Microscale Inertial Flow Enables Direct Separation of Nanoscale Extracellular Vesicles from Whole Blood for Gastric Cancer Proteomic Analysis

Extracellular vesicles (EVs) hold great potential as disease biomarkers due to their ubiquity, high stability, and rich biomolecular cargo, but their clinical translation hinges on simple yet efficient isolation. Here, immunomicrosphere-assisted inertial flow microfluidics (IM-IFM) is presented for high-throughput, high-purity EV isolation from whole blood, enabling subsequent proteomic profiling. The IM-IFM first converts nanoscale EVs into microscale PSMSs@EVs complexes using antibody-conjugated polystyrene microspheres (PSMSs) and then directly isolates EVs from whole blood based on inertial focusing effects. Experimental results demonstrate that this method can isolate EVs from 2 mL of whole blood within 1 h while minimizing nonspecific adsorption. Mass spectrometry-based proteomics confirmed the EV-specific origin of isolated proteins while demonstrating a similar depth of coverage and a superior performance in removing high-abundance plasma proteins comparable to that of the conventional UC method. Clinically, proteomic analysis of IM-IFM-isolated EVs from gastric cancer (GC) patient blood identified multiple GC-associated protein biomarkers such as POSTN, demonstrating its utility for noninvasive cancer diagnostics. This strategy combines the specificity of immunocapture with the efficiency of inertial microfluidics, offering great potential for advancing EV-based liquid biopsy and precision medicine applications.