Proximity Effect-Mediated DNA Self-Assembly for the Highly Specific Detection of Glioma-Derived Exosomes by Combining Multiple Exosomal Surface Markers

Exosomes that can cross the blood-brain barrier are promising biomarkers for glioma diagnosis, yet highly specific and sensitive detection of glioma-derived exosomes remains a challenge. Herein, a strategy of "proximity effect-mediated DNA self-assembly" has been proposed to achieve highly specific, sensitive, and flexible detection of glioma-derived exosomes. Exosomes are separated and enriched by CD63 aptamer-modified nanomagnetic beads; a pair of proximity probes simultaneously binds to PDPN and EGFR on the surface of exosomes, which will induce proximity effect-mediated DNA self-assembly with linker probes and the subsequent invertase-labeled signal amplification probes, thus converting one target exosome in the presence of multiple invertases. Benefiting from the dual signal amplification from nucleic acid self-assembly and enzymatic reaction, highly sensitive and flexible detection of glioma-derived exosomes can be achieved by using a portable blood glucose meter, with a limit of detection of 3 × 10⁴ particles/mL. Of note, the combined detection of multiple exosomal surface markers (CD63/PDPN/EGFR) based on proximity hybridization significantly improves the specificity of glioma-derived exosome detection, enabling efficient discrimination of glioma cells from normal microglia and various other tumor cells. Furthermore, the level of CD63/PDPN/EGFR-positive exosomes in glioma patients was significantly higher than that of healthy subjects (<i>P</i> < 0.0001); compared with the CD63/PDPN- and CD63/EGFR-positive exosomes (AUCs of 0.852 and 0.895), the detection of CD63/PDPN/EGFR-based exosomes provides a remarkably accurate diagnosis of glioma (AUC of 0.98). Additionally, this strategy can be easily extended to the detection of other disease-derived exosomes just by replacing the corresponding recognition units.