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

单分子光学、机械和电学联合探测装置的升级和更新

• 批准号: RTI-2019-00060
• 负责人: Reisner, Walter
• 金额: $ 10.64万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=663188
• 关键词: Upgrade; Renewal; Setup; Combined; Optical

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.

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领域。 纳米流体装置是芯片上充满流体的通道网络，尺寸约为 1-100 nm。 此类设备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。 单分子设备无需分子扩增即可进行基因组分析，因此分析最终可以在单分子、单细胞的基础上进行。 与应用目标齐头并进，阐明纳米大分子的物理原理可以实现更有效的设备设计。 最后，“人工”纳米流体结构可以模拟生物系统（如细胞和病毒）中的限制。 *****这项 RTI 提案旨在更新和升级 PI 小组中的核心显微镜设置，用于探测和控制纳米流体装置中的单分子 DNA。该装置结合了多色荧光照明、光阱和电传感，用于组合光学、机械和电学探测。 PI 的总部在蒙特利尔的洁净室设施中构建纳米流体设备，然后使用此设置操作设备。 核心设置现在已经有将近八年了。 由于我们 2/3 的激光器出现故障，该装置无法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。 我们将用更快的背照式 sCMOS 升级旧的 EMCCD 相机。 结合两个额外的工具将进一步支持通过该设置进行的研究，一个双通道膜片钳放大器和一个气动控制板，以将四个独立的压力输入应用于我们的设备。 ******这次更新和升级对于推动我们 NSERC 资助的研究项目和利用最新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；这些由气压驱动的膜可以可逆地偏转，为单分子创建纳米腔陷阱。 压力板将使膜驱动和分子传输自动化。 我们新的 AFM 纳米孔制造技术 (TCLB) 可以在膜上创建孔，从而产生纳米通道集成孔器件。 更新我们的多色成像能力并升级相机将使实验能够在陷阱和纳米孔接口设备中以高帧速率动态成像多种不同染色的单分子物质。 最后，通过纳米孔初创公司 Two-Pore-Guys，我们成功构建了能够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两孔器件进行实验至关重要，从而能够在孔处独立应用电压和电流传感。