laurie locascio the microfluidics project analytical chemistry divsion nist control and modulation...
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Laurie Locascio
The Microfluidics Project
Analytical Chemistry Divsion
NIST
Control and Modulation of Biochemical Reactions in Plastic
Microfluid Devices
Overview
• Fabrication of plastic microdevices
Imprinting
Laser ablation
• Biochemical separations in plastic microfluidic devices
• Characterization of surface chemistry
• Changing the surface of plastic devices
Imprinting Plastic
Si
3” Silicon TemplateRaised 3-D inverse
of microfluid channel
SEM of silicon template
Plastic
Imprinted Plastic
Imprinting: 1000-8000 lbsROOM TEMPERATURE OR
HEATED PROCESS
Martynova, L., Locascio, L.E. et.al. Anal. Chem. 1997, 69, 4783-4789.
Biochemical Assays in Plastic Microfluid Systems
Morphine-3-glucuronide/morphine Mab
DeviceAcrylic25 mchannel1 cm arm 400 V/cm
Morphine ImmunoassayChannel: High charge, fast EOF
High surface adsorption leads to sample loss and peak broadening
10
15
20
25
30
35
40
45
50
1 88 175 262 349 436 523 610 697 784 871 958
Pixels
Inte
ns
ity
Un
its
Isoelectric Focusing of ProteinsChannel: Low charge, low EOF
Detector+ -pH4 pH10
Morphine Mab
Isoelectric Focusing (IEF)
Fill channel with ampholyte solution and protein sample
Establish pH gradient and focus protein
pH=4 pH=10
-+
Some residual charge/adsorption causing peak broadeningChannel: Low surface charge, lower EOF
10
15
20
25
30
35
40
45
50
1 88 175 262 349 436 523 610 697 784 871 958
PixelsIn
ten
sit
y U
nit
spH 4 pH 6
Peaks 10 times broader than in capillary
Surface Interactions in Protein Separation
Surface Charge Density/Distribution
• Higher charge = high EOF• Greater protein adsorption
with high charge density, low buffer strength
• Peak dispersion caused by uneven charge distribution
Surface Roughness•High surface
roughness induces protein precipitation and aggregation
- - - - - -
- - - - - - -+ EOF
-+
+ ++ + ++++ +++ ++ + ++
EOF Mobility = Flow velocity/Field Strength
Chemical Mapping of Plastic Surfaces
• Labeling of charged groups with specific fluorescent probes
• Carboxylate and amine groups identified
• Carboxylate groups labeled with EDAC (ethyldimethylaminopropyl carbodiimide hydrochloride)/fluorescein
• Results measured by fluorescence microscopy
Room Temperature Imprinting
Measuring Surface Charge in Imprinted Channels
• Microchannel floor is uncharged in room T imprints• Wall charge varies with imprinting protocol
Brightfield Image
Fluorescence Image
Hot Imprinting
Surface Morphology: PMMA Channels
Hot Imprinted Channel Laser Ablated Channel
Sample Dispersion in Plastic Microchannels
20
40
60
80
100
120
0 50 100 150 200 250 300
OP4 6/5OP4/PDMS 7/7PDMS 7/7Quartz 6/8Pressure
Sam
ple
Wid
th (m
)
Distance (m)Distance (m)
Sam
ple
Wid
th (m
)
Note:PDMS highly variable
Why Surface Modification?
Reduce device variability
Improve measurement reproducibility
Reduce peak broadening
Improve detection limits
Polyelectrolyte Multilayers
• Facile construction• Reproducible surface chemistry• Control of EOF mobility• Change surface charge to prevent adsorption
- - - - - - - - - -- - - - - - - - - -Plastic Substrate
PEM
Alternating layers of positively and negatively charged polyelectrolytes held by electrostatic interaction
Polyelectrolytes
SO3-Na+
n
•HCl CH2NH2
CH2CH n
Polystyrene sulfonatePoly(allylamine hydrochloride)
• 15 min treatment of channel with 1 M NaOH at 50-60°C• 20 min treatment with polycation followed by polyanion• Alternating 5 min treatments with polycation and
polyanion solutions for desired number of layers
Chen, W.; McCarthy, T. J. Macromolecules 1997, 30, 78-86
EOF Mobility in PEM Treated PETG
-4.0E-04 -2.0E-04 0.0E+00 2.0E-04 4.0E-04 6.0E-04
EOF Mobility (cm2/V s)
Native Plastic
1 M NaOH
3 Layer PEM
14 Layer PEM
13 Layer PEM
4 Layer PEM
EOF Mobility in PEM Treated Plastics
-6.0E-04 -3.0E-04 0.0E+00 3.0E-04 6.0E-04
EOF Mobility (cm2/V s)
PS
PETG
Native Plastic
13 Layer PEM
14 Layer PEM
Surface Regeneration with PEMS
•Peaks broad but reproducible •Surface regenerable with application of final layer
0 100 200 300 400 500 600
Time (s)
Re
lati
ve
Vo
lta
ge A. Separations after continuous
channel use
B. Separations after PEM regeneration
Two sides of channel have opposite charge
+++
++
+ +
+ +
Controlling Flow with PEMS
PAH
H2OT-device in single plastic material
• Whole device first coated with PAH then PSS (negative charge)
• Device then treated with H2O or PAH on opposite sides of same channel
Cross Sectional View
Solution Flow +- +++ ++++++++ ++ +++++Split Flow Imaging
Fluorescent dye uncaged in microchannel
Electroosmosis moves the dye in opposite directions
Particle Distribution in Split Flow
-0.08-0.06-0.04-0.02
00.020.040.06
0 10 20 30 40 50 60
Distance across microchannel (m)
Vel
oci
ty (
cm/s
)
Acknowledgements
Dr. Susan Barker
Dr. David Ross
Dr. Emanuel Waddell
Dr. Tim Johnson
Dr. Michael Gaitan
Dr. Michael Tarlov
Maria I. Aquino
Jay XuDr. Cheng Lee
University of MarylandNIST
Conclusions
• Protein separations dependent on charge distribution and density
• Surface charge density can be modified by fabrication protocol
• Surface charge and charge density can be altered in a reproducible manner by PEMS
Flow Imaging
To measure the effect of substrate material and microchannel geometry on sample dispersion
No distortion of the plug caused by the sample “injection” process
Paul, P. H. et.al.Anal. Chem. 1998, 70, 2459-2467
Flow Profile: Electroosmotic Pumping
PMMA PDMS Quartz tubing
Measuring Surface Charge inPMMA Ablated ChannelsMicrochannels laser ablated under nitrogen with varying
ablation power
15 J 25 J 40 J
Microchannel Laser Ablation
Eximer Laser(Kr, Fl, Neon balance)
248 nmFocussing Optics
Programmable stagevacuum chuck
Process GasVacuum
Channel
Altering Ablation Conditions to Affect Surface Charge
PETG ablated in air PETG ablated under O2
Surface charge density varies with process gas