Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Advanced Biomedical Instrumentation Centre, Hong Kong Science Park, Shatin, New Territories, Hong Kong, China.
Anal Chem. 2024 Oct 15;96(41):16134-16144. doi: 10.1021/acs.analchem.4c02429. Epub 2024 Oct 3.
Detection and quantification of viral particles (VPs) facilitate both diagnostics of pathogenic viruses and quality control testing of virus-based products. However, existing technologies fail to afford concurrent ultrasensitive detection and large-scale absolute quantification of VPs. Here, we propose a digital Microgel-in-Droplet enzyme-linked immunosorbent assay (ELISA) system that enables the processing and monitoring of millions of ELISA reactions at the single-VP level by incorporating droplet microfluidics with sandwich ELISA. Upon validating the microfluidic workflow and optimizing ELISA parameters, we demonstrate ultrasensitive VP detection at a limit of detection of 56 PFU/test. Leveraging a fluorescence-based screening platform, we further realize high-throughput digital counting of VPs with a linear detection range of 500-64 000 PFU/test. The precision is comparable to that of the gold standard, the plaque assay, across a wide range of virus concentrations. We anticipate that our system will provide a novel paradigm for the absolute enumeration of various types of viral particles.
病毒颗粒（VPs）的检测和定量既有助于致病性病毒的诊断，也有助于病毒产品的质量控制检测。然而，现有的技术无法同时实现对 VPs 的超灵敏检测和大规模绝对定量。在这里，我们提出了一种数字微凝胶-液滴酶联免疫吸附测定（ELISA）系统，通过将液滴微流控技术与夹心 ELISA 相结合，能够在单个 VP 水平上处理和监测数百万个 ELISA 反应。在验证微流控工作流程并优化 ELISA 参数后，我们证明了在检测限为 56 PFU/test 的情况下具有超灵敏的 VP 检测能力。利用基于荧光的筛选平台，我们进一步实现了 VP 的高通量数字计数，其线性检测范围为 500-64 000 PFU/test。其精度与金标准——空斑测定法——在广泛的病毒浓度范围内相当。我们预计，我们的系统将为各种类型的病毒颗粒的绝对计数提供新的范例。
