Kühnemund Malte, Witters Daan, Nilsson Mats, Lammertyn Jeroen
Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Box 815, 751 08 Uppsala, Sweden.
Lab Chip. 2014 Aug 21;14(16):2983-92. doi: 10.1039/c4lc00348a. Epub 2014 Jun 17.
We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rolling circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products μL(-1) as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.
我们展示了一种基于锁式探针介导的DNA检测和等温环到环扩增（C2CA）的新型数字微流控核酸扩增概念。该检测平台结合了两种数字方法。首先，对用作微反应室的液滴进行数字微流控操作，并在这些液滴之间穿梭磁性颗粒，有助于复杂固相多步检测的整合。我们展示了一种在数字微流控芯片上针对超顺磁性颗粒的优化新型颗粒提取和转移方案，该方案可实现近100%的提取效率，确保高检测性能。其次，数字单分子检测所需的分隔通过简单的分子生物学方法解决，避免了大多数（如果不是全部）其他数字核酸检测方法所需的复杂微加工程序。为此，锁式探针在严格依赖靶标的连接反应中环化，并通过两轮滚环扩增进行扩增，包括一个中间消化步骤。每个检测到的靶分子反应会产生数百个直径为500 nm的可单独计数的DNA纳米球。我们证明，集成的小型数字微流控C2CA产生的C2CA产物数量与芯片外试管对照实验中每微升（μL(-1)）的数量一样高，表明检测性能高且无信号损失。低至1 aM的合成铜绿假单胞菌DNA被检测到，线性动态范围超过4个数量级，高达10 fM，证明其非常适合传染病诊断。
