远场光学超分辨成像技术研究DNA单分子穿越纳米通道的流动特性

To exactly manipulate the DNA molecules moving uniformly inside nanochannels with a fully stretched length has significant impact in the realizations of ultra-fast DNA sequencing of nanopore technique, biochemical molecular sensor and lab-on-a-chip with high integration and intellectualization. Because of the lack of effective technology for measuring the multiphysical quantities with required simultaneous high spatial and temporal resolutions, the research of nanofluidics had been limited to the theory and simulation. In this project, one kind of far-field optical super-resolution imaging analysis system is innovated, which integrated the Stimulated emission depletion (STED) nano/microscopy technology, the Laser-induced fluorescence photobleaching anemometer (LIFPA) technology, the laser spot array light source technology and the two-color fluorescence imaging technology. The system can achieve an unprecedented ultrahigh spatial resolution about 20nm and high temporal resolution about 1μs to 500ms. By combining a micro current meter with femto-ampere resolution and constant-voltage DC source with microvolt resolution, we will experimentally study and characterize the immigration and conformation of DNA molecule with the influencing factors, i.e., radius and shape of the channel, PH value and concentration of buffer solution, length and sequence of DNA, when the DNA molecule is driven to transport through the nanochannel with an external electric field force. Under consideration of the overlapping electrical double layer, van der Waals force, Zeta potential around the DNA molecule, a non-continuous flow model will be employed to theoretically elucidate the electrodynamic properties and the detailed mechanism of biomolecules such as DNA molecule passing through the nanochannel.

精准操纵DNA分子在纳通道内均匀拉伸、匀速运动，对于实现纳米孔全基因测序技术、分子生化传感器与芯片实验室的高度集成及实用化等具有重要的科学意义和实际价值。本项目针对目前纳通道流体物理特性尚无同时具备高空间、高时间分辨率的有效测量方法与技术，基于自主研建的结合了激光受激辐射损耗成像技术、激光诱导荧光漂白测速技术的远场光学超分辨成像显微分析系统，发展激光点阵列光源技术、双色荧光成像技术，获得空间分辨率~20nm、单幅成像时间1μs~500ms；进而结合微伏精度恒压直流源、飞安精度电流测试仪，研究在外电场力作用下，DNA分子穿越形状与大小不同的纳通道时，其位置迁移、构象变化及速度矢量等特性与纳通道性质、DNA长度及排序、缓冲液性质等之间的定量关系；联合非连续流理论，考虑重叠电双层、范德瓦尔斯力、Zeta电势等因素，阐明DNA分子穿越纳通道的电动力学特性，揭示其在纳通道内匀速运动的详细机制。

准确理解DNA分子在外电场作用下，穿越微纳流体通道的动力学规律、详细机理，进而精准操纵单个DNA分子在纳通道内均匀拉伸、均匀运动，对于实现纳米孔全基因测序技术、分子生化传感器与芯片实验室的高度集成及实用化等具有重要的科学意义和实际价值。.本项目基于单点扫描 STED 超分辨成像技术、激光诱导荧光漂白测速LIFPA技术，利用相位图条状分割方法，结合德拜衍射积分理论，通过空间光调制器、在高NA物镜的焦平面形成形状均匀、位置灵活6维可调的点光斑阵列；解决基于点阵列 STED 超分辨成像技术的纳通道流体流速测量仪（nLIFPV）所涉及的关键难题，实现成像空间分辨率~20nm，单幅成像时间 1µs~500ms。搭建了双色（405nm/532nm、470nm/640nm）超分辨成像STED系统，优化光路，实现了系统的紧凑、小型化，开展相关配套量子点（半导体量子点、碳量子点）染料的筛选、研发；系统研究荧光染料Coumarin102的性质；将ATTO390染色用于神经母瘤细胞的研究，分辨率达到了120nm。.本项目基于LIFPA研究微米通道绝缘壁面电双层附近的线性振荡电渗流在弱交流电场下的流动响应，发现在远离电极表面并平行于电场的侧壁面处，存在混沌状态的交流电场电渗流；基于飞安级微电流监测仪，采用三电极系统，实时监测外电场作用下，缓冲液TBE穿越亚微米通道（内径200nm）时的电流-电压特性，发现当电场强度低于临界值6.7V/m时，溶液在通道内近壁面表现为非牛顿流体特性，而且有剪切增稠现象；研究λ-DNA分子在混合有金属离子（K+和Na+， Mg2+和Ca2+）情况下通过微毛细管（5mm，10mm，50mm等）时的电流-电压关系，提出利用指数的幂律模型评估极化对施加电压的敏感性，这对于理解、设计基于电泳原理的 DNA 检测方法及提高检测和操控微流体中单个 DNA 分子的准确性具有重要意义。.利用单分子荧光成像系统，研究DNA分子在电场作用下，进出微米通道端口的动力学特性，发现DNA分子在穿越微米通道时所发生反转、往复、旋转等现象，主要原因是DNA分子穿越通道时，除了受轴向电泳力、电渗力、液体反压差力等，还受萨夫曼力和马格努斯力的作用，这为后续研究生物分子、药物分子/粒子等与DNA分子作用，并基于可视化微通道技术揭示作用机制，研发生化分子传感芯片、临床检测仪器等，奠定了基础。