Proximity hybridization-mediated fluorescence resonance energy transfer for highly specific detection of tumor-derived exosomes: Combining multiple exosomal surface markers

Highly specific detection of tumor-derived exosomes is of great significance to improve the accuracy of diagnosis of cancer. Herein, based on the proximity hybridization-mediated fluorescence resonance energy transfer (FRET), a novel strategy for highly specific detection of tumor-derived exosomes was proposed by combining detection of multiple exosomal surface markers. Exosomes were enriched and separated by CD63 aptamer-functionalized nanomagnetic beads; a pair of FAM-labeled proximity probes simultaneously bind to EGFR and EpCAM on exosomes surface, which could mediate the formation of a stable DNA self-assemble complex with TAMRA-labeled signal probes through proximity hybridization, and then trigger the FRET between FAM and TAMRA to achieve highly specific detection of exosomes co-expressed CD63/EGFR/EpCAM with a LOD of 400 particles/μL. The relative standard deviation was lower than 7.3% in 50% UC-FBS due to the ratiometric signal improving its anti-interference ability. Importantly, in addition to effectively distinguish exosomes derived from A549 cells and BEAS-2B cells, the feasibility of exosomes detection in human serum was verified, and the level of exosomes co-expressed CD63/EGFR/EpCAM in non-small cell lung cancer patients (n = 15) was significantly higher than that of healthy people (n = 15) (P < 0.001). Moreover, this strategy was universal for the detection of other tumor-derived exosomes just by replacing the corresponding aptamers.