Microfluidic digital focus assays for the quantification of infectious influenza virus

Quantifying infectious virus is essential for vaccine development, clinical diagnostics, and infectious disease research, but current assays are constrained by long turnaround times, high costs, and laborious procedures. To address these limitations, we present a digital focus assay employing an array of independent nanoliter cell cultures. The microfluidic platform allows cells in each nanowell to be inoculated with virus, followed by oil discretization to prevent cross-contamination. After incubation, infected cells are visualized through immunofluorescence staining, and a binary map of wells positive for viral antigen is generated by automated image analysis, allowing infectious viral titer to be calculated by statistical analysis. The platform requires significantly smaller sample and reagent volumes than conventional focus assays while enhancing assay automation and endpoint time flexibility. The technology is applied to the quantification of infectious influenza A using both model virus and clinical specimens, demonstrating the digital platform as an accurate, rapid, cost-effective, and convenient tool for viral load quantification with broad utility in clinical, pharmaceutical, and research applications.