Department of Biomedical Engineering , Technion - IIT , Haifa 32000 , Israel.
Russell Berrie Nanotechnology Institute , Technion - IIT , Haifa 32000 , Israel.
ACS Nano. 2019 Dec 24;13(12):14388-14398. doi: 10.1021/acsnano.9b07873. Epub 2019 Nov 26.
Solid-state nanopore sensing of ultralong genomic DNA molecules has remained challenging, as the DNA must be controllably delivered by its leading end for efficient entry into the nanopore. Herein, we introduce a nanopore sensor device designed for electro-optical detection and sorting of ultralong (300+ kilobase pair) genomic DNA. The fluidic device, fabricated in-silicon and anodically bonded to glass, uses pressure-induced flow and an embedded pillar array for controllable DNA stretching and delivery. Extremely low concentrations (50 fM) and sample volumes (∼1 μL) of DNA can be processed. The low height profile of the device permits high numerical aperture, high magnification imaging of DNA molecules, which remain in focus over extended distances. We demonstrate selective DNA sorting based on sequence-specific nick translation labeling and imaging at high camera frame rates. Nanopores are fabricated directly in the assembled device by laser etching. We show that uncoiling and stretching of the ultralong DNA molecules permits efficient nanopore capture and threading, which is simultaneously and synchronously imaged and electrically measured. Furthermore, our technique provides key insights into the translocation behavior of ultralong DNA and promotes the development of all-in-one micro/nanofluidic platforms for nanopore sensing of biomolecules.
超长基因组 DNA 分子的固态纳米孔传感一直具有挑战性，因为 DNA 必须通过其前端进行可控传递，才能有效地进入纳米孔。在此，我们引入了一种专为超长（300 千碱基对以上）基因组 DNA 的光电检测和分选而设计的纳米孔传感器装置。该流体装置采用硅刻蚀工艺制造，并与玻璃阳极键合，使用压力诱导的流动和嵌入式柱阵列实现可控的 DNA 拉伸和传递。可以处理极低浓度（50 fM）和小体积（∼1 μL）的 DNA。该装置的低高度轮廓允许高数值孔径、高放大倍数的 DNA 分子成像，这些分子在远距离仍保持聚焦。我们展示了基于序列特异性缺口翻译标记和高帧率相机成像的选择性 DNA 分选。纳米孔通过激光刻蚀直接在组装好的装置中制造。我们表明，超长 DNA 分子的解旋和拉伸允许高效的纳米孔捕获和穿线，同时进行同步成像和电测量。此外，我们的技术为超长 DNA 的迁移行为提供了关键的见解，并推动了用于生物分子纳米孔传感的一体式微纳流控平台的发展。
