A pneumatically actuated droplet microfluidics platform coupled with CRISPR/Cas12a enables amplification-free detection of SARS-CoV-2

Amplification-free digital detection of SARS-CoV-2 via CRISPR/Cas12a. • Pneumatically actuated droplet microfluidics enables uniform ∼30 μm droplets. • Rapid detection of SARS-CoV-2 with demonstrated performance in clinical samples. • Offers a simple, portable method for rapid virus detection. Outbreaks of respiratory viruses, including severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), Influenza A (IAV), and Influenza B (IBV) viruses, present a formidable challenge to global public health, driving the urgent need for rapid, sensitive, and deployable diagnostic tools. Here, we present an amplification-free digital nucleic acid detection platform that synergistically integrates a pneumatically actuated droplet microfluidics platform (PADMP) with CRISPR/Cas12a. PADMP enables the precise generation of highly uniform, monodisperse microdroplets (∼30 μm), within which an optimized Cas12a reaction is compartmentalized. This digital partitioning strategy exponentially increases the local target concentration and facilitates single-molecule detection without pre-amplification. Through systematic optimization of droplet stability, surfactant formulation, and Cas12a assay kinetics, the PADMP-Cas12a platform achieves a detection limit (LOD) of 100 pM for synthetic SARS-CoV-2 N gene targets within 40 min. It demonstrates exceptional specificity, showing no cross-reactivity with non-target respiratory viruses (IAV, IBV, SARS-CoV, MERS-CoV, HRSV). The platform successfully detected SARS-CoV-2 in clinical positive samples, with mean droplet fluorescence intensity negatively correlated with Ct values. By combining the advantages of droplet microfluidics with the programmable recognition and trans -cleavage activity of CRISPR/Cas12a, this platform provides a rapid, amplification-free, and highly specific digital detection pathway for RNA viruses, marking an important advance toward high-throughput and point-of-care testing (POCT) for molecular diagnostics. Download: Download high-res image (127KB) Download: Download full-size image