Mauk Michael G, Liu Changchun, Sadik Mohamed, Bau Haim H
Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, 220 South 33rd Street, 107 towne building, Philadelphia, PA, 19104-6315, USA.
Methods Mol Biol. 2015;1256:15-40. doi: 10.1007/978-1-4939-2172-0_2.
Molecular (nucleic acid)-based diagnostics tests have many advantages over immunoassays, particularly with regard to sensitivity and specificity. Most on-site diagnostic tests, however, are immunoassay-based because conventional nucleic acid-based tests (NATs) require extensive sample processing, trained operators, and specialized equipment. To make NATs more convenient, especially for point-of-care diagnostics and on-site testing, a simple plastic microfluidic cassette ("chip") has been developed for nucleic acid-based testing of blood, other clinical specimens, food, water, and environmental samples. The chip combines nucleic acid isolation by solid-phase extraction; isothermal enzymatic amplification such as LAMP (Loop-mediated AMPlification), NASBA (Nucleic Acid Sequence Based Amplification), and RPA (Recombinase Polymerase Amplification); and real-time optical detection of DNA or RNA analytes. The microfluidic cassette incorporates an embedded nucleic acid binding membrane in the amplification reaction chamber. Target nucleic acids extracted from a lysate are captured on the membrane and amplified at a constant incubation temperature. The amplification product, labeled with a fluorophore reporter, is excited with a LED light source and monitored in situ in real time with a photodiode or a CCD detector (such as available in a smartphone). For blood analysis, a companion filtration device that separates plasma from whole blood to provide cell-free samples for virus and bacterial lysis and nucleic acid testing in the microfluidic chip has also been developed. For HIV virus detection in blood, the microfluidic NAT chip achieves a sensitivity and specificity that are nearly comparable to conventional benchtop protocols using spin columns and thermal cyclers.
基于分子（核酸）的诊断测试相比免疫测定法具有许多优势，特别是在灵敏度和特异性方面。然而，大多数现场诊断测试都是基于免疫测定法的，因为传统的基于核酸的测试（NATs）需要大量的样本处理、训练有素的操作人员和专门的设备。为了使NATs更便捷，尤其是用于即时诊断和现场检测，已经开发出一种简单的塑料微流控盒（“芯片”），用于对血液、其他临床标本、食品、水和环境样本进行基于核酸的检测。该芯片结合了通过固相萃取进行核酸分离；等温酶促扩增，如LAMP（环介导等温扩增）、NASBA（基于核酸序列的扩增）和RPA（重组酶聚合酶扩增）；以及对DNA或RNA分析物的实时光学检测。微流控盒在扩增反应室中集成了一个嵌入式核酸结合膜。从裂解物中提取的目标核酸被捕获在膜上，并在恒定的孵育温度下进行扩增。用荧光团报告分子标记的扩增产物由LED光源激发，并通过光电二极管或CCD探测器（如智能手机中可用的探测器）进行原位实时监测。对于血液分析，还开发了一种配套的过滤装置，用于从全血中分离血浆，为微流控芯片中的病毒和细菌裂解以及核酸检测提供无细胞样本。对于血液中的HIV病毒检测，微流控NAT芯片的灵敏度和特异性几乎与使用旋转柱和热循环仪的传统台式检测方法相当。
