Density-shift Immunomagnetic Separation for Pathogen Retrieval from Complex Media

In industries like food production, pathogenic bacteria detection is required for producers to confidently send products to market without the risk of costly product recalls. However, rapid, reliable detection remains an unmet need. Conventional methods for detecting bacteria rely on time-consuming culture and emerging rapid tests struggle to achieve high sensitivity when the sample has a background of nontarget cells, proteins, and detritus. Reducing heterogeneity, removing inhibitory substances, and concentrating target cells by sample preparation could enable more rapid detection of pathogens from complex media, such as food samples. This thesis describes a novel immunomagnetic bead-based sample preparation method called density-shift immunomagnetic separation (DIMS). The DIMS method uses beads to capture target pathogenic bacteria and spatially separate them from both the background components of the sample and the unbound magnetic beads by centrifugation through a density media bi-layer, and then the spatially separated target bacteria are magnetically concentrated to a surface where they can be directly imaged. The purified, concentrated target bacteria can be analyzed in downstream analyses. In the first part of this thesis, the theoretical framework of DIMS is described and criteria are developed to select an immunomagnetic bead and density media to enact DIMS. In the second part of the thesis, procedures were developed to implement DIMS in a laboratory setting, using commercially available components. In addition, models for centrifugal separation and magnetic concentration were described. In the third part of the thesis, DIMS is demonstrated in a bead-bead system that models viable but non-culturable bacteria behavior. Capture of target particles was achieved with a probability of detection 50% limit of detection (50%) of 10 target particles per milliliter. In the fourth part of the thesis, we implemented DIMS in a system with Salmonella enterica in buffer and simulated spinach rinse contaminated with Escherichia coli. DIMS in buffer with S. enterica yielded an 50% of 90 CFU/mL. We demonstrated multiple detection techniques: The captured bacteria were plated, fluorescently imaged, and observed during miniature culture to identify colony formation at a single cell level. Using DIMS, pathogenic bacteria can be isolated and observed in less than two hours, a significant improvement over FDA-regulated testing that can take up to seven days to return a positive result.Ph.D