Simple and high-efficiency extraction and analysis of exosomes from body fluids using zirconium (IV)-immobilized...

Exosomes (EXOs) carry abundant biological information from their parent cells and thereby are considered a potential source of disease biomarkers, but how to extract high-purity EXOs from body fluids in a simple and effective manner remains explored during screening biomarkers. Herein, a zirconium (IV)-immobilized supermacroporous restricted-access adsorbent was created to address this issue. Scanning electron microscope (SEM), laser scanning confocal microscope (LSCM) and nanoparticle tracking analysis (NTA) were used to validate the restricted-access mechanism of the adsorbent, by which EXOs are captured by Zr⁴⁺ sites inside supermacropores while particles larger than EXOs are excluded in the solution. The established solid-phase extraction method (SPE) was employed to directly treat cell-free culture, urine and plasma that were obtained via only one-step low-speed centrifugation. In terms of particle-to-protein ratios and particle size distribution, the quality of captured EXOs is much better than that with control adsorbent and "gold standard" ultracentrifugation method. By using the SPE coupled with LC-MS/MS analysis, a total of 898 proteins are identified from urinary EXOs of prostate cancer (PCa) patients and healthy volunteers, among which 68 proteins belong to the top 100 EXO marker proteins. Compared with healthy volunteers, 227 upregulated and 102 downregulated proteins are identified in PCa group, among which 155 proteins are related to PCa, demonstrating the clinical potential of SPE method in the tumor biomarker screening.