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

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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.