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State-of-the-art probes

State-of-the-art probes. Alan Bigelow. Alternative sensing methods Real-time, single-cell analysis techniques. Outline. Miniature ion-selective single-cell probes Collaboration with the Biocurrents Research Lab at Woods Hole Probe positioner and manipulator

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State-of-the-art probes

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  1. State-of-the-art probes Alan Bigelow • Alternative sensing methods • Real-time, single-cell analysis techniques

  2. Outline • Miniature ion-selective single-cell probes Collaboration with the Biocurrents Research Lab at Woods Hole • Probe positioner and manipulator • Laser excited single-cell optical nanosensors Collaboration with Tuan Vo-Dihn • Kambiz Pourrezaei collaboration • A Surface-Enhanced Raman Scattering Nano-Needle for Cellular Measurements • Carbon Nanotube Cellular Endoscopes • Automated Microscope Observation Environment for Biological Analyses (AMOEBA)

  3. Miniature Ion-Selective Single-Cell Probes These probes are used to study changes of inflows or outflows of small molecules from individual living cells, in response to spatially-defined damage 1 mm 1 mm 1mm

  4. Making Probes

  5. Laser-Based Micropipet Pulling Device (Model P-2000; Sutter Industries)

  6. Glass Microelectrode Copper Wire Graphite Epoxy Paste Epoxy Carbon Fiber O-Phenylenediamine Nafion The Woods-Hole team have developed sensors for a variety of molecules, such as nitric oxide:

  7. Getting these single-cell probes into position, efficiently and reproducibly....A non-trivial task!

  8. Offset Hinge: probe positioning system

  9. Other manipulations using the offset hinge mount • Cell micro-injection • Single cell harvesting • Optical fiber based Raman spectroscopy • Orientation of medaka embryos

  10. Nanobiosensors Collaboration with Tuan Vo-Dinh Advanced Biomedical Science and Technology Group Life Science Division Oak Ridge National Laboratory

  11. Nano-biosensor tip • Pulled nano-sensors have tip diameters of approximately 40-50 nm • Final coated fibers are approximately 200 nm diameter • Antibody coated tips for specificity in binding • Nanometer diameter tip provides near-field excitation Sensor inside cell

  12. Metalic coating of probe end to prevent leakage of the excitation light Gold, Aluminum, or Silver

  13. Scanning Electron Microscope Images of a Nanofiber Before Metal Coating (tip diameter ~50nm) After Metal Coating (tip diameter 250-300nm)

  14. Nano-probe attachment

  15. Automated Microscope Observation Environment for Biological Analyses (AMOEBA)

  16. Environment Control User Requests: Physiological conditions Control temperature (e.g. 37 ± 0.5 ºC) Control medium concentrations (CO2, pH, oxygen, etc.) Initial Solutions: • Air-CO2 mixture: allows accurate particle count; limited time • Heater ring: Maintains temperature; cell medium evaporates

  17. AMOEBA Flow system for temperature-controlled medium exchange Flexible, user-friendly, modular design offers: • Medium aspiration, replacement, and collection • Multiple dispensers to change medium type during experiment • Additive introduction, such as trypsin to remove cells • Sensor insertion to monitor absorbed gas • Microfluidics compatibility: Lab-on-a-chip for in-line analysis

  18. “Flow” Diagram Example Reservoir I Additive Inlet Reservoir II Pump Heater / Cooler Reservoir III Hinge mount Microbeam Dish Lab-on-a-chip Dispenser

  19. Proof of Principle Cells were observed for 2 hours with circulating medium at 37 ± 0.5 ºC.

  20. Proof of Principle System included heated-window cap, to assist heating control.

  21. Lab-in-a-Box Sensor • Assemble your own system from modules. • Automation is computer controlled. • AMOEBA is flexible and has potential use in labs across the country and the world.

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