Multiplex Digital\nNucleic Acid Analysis by a LAMP–Argonaute\nCoupling Assay via a Parallel Droplet Fusion SlipChip

Multiplex\ndigital nucleic acid analysis (NAA) allows\nthe precise\nquantification of multiple target nucleic acids with single-molecule\nsensitivity, making it highly appealing for life science research\nand clinical diagnostics. Nucleic acid-guided endonucleases, such\nas CRISPR, have demonstrated great potential in digital NAA. However,\nperforming multiplex digital NAA with an endonuclease remains challenging.\nThe thermophilic Argonaute protein (Ago) enables specific targeting\nof multiple sequences by a single enzyme, exhibiting superior potential\nin multiplex detection. Here, we developed a multiplex digital NAA\nby coupling nucleic acid amplification and Ago-specific detection\nusing parallel droplet fusion facilitated by a SlipChip. The SlipChip\ncan generate a series of droplets to perform multiplex digital loop-mediated\nisothermal amplification (LAMP), followed by a series of droplets\ncontaining Ago reagents for parallel mixing and reactions, resulting\nin three distinct digital fluorescence signals (FAM, ROX, and Cy5)\ncorresponding to each specific target sequence. We performed viral\nload analysis of respiratory viruses, including influenza A, influenza\nB, and SARS-CoV-2, within 60 min. In addition, we used this digital\nLAMP-Ago assay to analyze viral loads in 34 clinical samples. The\nsystem provides a multiplex digital NAA capable of precise nucleic\nacid quantification with high sensitivity and specificity.