Single extracellular vesicle detection assay identifies membrane-associated α-synuclein as an early-stage biomarker in Parkinson's disease.

Accurate diagnosis of early Parkinson's disease requires platforms suitable for detecting minute amounts of neuronally derived biomarkers in the massive protein excess of easily accessible biofluids such as blood. Here, we describe an on-chip droplet-confined fluorescence reporting assay that identified α-synuclein on the membrane of L1CAM+ extracellular vesicles (EVs) immunocaptured from human serum and corroborate this finding by super-resolution direct stochastic optical reconstruction microscopy (dSTORM) microscopy. Using conditioned media from neuroblastoma cells expressing α-synuclein mutants or patient-derived induced pluripotent stem cell (iPSC) neurons with α-synuclein gene triplication, we found that association of α-synuclein with the L1CAM+ EV surface is increased under pathological conditions. Accordingly, this readout, as measured by the droplet-based assay, is an improved predictive biomarker in the prodromal phase (area under the receiver operating characteristic curve [AUC] = 0.93) or diagnostic biomarker in the clinical phase (AUC = 0.95) of Parkinson's disease. More broadly, our platform will simplify the assessment of EV membrane proteins and facilitate their application as diagnostic biomarkers across diverse clinical indications.