Immunomagnetic hydrogel nanofibrils for exosomes isolation

Exosomes (EXOs) are promising biomarker sources for liquid biopsy. Due to their low abundance in biofluids and the presence of non-target vesicle proteins during the isolation process, traditional methods such as ultracentrifugation (UC) are time-consuming, yield low purity, and can cause damage to EXOs. Achieving rapid, high-purity isolation remains a significant challenge. The magnetic beads (MBs) method is the most effective way to enhance EXO purity, but existing MBs face bottlenecks, including poor suspension stability, limited surface functionalization, slow magnetic response, and increased adsorption of interfering contaminants, ultimately leading to the loss of critical proteomic information.

In this study, a novel strategy for EXO separation was proposed based on the specific interaction between the tetraspanin protein CD63 on EXOs and CD63-targeted aptamers functionalized on immunomagnetic hydrogel nanofibrils (IMHNFs). The IMHNFs showed streamlined magnetic responsivity with a responsivity rate 8-fold faster than that of conventional magnetic beads and exhibited good suspension stability. The IMHNFs enabled rapid EXO isolation from biofluids within 30 min, faster than UC. Furthermore, IMHNFs offer universality for multiple biofluids, higher purity (3.28-8.05-fold compared with UC), lower pollution protein, richer proteomic information, and higher signal intensity (the signal intensity of the specific protein CD63 in urine-derived EXOs was over 10-fold that achieved with UC). Notably, 332 upregulated proteins were identified in urine-derived EXOs from prostate cancer (PCa) patients compared with normal controls, covering 61 PCa biomarkers confirmed and published in the literature, demonstrating the excellent potential of this method for future clinical research and applications.

The proposed IMHNFs method exhibited significant advantages in magnetic separation performance (rapid magnetic response and good suspension stability), high-purity EXO enrichment, and proteomic analysis-especially its universality in various biofluids. It not only provides new insights into immunomagnetic separation technology but also contributes to advancing EXO development in liquid biopsy.