"Paper Machine" for Molecular Diagnostics
"Paper Machine" for Molecular Diagnostics
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DOI：
10.1021/acs.analchem.5b00411
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发表时间：
2015-08-04
影响因子：
7.4
通讯作者：
Whitesides, George M.
中科院分区：
文献类型：
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作者：
Connelly, John T.;Rolland, Jason P.;Whitesides, George M.
基于引物引发的特定核酸序列扩增的临床测试实现了高水平的灵敏度和特异性。尽管具有这些理想的特性，但这些测试尚未充分发挥其潜力，因为它们的复杂性和费用限制了它们在集中实验室中的实用性。本文描述了一种将样品制备和环介导等温扩增 (LAMP) 与使用手持式紫外线源和拍照手机进行终点检测集成在一起的设备。该原型设备集成了纸微流体（以实现流体处理）和多层结构或造纸机，允许中心图案纸条滑入和滑出流体路径，从而允许在手持式一次性设备中引入样品、洗涤缓冲液、扩增预混液和检测试剂，以最少的移液量，用于资源有限环境中的护理点使用。该装置形成动态密封，可防止在 65 摄氏度孵育 1 小时期间蒸发。该间隔足以允许大肠杆菌 malB 基因的 LAMP 反应以 1 个双链 DNA 靶拷贝的分析灵敏度进行。本文从掺有完整活大肠杆菌细胞的人血浆开始，演示了样品制备与 LAMP 扩增和终点检测的完全集成，检测限为 5 个细胞。此外，它表明用于制备样品的方法能够从通常通过指尖采血获得的样品体积中浓缩 DNA。
Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a paper machine, that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65 degrees C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 double-stranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.