DNA单分子穿越内径<10nm纳米通道机制的实时可视化研究

To clearly understand the detailed mechanism of DNA molecule transporting through the nanochannel with diameter less than 10nm under external electric fields has an important practical value and scientific significance for accomplishing the ultra-fast nanopore DNA (whole genome) sequencing technology, the high integration of nano-biochemical sensor and analyzer, and Lab-on-a-chip technology. In this project, a microsecond pulse switch and a constant voltage DC source with microvolt precision and self-made micro-nanofluidic system will be used to induce single DNA molecule into the functional nanochannels which have different shapes, e.g., curved "S", inner diameter gradually changed "wedge", long and thin "straight line". The lengths of these non-straight shape channels are bout 0.5~1mm, and the smallest inner diameter is about 2nm. The lengths of these continuous smooth straight channels are more than 10mm and their inner diameters are not more than 10nm. Then, combined with the single molecule fluorescence imaging technology, EMCCD and a micro current meter with femto-ampere precision, the single DNA molecule movement velocity and conformational changes as it moving along these nanochannels will be real-time visual monitored. And then, the non continuous flow theory is employed to analyze the factors such as the overlapping electrical double layer and van der Waals force and PH value of solution and size of nanochannel which influence the DNA molecule biophysics and dynamics properties when DNA passing through the nanochannel. Eventually, the mechanism of DNA molecule transporting through the nanochannels with diameter about 2nm to 10nm will be explained in detail.

清楚理解DNA分子在外电场作用下，穿越内径<10nm纳米通道的详细机理，对于实现DNA分子（全基因组）纳米孔超快测序，实现纳米生化传感器、分析器、生物芯片实验室的高度集成和实用化等有重要的实际价值和科学意义。本项目利用微秒精度脉冲开关、微伏精度恒压直流源，结合自行研制的微纳流控系统，有效引导DNA单分子匀速穿越长度0.5~1mm、内径最小~2nm的具有不同形状与功能的弯曲"S形"、内径渐变"楔形"纳米通道，以及内径2~10nm、长度>10mm的连续光滑"直线形"纳米通道等；并结合单分子荧光成像技术、EMCCD和飞安级精度微电流测试仪等技术实时可视化监测DNA分子穿越通道时其运动速度、构象变化等规律；再结合非连续流理论分析重叠电双层、范德瓦尔斯力、溶液PH值、通道内径大小等因素对DNA分子生物物理性质、动力学性质改变的影响，阐明DNA分子穿越内径<10nm至~2nm纳米通道的详细机制。

清楚理解DNA分子在外电场作用下，穿越微米、纳米通道（内径可<10nm）的详细机理，对于实现DNA分子（全基因组）纳米孔超快测序，实现纳米生化传感器、分析器、生物芯片实验室的高度集成和实用化等有重要的实际价值和科学意义。本项目利用微秒精度脉冲开关、微伏精度恒压直流源，结合自行研制的微纳流控系统，有效引导DNA单分子匀速穿越长度0.5~1mm、内径最小~2nm的具有不同形状与功能的弯曲"S形"、内径渐变"楔形"纳米通道，以及内径2~10nm、长度>10mm的连续光滑"直线形"纳米通道等；并结合单分子荧光成像技术、EMCCD和飞安级精度微电流测试仪等技术实时可视化监测DNA分子穿越通道时其运动速度、构象变化等规律，再结合非连续流理论分析重叠电双层、范德瓦尔斯力、溶液PH值、通道内径大小等因素对DNA分子生物物理性质、动力学性质改变的影响。本课题系统研究了特征电流信号与通道大小、缓冲液浓度、Ph值、样品种类、DNA分子长度、以及外加电场矢量特性等因素的对应关系；在此基础上，还发现了几种新现象：非连续电场力作用下，DNA分子穿越微通道时，电流的时间特性曲线有6种，统计分析表明所有正向的电流响应出现的概率大于相对应的负向电流响应；当辅助电极有效面积大于工作电极时，电流响应随外加电压的增加由负向反转到正向，该现象是由电极表面电荷积累产生的屏蔽场与外加电场之间的竞争效应引起的；DNA分子穿越微纳通道时的运动方向也存在反转现象，即，当外加电场小于阈值电场时，DNA分子在通道内的运动方向与电场方向一致，而当外加电场大于阈值电场时，其运动方向与电场方向相反。这些新现象及其机制，对于阐明DNA分子穿越微纳米通道的详细机制具有重要意义，而且，对于微纳流控芯片以及生物传感器的微型化、精确化具有实际应用价值，能用于精准操纵生物单分子，进而揭示生物大分子、药物分子之间作用机理。