Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification.
Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification.
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DOI：
10.3390/microarrays4040474
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发表时间：
2015-10-20
期刊：
影响因子：
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通讯作者：
Bau HH
中科院分区：
文献类型：
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作者：
Mauk MG;Liu C;Song J;Bau HH
描述了用于快速（<60分钟），低成本，方便，现场可部署的序列特异性核酸扩增测试（NAATs）的微流体组件和系统。从血液样本中定量HIV病毒载量的微流体点护理（POC）诊断测试可作为讨论“芯片实验室”NAAT设备的技术问题和能力的代表性和指发性示例。一种便携式、小型化的POC NAAT，其性能可与临床实验室中基于传统PCR（聚合酶链反应）的测试相比较，可以通过一个一次性的、手掌大小的塑料微流控芯片来实现，其中：(1)利用嵌入的多孔硅玻璃纤维或纤维素结合相（“膜”）从相对大（~mL）体积的样品裂解物中提取核酸（NAs），以流动过滤模式捕获样品NAs；(2)膜上捕获的NAs被等温（~65℃）扩增；(3)通过实时荧光检测监测扩增子的产生，例如使用智能手机CCD相机作为低成本检测器；(4)将石蜡包封的冻干试剂预先储存在芯片中，用于温度活化释放。检测限（LOD）优于103个病毒/样品。一个改进的芯片与导管承载扩散模式放大过程提供了一个简单的可视化指标，容易定量样品NA模板。此外，描述了一种用于从全血中提取血浆的配套微流体装置，该装置无需离心机即可产生无细胞血浆，用于基于芯片的分子诊断。扩展到无数的相关应用，包括，例如，食品检测，癌症筛查和昆虫基因分型简要调查。
Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.