Clockwork PCR including sample preparation
Clockwork PCR including sample preparation
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DOI：
10.1002/anie.200705016
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Hsieh, Tseng-Ming
中科院分区：
文献类型：
--
作者：
Pipper, Juergen;Zhang, Yi;Hsieh, Tseng-Ming
除了少数例外，目前可用的微全分析系统（μ塔斯）未能实现将多个实验室操作小型化到单个芯片上的理想。[1]这些系统在芯片外执行样品制备，并且仅追求单一功能。[2]因此，最终的挑战是将真实世界生物样品的处理与下游应用相结合。[3]为此，在平面上操纵单个液滴为μTAS提供了一个有吸引力的选择。[4]在本文中，我们将含有表面官能化的超顺磁性颗粒的自由液滴转化为具有（亚）微升（μL）体积的虚拟μTAS。除了作为用于在磁场中致动液滴的力介质之外，超顺磁性颗粒还用作实验室或（生物）化学过程的连续性能的固体支持物。根据其特定的任务，液滴暂时成为泵，阀，混合器，提取器或热循环仪。在自动化实验中，从25 μL血液中分离30个绿色荧光蛋白（GFP）转染的THP-1细胞，100倍预浓缩，纯化，裂解，并进行靶向转染载体的实时PCR（RT-PCR），所有这些都在17分钟内完成。通过在不同的温度区上顺时针旋转液滴，在时间-空间转换下在一次性基底上发生8 s的快速热循环。其他基于PCR的（生物）检测格式易于适应，这使得μTAS成为分散式诊断的有吸引力的候选产品。大多数台式热循环仪依赖于热电加热的金属块，金属块上装有最多50 μL PCR混合物的塑料管。这种设置导致高热质量，并且PCR运行时间（通常为数小时）受到低加热和冷却速率的限制。缩小规模和/或利用高导热材料可以克服这些限制，基于芯片的（亚）微型PCR可以在几分钟内完成这项工作。[5]有两种方式进行芯片上PCR。[6-9]在时域中，将固定PCR混合物在三个温度之间热循环。
With a few exceptions, the micro total analysis systems (μTASs) currently available have failed to live up to the ideal of the miniaturization of multiple laboratory operations onto a single chip.[1] These systems perform sample preparation off chip and only pursue a single function.[2] Hence, the definitive challenge is to interface the processing of real-world biological samples with downstream applications.[3] To this end, the manipulation of individual droplets on a planar surface offers an attractive option for a μTAS.[4] Herein, we transform a free droplet containing surface-functionalized superparamagnetic particles into a virtual μTAS with a (sub) microliter (μL) volume. Aside from being force mediators for actuating the droplet in a magnetic field, the superparamagnetic particles serve as a solid support for the sequential performance of laboratory or (bio) chemical processes. Depending on its particular task, the droplet temporarily becomes a pump, valve, mixer, extractor, or thermocycler. In an automated experiment, 30 green-fluorescent protein (GFP) transfected THP-1 cells are isolated from 25 μL of blood, 100-fold preconcentrated, purified, lysed, and subjected to a realtime PCR (RT-PCR) targeting the transfection vector, all within 17min. Fast thermocycles of 8s take place on a disposable substrate under time–space conversion by rotating the droplet clockwise over different temperature zones. Other PCR-based (bio) assay formats are easily adaptable, which makes this μTAS an attractive candidate for decentralized diagnostics.Most bench-scale thermocyclers rely on a thermoelectrically heated metal block holding plastic tubes containing up to 50 μL of PCR mixture. This setup results in a high thermal mass and the PCR run time—typically hours—is limited by the low heating and cooling rates. Downscaling and/or the utilization of highly heat-conductive materials can overcome these limitations and a chip-based (sub) microscale PCR can perform the job within minutes.[5] There are two ways of conducting an on-chip PCR.[6–9] In the time domain, a stationary PCR mixture is thermocycled between three