mRNA Expression Profiling from Extracellular Vesicles (EVs): Generating a Rapid Diagnostic for Stroke

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Project Summary/Abstract Stroke is a disorder of the brain by which a part loses its blood supply and the affected region rapidly progresses to death if blood flow is not restored in time. Treatments to restore blood flow after acute ischemic stroke (AIS) are effective if administered <4.5 h from the onset of the stroke event. But, due in part to the lack of an in vitro diagnostic test for AIS, imaging (CT – clinical sensitivity ~26%) at the attending hospital is required for diagnosis. As a result, <7% of AIS patients receive treatment. Therefore, a critical need exists to develop strategies for diagnosing stroke in near real time that potentially can allow for point-of-care testing (POCT). One approach is a peripheral blood test using markers that quickly respond to changes in the brain induced by stroke. The proposed project will develop innovative technologies that uses peripheral blood markers for diagnosing stroke syndromes (AIS and hemorrhagic stroke) in near real time (~31 min for sample-to-answer) with an LOD of ~0.03 ng (total RNA). The research team has found that alterations in mRNA expression secured from white blood cell subsets can be used for stroke diagnosis and appear rapidly in peripheral blood following a stroke event. Resulting from a prior R01, CD15+ and CD8+ leukocytes were discovered as a predominant source of stroke- related mRNA biomarkers. This project seeks to realize the development of an innovative fluidic cartridge and the associated assay for the measurement of stroke-related RNA markers sourced from CD15 and CD8 expressing extracellular vesicles (EVs). The utility of EVs as a source of stroke-related RNA biomarkers is based on their rapid appearance and high abundance in plasma, potentially providing even faster stroke diagnosis compared to the cells from which they originate. This project will discover EV-RNA markers with high clinical sensitivity and specificity (>80%) for diagnosing ischemic and hemorrhagic stroke in <3 h from stroke onset. The cartridge, which consists of task-specific modules made from plastics via replication (i.e., injection molding) connected to a fluidic motherboard, will use the EV-RNA markers emanating from this project. Plasma will serve as the input from which surface-affinity selection of CD8 and CD15 EVs will occur using a specifically designed module. Following EV release from the capture surface via a photocleavable linker (cleaved using a blue-light LED), the cartridge will quantify the number of EVs selected using a label-free readout strategy. The fluidic cartridge also consists of a mixed-scale (nm → µm) module to read electrically copy numbers of stroke EV-RNA markers in a highly multiplexed fashion (>24 targets). This module will consist of in-plane nanopores made in a plastic via nano-injection molding and can identify RNAs using a distinct oligonucleotide primer pair querying a specific RNA using a solid-phase ligase detection reaction (spLDR). Single-molecule readout will allow for high analytical sensitivity to observe subtle changes in EV-RNA marker copy numbers. The utility of this cartridge will be evaluated in a clinical setting. Success of the project is leveraged by a strong multidisciplinary team, whom have a productive record of collaboration in developing stroke markers and diagnostic platforms for stroke.