Srimathi Siddharth Raghu, Ignacio Maxinne A, Rife Maria, Tai Sheldon, Milton Donald K, Scull Margaret A, DeVoe Don L
Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, USA.
Fischell Institute for Biomedical Devices, University of Maryland, College Park, USA.
Lab Chip. 2025 Apr 8;25(8):2004-2016. doi: 10.1039/d4lc00940a.
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.
定量检测感染性病毒对于疫苗开发、临床诊断和传染病研究至关重要，但目前的检测方法受到周转时间长、成本高和操作繁琐的限制。为了解决这些局限性，我们提出了一种数字聚焦检测方法，该方法采用一系列独立的纳升细胞培养体系。微流控平台允许在每个纳升孔中接种病毒感染细胞，随后通过油相离散化防止交叉污染。孵育后，通过免疫荧光染色观察感染细胞，并通过自动图像分析生成病毒抗原阳性孔的二元图，从而通过统计分析计算感染性病毒滴度。该平台所需的样品和试剂体积比传统聚焦检测方法显著更小，同时提高了检测自动化程度和终点时间灵活性。该技术应用于使用模型病毒和临床标本对甲型流感病毒的感染性进行定量检测，证明该数字平台是一种准确、快速、经济高效且便捷的病毒载量定量工具，在临床、制药和研究应用中具有广泛的用途。
