Micro/Nanofluidics for Isolation and Quantification of Extracellular Nucleic Acids and Extracellular RNA Carriers

In recent years, extracellular nucleic acids and extracellular RNA carriers have been demonstrated as promising biomarkers for cancer diagnostics, treatment, and prognostics. However, the lack of proper isolation and quantification tools still hinders the advances of related liquid biopsy technologies based on these biomarkers. High-efficiency RNA carrier isolation techniques must be accessible to researchers and doctors to truly understand and utilize them for clinical applications. The sensitivity and specificity of nucleic acid quantification also can be improved through the development of extraction methods with better yield and purity. A fully-automated point-of-care system can be envisioned by integrating all these steps sequentially into a turn-key microfluidic system. We address these challenges through the development and integration of multiple micro/nanofluidic devices for isolation of carriers and nucleic acids, downstream lysis and quantification. Sensitivity, selectivity, and throughput of isolation and diagnostic technologies are enhanced with optimized non[1]equilibrium mechanisms and non-uniform structures. In Chapter 1, I review current extracellular RNA carrier isolation and nucleic acids purification/quantification technologies. In Chapter 2, I demonstrate our solution to overcome the current inefficient size-based EV isolation by asymmetric nanoporous membrane (ANM) ultrafiltration and explain how the symmetry-breaking geometry of the nanopore combining with shear force increase the yield and throughput. In Chapter 3, I will further utilize the asymmetric nanoporous membrane (ANM) for purification of both retrovirus and SARS-CoV-2. I demonstrate our technology, along with qRT-PCR improves the virus detection sensitivity by more than 30-fold compared to the conventional RNA extraction-based method. In Chapter 4, a novel electroplated magnetic nanoporous membrane (MNM) with heterogeneous superparamagnetic nano-junction is fabricated to improve the throughput and yield of immunocapture. In Chapter 5, an ionic transistor is designed to control the non-equilibrium depletion front for extraction of highly-charged biomolecules like nucleic acids. Both extracellular DNAs and RNAs can be isolated effectively without bias in length and concentration. In Chapter 6, I integrate surface acoustic wave lysis, depletion-induced concentration, and ion-exchange membrane sensors into a microfluidic platform for plasma EV-miRNA sensing. The various micro/nanofluidic modules developed in my thesis, plus their parallel electronic modules, enable a new turn-key integrated platform for isolating and quantifying extracellular nanocarriers and their disease biomarker (RNA and protein) cargoes. In the last chapter, I summarize their functionalities, how they can be further improved and the appropriate integration strategies for different applications.