Multiplex Digital Nucleic Acid Analysis by a LAMP-Argonaute Coupling Assay via a Parallel Droplet Fusion SlipChip.

Multiplex digital nucleic acid analysis (NAA) allows the precise quantification of multiple target nucleic acids with single-molecule sensitivity, making it highly appealing for life science research and clinical diagnostics. Nucleic acid-guided endonucleases, such as CRISPR, have demonstrated great potential in digital NAA. However, performing multiplex digital NAA with an endonuclease remains challenging. The thermophilic Argonaute protein (Ago) enables specific targeting of multiple sequences by a single enzyme, exhibiting superior potential in multiplex detection. Here, we developed a multiplex digital NAA by coupling nucleic acid amplification and Ago-specific detection using parallel droplet fusion facilitated by a SlipChip. The SlipChip can generate a series of droplets to perform multiplex digital loop-mediated isothermal amplification (LAMP), followed by a series of droplets containing Ago reagents for parallel mixing and reactions, resulting in three distinct digital fluorescence signals (FAM, ROX, and Cy5) corresponding to each specific target sequence. We performed viral load analysis of respiratory viruses, including influenza A, influenza B, and SARS-CoV-2, within 60 min. In addition, we used this digital LAMP-Ago assay to analyze viral loads in 34 clinical samples. The system provides a multiplex digital NAA capable of precise nucleic acid quantification with high sensitivity and specificity.