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Integration of Chip-based Sample Preconcentration into the µChemLabTM/CB
Analysis Platform
Victoria A. VanderNoot, Boyd Wiedenman, YolandaFinstchenko and Julia Fruetel
Sandia National Laboratories
P.O. Box 969, Livermore, CA 94551-0969
HPCE 2003San Diego, CA
Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company,for the United States Department of Energy under contract DE-AC04-94AL85000.
dmlollSAND2003-8005P
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µChemLab™ Separation PlatformAerosol
Collector
WaterAnalysis
SamplePrep
µChemLab™/CBLiquid Analysis
Module
Dual Channel Microchip Separations
Laser Induced Fluorescence
Detection
Microfluidic Delivery
•Application of electric fields moves fluids
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µChemLab™ Separation Platform
Two Channel DeviceCapillary Gel Electrophoresis (CGE) Channel
Beckman 14-200 SDS GelReversed polarity
Capillary Zone Electrophoresis (CZE) Channel10 mM phytic acid, pH 9.5 containing 2 mM DAPS (zwitterionic detergent)Normal polarity
Fluorescamine labelingFluorogenic dyeFast-reacting, amine specificEx/Em 390 nm/480nm
Detection limitsnM for CGE
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Approaches to Preconcentration
• Solid Phase Extraction (on-going efforts for the miniaturization of SPE)
• Electrokinetic Trapping (P411-T: Novel Miniaturized ProteinPreconcentrator Based On Electrokinetic Trapping, Anup K. Singh; Daniel J. Throckmorton; Brian J. Kirby)
• Salt-Bridge[1] J. Khandurina, T.E. McKnight, S.C. Jacobson, L.C. Waters, R.S. Foote, J.M. Ramsey, Analytical Chemistry 72 (2000) 2995-3000[2] R. S. Foote, J. Khandurina, S. C. Jacobson, J. M. Ramsey, Preconcentrationof Proteins on Microchips for Enhanced Detection, Poster presentation at HPCE 2001
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Integration into the µChemLabTM/CB
Silicate layer Approach:Cover glass
Conduction via very small bore channels
Sol-gel silicate layer allows current to pass
Etched channels
Large molecules (i.e. proteins) retained
Cover glassAlternative Approach:
•Eliminate silicate layer
•Makes use of narrow gap and natural surface roughness
Large molecules (i.e. proteins) retained
Etched channels
Jean Pierre Alaire, Stephen C. Jacobson, B. Scott Broyles, Timothy E. McKnight, Christopher T. Culbertson and J. Michael Ramsey, ElectroosmoticallyInduced Hydraulic Pumping on Microchips. µTAS 2001. J. Michael Ramsey and A. van den Berg. ed.s, (Kluwer Academic, Amsterdam), (2001), pp. 127-128.
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Chip Layout
W
P1
P2
S SW
B
Fill
Close-up of the Preconcentrator
Region
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µChemLab™ Separation Platform
Two Channel DeviceCapillary Gel Electrophoresis (CGE) Channel
Beckman 14-200 SDS GelReversed polarity
Capillary Zone Electrophoresis (CZE) Channel10 mM phytic acid, pH 9.5 containing 2 mM DAPS (zwitterionic detergent)Normal polarity
Fluorescamine labelingFluorogenic dyeFast-reacting, amine specificEx/Em 390 nm/480nm
Detection limitsnM for CGE
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Imaging Normal and PreconcentratedInjections in CGE
P2
SW S
P1
Normal Mode P1 PreconcentrationMode
S P1
P2 PreconcentrationMode
S P2S SW
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CGE Separations Using Normal and Preconcentrated Injections
1000
1500
2000
2500
3000
3500
4000
4500
5000
0 50 100 150 200 250 300
Time (s)
Fluo
resc
ence
Sig
nal (
AU
)
No preconcentration
60 s preconcentration
20 nM Lactalbumin,20 nM Ovalbumin
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Peak Areas in CGE Separations Increase as a Function of Preconcentration Duration
800
1300
1800
2300
2800
3300
3800
4300
4800
5300
0 50 100 150 200 250 300 350
Time (s)
Fluo
resc
ence
No preconcentration
60 s preconcentration
50 nM Lactalbumin,50 nM Ovalbumin
120 s preconcentration
***
***
***
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Sub-Nanomolar Detection Limits Using CGE Separations and Preconcentration
2100
2300
2500
2700
2900
3100
3300
3500
3700
3900
4100
0 100 200 300Time (s)
Fluo
resc
ence
500 pM LAC500 pM OVA
LAC
OVA
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Characterization of Performance as a Function of Gap Width
Gap width• Isotropic etch depth used to generate
gaps of different widths • For comparison purposes, all
preconcentrations were held to 60s
Cover glass
variable etch depth
00.5
11.5
22.5
33.5
44.5
7 9 11 13 15 17 19Gap Width (µm)
Pre
conc
entra
tion
Cur
rent
(µ
A)
02468
1012141618
7 9 11 13 15 17 19Gap Width (µm)
Appr
oxim
ate
Prec
once
ntra
tion
Fact
or
*Single Batch Data *Single Batch Data
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µChemLab™ Separation Platform
Two Channel DeviceCapillary Gel Electrophoresis (CGE) Channel
Beckman 14-200 SDS GelReversed polarity
Capillary Zone Electrophoresis (CZE) Channel10 mM phytic acid, pH 9.5 containing 2 mM DAPS (zwitterionic detergent)Normal polarity
Fluorescamine labelingFluorogenic dyeFast-reacting, amine specificEx/Em 390 nm/480nm
Detection limitsnM for CGE
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Preconcentration Using CZE
Elimination of EOF is required• no bulk flow of fluid through the gap
∴residual EOF could lead to pressure generation
Coating with linear polyacrylamide
O NH2
( )n
O
Si
OO
O
O
Si
OO O
Si
OO O
( )m n>>m
O
CONH2CONH2CONH2
O O
CONH2CONH2CONH2
O O
CONH2CONH2
O
CONH2 CONH2CONH2CONH2 CONH2CONH2CONH2 CONH2
SAM applicationUV light polymerization
•Julie Fruetel, Victoria VanderNoot, Jay West, Brian Kirby, Ernest Hasselbrink and Timothy Shepodd, Laser-polymerized thin-film coating for protein analysis by CGE in a microchip, HPCE 2002
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Imaging Normal and PreconcentratedInjections in CZE
P2
SW S
P1
Normal Mode P1 Preconcentration ModeS SW S P1
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CZE Separations Using Normal and Preconcentrated Injections
1000
1200
1400
1600
1800
2000
2200
2400
2600
0 100 200 300 400Time (s)
Rel
ativ
e Fl
uore
scen
ce
No Preconcentration
60 s Preconcentration
Running buffer: 5 mM phosphate, pH 8 containing 5 mM CAS-U zwitterionic detergentReverse polarity
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Summary
•Preconcentration has been incorporated in the chip design with no additional processing steps during chip fabrication
•Successfully demonstrated with both CGE and CZEPreconcentration factors of 10-20x are routinely achievable in only 60s
Coating and/or elimination of EOF is essential for CZE separations
Buffer conditions in CZE will need to be optimized to achieve both good separation efficiency and the elimination of EOF
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AcknowledgementsRobert S. Foote and J. Michael Ramsey at Oak Ridge National Labs for their valuable insights
&Nicole Baryla and Charles A. Lucy for supplying us with a sample of CAS-U zwitterionic detergent
Brian HollidayBrent Horn
Susan JamisonBill Kleist
Ron RenziGeorge Sartor
George Schubert
Isaac ShokairJamie Stamps
Mary Clare StoddardVictoria VanderNoot
Jay WestBoyd Wiedenman
Dan Yee
FundingDept. of Energy Chemical and Biological National Security Program
Gabriela ChiricaWen-Yee ChoiMark ClaudnicEvelyn CruzScott Ferko
Yolanda FintschenkoJulie Fruetel
The µChemLab Technical Team
Integration of Chip-based Sample Preconcentration into the µChemLabTM/CB Analysis PlatformµChemLab™ Separation PlatformApproaches to PreconcentrationChip LayoutImaging Normal and Preconcentrated Injections in CGESub-Nanomolar Detection Limits Using CGE Separations and PreconcentrationCharacterization of Performance as a Function of Gap WidthPreconcentration Using CZEImaging Normal and Preconcentrated Injections in CZESummaryAcknowledgements