High-Flux Chip for Sensitive Profiling of Small Extracellular Vesicle Proteins by Cas9/Switch-sgRNA Complex-Mediated Proximity Cleavage Assay.

Small extracellular vesicles (sEVs) play pivotal roles in modulating the pathological processes of various diseases and have emerged as promising biomarkers for disease diagnosis, including acute spinal cord injury and cancers. This is attributed to their ability to transport multiple proteins that reflect the molecular signatures of their parent cells. The evaluation of surface proteins presents a robust strategy for identifying a comprehensive set of biomarkers. In this study, we developed a high-throughput device capable of characterizing surface proteins on intact sEVs. Our approach employs CD63 antibodies immobilized on a 96-well plate and a CD9 aptamer integrated into switch-sgRNA, facilitating the efficient capture of intact sEVs and enabling subsequent surface protein profiling. The system utilizes a proximity cleavage assay mediated by the Cas9-nickase/switch-sgRNA complex and an identity probe, combined with DNA polymerase-assisted chain extension and displacement, to achieve highly specific and precise identification of target proteins. The DNA polymerase-mediated chain extension and displacement mechanism within the wells generates multiple G-rich sequences, which facilitate Thioflavin T (ThT)-based label-free signal amplification. This innovative design allows the high-throughput chip to profile the surface protein EpCAM on intact sEVs with exceptional sensitivity, achieving a remarkably low detection limit of 3.5 particles/μL. Moreover, the chip has been successfully applied to identify surface markers including EpCAM, PTK7, PDGF, and PSMA on sEVs derived from various biological samples, demonstrating its significant potential for high-throughput biomarker discovery and analysis.