# Exosome Biomarker Profiling Using a Paper-Based Vertical Flow Assay

## TL;DR

A low-cost paper-based test is developed to detect and profile exosomes from breast cancer cells, offering a simpler alternative to expensive methods.

## Contribution

A paper-based vertical flow assay is introduced for exosome biomarker profiling with visual and smartphone-compatible readout.

## Key findings

- The VFA detects exosomes at ~6 × 10⁷ µL⁻¹ in under 20 minutes.
- The assay reliably profiles exosomal surface proteins CD9, CD63, CD81, and EGFR1.
- Results are consistent with bead-based flow cytometry.

## Abstract

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.