Na no and Microtechnologies of hybrid bioelectronic systems (Lecture 2- nano-topography)

1. Nano and Microtechnologies of hybrid bioelectronic systems(Lecture 2- nano-topography) Dr. Yael Hanein

2. CellPatterning Approaches • Direct protein lithography • Micro-contact printing/micro fluidics • Proteins • SAMs • Dry lithography • Patterned polymers • Temperature sensitive polymers • Nano-topography • Ordered nano-patterning • Disordered nano-patterning • Wells

3. Approaches (V) : Nanotopography • Ancient methods • Micro-methods • Silicon pillars • Silicon grass • Nano-methods • Carbon nanotubes

4. Approaches (V) : Nanotopography Thermally grown SiO2 Resist Exposure, development RIE, CHF3: oxide etch Photoresist removal RIE: Cl2, BCl3 Si etch HF: Oxide removal

5. Approaches (V) : Nanotopography http://www.hgc.cornell.edu/neupostr/lrie.htm

6. Approaches (V) : Nanotopography http://www.wadsworth.org/divisions/nervous/nanobio/DG06.htm

7. Approaches (V) : Nanotopography RIE: Cl2,CF4,O2 Photoresist Wet etching: HF, nitric acid, H2O Resist removal, Cleaning Craighead

8. Approaches (V) : Nanotopography LRM55 Astroglial cells – prefer smooth surfaces Cortical astrocytes – Preferred rough surface

9. Culture of neural cells on silicon wafers with nano-scale surface topograph • Y.W. Fan et all, “Culture of neural cells on silicon wafers with nano-scale surface topograph” : • Si surfaces with variable roughness (without surface treatment) • Morphology of adherent cells remarkably differs on differently rough surfaces • Larger contact area? doesn’t explain the decline in cell adhesion after a certain Ra value! • (Can you really change Ra without changing other parameters?!)

10. Cells respond to surface topography The mechanisms involving cell adhesion and migration on surfaces is poorly understood Extremely important in the field of tissue engineering and biomaterials Important in lab-on a chip/micro bio-sensors Cells and nanotopography

11. Cells React to Nanoscale Order and Symmetry inTheir SurroundingsA. S. G. Curtis*, N. Gadegaard, M. J. Dalby, M. O. Riehle, C. D. W. Wilkinson, and G. Aitchison

12. Methods Arrays of nano-pits were prepared in a three-step process: • Electron Beam Lithography • Nickel die fabrication • Hot embossing into polymers

13. Electron Beam Lithography Positive resist ZEP 520A coating on silicon EBL of pits, with diameter of 35, 75, 120 nm Development

14. Nickel die fabrication 100 nm thick resist with nanopits Sputter coating of a 50nm Ni-V laye Electroplating of Ni to a thickness of 300 mm Nickel Die

15. Hot embossing into polymers Polymeric replicas were made by embossing the nickel die in a heated polymethylmethacrylate (PMMA) or polycaprolactone (PCL) sheets

16. Cell Cultures Primary human fibroblasts (connective tissue cells)/ rat epithenon cells were seeded on patterned PCL or PMMA • Short term experiments: measurements taken at intervals from 2-24 hr • Long term experiments: cells cultured for up to 71 days counting no of adherent cells and measuring their orientation

17. Human fibroblast cells grown on PCL Adhesion on spaced nanopatterened areas is much lower than on planar areas, but on the smallest closest spaced pits is the same as on the planar area! Rat epitenon cells grown on PCL surfaces for 24 h

18. Many cells possess surface nanometric features Filopodia and microspikes may be the organelle whose major function is to explore nanofeatures around the cell It is interesting to note that the filopodia follows the nanopattern, and seems to be directed by it

19. Reaction of cells to different symmetries • CathrineC. Berry et all, “The influence of microscale topography on fibroblast attachment and motility”: fibroblasts were grown on arrays of pits, 7, 15 and 25 diameter, 20 and 40 mm spacing • Cells “prefer” entering the larger diameter pits, meaning they might be sensitive to differences in radius of curvature • The smallest pits allow the highest proliferation rate and the highest migration rate of a single cell

20. Orientation is nonrandom • On orthogonal patterns :cells show preference of 90° separated orientations • On hexagonal patterns: cells show preference of 120° separated orientations Cells can distinguish between symmetries???

21. Fredrick Johansson et al, “Axonal outgrowth on nano-imprinted patterns” • Investigated guidance of axons on patterns of parallel grooves of PMMA, with depths of 300nm, widths of 100-400 nm and distance between grooves 100-1600 nm. -axons display contact guidance on all patterns -preferred to grow on edges and elevations in the patterns rather than in grooves- this may be due to edge effects, as concentration of charges

22. How cells sense ORDER and SYMMETRY of surfaces? What makes cells adhere to surfaces? Why do differences in diameters and spacing of micro and nano features have such dramatic effect on cell adhesion?

23. Two possible explanations The effect is caused by the nonliving surfaces alone Nanofeatures are known to affect orientations in nonliving systems It is unknown whether nanofeatures affect protein adsorption on the nanoscale, (exposure to protein rich culture media- showed no difference) The effect is caused by interaction of cellular processes and interfacial forces

24. Types of nano-topography Ordered conducting grooves Ordered insulating grooves Perturbed ordered insulating grooves Nano topography Random nano-topography insulating substrate Rough conducting substrate

25. Symmetry Ra

26. Carbon nanotube based neuro-chips for engineering and recording of cultured neural networks

27. Recording from cultured neural networks Ben-Jacob, TAU Gross, UNT Bauman, URos Fromherz, MPI

28. Multi electrode arrays Large electrically active networks, Long term (weeks), Relevant biological activity BUT Large electrodes, Poor sealing, Average (many neurons) signal, Poor electrode-cell coupling, Random networks E. Ben-Jacob

29. Multi electrode arrays

30. Outline • How can we make better/new MEA • How do we manipulate cells on substrates? • Properties of our new MEAs

31. Signal fidelity ~ Noise Rseal Rspread Chd Soma Csh Re Ce Rmet Re Rspread Chd Rmet Csh Ce Rseal How can we make better/new MEA? Kovacs, 1994

32. Cell-substrate interactions Wong et al. Surface chemistry 2004

33. Nano-topography Craighead, Cornell Hu et al. Mattson et al. J. Mol. Neurosci 2000

34. Electronic properties (CNTs) zigzag armchair

35. Carbon nanotube multi-electrode arrays Carbon nanotubes • Biocompatible • Super capacitors • Compatibility with micro fabrication CNT electrodes • Self-cell-organization • Network engineering • Excellent recording

36. CNT based MEA Mo electrodes SOG passivation RIE etch PDMS stencil CNTs

37. NN on CNT islands

38. Engineered Networks Tension competes with adhesion to surface

39. Neuronal tissue on CNT electrodes

40. Electrode Capacitance Cyclic voltammetry Specific capacitance 137 mM NaCl, 2.7mM KCL, pH 7.4 at 25ºC DC Electrochemical Performances Comparable to Commercial MEA

41. Electrical activity (patch clamp) Stimulated electrical activity

42. Electrical activity (CNTs) Spontaneous electrical activity

43. Cell-surface interaction Mo electrode Craighead, Cornell

44. Summary • CNT are excellent substrates for neuronal growth • Self-organization of neurons • Engineered networks • Very good recording properties

45. Approaches (V) : Topography Peter Fromherz, Max Planck Institute

46. Approaches (V) : Topography Fromherz (http://www.biochem.mpg.de/mnphys/)

47. CNT FET Bio-sensors

48. Approaches (V) : Topography

49. Approaches (V) : Topography

50. Approaches (VII) : Wells Pine, Caltech