Upgrade and Renewal of Setup for Combined Optical, Mechanical and Electrical Probing of Single Molecules

PI的研究项目横跨纳米技术和软物质/生物物理学领域，致力于发展纳米流体学领域。纳米流控器件是在尺寸为1-100纳米的芯片上由充满流体的通道组成的网络。这种设备可以分析和操纵单分子。例如，纳米通道可以扩展DNA；纳米孔可以电感应DNA。单分子设备不需要分子扩增就可以进行基因组分析，因此分析最终可以在单分子、单细胞的基础上进行。与应用目标携手并进，阐明纳米限制大分子的物理导致更有效的器件设计。最后，“人工”纳米流体结构可以模拟生物系统中的禁闭，如细胞和病毒。******该RTI提案旨在更新和升级PI小组的核心显微镜装置，用于探测和控制纳米流体装置中的DNA单分子。该装置结合了多色荧光照明、光学陷阱和电传感，用于光学、机械和电探测。PI的HQP在蒙特利尔的洁净室设施中构建纳米流体装置，然后用这个装置操作这些装置。核心装置现在已经差不多有8年的历史了。由于2/3的激光器失效，该设置不能再以足够高的帧速率执行多色荧光照明。我们将用新的多led照明系统取代旧的激光TIRF。我们将把旧的EMCCD相机升级为速度更快的背光sCMOS。结合两个额外的工具将进一步使研究能够通过设置进行，一个双通道膜片钳放大器和一个气动控制板，为我们的设备施加四个独立的压力输入。******这种更新和升级对于推动我们NSERC资助的研究计划和利用最近的技术突破至关重要。我们已经成功地将纳米级膜与纳米通道连接起来；这些膜由气压驱动，可以可逆地偏转，为单个分子创造纳米腔陷阱。压力板将自动驱动膜和分子运输。我们的新AFM纳米孔制造技术（TCLB）可以在膜上形成孔，从而产生纳米通道集成孔器件。更新我们的多色成像能力和升级相机将使实验中多个不同染色的单分子物种可以在陷阱和纳米孔接口设备中以高帧率动态成像。最后，通过纳米孔启动公司Two-Pore-Guys，我们已经成功地构建了通过两个孔同时进行DNA易位的设备。双通道放大器对于使用双孔器件进行实验至关重要，可以在孔处独立应用电压和电流传感。

The PI's research program, straddling the fields of nanotechnology and soft-matter/biophysics, is devoted to developing the field of nanofluidics. Nanofluidic devices are networks of fluid-filled channels on a chip with dimensions ~1-100 nm. Such devices can analyze and manipulate single-molecules. For example, nanochannels can extend DNA; nanopores can sense DNA electrically. Single-molecule devices perform genomic analysis without requiring molecular amplification, so that analysis could ultimately be performed on a single molecule, single-cell, basis. Hand in hand with application goals, elucidating the physics of nanoconfined macromolecules leads to more effective device design. Finally, ‘artificial' nanofluidic structures can model confinement in biological systems, like cells and viruses.******This RTI proposal is designed to renew and upgrade a core microscopy setup in the PI's group for probing and controlling single-molecules of DNA in nanofluidic devices. This setup combines multi-color fluorescence illumination, an optical trap and electrical sensing for combined optical, mechanical and electrical probing. The PI's HQP construct nanofluidic devices at clean-room facilities in Montreal and then operate the devices with this setup. The core setup is now almost eight years old. Due to the failure of 2/3 of our lasers, the setup can no longer perform multi-color fluorescence illumination with sufficiently high frame rates. We will replace our old laser TIRF with a new multi-LED illumination system. We will upgrade our old EMCCD camera with a faster back-illuminated sCMOS. Incorporating two additional tools will further enable research performed with the setup, a two-channel patch clamp amplifier and a pneumatic control board to apply four independent pressure inputs to our devices. ******This renewal and upgrade is critical for driving our NSERC funded research program and exploiting recent technology breakthroughs. We have successfully interfaced nanoscale membranes to nanochannels; these membranes, actuated by pneumatic pressure, can be reversibly deflected to create nanocavity traps for single molecules. The pressure-board will automate membrane actuation and molecule transport. Our new AFM nanopore fabrication technology (TCLB) can create pores in the membranes giving rise to nanochannel integrated pore devices. Renewing our multi-color imaging capability and upgrading the camera will enable experiments where multiple differentially stained single-molecules species can be dynamically imaged in the traps and nanopore-interfaced devices at high frame rates. Finally, with nanopore startup Two-Pore-Guys, we have succeeded in building devices enabling simultaneous DNA translocation through two-pores. The two-channel amplifier will be critical for performing experiments with the two-pore devices, enabling independent application of voltage and current sensing at the pores.