State-of-the-art probes

Alan Bigelow

• Alternative sensing methods• Real-time, single-cell analysis techniques

1. Miniature ion-selective single-cell probesCollaboration with the Biocurrents Research Lab at Woods Hole

2. Probe positioner and manipulator

3. Laser excited single-cell optical nanosensorsCollaboration with Tuan Vo-Dihn

4. Kambiz Pourrezaei collaboration1. A Surface-Enhanced Raman Scattering Nano-Needle for Cellular

Measurements

2. Carbon Nanotube Cellular Endoscopes

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

Outline

1 mm

1m 1 m

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

Making Probes

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

Graphite Epoxy Paste

Glass Microelectrode

O-Phenylenediamine

Copper Wire

Carbon Fiber

Nafion

Epoxy

The Woods-Hole team have developed sensors for a variety of molecules, such as nitric oxide:

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

• Cell micro-injection

• Single cell harvesting

• Optical fiber based Raman spectroscopy

• Orientation of medaka embryos

Nanobiosensors

Collaboration with Tuan Vo-DinhAdvanced Biomedical Science and Technology Group

Life Science DivisionOak 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 Imagesof 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 conditionsControl 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

Reservoir II

Reservoir III

PumpHeater / Cooler

Lab-on-a-chip

Dispenser

Microbeam Dish

Hinge mount

AdditiveInlet

Cells were observed for 2 hours with circulating medium at 37 ± 0.5 ºC.

Proof of Principle

Proof of Principle

System included heated-window cap, to assist heating control.

Lab-in-a-Box• 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.

Sensor