Multiplex Liquid Biopsy Platform for Fractionation of Heterogeneous Vesicles and Precision Analyses of Their RNA Cargo
Hsueh-Chia Chang,
Bayer Professor of Engineering / Director,
University of Notre Dame
Extracellular RNAs in blood are promising circulating biomarkers. However, as they can be easily degraded, extracellular RNAs must form complexes with proteins or are encapsulated by liposomes, exosomes or microvesicles to remain stable. As a result, stable RNA biomarkers, like mRNA and miRNA, are carried by a heterogeneity of nanocarriers in blood with wide size, electrophoretic mobility and isoelectric point distributions. Due to their different biogenesis, these carriers have specific surface proteins and are often selective in their choice of RNA cargoes. To offer more precise quantification of the circulating RNA biomarkers, we have developed a suite of microfluidic modules that can fractionate the carriers by size, surface proteins and isoelectric point. Size-based separation is achieved by an asymmetric nanoporous membrane whose conic pores reduce hydrodynamic resistance and vesicle fusion. Extensive rinsing can be carried out to remove free-floating contaminating proteins like albumin. We also developed several lysing modules that can lyse the vesicles and dissociate the complexes non-chemically, such that low-yield extraction can be eliminated as an intermediate step. We integrate these modules with our membrane sensor, digital PCR module and nanopore sensor for RNA identification and quantification to achieve an integrated multiplexed platform for precise profiling of RNA cargoes in fractionated carriers. Some of the upstream modules can also be integrated into a pretreatment platform for NGS.
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