Department of Mechanical Engineering, University of Washington, Stevens Way, Box 352600, Seattle, Washington, 98195, USA.
Department of Chemical Engineering, University of Washington, Seattle, Washington, USA.
Lab Chip. 2024 Oct 9;24(20):4879-4891. doi: 10.1039/d4lc00592a.
Isothermal nucleic acid amplification tests (NAATs) are a vital tool for point-of-care (POC) diagnostics. These assays are well-suited for rapid, low-cost POC diagnostics for infectious diseases compared to traditional PCR tests conducted in central laboratories. There has been significant development of POC NAATs using paper-based diagnostic devices because they provide an affordable, user-friendly, and easy to store format; however, the difficulties in integrating separate liquid components, resuspending dried reagents, and achieving a low limit of detection hinder their use in commercial applications. Several studies report low assay efficiencies, poor detection output, and poorer limits of detection in porous membranes compared to traditional tube-based protocols. Recombinase polymerase amplification is a rapid, isothermal NAAT that is highly suited for POC applications, but requires viscous reaction conditions that has poor performance when amplifying in a porous paper membrane. In this work, we show that we can dramatically improve the performance of membrane-based recombinase polymerase amplification (RPA) of HIV-1 DNA and viral RNA by employing a coin cell-based vibration mixing platform. We achieve a limit of detection of 12 copies of DNA per reaction, nearly 50% reduction in time to threshold (from ∼10 minutes to ∼5 minutes), and an overall fluorescence output increase up to 16-fold when compared to unmixed experiments. This active mixing strategy enables reactions where the target and reaction cofactors are isolated from each other prior to the reaction. We also demonstrate amplification using a low-cost vibration motor for both temperature control and mixing, without the requirement of any additional heating components.
等温核酸扩增测试（NAATs）是即时检测（POC）诊断的重要工具。与在中央实验室进行的传统 PCR 测试相比，这些检测方法非常适合用于传染病的快速、低成本 POC 诊断。已经有许多使用基于纸张的诊断设备的即时检测 NAAT 得到了显著发展，因为它们提供了一种经济实惠、用户友好且易于存储的格式；然而，将单独的液体成分集成、重新悬浮干燥试剂以及实现低检测限的困难阻碍了它们在商业应用中的使用。几项研究报告称，与传统的基于管的方案相比，多孔膜中的分离效率低、检测输出差且检测限较差。重组酶聚合酶扩增是一种快速的等温 NAAT，非常适合即时检测应用，但需要粘性的反应条件，在多孔纸膜中扩增时性能较差。在这项工作中，我们展示了我们可以通过使用硬币电池为基础的振动混合平台，极大地提高基于膜的重组酶聚合酶扩增（RPA）检测 HIV-1 DNA 和病毒 RNA 的性能。我们实现了每个反应检测下限为 12 个 DNA 拷贝，达到阈值的时间减少了近 50%（从约 10 分钟减少到约 5 分钟），荧光输出总体增加了 16 倍，与未混合的实验相比。这种主动混合策略可以使反应在反应之前将目标和反应辅助因子彼此隔离。我们还展示了使用低成本振动马达进行温度控制和混合的扩增，而不需要任何额外的加热组件。
