1. Project Update June 22, 2006 ME342A

2. Project Goal Design a bioMEMs substrate to apply and measure electromechanical forces in the differentiation of human embryonic stem cell- derived (hESC)-cardiac myocytes (CM) Undifferentiated hESCs-Fluc-eGFP (DAPI nuclear stain) hESC-CMs organized in embryoid body bioMEMS device Contractility Electrophysiology Mechanical force

3. BioMEMS: Engineering Specs Device RequirementTarget Value 1. Apply mechanical strainUp to 10% 2. Apply electric field~O(1) V/cm 3. Measure electric potential (ECG)100μV—1mV 4. Area of mechanical deformationA < 1cm 2 5. Size of electrodesdiameter = 20μm 6. Inter-electrode spacingspacing = 250μm 7. Area of cell cultureA > 1cm 2 8. Thickness of substratet < 1mm

4. BioMEMS: Device Design Poly(dimethylsiloxane) (PDMS): A biocompatible elastomeric polymer with low water permeability Quartz: Optically transparent substrate Gold: Biocompatible thin film electrodes Indium-Tin Oxide (ITO): Transparent thin film conducting electrodes traces A. Unstrained state B. Strained state

5. BioMEMS: Loading Curves Young’s Modulus PDMS E = 500kPa Thickness = 50um Membrane length = 1cm Loading post length = 0.7cm

6. 1. Double polished quartz wafer ~ 500μm BioMEMS: Fabrication Quartz

7. 2. Laser cut alignment marks & pressure channels (frontside wafer) BioMEMS: Fabrication Quartz Channels etched to apply suction pressure to PDMS substrate

8. 3. Laser cut channels to connect to pressure lines (backside wafer) BioMEMS: Fabrication Quartz Channels backside to connect to vacuum source

9. 3a. Laser cut channels to connect to pressure lines (backside wafer) Alternative Step—Replace 3 & 4 Quartz Channels frontside etch to connect to vacuum source *will require punch holes in PDMS layer, so need alignment marks on PDMS layer for this interface…same as uFluidic interconnect

10. 4. Bond a second quartz wafer to the first quartz wafer BioMEMS: Fabrication Quartz Channels backside to connect to vacuum source

11. 5. Fill with sacrificial layer—acrylate or agaraose. Squeeqy off. BioMEMS: Fabrication Quartz Sacrificial later Channels backside to connect to vacuum source

12. 6. Spin photoresist and expose area for second sacrificial layer (loading posts and vacuum channel are covered). BioMEMS: Fabrication Quartz Sacrificial later Photoresist Channels backside to connect to vacuum source

13. 7. Cast second sacrificial layer of acrylate BioMEMS: Fabrication Quartz Sacrificial later Photoresist Channels backside to connect to vacuum source

14. 8. Strip photoresist (should remove sacrificial layer from alignment marks here) and plasma surface area for PDMS BioMEMS: Fabrication Quartz Sacrificial later Photoresist Channels backside to connect to vacuum source

15. 9. Spin PDMS BioMEMS: Fabrication Quartz Sacrificial later Photoresist PDMS

16. 10. Spin photoresist BioMEMS: Fabrication Quartz Sacrificial later Photoresist PDMS

17. 10. Ebeam 20nm Ti (adhesion layer for gold and traces for electrodes) BioMEMS: Fabrication Quartz Sacrificial layer Photoresist PDMS Titanium

18. 11. Ebeam 150nm gold film (actual stretchable traces—geometry) BioMEMS: Fabrication Quartz Sacrificial layer Photoresist PDMS Titanium Gold

19. 12. Strip and pattern photo resist for BioMEMS: Fabrication Quartz Sacrificial layer Photoresist PDMS Titanium Gold

20. 13. Strip and pattern photo resist for electrodes, gauges, contact pads BioMEMS: Fabrication Quartz Sacrificial layer Photoresist PDMS Titanium Gold

21. 14. Ebeam gold electrodes BioMEMS: Fabrication Quartz Sacrificial layer Photoresist PDMS Titanium Gold

22. Passivation layer 15. Strip photoresist and passivate BioMEMS: Fabrication Quartz Sacrificial layer Photoresist PDMS Titanium Gold

23. Passivation layer 16. Dissolve sacrificial layer BioMEMS: Fabrication Quartz Sacrificial layer Photoresist PDMS Titanium Gold

24. BioMEMS: Stretchable Electrodes C. S. Park, M. Maghribi Characterizing the Material Properties of Polymer-Based Microelectrode Arrays for Retinal Prosthesis

25. Stimulation Electrodes Goal: To pattern gold electrodes within a flow chamber for selectively stimulating hESCs –Electrodes 100μm x 5000μm (10 per well) –Interelectrode distance 1000μm –Contacts pads 2mm x 2mm (10 per well) Polished glass wafers 1 mm thick

26. BioMEMS: Strain gauge Need a strain gauge and a reference strain gauge for every deformable area.

27. Strain gauge design Length (L = 1 mm) Trace width (w = 50 um) Distance between turns (p = 450 um) Number of turns (t = 3—38) Thickness of gold electrodes ~several hundred nm

28. Mechanical Strain Goal: To apply cyclic mechanical strain to hESC precursor cells and observe differentiation

29. Next Steps QFD write-up for Beth Refine process cartoons Define geometry of membrane and electrodes ANSYS analysis of membrane and electrode deformation Define redundant layers—ie, cover up alignment marks w/ foil Creation of Ledit mask Selection of machines Training