Rotationally-driven lab-on-a-disc (LoaD) microfluidic systems are among the most promising methods for realizing complex nucleic acid (NA) testing at the point-of-need (PoN). However, despite significant advancements in NA amplification methods, very few sample-to-answer centrifugal microfluidic platforms have been realized due, in part, to a lack of on-disc sample preparation. In many instances, NA extraction (NAE) and/or lysis must be performed off-disc using conventional laboratory equipment and methods, thus tethering the assay to centralized facilities. Omission of in-line cellular lysis and NAE can be partially attributed to the nature of centrifugally-driven fluidics. Since flow is directed radially outward relative to the center of rotation (CoR), the number of possible sequential unit operations is limited by the disc radius. To address this, we report a simple, practical, automatable, and easy-to-implement method for inward fluid displacement (IFD) compatible with downstream nucleic acid amplification tests (NAATs). This approach leverages carbon dioxide (CO₂) gas generated from on-board acid-base neutralization to drive liquid from the disc periphery towards the CoR. Large architectural features or highly corrosive chemicals required in other approaches were replaced with safe-to-handle IFD reagents that maintained their reactivity for at least six months of storage on-disc. Further, spatiotemporal control over neutralization initiation and containment of the resultant pneumatic pressure head was reliably achieved using a single diode for both laser-actuated valve opening and channel sealing, which eliminated the need for manual intervention (e.g., taping over vents) required in other IFD methods. Following initial characterization via dye recovery studies, we demonstrated for the first time that CO₂-driven displacement does not inhibit downstream NAATs; NAs isolated direct-from-swab on disc were compatible with both ‘gold standard’ polymerase chain reaction (PCR) techniques and loop-mediated isothermal amplification (LAMP). The IFD approach described here stands to significantly ease integration of an increased number of sequential on-board processes, including cellular lysis, nucleic acid extraction, amplification, and detection, to greatly lower barriers towards automatable sample-to-answer LoaDs amenable for use on-site operation by non-technical personnel.

旋转驱动的盘上实验室 (LoaD) 微流体系统是在需要点 (PoN) 实现复杂核酸 (NA) 测试的最有希望的方法之一。然而，尽管 NA 扩增方法取得了显着进步，但很少有样品到答案的离心微流体平台被实现，部分原因是缺乏盘上样品制备。在许多情况下，NA 提取 (NAE) 和/或裂解必须使用传统的实验室设备和方法在光盘外进行，从而将检测与集中设施联系起来。在线细胞裂解和 NAE 的省略可部分归因于离心驱动流体的性质。由于流动相对于旋转中心 (CoR) 径向向外引导，因此可能的顺序单元操作的数量受到圆盘半径的限制。为了解决这个问题，我们报告了一种简单、实用、可自动化且易于实施的向内流体置换 (IFD) 方法，该方法与下游核酸扩增测试 (NAAT) 兼容。这种方法利用二氧化碳（CO₂ ) 机载酸碱中和产生的气体，用于将液体从圆盘外围驱向 CoR。其他方法所需的大型结构特征或高腐蚀性化学品被安全处理的 IFD 试剂所取代，这些试剂在盘上存储至少六个月时保持其反应性。此外，使用用于激光驱动的阀门打开和通道密封的单个二极管可靠地实现了对中和启动和所产生的气压头的遏制的时空控制，这消除了其他需要人工干预（例如，在通风口上贴胶带）的需要。 IFD 方法。在通过染料回收研究初步表征之后，我们首次证明了 CO ₂-驱动的位移不会抑制下游的 NAAT；NAs 直接从盘上的拭子中分离出与“黄金标准”聚合酶链式反应 (PCR) 技术和环介导等温扩增 (LAMP) 兼容。此处描述的 IFD 方法可以显着简化越来越多的连续机载过程的集成，包括细胞裂解、核酸提取、扩增和检测，从而大大降低适用于- 由非技术人员进行现场操作。