Chitosan-Modified Filter Paper for Nucleic Acid Extraction and "in Situ PCR" on a Thermoplastic Microchip
Chitosan-Modified Filter Paper for Nucleic Acid Extraction and "in Situ PCR" on a Thermoplastic Microchip
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DOI：
10.1021/acs.analchem.6b04882
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发表时间：
2017-03-21
影响因子：
7.4
通讯作者：
Liu, Peng
中科院分区：
文献类型：
--
作者：
Gan, Wupeng;Gu, Yin;Liu, Peng
利用两种不同的DNA捕获机制：长链DNA分子与滤纸纤维的物理缠结和DNA在壳聚糖改性滤纸上的静电吸附，成功地研制了嵌入壳聚糖-DNA改性Fusion 5滤纸(从GE Healthcare购买)的塑料微流控装置，用于DNA的提取和浓缩。这种新方法不仅通过将这两种捕获机制协同结合在一起，在pH为5时提供了高的DNA提取效率，而且在pH>；8时，由于DNA与纤维的缠绕而抵抗了DNA从过滤器中的洗脱。因此，可以将聚合酶链式反应缓冲液直接装载到萃取室中进行原位聚合酶链式反应，捕获的DNA用于下游分析而不会造成任何损失。我们证明，在一个芯片中，直径3 mm的滤盘对1(562)人基因组DNA和噬菌体lambda-DNA的捕获效率分别为98%和95%。用去离子水、聚合酶链式反应混合液和TE缓冲液洗涤不能洗脱捕获的DNA。此外，滤盘可以从稀释的样本(0.05 ng/亩L)中浓缩62%的lambda-DNA，提供30倍以上的浓缩倍数。最后，开发了一种简单的两室结构的芯片上细胞裂解、DNA提取和血液中15-plex短串联重复序列扩增的微设备。这种DNA提取与“原位聚合酶链式反应”相结合，在完全集成的微系统中具有巨大的潜力，用于快速、接近患者的核酸检测。
Plastic microfluidic devices with embedded chitosan-DNA modified Fusion 5 filter paper (unmodified one purchased from GE Healthcare) have been successfully developed for DNA extraction and concentration, utilizing two different mechanisms for DNA capture: the physical entanglement of long-chain DNA molecules, with the fiber matrix of the filter paper and the electrostatic adsorption of DNA to the chitosan-modified filter fibers. This new method not only provided a high DNA extraction efficiency at a pH of 5 by synergistically combining these two capture mechanisms together, but also resisted the elution of DNA from filters at a pH > 8 due to the entanglement of DNA with fibers. As a result, PCR buffers can be directly loaded into the extraction chamber for '' in situ PCR '', in which the captured DNA were used for downstream analysis without any loss. We demonstrated that the capture efficiencies of a 3-mm-diameter filter disc in a microchip were 98% and 95% for 1(562 human genomic DNA and bacteriophage lambda-DNA, respectively. The washes with DI water, PCR mixture, and TE buffer cannot elute the captured DNA. In addition, the filter disc can enrich 62% of lambda-DNA from a diluted sample (0.05 ng/mu L), providing a concentration factor more than 30-fold. Finally, a microdevice with a simple two-chamber structure was developed for on-chip cell lysis, DNA extraction, and 15-plex short tandem repeat amplification from blood. This DNA extraction coupled with '' in situ PCR '' has great potential to be utilized in fully integrated microsystems for rapid, near-patient nucleic acid testing.