Low-MESF fluorescent reference particles for flow cytometry analysis of extracellular vesicles

Extracellular vesicles (EVs) hold strong potential as non-invasive biomarkers for early detection of diseases. However, their clinically relevant quantitative flow cytometric (FCM) analysis remains limited by the lack of suitable calibration particles with low single-particle fluorescence values. Although fluorescent labeling of silica particles is well established, traditional approaches typically yield heterogeneous fluorophore distributions in the low MESF (Molecules of equivalent soluble fluorophore) range, which is required for single-EV fluorescence calibration. To this end, we introduce a labeling strategy that overcomes this limitation by using fluorescently tagged silica-binding peptides (R5-FITC) to produce submicron silica particles with homogeneous low-level fluorophore coverage. Silica particles of three different sizes were synthesized and labeled in a fast and straightforward manner. After comprehensive characterization, FCM investigations revealed that the resulting dim fluorescent silica particles (as low as 278 MESF with narrow distribution) are suitable for calibrating the fluorescence intensity of EVs isolated from red blood cells. Our findings show that fluorescent particles resembling EVs in both size and fluorescence can improve calibration accuracy and foster standardization in FCM-based EV characterization.