Division of Chemistry and Chemical Engineering, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.
Anal Chem. 2013 Nov 19;85(22):11129-36. doi: 10.1021/ac4030413. Epub 2013 Nov 7.
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 2-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 min change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R(2) = 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.
定量生物分析测量通常以动力学格式进行，并且已知对影响动力学本身或动力学测量的扰动不具有稳健性。我们假设，在“数字”（单分子）格式下进行相同的测量将显示出对这种扰动的更高稳健性。在这里，我们研究了在资源有限环境（LRS）下使用反转录环介导扩增（RT-LAMP）进行 HIV-1 RNA 分子定量测量时，温度和时间波动对扩增反应的稳健性。使用 dRT-LAMP 化学计数分子的数字格式即使在温度变化 6°C（p 值= 6.7×10（-7））的情况下，也可以检测到 HIV-1 RNA 浓度的 2 倍变化，而传统的动力学（实时）格式则无法检测到（p 值= 0.25）。数字分析也对反应时间的 20 分钟变化、使用消费级手机相机获得的较差成像条件以及对这些图像进行自动化基于云的处理具有鲁棒性（在 100 倍动态范围内，R（2）= 0.9997 与真实计数相比）。还可以使用闪光灯作为激发源，通过手机摄像头对多重 PCR 扩增的荧光输出进行成像。已经开发了许多基于有机、无机和生化反应的非线性扩增方案，但它们的稳健性尚不清楚。这项工作意味着这些化学物质在数字格式中而不是动力学格式中可能具有更高的稳健性。它还呼吁进行理论研究，以预测这些化学物质的稳健性，更一般地，设计稳健的反应架构。我们在这里使用的 SlipChip 和其他数字微流控技术已经存在，以能够对这些预测进行测试。这种工作可能导致鉴定或创建稳健的扩增化学物质，从而在 LRS 下实现快速和精确的定量分子测量。此外，它可能提供描述数字格式中化学和生物网络稳健性的更一般原则。
