Dysprosium(III)–Nanopolymer Functionalized Array for Capture of Extracellular Vesicles and In Situ Phosphoprotein Analysis

Abstract The analysis of extracellular vesicle (EV) phosphoproteins holds great promise for biomarker discovery but is hindered by the multi-step and inefficient nature of conventional methods. Here, we develop a novel dysprosium(III)–nanopolymer (PEI–Dy3+) functionalized array for the simultaneous capture and in situ (on-membrane capture, lysis, and detection without sample transfer) phosphoprotein analysis of EVs. This platform leverages the high affinity of Dy3+ for phosphate groups to enable an integrated workflow for EV capture and phosphoprotein enrichment on the array, followed by on-membrane detection. The array exhibited excellent analytical performance with high phosphopeptide specificity, uniform EV capture, and robust 15 day stability. Furthermore, it outperformed ultracentrifugation by achieving higher EV purity (76.3% vs 63.6% for UC and 68.0% for ADSP) and detecting more EV proteins (1296 vs 520 for UC). When applied to an Alzheimer’s disease (AD)-like damaged cell model, the array enabled LC–MS/MS-based quantification of 107 significantly altered phosphoproteins from Aβ25–35 stimulated SH-SY5Y cells culture medium, revealing disease-relevant pathways and kinase activities. Finally, the platform enabled in situ detection of AD-related EV phosphoproteins, including APP and SEPTIN2 in cell models, and further validated APP in a pilot clinical cohort of AD patients and healthy controls. Overall, the PEI–Dy3+ functionalized array facilitates the capture of EVs and subsequent in situ analysis of their phosphoproteins, thereby providing a novel and efficient analytical tool for EV phosphoprotein analysis.