Extracellular vesicles (EVs) play an important role in intercellular communication. Recently, there has been increasing interest in EVs as potential diagnostic biomarkers and therapeutic vehicles. However, the molecular properties and cargo information of EV subpopulations have not yet been fully investigated due to lack of reliable and reproducible EV separation technology. Current approaches have faced difficulties with efficiently isolating EVs from biofluids, especially subpopulations of small EVs. Here, we report an EV isolation method based on a size-selective microfluidic platform (ExoSMP) via nanomembrane filtration and electrophoretic force. This unique platform offers an enhanced approach to sorting a heterogeneous population of EVs based on size, with the additional advantages of being label-free and low-cost, and featuring a short processing time (<1 h), and convenient integration with downstream analysis. In this research, we used ExoSMP to demonstrate the isolation of cancer-derived small EVs (30-120 nm) with high recovery (94.2%) and reproducibility at an optimum sample flow rate. Furthermore, we investigated isolation of EV subpopulations by altering nanomembrane combinations with different pore size combinations (50 and 100 nm, 30 and 100 nm, 30 and 200 nm, and 30 and 50 nm). This ExoSMP technique can serve as a standardized EV isolation/separation tool, facilitating the clinical prospects of EVs and opening up a new avenue for future point-of-care applications in liquid biopsies.
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