Micro-Fluidic Device for Antigen Discovery By: Khine Lwin August 23, 2007 Graduate Student: Armando Tovar Professor: Dr. Abraham Lee

Background Antigen: a foreign molecule that triggers an immune response upon entering the body Antibody: a protein that attaches to antigens, tags them as foreign, and neutralizes them Binding-specific Obtained from Wikipedia.org

Purpose Create a micro-fluidic device that can detect diseases quickly and efficiently Faster than ELISA (takes about 24 hours) Simple design Diagnostics: Detect serum concentrations West Nile, HIV, Smallpox, bioterrorist threats, etc. Detect food allergens Device consists of: Ti/Au electrode array Micro-channel created using polydimethylsiloxane (PDMS) H3L Proteins immobilized on electrode array H3L, an intracellular mature virion envelope protein. Figure 1. Micro-fluidic Device

Device Fabrication Photolithography Etch Au/Ti electrodes onto glass Protein Spotting Place droplets of H3L proteins onto electrode tips PDMS PDMS is used to create a micro-channel which will be sealed on top of the electrode gap

Photolithography Figure 2. Photolithography Process

Protein Immobilization Surface Printing Tip (SPT) Nano Ware Nano eNabler Printing Process

System Equivalent Circuit Function Generator Current Amplifier DMM Flow R DAQ Computer AC Device Equivalent Circuit Faradic Cage Figure 5. Flowchart of System

Channel Cs Bulk Solution Rs Double Layer Antibody Diffusion layer CDL Antigen All of these components affect impedance CIL Immobile layer

Double Layer CDouble Layer

Optimization

Experimental Procedure Solution Flow Rate (µL/min) Duration (min) Data TTBS Washing Buffer 40 5 Wash 5 Measurements Flow turned on/off for 50 seconds (x2) Blocking Buffer 10 12 TTBS Primary Antibodies 5 20 Secondary Antibodies DI H20 Manual Approx. 3 Figure 2. Flow chart of system Table 1. Protocol for Testing Micro-fluidic Device 11

Results Talk about misalignment Figure 6. Signal Response as Flow is Turned On and Off

Results

Confirmation of Antigen-Antibody Binding

PDMS Current Design 1x .5 mm Electrode Tips 200 µm PDMS channel 10 µm gap between the Electrode Tips Figure 4. PDMS Channel Sealed on top of Electrode Tips

Future Work Improve LabVIEW program Further research how channel alignment affects impedance Improve methods for fluorescently tagging antibodies to confirm antigen-antibody binding Antibodies are fluorescently tagged to not only track antigen-antibody binding, but also to prove that the two entities are bound together.

Special Thanks Dr. Abraham Lee Armando Tovar BioMint Lab IM-SURE National Science Foundation Said Shokair Jason Choi & Lillian Shido Any Questions?

Optimization Cont’d

LabVIEW Current Program: Prompt user to save file Graph voltage root mean square Extract data and calculate admittance Save data to spreadsheet in Excel Figure 7. User-interface of current LabView program 19