The ALIVE project addressed the urgent need for more efficient, selective, and scalable methods for the separation and analysis of extracellular vesicles (EVs)-nanoscale carriers of biomolecules involved in cell-to-cell communication and promising biomarkers for disease diagnostics. Traditional techniques struggle to separate EVs from similar particles such as lipoproteins, particularly in clinical samples like blood plasma. ALIVE proposed an innovative electrophoretic strategy-depletion-zone isotachophoresis (dzITP), to fractionate EV subpopulations based on their electrophoretic mobility. Set against the backdrop of a growing demand for liquid biopsy tools, precision diagnostics, and affordable healthcare, the project aimed to develop a miniaturized, reusable chip platform capable of performing high-resolution EV separation. It combined electrokinetic technologies with mass spectrometry to enable lipidomic fingerprinting of EVs, offering new possibilities for disease biomarker discovery, particularly in liver-related disorders such as NAFLD. Through a series of proof-of-concept experiments, ALIVE demonstrated the capability of dzITP to separate EVs from lilpoproteins, protein and dye residues, as well as among EV subtypes. The integration of precise fraction retrieval methods, including electroosmotic flow control and segmented flow, opened avenues for downstream lipidomic profiling. The long-term goal is to translate this technology into a robust, scalable, and cost-effective diagnostic platform with direct applications in precision medicine and clinical research.
