Rolling Circle Amplification-Assisted Flow Cytometry Approach for Simultaneous Profiling of Exosomal Surface Proteins

Exosomes that carry multiple proteins from the originating cells are known as emerging biomarkers for tumor diagnostics. However, it is still technically challenging to accurately evaluate subtle differences of exosomal membrane proteins. Here, we developed a rolling circle amplification (RCA)-assisted flow cytometry approach (FCA) to simultaneously profile surface proteins and quantify exosomes. In this work, specific anti-CD63 antibody-conjugated magnetic beads were first utilized to capture exosomes. Then, the captured exosomes were bound with DNA primers, which comprise exosomal surface protein-specific recognition aptamers. The RCA reaction generates repeat DNA sequences for fluorescent probe hybridization. Finally, a conventional flow cytometer was introduced to phenotype exosomal protein markers. Such a sensitive RCA-assisted FCA displays an excellent detection limit of 1.3 × 10⁵ exosome/mL. The variable composition of four protein markers on different cell-derived exosomes was sensitively detected through changing the protein-recognition sequence of the DNA primer, which reveals a heterogeneous pattern. Exosomes from different cell sources could be distinguished by the abundance difference of multiple surface proteins. Furthermore, the developed RCA-assisted FCA enabled quantitative analysis of blood samples from lung cancer patients, indicating its potential for early clinical diagnosis and prognosis of cancer.