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

PI的研究计划，跨越纳米技术和软物质/生物物理学领域，致力于发展纳米流体领域。 纳米流体装置是芯片上的流体填充通道的网络，尺寸为约1-100 nm。 这种装置可以分析和操纵单分子。 例如，纳米通道可以延伸DNA;纳米孔可以电感应DNA。 单分子装置在不需要分子扩增的情况下进行基因组分析，使得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，阐明纳米限制的大分子的物理导致更有效的器件设计。 最后，“人造”纳米流体结构可以模拟生物系统中的限制，如细胞和病毒。该RTI提案旨在更新和升级PI组的核心显微镜设置，用于探测和控制纳米流体设备中的DNA单分子。该装置结合了多色荧光照明、光阱和电感测，用于组合光学、机械和电学探测。 PI的HQP在蒙特利尔的洁净室设施中构建纳米流体设备，然后使用该设置操作设备。 核心设置现在已经快八年了。 由于2/3的激光器出现故障，该装置无法再以足够高的帧速率进行多色荧光照明。 我们将用新的多LED照明系统取代旧的激光TIRF。 我们将升级旧的EMCCD相机，使用速度更快的背照式sCMOS。 增加两个额外的工具将进一步使研究与设置，一个双通道膜片钳放大器和一个气动控制板应用四个独立的压力输入到我们的设备。** 这次更新和升级对于推动我们的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连接到纳米通道上;这些膜由气动压力驱动，可以可逆地偏转，为单个分子创建纳米腔陷阱。 压力板将自动化膜驱动和分子运输。 我们新的AFM纳米孔制造技术（TCLB）可以在膜中产生孔，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，多个差异染色的单分子物种可以动态成像的陷阱和纳米孔接口设备在高帧速率。 最后，通过纳米孔创业公司Two-Pore-Guys，我们成功地构建了能够同时通过两个孔进行DNA易位的设备。 双通道放大器对于使用双孔装置进行实验至关重要，从而能够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.