Exosome Biomarker Profiling Using a Paper-Based Vertical Flow Assay

Exosomes are nanoscale extracellular vesicles that carry valuable biomolecular information. However, their characterization still depends on complex and costly techniques such as flow cytometry. In this study, a paper-based Vertical Flow Assay (VFA) specifically designed for the detection and profiling of exosomes derived from metastatic breast cancer cell lines is presented. The assay operates in an ELISA-like format, targeting exosomal surface proteins (CD9, CD63, CD81, and EGFR1) with specific antibodies and a secondary antibody conjugated to alkaline phosphatase. Upon reaction with the NBT/BCIP substrate, an insoluble indigo precipitate forms on the nitrocellulose membrane, generating a visual signal that can be further quantified by smartphone imaging. The VFA was optimized for membrane type, pore size, and blocking agents, reaching a detection limit of ~6 × 10⁷ exosomes µL^-1 in less than 20 min. Comparative studies with bead-based flow cytometry confirmed consistent biomarker expression profiles, demonstrating the reliability of the method. By enabling exosome biomarker profiling in a simplified and low-cost format, this approach provides a promising alternative to flow cytometry and other applications required for exosome characterization.