Isolation of bacterial extracellular vesicles from raw samples using a portable microstructured electrochemical device.

Bacterial extracellular vesicles (EVs) are nanosized vesicles released by both Gram-negative and Gram-positive bacteria, playing critical roles in microbial communication, host-pathogen interactions, and immune modulation. Despite their significance in research and clinical applications, conventional isolation methods, such as ultracentrifugation (UC), are often slow, labor-intensive, and susceptible to contamination. In this study, we evaluated a novel portable microstructured electrochemical device (PMED) designed for rapid and selective bacterial EV isolation directly from biological samples. Using immunoaffinity-based capture and voltage-triggered release, the device-isolated EVs from Gram-negative Escherichia coli (E. coli), Gram-positive Lactobacillus fermentum (Lb. fermentum) culture supernatants and from urine samples spiked with E. coli , showing superior purity compared to UC. Characterization through nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and Western blot confirms enhanced selectivity and reduced contaminants. Functional assays demonstrated that device-isolated Lb. fermentum EVs selectively activated Toll-like receptor 4 (TLR4) without triggering TLR2, unlike UC-isolated EVs, suggesting a more refined immunomodulatory effect. These findings highlight the device's translational potential for EV-based diagnostics, particularly for noninvasive urinary tract infection detection, and its broader applications in studying bacterial communication and immune regulation.