连续流动式聚合酶链式反应芯片的设计进展

介绍了PCR的概念以及微型PCR芯片的优点，着重介绍连续流动式PCR(continuous-flow PCR)芯片／微装置的一般原理、串行流动及其设计进展。     

连续流动式聚合酶链式反应生物芯片/微装置中脱氧核糖核酸样品的驱动控制技术进展

介绍国内外连续流动式聚合酶链式反应生物芯片／微装置中脱氧核糖核酸样品的驱碧控制技术进展，主要包括恒流泵（注射泵驱动和蠕动泵驱动）、旋转泵驱动、磁流体动力驱动以及自然对流驱动等。并对这几种驱动方式的优缺点作简要的讨论（引用文献43篇）。     

微流控振荡流反转录聚合酶链式反应快速检测烟草花叶病毒

建立了一种基于毛细管的振荡流反转录聚合酶链式反应(RT-PCR)的微流控装置检测烟草花叶病毒(TMV)的新方法.根据TMV移动蛋白基因设计一对PCR引物,对粗提纯TMV颗粒直接进行RT-PCR.用0.025%小牛血清蛋白(BSA)对反应毛细管内壁进行静力学和动力学钝化,提高了PCR效率.在此微流控装置上进行RT-PCR,流速为70 μL/min时, 检出限为0.01 μg cDNA;可以在17 min内成功扩增出179 bp的目的DNA片段.     

激光技术在聚合酶链式反应微流控芯片中的应用

评述了激光技术在聚合酶链式反应(polymerase chain reaction,PCR)微流控芯片领域中的应用进展,包括激光技术在PCR微流控芯片微加工、微流体物理参数测量、温度循环控制以及芯片上在线产物检测(包括荧光实时定量/终点和毛细管电泳检测)中的应用。最后,展望了激光技术在未来基于PCR的微全分析系统(micro-total analysis system,μTAS)中的新应用。     

液滴微流控系统在数字聚合酶链式反应中的应用研究进展

数字聚合酶链式反应( PCR)技术近年来发展迅速。与以实时荧光定量PCR为代表的传统PCR技术相比,数字PCR技术显著提高了定量分析的精确度和灵敏度。数字PCR的快速发展与近年来微流控技术在数字PCR技术中的广泛应用有着密切的联系。早期的研究和商业化产品使用的是大规模集成流路微流控芯片,加工过程复杂且价格高昂。近年来,液滴微流控芯片被应用到数字PCR技术中,它可以在短时间内产生102~107个微液滴,每一个微液滴都是最多只含有一个目的基因片段的PCR反应器。 PCR扩增后,通过对单个微液滴的观察计数,就可以获得绝对定量的分析数据。本文综述了不同种类的液滴微流控系统在数字PCR技术中的应用,以及液滴数字PCR微流控芯片在生物、医药、环境等领域的应用。     

微流控PCR芯片的研究进展

基因是人类的遗传密码,人类个体之间只有万分之一的基因不相同,却导致了人与人之间丰富的差异.了解这种差异对于科学研究具有巨大的应用价值.PCR是基因研究中常用的手段之一,但传统PCR仪存在反应时间长、能量消耗大、不便于集成与携带等缺陷,微流控技术与PCR结合可以有效缩小反应体系,提高反应效率,且易于集成化与微型化.本文按照微流控PCR芯片的结构分类,详细介绍了微池型、连续流动型PCR芯片,以及电泳、荧光、电化学和DNA杂交阵列等检测方法,并在最后进行了总结与展望.     

单温控模块空间构型连续流PCR结构研究

提出了一种新型空间构型连续流PCR结构,该结构将传统二维平面逶迤通道结构转化至三维空间,并基于单温控模块,构筑变性、退火/延伸2个温区,实现符合PCR扩增要求的试样空间温度分布。通过有限元方法详细分析了空间尺寸、通道直径和流体速度对通道内流体温度分布的影响规律,分析表明当该结构几何尺寸为54 mm×58 mm×28 mm,对应壁厚为2 mm×10 mm×2 mm,流体通道直径为1 mm,流体速度为0.0005 m/s时,可实现94.5℃的变性区温度分布和65.5℃的退火/延伸区温度分布,满足核酸分子扩增的温度需求。     

连续流动式PCR芯片相关技术研究进展

微电子机械系统(microeletromechanical system,MEMS)技术的兴起及其在生物化学领域的广泛应用,推动了聚合酶链式反应(polymerase chain reaction,PCR)热循环装置越来越微型化,各种PCR微芯片／装置被开发。本文主要介绍了基于MEMS技术的连续流动式PCR(continuous—flow PCR)芯片／微装置的相关技术,包括基底材料的选择、通道表面钝化技术、微细加工技术、封接技术以及系统检测技术等,最后简单介绍目前实验室的研究工作。     

智能数字聚合酶链式反应系统的开发与应用验证

数字聚合酶链式反应(digital polymerase chain reaction,dPCR)技术可以针对低浓度的目标核酸分子实现精确的绝对定量检测,在各类疾病的检测与治疗方面有着极大应用价值. 针对目前商业数字PCR仪造价昂贵、体积庞大等缺点,基于智能手机与微流控芯片,设计开发了一种低成本、高集成的智能数字PCR设备. 介绍了硬件系统的制作以及整机的整合搭建过程. 采用PID算法,结合温控电路与半导体制冷片等硬件,进行了PCR温度循环的精准控制. 最后,采用自适应阈值分割法对采集到的荧光图像进行了处理,并依据泊松分布的规律对统计结果进行了校正,完成了对PCR反应后采集到荧光图像的结果分析与检测.     

变性无胶毛细管筛分电泳分析聚合酶链式反应产物

在分离Ｙ染色体性别决定区,人１１号染色体短β－珠蛋白基因部分序列扩增产物和ＰＢＲ３２２／ＨａｅⅢ标准片段的混合样品时,ＳＲＹ和１０８／Ｂ的迁移时间均产生较大的偏差,在尿素变性存在的无胶筛分体系中迁移时,这种迁移时间的偏差得以消除,这意味着尿素性剂的存在消除了ＤＮＡ空间结构对迁移的影响,ＤＮＡ在此筛分介质中的迁移只与其片段长度有关。     

A Universal Polymerase Chain Reaction Developer

The versatility of PCR, the gold standard for amplification of DNA targets, is hampered by the laborious, multi‐step detection based on gel electrophoresis. We propose a one‐step, one‐tube method for the rapid (5 min) naked‐eye detection of PCR products, based on controlled aggregation of gold nanoparticles. Our method is universal, instrument‐free, and ultra‐sensitive, as it could detect as low as 0.01 zeptomoles of HIV template DNA in an excess of interfering human genomic DNA.     

One-Step Nucleic Acid Purification and Noise-Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow-Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point-of-care diagnostics. Currently, nucleic acid (NA) purification remains time-consuming and labor-intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one-step, liquid-phase NA purification that is simpler and faster than conventional solid-phase extraction. By further re-concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non-specific amplification caused by non-optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non-optimal PCR designs, which is 10- and 1000-fold fewer than those of the standard bench-top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point-of-care diagnosis.     

Designer Extracellular Matrix Based on DNA–Peptide Networks Generated by Polymerase Chain Reaction

Cell proliferation and differentiation in multicellular organisms are partially regulated by signaling from the extracellular matrix. The ability to mimic an extracellular matrix would allow particular cell types to be specifically recognized, which is central to tissue engineering. We present a new functional DNA‐based material with cell‐adhesion properties. It is generated by using covalently branched DNA as primers in PCR. These primers were functionalized by click chemistry with the cyclic peptide c(RGDfK), a peptide that is known to predominantly bind to αvβ3 integrins, which are found on endothelial cells and fibroblasts, for example. As a covalent coating of surfaces, this DNA‐based material shows cell‐repellent properties in its unfunctionalized state and gains adhesiveness towards specific target cells when functionalized with c(RGDfK). These cells remain viable and can be released under mild conditions by DNase I treatment.     

乙肝病毒聚合酶链反应扩增产物的毛细管电泳测定

本文用毛细管电泳对乙肝病毒聚合酶反应扩增产物进行了检测，并用这一方法和酶联免疫吸附实验以及ＰＣＲ与传统电泳联用的方法进行比较，探讨了用高效，快速，微量，易自动化的毛细管电泳代替传统检测方法的可能性，取得了令人满意的结果。     

One‐Step Nucleic Acid Purification and Noise‐Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow‐Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point‐of‐care diagnostics. Currently, nucleic acid (NA) purification remains time‐consuming and labor‐intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one‐step, liquid‐phase NA purification that is simpler and faster than conventional solid‐phase extraction. By further re‐concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non‐specific amplification caused by non‐optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non‐optimal PCR designs, which is 10‐ and 1000‐fold fewer than those of the standard bench‐top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point‐of‐care diagnosis.