August 29, 2007 Centrifugal (CD) Microfluidic Solid-Phase Extraction By Dane Van Domelen, 2007 IM-SURE Fellow Faculty Mentor: Professor Marc J. Madou Graduate Student: Robert A. Gorkin
CDs as a Microfluidic Platform • Centrifugal force vs. surface tension for many fluidic functions • Valving • Mixing • Sample splitting • Fluid separation • Decanting • Chambers and microchannels tailored for desired fluid movement
Specialized CDs: A 5-Layer Design Polycarbonate discs (0.6 mm thick) Adhesive layers (100 µm) Polycarbonate disc (1.2-3 mm)
BioMEMS Research Goal • Translate entire disease testing process to CDs • Automation • Speed • Price • Must demonstrate CD-based extraction, purification, amplification, and detection
Cell Lysis Accomplished Spring 2007 From “A Novel, Compact Disk-Like Centrifugal Microfluidics System for Cell Lysis and Sample Homogenization” by Kido et al. • Extraction efficiency comparable to conventional lysis methods • Lyses 70 µL samples
New Focus: Purification / Amplification of Target DNA (Group B Streptococcus) • 50 µL lysate from cell lysis CD • Trace amounts of GBS • Overwhelming amount of healthy DNA and other cellular material • GBS must be isolated and purified to a detectable level • A. Amplification by polymerase chain reaction • Primers, Taq polymerase, nucleotides, heating/cooling • B. Isolation and concentration by solid-phase extraction
Solid-Phase Extraction • Purifying technique to isolate and concentrate specific DNA 1. Fluidic sample is introduced to a solid phase (beads). DNA-bead binding occurs. 2. Rinse solution is added to wash away remaining sample. 3. Small volume of heated dH20 added to denature captured DNA and elute from beads to optical chamber; high GBS concentration. 4. Detection to determine whether blood sample is GBS positive.
IM-SURE Project: SPE on CDs • Large functionalized beads (300 µm to 1200 µm in diameter) as solid phase • Dowex Mac-3 ion exchange resin • Two SPE mechanisms • Slow-tilt rotation • Flow through a bead column • Translate both techniques to CD designs for testing
Mixing CD to Mimic Slow-Tilt Rotation • Tangential acceleration for sample-bead mixing • Spin one way at a high acceleration • Once a certain velocity is reached, decelerate and spin the opposite way • Repeat
Final CD Mixing Design Sample Loading Hole Mixing Chamber Air Venting Holes Microchannels Coriolis Valve Elution Chamber Waste Chamber
Column Flow-Through CD • Goal: Gradual fluid flow through a column of tightly packed beads ORIGINAL DESIGN • Flow rates too high (> 50 µL/min) • Problems with Coriolis valve
Experimental Column CD Designs • Angled channels • Much slower initial flow rate (<1 µL/min) • Once channel fills, 25x increase in flow rate • Channel only holds 1 µL
Spiral CD (video) • Long microchannel, 26 cubic millimeters in volume • Consistent 1-2 µL/min flow rates during first test • Only 1 test per CD
Column Design Based on Microchannel Flow Rate Equation • From previous bioMEMS publication: Flow Rate Through a Microchannel: U = D2hρω2rΔr/32µL L = length of microchannel Δr = radial extent of the fluid ω = angular velocity of CD ω L Δr
Formula-Based Column CD L = 29.5 mm Introduction of intermediate chamber allows ∆r calibrations Δr = 8.75 mm
Very Slow Flow Rates Observed Initial Filling of Intermediate Chamber Empting to Waste Chamber [Click to Play]
Excellent Flow Rate Control Volumetric Flow Rate (uL/min) Rotating Speed (RPM) - Three trials per data point - Error bars represent mean +/- one standard deviation
Unexpected Mixing CD Testing Results [10 minute sample/bead mix, varied wash times] • Expected: • Strong fluorescence intensity for specific DNA • Dwindling intensity for nonspecific as wash times increase • Actual: • Fluorescence observed, but no apparent distinction between specific and nonspecific intensity Fluorescence comparison after 10 minute wash (5x) SPECIFIC NONSPECIFIC
Fluorescence Intensity vs. Wash Times Approximately equivalent decrease in fluorescence intensity for specific and nonspecific oligos.
A Bead Adsorption Problem • Off-the-CD Bead Experiment • Functionalized vs. unfunctionalized beads • Traditional slow-tilt rotation in test tube for 10 minutes; washes in 6-minute intervals Unfunctionalized Functionalized Student’s t-test: No significant difference between sample means (n = 3) Conclusion: Beads adsorb DNA; no specificity for nucleotide sequence
Research Progress, Conclusions • Two CD designs optimized for SPE • Functionalized beads proven inefficient • Future Experiments for DNA capture vs. mixing times, flow rates, wash times, wash volumes, etc. • Is CD-based solid-phase extraction sensitive enough to detect realistic GBS concentrations?
Thanks for a Great Summer • Professor Marc Madou and Jim Zoval for mentoring • Robin Gorkin for support, enthusiasm, and helpfulness • Said Shokair, IM-SURE program director, for excellent involvement and ping pong competition • National Science Foundation • Jonathan Siegrist, Nahui Kim, BioMEMS Laboratory