Detection of ferrous ions in extracellular vesicles at the single-particle level by nano-flow cytometry

Iron, particularly redox-active ferrous ions (Fe²⁺), is essential for biological processes. Despite their pivotal roles, analysis of Fe²⁺ ions within individual extracellular vesicles (EVs) has been hindered by the ultralow Fe²⁺ content and substantial heterogeneity of EVs. To address this, we developed a novel approach by integrating an Fe²⁺-specific fluorescent chemosensor (Ac-FluNox) with nano-flow cytometry (nFCM) for precise single-EV Fe²⁺ mapping. Method specificity to Fe²⁺ was validated via Fe²⁺-loaded liposomal models at the single-particle level. Comprehensive profiling of Fe²⁺ distributions in HT-1080-derived EVs under varying ferroptotic stress conditions revealed the striking heterogeneity in Fe²⁺ loading among EVs and a strong positive correlation between EV Fe²⁺ levels and their parental cells. Notably, we identified an EV-mediated Fe²⁺ export mechanism that functionally parallels to ferroportin (FPN)-dependent iron efflux, suggesting EVs may serve as a compensatory iron-release pathway during FPN inhibition. The nFCM platform achieved superior detection sensitivity with high throughput (up to 10⁴ particles per min), providing a powerful analytical tool for investigating EV heterogeneity and Fe²⁺-mediated regulatory networks in iron homeostasis and ferroptosis-related pathologies.