Department of Mechanical Engineering, University of Washington, Seattle, WA, USA.
Department of Chemical Engineering, University of Washington, Seattle, WA, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jan 15;1163:122494. doi: 10.1016/j.jchromb.2020.122494. Epub 2020 Dec 13.
Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 10 copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.
核酸扩增检测（NAAT）是传染病诊断和监测的重要工具。NAAT 的一个关键程序步骤是样品制备：在核酸扩增和检测之前，从粗生物样品中分离和纯化靶核酸。传统上，样品制备是通过液相等或固相萃取来完成的，这两种方法都需要经过多次训练有素的用户操作和大量的实验室设备。与样品制备相关的挑战限制了 NAAT 即时诊断在资源有限环境中的传播，包括低收入和中等收入国家。我们报告了一种基于纸张的设备，用于使用等速电泳（ITP）从全血中纯化核酸，用于即时护理 NAAT。我们展示了从大量（33µL）全人血样中成功提取和纯化靶核酸，无需移动部件，用户操作步骤也很少。我们的设备利用基于纸张的缓冲剂储液器完全容纳液体 ITP 缓冲剂，不需要复杂的填充程序，而是依靠集成的纸张膜的自然吸液作用。我们通过过滤在设备上进行血液分离，从样品中去除白细胞和红细胞，然后对天然内源性血浆蛋白进行集成的纸上蛋白水解消化，以实现成功的等速电泳提取。基于纸张的等速电泳纯化和浓缩直接添加到重组酶聚合扩增（RPA）反应中的靶核酸。我们显示了输入拷贝浓度低至 3×10 拷贝/ mL 输入血液的一致扩增，提取和纯化仅需 30 分钟。通过采用纸张架构，我们能够将这些过程整合到一个单一、稳健、低成本的设计中，从而能够直接处理大量血液，唯一的中间用户步骤是去除和添加胶带。我们的设备代表了在即时护理点进行核酸扩增检测的简单、完全集成的样品制备系统的一个步骤。
