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

Yu-He Liu; Yi-Chien Chen; Li-Hsien Ho; Zhi-Yang Wei; Chia-Hung Chen; Yun-Hsien Chung; I-Hsuan Chou; Tao-Yun Yen; Wei-Chun Lan; Hsien-Wen Yao; Peng-Wei Hsu; Chen-Han Huang; Hsing-Ying Lin

doi:10.1117/12.3007901

20 September 2023 Crafting a streamlined isothermal amplification assay for sensitive and rapid nucleic acid detection of glioblastoma

Yu-He Liu, Yi-Chien Chen, Li-Hsien Ho, Zhi-Yang Wei, Chia-Hung Chen, Yun-Hsien Chung, I-Hsuan Chou, Tao-Yun Yen, Wei-Chun Lan, Hsien-Wen Yao, Peng-Wei Hsu, Chen-Han Huang, Hsing-Ying Lin

Author Affiliations +

Proceedings Volume 12608, Biomedical Imaging and Sensing Conference; 126080N (2023) https://doi.org/10.1117/12.3007901
Event: Optics and Photonics International Congress, 2023, Yokohama, Japan

Abstract

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.

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Yu-He Liu, Yi-Chien Chen, Li-Hsien Ho, Zhi-Yang Wei, Chia-Hung Chen, Yun-Hsien Chung, I-Hsuan Chou, Tao-Yun Yen, Wei-Chun Lan, Hsien-Wen Yao, Peng-Wei Hsu, Chen-Han Huang, and Hsing-Ying Lin "Crafting a streamlined isothermal amplification assay for sensitive and rapid nucleic acid detection of glioblastoma", Proc. SPIE 12608, Biomedical Imaging and Sensing Conference, 126080N (20 September 2023); https://doi.org/10.1117/12.3007901

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KEYWORDS

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Nucleic acids

Biopsy

Profiling

Cancer detection

Data modeling

Liquids

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