Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.
Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.
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DOI：
10.1021/acsnano.5b07338
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发表时间：
2016-03-22
期刊：
影响因子：
17.1
通讯作者：
Ismagilov RF
中科院分区：
文献类型：
--
作者：
Rodriguez-Manzano J;Karymov MA;Begolo S;Selck DA;Zhukov DV;Jue E;Ismagilov RF
数字单分子技术正在扩大诊断能力，通过直接计数单分子，实现对目标的超灵敏定量，例如HIV和丙型肝炎感染中的病毒载量。用任何未经修改的手机摄像头都可以捕捉到的强大的视觉读数取代荧光读数，将有助于诊断测试的全球分布，包括在资源有限的环境中，需求最大。本文描述了一种用于开发用于RNA和DNA的数字单分子扩增的视觉读出系统的方法，通过（i）选择与标准移动的电话的光谱灵敏度兼容的比色扩增指示剂染料，以及（ii）识别最佳比率度量图像-用于所选染料的过程，以实现对照明条件和相机硬件稳健的读出，并提供明确的定量结果，即使是色盲用户。我们还分析了这种方法的局限性，并提供了一种微流体方法，可用于扩大动态范围和提高反应性能，允许在低至5 nL的体积下进行超灵敏的定量测量。我们使用基于SlipChip的数字单分子等温扩增以λDNA作为模型，丙型肝炎病毒RNA作为临床相关靶标来验证这种方法。在明智选择的指示剂染料和比率图像处理与滑动芯片技术的存在下的等温扩增化学的创新组合允许使用未修改的手机相机在纳升体积中的单个核酸分子的序列特异性视觉读出。当与集成样品制备和核酸扩增的设备配对时，这种与硬件无关的方法将提高定量诊断和环境测试的可负担性和分布。
Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.