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Exosomes (EXOs) have been proven as biomarkers for disease diagnosis and agents for therapeutics. Great challenge remains in the separation of EXOs with high-purity and low-damage from complex biological media, which is critical for the downstream applications. Herein, we report a DNA-based hydrogel to realize the specific and nondestructive separation of EXOs from complex biological media. The separated EXOs were directly utilized in the detection of human breast cancer in clinical samples, as well as applied in the therapeutics of myocardial infarction in rat models. The materials chemistry basis of this strategy involved the synthesis of ultralong DNA chains via an enzymatic amplification, and the formation of DNA hydrogels through complementary base-pairing. These ultralong DNA chains that contained polyvalent aptamers were able to recognize and bind with the receptors on EXOs, and the specific and efficient binding ensured the selective separation of EXOs from media into the further formed networked DNA hydrogel. Based on this DNA hydrogel, rationally designed optical modules were introduced for the detection of exosomal pathogenic microRNA, which achieved the classification of breast cancer patients versus healthy donors with 100% precision. Furthermore, the DNA hydrogel that contained mesenchymal stem cell–derived EXOs was proved with significant therapeutic efficacy in repairing infarcted myocardium of rat models. We envision that this DNA hydrogel–based bioseparation system is promising as a powerful biotechnology, which will promote the development of extracellular vesicles in nanobiomedicine.

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