1-Million droplet array with wide-field fluorescence imaging for digital PCR
1-Million droplet array with wide-field fluorescence imaging for digital PCR
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DOI：
10.1039/c1lc20561g
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发表时间：
2011-01-01
期刊：
影响因子：
6.1
通讯作者：
Lee, Abraham P.
中科院分区：
文献类型：
--
作者：
Hatch, Andrew C.;Fisher, Jeffrey S.;Lee, Abraham P.
数字液滴反应器可用作化学和生物容器，以将试剂离散成皮升或纳升体积，用于分析单细胞、生物体或分子。然而，大多数基于DNA的测定需要处理几十微升数量级的样品，并且含有少至一个到多达数百万个待检测的片段。提出了一种微滴微流控平台和荧光成像装置，通过在芯片上集成多个高通量微滴处理方案，更好地满足高通量和高动态范围的需求。该设计能够在2-7分钟内产生超过100万个单分散的50皮升液滴，然后在大型观察室中自组装成高密度三维球体包装配置，用于可视化和分析。然后，该设备进行芯片上聚合酶链反应（PCR）扩增和荧光检测，以数字量化样品的核酸含量。使用低成本的2100万像素数码相机和微距镜头分别在1倍至0.85倍放大率下捕获宽视场荧光图像，视野为8-12 cm（2）。我们展示了终点和实时成像能力，以执行整个液滴阵列的芯片上定量数字PCR分析。与以前的工作相比，这种高度集成的设计使芯片上数字化反应器的数量增加了100倍，同时具有用于基于数字PCR的测定的荧光成像。
Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1x to 0.85x magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.