String-Powered Microfluidic Chip Integrating Heparin-Mediated One-Pot RT-RPA/CRISPR-Cas12a for Multiplex Detection of HFMD Viruses

作者：Z Chen, Kang Mao, Meng Hanqing, Chen Gao, Haoran Lv, Xiqing Li, Qiuda Zheng, Zhugen Yang, Ibrahim Ahmed Hamza, C Tu, Hailong Zhang · 发表于：Analytical Chemistry · 年份：2026 · DOI：10.1021/acs.analchem.6c00663 · 被引用次数：4 · 研究领域：Viral Infections and Immunology Research、CRISPR and Genetic Engineering、Respiratory viral infections research

Hand, foot, and mouth disease (HFMD) is a common childhood infection caused by enteroviruses, which exhibit distinct regional and seasonal epidemiological patterns. Wastewater-based epidemiology is a crucial tool for monitoring population infection dynamics and viral subtype distribution. However, the lack of effective on-site viral detection methods limits timely early warning and effective surveillance of infectious disease outbreaks. This study developed a one-pot RT-RPA/CRISPR-Cas12a assay-based, string-powered flywheel microfluidic chip for the multiplex detection of HFMD viruses in wastewater. First, by leveraging the regulatory effect of heparin sodium on CRISPR/Cas12a activity, a one-pot RT-RPA/CRISPR-Cas12a system was constructed to detect four major subtypes of HFMD virus (EV-A71, CV-A16, CV-A6, and CV-A10). Subsequently, this method was integrated into a pull-wire, flywheel-type, dual-axis centrifugal microfluidic chip, named the Heparin-Inhibited CRISPR-Associated System Chip (HICAS-Chip), enabling integrated enrichment, purification, elution, and multiplexed detection. The HICAS-Chip allowed visual detection of nucleic acids at 10 aM sensitivity within 1 h, corresponding to the sensitivity of the one-pot RT-RPA/CRISPR-Cas12a assay. During a year-long wastewater monitoring program in Guiyang City, China, the HICAS-Chip identified EV-A71 and CV-A10 as the predominant circulating subtypes, with incidence peaks observed in June, November, and December. The wastewater...