A CRISPR-assisted passive microfluidic chip for rapid, visual detection of multiple respiratory viruses

a-crispr-assisted-passive-microfluidic-chip-for-rapid,-visual-detection-of-multiple-respiratory-viruses
A CRISPR-assisted passive microfluidic chip for rapid, visual detection of multiple respiratory viruses

References

  1. Guo, X. et al. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature 579 (7799), 427–432. https://doi.org/10.1038/s41586-020-2078-2 (2020).

    Google Scholar 

  2. Malay, A. D. et al. An ultra-stable gold-coordinated protein cage displaying reversible assembly. Nature 569 (7756), 438–442. https://doi.org/10.1038/s41586-019-1185-4 (2019).

    Google Scholar 

  3. Murdoch, C. C. & Skaar, E. P. Nutritional immunity: the battle for nutrient metals at the host-pathogen interface. Nat. Rev. Microbiol. 20 (11), 657–670. https://doi.org/10.1038/s41579-022-00745-6 (2022).

    Google Scholar 

  4. Lucas, C. et al. Longitudinal analyses reveal immunological misfiring in severe COVID-19. Nature 584 (7821), 463–469. https://doi.org/10.1038/s41586-020-2588-y (2020).

    Google Scholar 

  5. Zhao, C. et al. Characterising the asynchronous resurgence of common respiratory viruses following the COVID-19 pandemic. Nat. Commun. 16 (1), 1610. https://doi.org/10.1038/s41467-025-56776-z (2025).

    Google Scholar 

  6. Zhao, X., Zhu, X., Wang, J., Ye, C. & Zhao, S. The epidemiological analysis of respiratory virus infections in children in Hangzhou from 2019 to 2023. Virus Res. 355, 199558. https://doi.org/10.1016/j.virusres.2025.199558 (2025).

    Google Scholar 

  7. Li, J. et al. Disease burden and epidemiological characteristics of common respiratory pathogens in children with respiratory tract infections in Guangzhou, 2017–2022. Chin. J. Infect. Control. 22 (1), 44–51. https://doi.org/10.12138/i.issn.1671-9638.20232814 (2023).

    Google Scholar 

  8. Tombuloglu, H. et al. Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N, RdRP and human RP genes. Sci. Rep. 12 (1), 2853. https://doi.org/10.1038/s41598-022-06977-z (2022).

    Google Scholar 

  9. Rong, G. et al. COVID-19 diagnostic methods and detection techniques. Encyclopedia Sens. Biosens. 17–32. https://doi.org/10.1016/B978-0-12-822548-6.00080-7 (2023).

  10. Pike, A. M., Friend, C. M. & Bell, S. P. Distinct RPA functions promote eukaryotic DNA replication initiation and elongation. Nucleic Acids Res. 51 (19), 10506–10518. https://doi.org/10.1093/nar/gkad765 (2023).

    Google Scholar 

  11. Xu, T. et al. Deep learning-enhanced hand-driven microfluidic chip for multiplexed nucleic acid detection based on RPA/CRISPR. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 12 (21), e2414918 https://doi.org/10.1002/advs.202414918 (2025).

  12. Zheng, F. et al. A highly sensitive CRISPR-Empowered surface plasmon resonance sensor for diagnosis of inherited diseases with Femtomolar-Level Real-Time Quantification. Advanced science (Weinheim. Baden-Wurttemberg Germany). 9 (14), e2105231. https://doi.org/10.1002/advs.202105231 (2022).

    Google Scholar 

  13. Chen, Y. et al. Ultrasensitive and specific clustered regularly interspaced short palindromic repeats empowered a plasmonic fiber tip system for Amplification-Free Monkeypox virus detection and genotyping. ACS Nano. 17 (13), 12903–12914. https://doi.org/10.1021/acsnano.3c05007 (2023).

    Google Scholar 

  14. Wang, L. et al. Rapid and ultrasensitive detection of Mpox virus using CRISPR/Cas12b-empowered graphene field-effect transistors. Appl. Phys. Reviews. 10 (3). https://doi.org/10.1063/5.0142494 (2023).

  15. Wu, C. et al. CRISPR-Cas12a-Empowered electrochemical biosensor for rapid and ultrasensitive detection of SARS-CoV-2 delta variant. Nano-micro Lett. 14 (1), 159. https://doi.org/10.1007/s40820-022-00888-4 (2022).

    Google Scholar 

  16. Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9 (6), 467–477. https://doi.org/10.1038/nrmicro2577 (2011).

    Google Scholar 

  17. Boonbanjong, P., Treerattrakoon, K., Waiwinya, W., Pitikultham, P. & Japrung, D. Isothermal amplification technology for disease diagnosis. Biosensors 12 (9), 677. https://doi.org/10.3390/bios12090677 (2022).

    Google Scholar 

  18. Sashital, D. G. Pathogen detection in the CRISPR-Cas era. Genome Med. 10 (1), 32. https://doi.org/10.1186/s13073-018-0543-4 (2018).

    Google Scholar 

  19. Kang, Y. et al. CRISPR-Cas12a-Based aptasensor for On-Site and highly sensitive detection of Microcystin-LR in freshwater. Environ. Sci. Technol. Environ. Sci. Technol. 56 (7), 4101–4110. https://doi.org/10.1021/acs.est.1c06733 (2022).

    Google Scholar 

  20. Li, S. Y. et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell. Discovery. 4, 20. https://doi.org/10.1038/s41421-018-0028-z (2018).

    Google Scholar 

  21. Dronina, J., Samukaite-Bubniene, U. & Ramanavicius, A. Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review). J. Nanobiotechnol. 20 (1), 41. https://doi.org/10.1186/s12951-022-01246-7 (2022).

    Google Scholar 

  22. Wang, B. et al. Cas12aVDet: A CRISPR/Cas12a-Based platform for rapid and visual nucleic acid detection. Anal. Chem. 91 (19), 12156–12161. https://doi.org/10.1021/acs.analchem.9b01526 (2019).

    Google Scholar 

  23. Fozouni, P. et al. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy. Cell 184 (2), 323–333e9. https://doi.org/10.1016/j.cell.2020.12.001 (2021).

    Google Scholar 

  24. de Dieu Habimana, J. et al. A rationally designed CRISPR/Cas12a assay using a multimodal reporter for various readouts. Anal. Chem. 95 (31), 11741–11750. https://doi.org/10.1021/acs.analchem.3c01876 (2023).

    Google Scholar 

  25. Jia, Z., Zhang, Y., Zhang, C., Wei, X. & Zhang, M. Biosensing intestinal alkaline phosphatase by pregnancy test strips based on Target-Triggered CRISPR-Cas12a activity to monitor intestinal inflammation. Anal. Chem. 95 (37), 14111–14118. https://doi.org/10.1021/acs.analchem.3c03099 (2023).

    Google Scholar 

  26. Li, Q. N. et al. Low-Background CRISPR/Cas12a sensors for versatile Live-Cell biosensing. Anal. Chem. 95 (42), 15725–15735. https://doi.org/10.1021/acs.analchem.3c03131 (2023).

    Google Scholar 

  27. Liu, L. et al. Generation and application of a novel high-throughput detection based on RPA-CRISPR technique to sensitively monitor pathogenic microorganisms in the environment. Sci. Total Environ. 838 (Pt 2), 156048. https://doi.org/10.1016/j.scitotenv.2022.156048 (2022).

    Google Scholar 

  28. Tian, B. et al. Tandem CRISPR nucleases-based lateral flow assay for amplification-free MiRNA detection via the designed locked RNA/DNA as fuels. Talanta 266 (Pt 1), 124995. https://doi.org/10.1016/j.talanta.2023.124995 (2024).

    Google Scholar 

  29. Yuan, J. et al. CRISPR-Cas12a-Mediated Hue-Recognition lateral flow assay for Point-of-Need detection of Salmonella. Anal. Chem. 96 (1), 220–228. https://doi.org/10.1021/acs.analchem.3c03753 (2024).

    Google Scholar 

  30. Zhang, H. et al. CRISPR-Cas12a based HSV DNA detection method using quantum dot-labeled immunochromatographic strips. Microchem. J. 207, 112117. https://doi.org/10.1016/j.microc.2024.112117 (2024).

    Google Scholar 

  31. Allen, A., Cooper, B. H., Singh, J., Rohs, R. & Qin, P. Z. PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding. Sci. Rep. 15 (1), 4930. https://doi.org/10.1038/s41598-025-87565-9 (2025).

    Google Scholar 

  32. Teo, S. M. et al. The infant nasopharyngeal Microbiome impacts severity of lower respiratory infection and risk of asthma development. Cell. Host Microbe. 17 (5), 704–715. https://doi.org/10.1016/j.chom.2015.03.008 (2015).

    Google Scholar 

  33. Zhou, H., Tsou, J. H., Chinthalapally, M., Liu, H. & Jiang, F. Detection and differentiation of SARS-CoV-2, Influenza, and respiratory syncytial viruses by CRISPR. Diagnostics (Basel Switzerland). 11 (5), 823. https://doi.org/10.3390/diagnostics11050823 (2021).

    Google Scholar 

  34. Ni, M., Xu, H., Luo, J., Liu, W. & Zhou, D. Simultaneous detection and differentiation of SARS-CoV-2, influenza A virus and influenza B virus by one-step quadruplex real-time RT-PCR in patients with clinical manifestations. Int. J. Infect. Diseases: IJID : Official Publication Int. Soc. Infect. Dis. 103, 517–524. https://doi.org/10.1016/j.ijid.2020.12.027 (2021).

    Google Scholar 

  35. Ho, Y. I. I., Wong, A. H. & Lai, R. W. M. Comparison of the cepheid Xpert xpress Flu/RSV assay to in-house Flu/RSV triplex real-time RT-PCR for rapid molecular detection of influenza A, influenza B and respiratory syncytial virus in respiratory specimens. J. Med. Microbiol. 67 (11), 1576–1580. https://doi.org/10.1099/jmm.0.000841 (2018).

    Google Scholar 

  36. Dou, Y. et al. Correction: A smartphone-based three-in-one biosensor for co-detection of SARS-CoV-2 viral RNA, antigen and antibody. Chem. Commun. (Camb., Engl). 58 (48), 6869. https://doi.org/10.1039/d2cc90184f (2022).

    Google Scholar 

  37. Najjar, D. et al. A lab-on-a-chip for the concurrent electrochemical detection of SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies in saliva and plasma. Nat. Biomedical Eng. 6 (8), 968–978. https://doi.org/10.1038/s41551-022-00919-w (2022).

    Google Scholar 

  38. Li, Y., Li, S., Wang, J. & Liu, G. CRISPR/Cas systems towards Next-Generation biosensing. Trends Biotechnol. 37 (7), 730–743. https://doi.org/10.1016/j.tibtech.2018.12.005 (2019).

    Google Scholar 

  39. Dincer, C., Bruch, R., Kling, A., Dittrich, P. S. & Urban, G. A. Multiplexed Point-of-Care Testing – xPOCT. Trends Biotechnol. 35 (8), 728–742. https://doi.org/10.1016/j.tibtech.2017.03.013 (2017).

    Google Scholar 

  40. Bruch, R. et al. CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free MiRNA diagnostics. Biosens. Bioelectron. 177, 112887. https://doi.org/10.1016/j.bios.2020.112887 (2021).

    Google Scholar 

  41. Broughton, J. P. et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat. Biotechnol. 38 (7), 870–874. https://doi.org/10.1038/s41587-020-0513-4 (2020).

    Google Scholar 

  42. Ali, Z. et al. iSCAN: an RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res. 288, 198129. https://doi.org/10.1016/j.virusres.2020.198129 (2020).

    Google Scholar 

  43. Sen, A. et al. Paper based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens. Biosens. Bioelectron. 257, 116292. https://doi.org/10.1016/j.bios.2024.116292 (2024).

    Google Scholar 

  44. Cao, H. et al. Paper device combining CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification for SARS-CoV-2 detection in wastewater. Environ. Sci. Technol. *56*. (18), 13245–13253. https://doi.org/10.1021/acs.est.2c03819 (2022).

Download references