Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection
Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection
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DOI：
10.1039/c4lc00348a
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发表时间：
2014-08-21
期刊：
影响因子：
6.1
通讯作者：
Lammertyn, Jeroen
中科院分区：
文献类型：
--
作者：
Kuhnemund, Malte;Witters, Daan;Lammertyn, Jeroen
我们展示了一种新的数字微流控核酸扩增概念，它是基于挂锁探针介导的DNA检测和等温环到环扩增（C2CA）。该分析平台结合了两种数字方法。首先，作为微反应室的液滴的数字微流体操纵和在这些液滴之间穿梭的磁性颗粒促进了复杂的固相多步测定的集成。我们在数字微流体芯片上展示了一种针对超顺磁性颗粒的优化新型颗粒提取和转移方案，该方案允许接近100%的提取效率，确保高检测性能。第二，数字单分子检测所需的区室化通过简单的分子生物学手段解决，避免了对大多数（如果不是全部）其他数字核酸检测方法所必需的复杂微加工过程的需要。为此目的，在严格的靶依赖性连接反应中环化锁式探针，并通过两轮腐环扩增（包括中间消化步骤）进行扩增。该反应导致每个检测到的靶分子产生数百个500 nm大小的单独可计数的DNA纳米球。我们证明，集成的小型化数字微流控C2CA的结果在同样高的C2CA产品μ L-1的芯片外管控制实验，表明高测定性能，没有信号损失。检测到低至1 aM的合成铜绿假单胞菌DNA，线性动态范围超过4个数量级，高达10 fM，证明其非常适合于传染病诊断。
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 rotting 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 mu L-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.