Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States.
Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, United States; Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States.
Colloids Surf B Biointerfaces. 2015 Feb 1;126:489-95. doi: 10.1016/j.colsurfb.2015.01.001. Epub 2015 Jan 10.
Amplification and detection of nucleic acid sequences within integrated microsystems is routinely conducted using the technique of droplet PCR, wherein the polymerase chain reaction (PCR) is performed in microscale water-in-oil droplets (nanoliter to picoliter volumes). During droplet PCR, interactions at the interface of the droplet tend to dominate. Specifically, adsorption of the polymerase at the droplet interface leads to inefficient amplification. To reduce polymerase adsorption, surfactants such as the silicone-based ABIL EM90 have been commonly used. However, these surfactants have been selected largely through trial and error, and have been only somewhat effective. For example, when using ABIL EM90, 8 times (8 ×) the manufacturer prescribed concentration of polymerase was necessary for amplification. In this report, we use the pendant drop technique to measure adsorption and loss of enzyme at droplet interfaces for various surfactant-oil combinations. Dynamic interfacial tension and surface pressure measurements showed that significant polymerase adsorption occurs when using ABIL EM90. In contrast, much lower polymerase adsorption is observed when using Brij L4, a nonionic surfactant with a C12 tail and an oxyethylene headgroup, which has not yet been reported for droplet PCR. These results correlate strongly with droplet PCR efficiency. Brij L4 enables highly efficient PCR at 2 × polymerase concentration, and still enables effective PCR at 1 × polymerase concentration. Overall, this work introduces a methodology for quantitatively assessing surfactants for use with droplet microreactors, and it demonstrates the practical value of this new approach by identifying a surfactant that can dramatically improve the efficiency of droplet PCR.
在集成微系统中扩增和检测核酸序列通常使用液滴 PCR 技术进行，其中聚合酶链反应 (PCR) 在微尺度的油水液滴（纳升到皮升级别）中进行。在液滴 PCR 过程中，液滴界面的相互作用往往占主导地位。具体来说，聚合酶在液滴界面的吸附会导致扩增效率降低。为了减少聚合酶的吸附，通常使用基于硅的 ABIL EM90 等表面活性剂。然而，这些表面活性剂主要是通过反复试验选择的，并且效果有限。例如，在使用 ABIL EM90 时，需要 8 倍（8×）制造商规定的聚合酶浓度才能进行扩增。在本报告中，我们使用悬滴技术测量各种表面活性剂-油组合在液滴界面处的酶吸附和损失。动态界面张力和表面压力测量表明，使用 ABIL EM90 时会发生显著的聚合酶吸附。相比之下，当使用 Brij L4 时，观察到的聚合酶吸附要低得多，Brij L4 是一种带有 C12 尾和乙氧基头的非离子表面活性剂，在液滴 PCR 中尚未报道过。这些结果与液滴 PCR 效率密切相关。Brij L4 可在 2×聚合酶浓度下实现高效 PCR，在 1×聚合酶浓度下仍能实现有效 PCR。总的来说，这项工作为定量评估用于液滴微反应器的表面活性剂引入了一种方法，并通过鉴定一种可以显著提高液滴 PCR 效率的表面活性剂，展示了这种新方法的实际价值。
