Transcript Microfluidic CD Platform for Point-of

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
1. Slow-tilt rotation
2. 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
Waste
Chamber
Elution
Chamber
Slow-Tilt Rotation on a CD (video)
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
ω
L
Δr
Δr = radial extent of the fluid
ω = angular velocity of CD
Formula-Based Column CD
Introduction of
intermediate
chamber allows
∆r calibrations
L = 29.5 mm
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
Flow Rate vs. Rotating Speed
Volumetric Flow Rate (uL/min)
60
50
40
30
20
10
0
200
250
300
350
400
450
500
550
600
650
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
40
35
Fluorescence Intensity
30
25
20
15
10
5
0
0
2
4
6
8
10
12
14
16
18
Wash Time (min.)
Specific Trial 1
Specific Trial 2
Specific Trial 3
Nonspecific Trial 1
Nonspecific Trial 2
Approximately equivalent decrease in fluorescence
intensity for specific and nonspecific oligos.
20
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
Functionalized
Unfunctionalized
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
Questions or comments?