Homogeneous fluorescence polarisation immunoassay for the quantification of extracellular vesicles from whole blood

Extracellular vesicles (EVs) are important biomarkers in disease diagnosis and monitoring. However, their quantification typically relies on laboratory instruments, limiting their application in point-of-care testing. In this study, we report a homogeneous fluorescence polarisation immunoassay for the quantification of EVs from whole blood using an antibody tracer. The assay uses a portable fluorescence polarisation analyser with microfluidic devices to simplify EV quantification after pretreatment. To reduce nonspecific binding of the tracer to the microfluidic channel walls, we employed polyethylene glycol-modified PDMS, which eliminates the need for blocking agents. Consequently, this study enabled homogeneous quantification using a portable fluorescence polarisation analyser, achieving a limit of detection of 5.0 × 10⁷ particles/mL and a limit of quantification of 6.6 × 10⁷ particles/mL. Overall, this approach provides a simple, and reagent-efficient method for EV quantification from complex biological matrices, highlighting the potential of fluorescence polarisation immunoassay as a practical tool for future EV-based clinical studies.