Engineering a tunable micropattern‐array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ

The molecular heterogeneity of extracellular vesicles (EVs) and the co‐isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single‐EV and particle (siEVP) protein and RNA assay ((siEVP)PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The (siEVP)PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single‐particle resolution, the (siEVP)PRA outperformed the sensitivities of bulk‐analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross‐validation. Despite the presence of single‐EV‐LP co‐isolates in serum, the (siEVP)PRA detected GBM‐associated vesicular RNA profiles in GBM patient siEVPs. The (siEVP)PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.