A 3D-printed, self-driven microfluidic sensor chip for point-of-care testing (POCT) of norovirus

Early diagnosis of norovirus (NV) is critical for effective prevention and control of outbreaks. Clustered regularly interspaced short palindromic repeats (CRISPR)-coupled isothermal amplification has been widely employed for the highly sensitive and specific detection of nucleic acids; however, optimizing the compatibility among multi-enzyme reaction systems remains a challenge. In this study, a compartmentalized, self-driven, 3D-printed microfluidic sensor device was developed for cascade isothermal amplification and CRISPR-mediated multistep reactions. This chip integrated the sample pretreatment process with glucose biosensing technology. By leveraging the digital quantification capability of a personal glucose meter (PGM) as an endpoint readout, the system detected NV nucleic acids with high sensitivity and specificity. A series of target RNA concentrations (0.1-10,000 fM) were quantified using the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR sensing method developed in this study. The relationship was linear between the logarithm of the NV target RNA concentration (log C) and the corresponding fluorescence intensity. The RT-RPA-CRISPR assay was further engineered into a microfluidic chip-based point-of-care testing (POCT) system, and the limit of detection of NV was about 60 copies. This integrated approach facilitates field-deployable diagnostics of viral and bacterial pathogens.