A simple approach for rapid and cost-effective quantification of extracellular vesicles using a fluorescence polarization technique.

Extracellular vesicles (EVs) are membrane-bound phospholipid vesicles actively secreted by all cells. As they carry specific markers expressed by their parental cells, EVs are utilized to identify specific cells via liquid biopsy. To facilitate EV-based clinical diagnosis, a fast and reliable method to count EVs is critical. We developed a method for rapid and cost-effective quantification of EVs which relies on the fluorescence polarization (FP) detection of lipophilic fluorescein probe, 5-dodecanoylamino fluorescein (C12-FAM). The alkyl tail of C12-FAM is specifically incorporated into the EVs, producing high FP values due to a slow diffusional motion. We quantified EVs derived from two cell lines, HT29 and TCMK1 using the new strategy, with good sensitivity that was at par with the commercial method. The new method involves minimal complexity and hands-on time. In addition, FP signaling is inherently ratiometric and is robust against environmental noise.