Acoustic Separation of Viruses and Small Extracellular Vesicles for Diagnostic Applications

Abstract

The isolation and molecular analysis of nanoscale bioparticles such as viruses and extracellular vesicles (EVs) are critical to advancing next-generation diagnostics, therapeutics, and vaccine development. However, conventional isolation techniques remain slow, labor-intensive, and inconsistent—hindering their practical use in clinical and research settings. This dissertation introduces a suite of acoustofluidic platforms that overcome these challenges by enabling rapid, high-purity, and scalable separation of viruses and small extracellular vesicles (sEVs) from complex biological fluids.To address the need for virus isolation, we developed the Bessel beam excitation separation technology (BEST), which utilizes non-diffractive, self-healing acoustic Bessel beams for continuous, high-resolution separation of viruses based on size. BEST achieves tunable cutoff thresholds and maintains high viral RNA yield, enabling efficient isolation of SARS-CoV-2 from saliva and MMLV from cell culture with minimal sample preprocessing. For neurodegenerative disease diagnostics, we created AHEADx, a compact and automated platform for isolating sEVs from plasma samples. AHEADx integrates acoustic Bessel beams, microelectrodes, and micropillar arrays to extract exosomal miRNAs in under 10 minutes—over 100 times faster than standard protocols—and achieves fivefold greater miRNA yield. In a clinical study, AHEADx distinguished Alzheimer’s disease patients from healthy controls with 100% accuracy using a four-miRNA panel, demonstrating its strong potential for early, non-invasive diagnosis. To enable broader clinical translation of sEV-based diagnostics, we developed PURE (Portable Ultrasonic Refinement for sEVs), a vacuum-free ultrafiltration platform that couples ultrasonic atomization and acoustic streaming to achieve efficient sEV purification. PURE removes over 98% of plasma protein contaminants while preserving sEV integrity and concentration. Direct RT-qPCR on PURE-purified sEVs enables miRNA-based detection of preeclampsia with significantly improved diagnostic performance, raising AUC values from ~50% to ~80% compared to total plasma RNA. Together, these acoustofluidic systems represent a major step toward practical, high-throughput, and high-fidelity bioparticle isolation, with broad applications across virology, neurodegenerative disease, and maternal health diagnostics.