Multiple Antibody‐Coated Gold Nanoparticle‐Based <scp>ExoAssay</scp> for Rapid Isolation of <scp>CNS</scp> ‐Specific Exosomes From Blood

In neurodegenerative diseases, brain-derived extracellular vesicles (EVs)/exosomes from blood offer a great opportunity to explore their contents for their utility as biomarkers. However, the conventional methodologies for the purification of EVs from complex biofluids have many limitations, restricting their clinical implementation. We aimed to optimize a direct, less time-consuming, affordable, and reliable nanowire-based method to isolate neuronal EVs from blood plasma. Here, we improved a simple and direct methodology using multiple antibody-coated magnetic nanowires for efficient and rapid isolation of neuronal EVs (ExoAssay) from human plasma. We characterized the isolated EVs and validated the protocol using multiple approaches, for example, nanoparticle tracking analysis (NTA), immunoblotting, and transmission electron microscopy (TEM). We purified round-shaped EVs with an average size of 116 nm. We identified the general markers of EVs including CD9, CD63, CD81, and Flotillin-1 and two neuronal EV markers L1-cell adhesion molecule (L1CAM) and neural cell adhesion molecule (NCAM) via immunoblotting. Interestingly, the levels of T-Tau and P-Tau were upregulated in EVs isolated from Alzheimer's patients (n = 30), in comparison with healthy controls. Furthermore, there were no significant differences between CSF- and EV-based Tau levels. The high-throughput mass-spectrometry analysis of isolated EVs revealed 280 proteins as significantly modified in Alzheimer's disease cases in comparison with controls. The presented nanotechnology-based methodology offers an innovative and efficient tool for EV-based biomarker investigations and clinical utility by simplifying the enrichment of CNS-originated exosomes from complex biological fluids. This methodology opens up the avenue for longitudinal monitoring of important disease-related proteins in the brain by analysis of brain-derived EVs from blood plasma using simple blood withdrawal.