Direct delivery of assay reagents to extracellular vesicles in liquid biopsies for biomarker analysis.
Review
Overview
abstract
Extracellular vesicles (EVs), present in blood as well as other biological fluids, encapsulate nucleic acid biomarkers used for diagnosis, prognosis and treatment monitoring of disease via minimally invasive liquid biopsy. EVs are a reliable source of biomarkers because their contents reflect the cells from which they are derived, and their lipid bilayer membranes protect nucleic acids from degradation. Previously, analyzing EVs in blood was difficult because of time-consuming, labor-intensive EV isolation methods. Here, we provide a protocol for an EV detection approach in which reagent-loaded liposomes fuse with EVs directly in patient blood to sensitively detect RNA within the EVs. In this 'liposome-EV fusion assay', antibodies capture EVs in blood, and reagent-loaded liposomes initiate liposome-EV fusion and CRISPR-based nucleic acid detection. We originally used this assay to detect EV-encapsulated viral RNA and accurately diagnose infectious diseases from patient plasma. It has since been adopted by many other research groups to detect mRNA, microRNA, DNA, DNA mutations and EV surface proteins in a variety of patient-derived tumor samples, incorporating enzymatic and nonenzymatic detection reagents and different diagnostic readouts. As a clinical and research tool, this approach has great potential for the diagnosis, treatment and study of cancer, infectious diseases and neurological dysfunction.
