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Investigating the Application of Size‐Exclusion Chromatography to Purify Bacterial Extracellular Vesicles

Sepsis is a leading cause of death as well as the most expensive condition to treat in U.S. Hospitals. Sepsis occurs when host proinflammatory response is unusually elevated, in part due to bacterial molecules secreted in bacterial extracellular vesicles (EVs). Due to their abundance, robustness, and unique characteristics between bacterial species, these bacterial EVs can potentially serve as a molecular diagnostic biomarker for Sepsis. Thus, considering the importance of diagnosing Sepsis accurately and early, a high‐purity purification technique is necessary for isolating bacterial EVs for further characterization. We propose that size exclusion chromatography (SEC) can be used to isolate bacterial EVs from the sera of sepsis patients. Additionally, we hypothesize that SEC columns packed with CL‐4B Sepharose beads will isolate bacterial EVs from heterogeneous samples with a finer resolution than columns packed with CL‐2B Sepharose beads. Previous literature has shown that CL‐2B SEC columns successfully separate human EVs from smaller particles according to size in heterogeneous samples. We have extended this work to investigate bacterial EV separation and CL‐4B Sepharose beads using three different systems: fluorescent beads and rhodamine, simulated sera, and bacterial EVs. Together, these preliminary experiments have shown that both CL‐2B and CL‐4B successfully separate bacterial EVs from smaller particles in heterogeneous samples. Future work will likely show that CL‐4B SEC columns separate bacterial EVs from other particles in patient samples with a higher resolution compared to CL‐2B beads due to their smaller pore sizes. Additional future studies will combine the use of these SEC approaches with ultrathin membranes in a single‐pass microfluidic device.