Capturing and Detecting of Extracellular Vesicles Derived from SingleEscherichia coliMother Cells

Cells have a phenotypic heterogeneity even in isogeneic populations. Differences in secretion of substances have been well-investigated with single mammalian cells. However, studies on the heterogeneity of secreted substances at the single-bacterial-cell level are challenging due to the small size, motility, and rapid proliferation of bacterial cells such as Escherichia coli . Here, we propose a microfluidic device to achieve an isolated culture of single bacterial cells and capture of extracellular vesicles (EVs) secreted from individuals. The device has winding channels to trap single rod-shaped E. coli cells at their entrances. Isolated single mother cells grew constantly up to 24 h, while their daughter cells were removed by flow. The flow carried EVs of the trapped cells along the channel, whose surface was rendered positively charged to electrostatically capture negatively charged EVs, followed by staining with a lipophilic dye to detect EVs by microscopy. Our results underline that the amounts of segregated EVs vary among cells. Moreover, individual responses to perturbation using a membrane-perturbing antibiotic were observed in growth dynamics and EV secretion of living-alone bacteria. The proposed method can be applied to detect other secreted substances of interest, possibly paving the way for elucidating unknown heterogeneities in bacteria.