Increased robustness of single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone versus real-time kinetic measurements.
Increased robustness of single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone versus real-time kinetic measurements.
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DOI：
10.1021/ac4030413
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发表时间：
2013-11-19
影响因子：
7.4
通讯作者：
Ismagilov, Rustem F.
中科院分区：
文献类型：
--
作者：
Selck, David A.;Karymov, Mikhail A.;Sun, Bing;Ismagilov, Rustem F.
定量生物分析测量通常以动力学形式进行，并且已知对于影响动力学本身或动力学测量的扰动并不稳健。我们假设以“数字”（单分子）格式进行的相同测量将显示出对此类扰动的更强的鲁棒性。在这里，我们研究了扩增反应（逆转录环介导扩增，RT-LAMP）在温度和时间波动的情况下的稳健性，当该反应用于在有限资源设置（LRS）下定量测量 HIV-1 RNA 分子时。尽管温度变化 6 °C，但使用 dRT-LAMP 化学计数分子的数字格式检测到 HIV-1 RNA 浓度发生了两倍变化（p 值 = 6.7×10−7），而传统的动力学（实时）格式则没有检测到（p 值 = 0.25）。数字分析对于 20 分钟的反应时间变化、消费者手机摄像头获得的较差成像条件以及这些图像的自动云处理也具有稳健性（R2 = 0.9997 与 100 倍动态范围内的真实计数相比）。多重 PCR 扩增的荧光输出也可以使用手机摄像头使用闪光灯作为激发源进行成像。已经开发了许多基于有机、无机和生化反应的非线性放大方案，但它们的鲁棒性尚不清楚。这项工作意味着这些化学物质在数字形式而不是动力学形式中可能更加稳健。它还需要理论研究来预测这些化学反应的稳健性，更普遍的是设计稳健的反应架构。我们在这里使用的滑动芯片和其他数字微流体技术已经存在，可以对这些预测进行测试。此类工作可能会导致识别或创建强大的扩增化学物质，从而能够在 LRS 下进行快速、精确的定量分子测量。此外，它可以提供更通用的原理来描述数字格式的化学和生物网络的稳健性。
Quantitative bioanalytical measurements are commonly performed in a kinetic format, and are known to not be robust to perturbation that affects the kinetics itself, or the measurement of kinetics. We hypothesized that the same measurements performed in a “digital” (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a two-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7×10−7), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20-minute change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R2 = 0.9997 vs. true counts over a 100-fold dynamic range). Fluorescent output of multiplexed PCR amplification could also be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification schemes based on organic, inorganic, and biochemical reactions have been developed but their robustness is not well understood. This work implies that these chemistries may be significantly more robust in the digital, rather than kinetic, format. It also calls for theoretical studies to predict robustness of these chemistries, and more generally to design robust reaction architectures. The SlipChip that we used here and other digital microfluidic technologies already exist to enable testing of these predictions. Such work may lead to identification or creation of robust amplification chemistries that enable rapid and precise quantitative molecular measurements under LRS. Furthermore, it may provide more general principles describing robustness of chemical and biological networks in digital formats.
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