State-of-the-art probes
Alan Bigelow
• Alternative sensing methods
• Real-time, single-cell analysis techniques
Outline
1. Miniature ion-selective single-cell probes
Collaboration with the Biocurrents Research Lab at Woods Hole
2. Probe positioner and manipulator
3. Laser excited single-cell optical nanosensors
Collaboration with Tuan Vo-Dihn
4. Kambiz Pourrezaei collaboration
1. A Surface-Enhanced Raman Scattering Nano-Needle for Cellular
Measurements
2. Carbon Nanotube Cellular Endoscopes
5. Automated Microscope Observation Environment for
Biological Analyses (AMOEBA)
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
1mm
1 mm
Making Probes
Laser-Based Micropipet Pulling Device (Model P-2000; Sutter Industries)
The Woods-Hole team have developed sensors
for a variety of molecules, such as nitric oxide:
Glass Microelectrode
Copper Wire
Graphite Epoxy Paste
Epoxy
Carbon Fiber
O-Phenylenediamine
Nafion
Getting these single-cell probes into position,
efficiently and reproducibly....
A non-trivial task!
Offset Hinge: probe
positioning system
Other manipulations using the
offset hinge mount
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Cell micro-injection
Single cell harvesting
Optical fiber based Raman spectroscopy
Orientation of medaka embryos
Nanobiosensors
Collaboration with Tuan Vo-Dinh
Advanced Biomedical Science and Technology Group
Life Science Division
Oak Ridge National Laboratory
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
Metalic coating of probe end to prevent leakage of the excitation light
Gold,
Aluminum,
or Silver
Scanning Electron Microscope Images
of a Nanofiber
Before Metal Coating
(tip diameter ~50nm)
After Metal Coating
(tip diameter 250-300nm)
Nano-probe attachment
Automated Microscope Observation
Environment for Biological Analyses
(AMOEBA)
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
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
“Flow” Diagram Example
Reservoir I
Additive
Inlet
Reservoir II
Heater / Cooler
Pump
Reservoir III
Microbeam Dish
Lab-on-a-chip
Dispenser
Proof of
Principle
Cells were observed for 2 hours with circulating medium at 37 ± 0.5 ºC.
Proof of Principle
System included heated-window cap, to assist heating control.
Lab-in-a-Box
• Assemble your own system from modules.
Sensor
• Automation is computer controlled.
• AMOEBA is flexible and has potential use
in labs across the country and the world.