High-Throughput Microfluidic Extraction of Platelet-free Plasma for MicroRNA and Extracellular Vesicle Analysis

Area of Science:

• Biomarker Discovery
• Microfluidics
• Extracellular Vesicles

Background:

• Cell-free RNAs and extracellular vesicles (EVs) are crucial liquid biopsy biomarkers but susceptible to preanalytical variability.
• Current isolation methods like centrifugation can introduce inconsistencies and platelet contamination.
• Standardized, high-throughput isolation is needed for reliable biomarker analysis.

Purpose of the Study:

• To develop a high-throughput, label-free inertial microfluidic device (ExoArc) for isolating platelet-free plasma and EVs.
• To reduce preanalytical variability and improve the yield and purity of isolated biomarkers.
• To validate the device for clinical applications in cancer and metabolic disease diagnostics.

Main Methods:

• Utilized a novel inertial microfluidic device (ExoArc) with a 500 nm cutoff for differential lateral migration and particle separation.
• Performed single-step isolation of platelet-free plasma, reducing platelet-associated miRNA contamination compared to centrifugation.
• Coupled ExoArc with size exclusion chromatography (SEC) for efficient EV isolation and integrated with RT-qPCR for miRNA analysis.

Main Results:

• ExoArc achieved a submicrometer size cutoff, effectively removing leukocytes, RBCs, platelets, and debris.
• A 7-miRNA panel identified using ExoArc demonstrated ~90% sensitivity for non-small cell lung cancer detection.
• EV isolation using ExoArc+SEC yielded ~10-fold higher than ultracentrifugation, with distinct miRNA profiles observed in T2DM subjects.

Conclusions:

• ExoArc provides a centrifugation-free, automated workflow for plasma and EV isolation, minimizing preanalytical variability.
• The device enables sensitive biomarker discovery and has potential for point-of-care diagnostics.
• ExoArc facilitates quality control in EV manufacturing and advanced transcriptomics analysis.