Control and Modulation of Biochemical Reactions in Plastic Microfluid Devices

1. Control and Modulation of Biochemical Reactions in Plastic Microfluid Devices Laurie Locascio The Microfluidics Project Analytical Chemistry Divsion NIST

2. Overview • Fabrication of plastic microdevices Imprinting Laser ablation • Biochemical separations in plastic microfluidic devices • Characterization of surface chemistry • Changing the surface of plastic devices

3. Imprinting Plastic Plastic Si SEM of silicon template 3” Silicon Template Raised 3-D inverse of microfluid channel Imprinting: 1000-8000 lbsROOM TEMPERATURE OR HEATED PROCESS Imprinted Plastic Martynova, L., Locascio, L.E. et.al. Anal. Chem. 1997, 69, 4783-4789.

4. Biochemical Assays in Plastic Microfluid Systems Morphine Immunoassay Channel: High charge, fast EOF Isoelectric Focusing of Proteins Channel: Low charge, low EOF - + pH4 pH10 Detector Morphine-3-glucuronide/ morphine Mab Morphine Mab Device Acrylic 25 mmchannel 1 cm arm 400 V/cm High surface adsorption leads to sample loss and peak broadening

5. + - Isoelectric Focusing (IEF) Fill channel with ampholyte solution and protein sample Establish pH gradient and focus protein pH=4 pH=10 pH 4 pH 6 Peaks 10 times broader than in capillary Some residual charge/adsorption causing peak broadening Channel: Low surface charge, lower EOF

6. - - - - - - - + + - EOF + + + + + + + + + + + + + + + + - - - - - - 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 EOF Mobility = Flow velocity/Field Strength • Surface Roughness • High surface roughness induces protein precipitation and aggregation

7. 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

8. Measuring Surface Charge in Imprinted Channels Room Temperature Imprinting Hot Imprinting Brightfield Image Fluorescence Image • Microchannel floor is uncharged in room T imprints • Wall charge varies with imprinting protocol

9. Surface Morphology: PMMA Channels Hot Imprinted Channel Laser Ablated Channel

10. Sample Dispersion in Plastic Microchannels Note: PDMS highly variable Sample Width (mm) Distance (mm)

11. Why Surface Modification? • Reduce device variability • Improve measurement reproducibility • Reduce peak broadening • Improve detection limits

12. Plastic Substrate - - - - - - - - - - PEM - - - - - - - - - - Polyelectrolyte Multilayers Alternating layers of positively and negatively charged polyelectrolytes held by electrostatic interaction • Facile construction • Reproducible surface chemistry • Control of EOF mobility • Change surface charge to prevent adsorption

13. •HCl CH2NH2 CH2CH n n SO3-Na+ Polyelectrolytes Poly(allylamine hydrochloride) Polystyrene sulfonate • 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. Macromolecules1997,30, 78-86

14. EOF Mobility in PEM Treated PETG

15. EOF Mobility in PEM Treated Plastics

16. Surface Regeneration with PEMS • Peaks broad but reproducible • Surface regenerable with application of final layer

17. H2O T-device in single plastic material PAH + + + + + + + + + Controlling Flow with PEMS • Whole device first coated with PAH then PSS (negative charge) • Device then treated with H2O or PAH on opposite sides of same channel Two sides of channel have opposite charge Cross Sectional View

18. - + + + + + + + + + + + + + + + + + + + Solution Flow Split Flow Imaging Fluorescent dye uncaged in microchannel Electroosmosis moves the dye in opposite directions

19. Particle Distribution in Split Flow

20. Acknowledgements NIST University of Maryland Dr. Susan Barker Dr. David Ross Dr. Emanuel Waddell Dr. Tim Johnson Dr. Michael Gaitan Dr. Michael Tarlov Maria I. Aquino Jay Xu Dr. Cheng Lee

21. 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

22. 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

23. Flow Profile: Electroosmotic Pumping PMMA PDMS Quartz tubing

24. Measuring Surface Charge inPMMA Ablated Channels Microchannels laser ablated under nitrogen with varying ablation power 15 mJ 25 mJ 40 mJ

25. Microchannel Laser Ablation Eximer Laser (Kr, Fl, Neon balance) 248 nm Focussing Optics Process Gas Vacuum Channel Programmable stage vacuum chuck

26. Altering Ablation Conditions to Affect Surface Charge PETG ablated in air PETG ablated under O2 Surface charge density varies with process gas