Combination of specific proteins as markers for accurate detection of extracellular vesicles using proximity ligation-mediated bHCR amplification.

As the molecular characteristics of extracellular vesicles (EVs) are closely related to the occurrence and progression of cancer, the detection of tumor-derived EVs provides a promising non-invasive tool for the early diagnosis and treatment of cancer. However, it would be difficult for most of the existing methods to avoid false positives because the obtained result declares the amounts of proteins, but cannot accurately reflect the protein sources, including EV proteins and interfering proteins, in the actual samples. In this manuscript, a robust, accurate, and sensitive fluorescent strategy for profiling EV proteins is developed by using the combination of specific proteins as markers (Co-marker). Our strategy relies on the Co-marker recognition-activated cascade bHCR amplification, which forms numerous G-quadruplex structures that are integrated with fluorescent dyes for signal transduction. Notably, the detection accuracy can be improved owing to the effective avoidance of false positives from interfering proteins or single protein markers. Moreover, by using the double-positive protein recognition mode, unpurified detection can be achieved that avoids time-consuming EVs purification procedures. With its capacities of accuracy, portability, sensitivity, high throughput, and non-purification, the developed strategy might provide a practical tool for EV identification and the related early diagnosis and treatment of cancer.