Control and Modulation of Biochemical Reactions in Plastic Microfluid Devices

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