An Ultrasensitive Strand Displacement Signal Amplification-Assisted Synchronous Fluorescence Assay for Surface Proteins of Small Extracellular Vesicle Analysis and Cancer Identification

Small extracellular vesicles (sEVs), often known as exosomes, are expected to be a promising biomarker for the early diagnosis of cancer because they carry enriched proteins that originated from parent cells. Profiling surface proteins of sEVs offers non-invasive access for the early diagnosis of cancer. However, it remains challenging to simultaneously detect surface proteins of sEVs with desired sensitivity. Herein, a dual color DNA nanodevice based on toehold-mediated DNA strand displacement signal amplification and the synchronous fluorescence technique has been developed for simultaneous analysis of surface proteins of sEVs with high sensitivity. As for the DNA nanodevice-based system, the nanoconjugates of aptamer-magnetic beads can recognize surface proteins of sEVs and lead to the release of single-stranded DNA. Then, the released DNA can trigger toehold-mediated DNA strand displacement for signal amplification. In this system, a CD63 aptamer and MUC1 aptamer were used as recognition elements for the detection of surface proteins of sEVs isolated from cancer cells. Under the optimal conditions, the corresponding proteins of sEVs were simultaneously determined with ultrasensitivity by the synchronous fluorescence method. Also, the detection limits of sEVs by two surface proteins were 67 particles/μL by CD63 and 37 particles/μL by MUC1. Of note, the as-constructed method can be applied to recognize sEVs from different tumor cell lines (SGC7901, HepG2, and MCF-7 cells). Furthermore, the system has been successfully applied to precisely identify cancer patients from healthy people by serum analysis. The strategy demonstrates great potential applications in the early diagnosis of cancer.