Introduction:: Exosomes are small extracellular vesicles (sEVs) released by nearly all types of cells, serving as important diagnostic biomarkers for cancers, Alzheimer's disease and other diseases. However, since they are small vesicle structures (30 ~ 150 nm in diameter), it’s difficult to isolate exosomes efficiently with intact structure and little contamination. Many methods have been applied to isolate exosomes from biofluids, but they all have their limitations. Ultracentrifugation (UC) is the gold standard in exosome isolation, but it is time-consuming and the product are mixed with lipoproteins. The on-chip acoustic tweezers could separate EVs from lipoproteins based on acoustic contrast factor difference, but the purity and throughput is limited. The EXODUS method combines the ultrafiltration with oscillation system and behaves well on dilute biofluid such as urine, tears and saliva, but it’s throughput for concentrated biofluid, such as plasma, is rather low (20 μL per test). Actually, the interactions between oscillating membranes and bioparticles during filtration is still not clear, blurring the applicability of the ultrafiltration method. Here we propose a mechano-electronic microplate (MEM) for parallel exosome isolation with high efficiency from human plasma.
Materials and Methods:: The MEM integrates anodic aluminum oxide (AAO) membranes into a microplate design. The operator only needs pipette to push and pull the sample from the wells on the microplate. The microplate is placed on a 3D-printed vacuum box with hole array, which could generate negative pressure below each well to launch the filtration. During filtration, the PZT ring under AAO would stimulate the membrane to avoid clogging and facilitate the small proteins to move through the membrane, while the exosomes with larger size would stay above the membrane. To address the heat management problem after integration, the MEM adapted a self-cooling design, using the negative pressure below the membrane as a natural cooling pump. The coolant air would flow through the microchannels on the O-ring below the PZT into the vacuum box due to pressure difference, leading to an efficient passive cooling during filtration. The purity of product is checked by BCA tests, and the yield is checked by fluorescent Nanoparticle Tracking Analysis (NTA). The structure of exosomes are examined by Transmission Electron Microscopy (TEM).
Results, Conclusions, and Discussions:: For a wide range of input volume (20 uL to 1 mL), the MEM could remove 96% protein contaminants from plasma with in less than 1 hour, with the exosome yield as high as 80%. The exosomes still maintain intact structure after the filtration. With the MEM, the biologists only need to spend a few dollars per sample to isolate high-quality exosomes, just with pipettes, and spend less than 1 hour for multiple samples, providing a significant technology for exosome research and applications.
