A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

Extracellular vesicles (EVs) are released by diverse cell types in biofluids and are increasingly studied in liquid biopsies as diagnostic and prognostic biomarkers for various diseases, including cancer. Typically, the analysis of EV-associated biomarker characteristics such as size and surface markers requires pre-purification from large volumes of complex biofluids, leading to longer turnaround times and more technical variability. To overcome these limitations, we developed a one-step workflow that combines size-based separation with size and surface marker characterisation of EVs in minute volumes from different biological fluids. We coupled a multi-angle light scattering detector (MALS) and a fluorescent light detector (FLD) in-line with the asymmetrical flow field-flow fractionation (AF4) equipment. The AF4-MALS-FLD workflow was optimised to enable the analysis of EV surface markers CD9, CD63 and CD81, and cancer biomarkers PSMA, EpCAM and HER2 in samples of increasing complexity, including purified EV preparations, cell culture supernatant, urine and blood plasma. Proof-of-concept was gained for the detection of PSMA-positive EVs in urine from prostate cancer patients and discrimination of breast cancer patients from healthy donors by quantifying EpCAM- or HER2-positive EVs in blood plasma. In conclusion, using low-volume biofluids, the one-step AF4-MALS-FLD workflow holds potential for fast and robust EV biomarker detection.