Crafting a streamlined isothermal amplification assay for sensitive and rapid nucleic acid detection of glioblastoma

Development of liquid biopsies for non-invasive tumor characterization techniques shows a great promise for real-time monitoring of tumor recurrence, progression, and treatment response. To better assess the various tumor evolution, cellular heterogeneity, consequent drug-resistance mechanisms, it is critical to screen specific proteins and nucleic acids from biofluid-derived extracellular vesicles (EVs). We are developing a rapid integrated ultra-sensitive digital bead-based sensor enabling evaluation of molecular profiling of EVs in glioblastoma (GBM). The tumor relevant nucleic acid information in EVs is enriched by antibody-specific magnetic beads and amplified by reverse transcription recombinase polymerase amplification using the CRISPR/Cas13a system with around twenty-five thousand droplets. Detection results are analyzed by a miniaturized portable imaging device. The niches of our developing technique include: (a) the streamlined sample preparation process and miniaturized detection system enable the sensitive detection of important mRNAs, EGFR, EGFRvIII, IDH1wt, IDH1 R132H and GAPDH, in EVs within 1.5 hours; (b) the fluorescent signal-to-noise ratio (SNR) in each micrometer sized droplet is significantly enhanced via using a simple Cas13a assay and triggering greater than 10⁴ turnovers of fluorescent reporters on a high-throughput microfluidic chip; (c) real-time image data are transferred to a cloud-based server and classified using several trained YOLOv5 models; (d) the RNAomics results are displayed on the Raspberry Pi touchscreen. This approach improves the speed of GBM diagnosis and treatment decision-making in using liquid biopsy.