS). Two types of heterofunctional particles were tested: Janus particles, defined as a particle with two spatially segregated functional proteins responsible for both cell binding and secreted protein collection, and particles that display mixed functional proteins. This dissertation discovers: 1) an optimized protocol to collect low concentration of soluble antibodies onto particles of different sizes and materials; 2) the design of a fabrication process to create heterofunctional particles displaying both the targeting and collecting molecules, and a protocol to isolate specific protein secreting cells; and 3) the demonstration of the particle platform to not only target specific cells but also to simultaneously collect secreted proteins to identify and isolate both high and low protein producing cells. The innovations of this study include the ability to characterize millions of cells for cell surface markers, protein secretion, as well as the ability to multiplex detection of two types of secreted protein to be simultaneously assessed. This work demonstrates that H1 influenza virus (H1IV)-specific antibody secreting cells (ASCs) can be identified and collected from blood of infected and/or vaccinated patients by the particles using an antigen specific H1IV fluorescently labeled tetramer By changing the targeting and collector molecules on the beads, the workflow was easily adapted to isolate other high and low producing cells, including IgG-producing hybridoma and VEGF-secreting human mesenchymal stem cells (hMSCs)Ph.D