Wash-Free Isolation and Quantification of Tumor-Derived Exosomes via In Situ-Formed Hydrogel

The isolation and detection of exosomes as tumor markers are of vital importance for the early diagnosis, therapeutic monitoring, and mechanistic studies of tumors. Here, exosomes derived from breast cancer cells were chosen as model targets, and a wash-free, enzyme-free, handheld mini centrifugation method based on hydrogels was developed to effectively isolate and detect breast cancer exosomes. Dual aptamers (CD63-T1 and EpCAM-T2) were employed to specifically recognize and capture breast cancer exosomes. This specific recognition triggered the formation of hybridization chain reaction (HCR) nanostructures on the captured exosomes through the interaction of hairpin 1 and the alginate complex (H1-Alg) and hairpin 2 (H2-Cy3). The interaction of Ca²⁺ and alginate enabled the in situ formation of a hydrogel on the exosome surface. Subsequent low-speed centrifugation using a handheld mini centrifuge facilitated the efficient isolation of the exosomes, thereby eliminating the need for tedious washing steps. Utilizing the classical chelation reaction of ethylene diamine tetraacetic acid (EDTA) with Ca²⁺, the hydrogel can be rapidly cleaved for enzyme-free release of exosomes. The method demonstrated excellent capture and release efficiencies of approximately 85% and 98%, respectively, for specific cancerous exosomes. Notably, the exosomes isolated by the hydrogel system retained excellent biological activity, making them suitable for further analysis and potential applications. Meanwhile, the highly sensitive detection of breast cancer exosomes based on this strategy could also be achieved with a lower limit of detection as low as 3.2 × 10³ particles/mL. This work provides a novel and cost-effective strategy for the effective isolation and detection of tumor-derived exosomes, which can help to promote the subsequent application of exosomes in research.