Commercial Strip-Inspired One-Pot CRISPR-Based Chip for Multiplexed Detection of Respiratory Viruses

The absence of sensitive, multiplexed, and point-of-care assays poses a critical obstacle in promptly responding to emerging human respiratory virus (HRV) pandemics. Herein, RECOGNIZER (re-building commercial pregnancy strips via large-size nanoflowers), an innovative one-pot CRISPR assay, is presented that employs commercially available strips to identify several types of HRVs. The superiority of the RECOGNIZER assay mainly relies on two aspects: (i) DNA nanoflowers possessing a high surface-to-volume ratio and well-defined surface allow for a considerable probe loading density and minimized non-specific interaction, achieving an impressive signal-to-noise proportion exceeding tenfold at 1 nM target. (ii) The design of the one-pot reaction, multi-channel chip, and custom-made app enables the rapid, sample-to-answer, and multiplexed analysis of four HRVs in 25 min. This assay demonstrates a sensitivity of 5.42 pM for synthetic SARS-CoV-2 RNA and 10 copies µL^-1 for SARS-CoV-2 plasmids after pre-amplification. Finally, the proposed approach indicated 100% accuracy in 50 clinical swab samples, demonstrating the robust performance in distinguishing SARS-CoV-2 from other HRVs. The versatility and scalability of RECOGNIZER renders it a user-friendly platform for virus infection monitoring, offering significant potential for improving pandemic response efforts.