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

2. Nano and Microtechnologies of hybrid bioelectronic systems Cell Patterning 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. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Nanotopography Ancient methods Micro-methods –Silicon pillars –Silicon grass Nano-methods –Carbon nanotubes

4. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Nanotopography Thermally grown SiO2 Resist Exposure, development RIE, CHF3: oxide etch Photoresist removal RIE: Cl2, BCl3 Si etch HF: Oxide removal

5. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Nanotopography http://www.hgc.cornell.edu/neupostr/lrie.htm

6. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Nanotopography http://www.wadsworth.org/divisions/nervous/nanobio/DG06.htm

7. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Nanotopography Craighead RIE: Cl 2,CF 4,O 2 Photoresist Wet etching: HF, nitric acid, H 2 O Resist removal, Cleaning

8. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Nanotopography LRM55 Astroglial cells – prefer smooth surfaces Cortical astrocytes – Preferred rough surface

9. Nano and Microtechnologies of hybrid bioelectronic systems 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. Nano and Microtechnologies of hybrid bioelectronic systems Cells and nanotopography 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

11. Nano and Microtechnologies of hybrid bioelectronic systems Cells React to Nanoscale Order and Symmetry in Their Surroundings A. S. G. Curtis*, N. Gadegaard, M. J. Dalby, M. O. Riehle, C. D. W. Wilkinson, and G. Aitchison

12. Nano and Microtechnologies of hybrid bioelectronic systems Methods Arrays of nano-pits were prepared in a three-step process: Electron Beam Lithography Nickel die fabrication Hot embossing into polymers

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

14. Nano and Microtechnologies of hybrid bioelectronic systems 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 m Nickel Die

15. Nano and Microtechnologies of hybrid bioelectronic systems Hot embossing into polymers Polymeric replicas were made by embossing the nickel die in a heated polymethylmethacrylate (PMMA) or polycaprolactone (PCL) sheets

16. Nano and Microtechnologies of hybrid bioelectronic systems Cell Cultures Primary human fibroblasts (connective tissue cells)/ rat epithenon cells were seeded on patterned PCL or PMMA 1.Short term experiments: measurements taken at intervals from 2-24 hr 2.Long term experiments: cells cultured for up to 71 days counting n o of adherent cells and measuring their orientation

17. Nano and Microtechnologies of hybrid bioelectronic systems 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 Human fibroblast cells grown on PCL

18. Nano and Microtechnologies of hybrid bioelectronic systems 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. Nano and Microtechnologies of hybrid bioelectronic systems Reaction of cells to different symmetries Cathrine C. 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  m spacing 1.Cells “prefer” entering the larger diameter pits, meaning they might be sensitive to differences in radius of curvature 2.The smallest pits allow the highest proliferation rate and the highest migration rate of a single cell

20. Nano and Microtechnologies of hybrid bioelectronic systems On orthogonal patterns :cells show preference of 90° separated orientations On hexagonal patterns: cells show preference of 120° separated orientations Orientation is nonrandom Cells can distinguish between symmetries???

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

23. Nano and Microtechnologies of hybrid bioelectronic systems 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. Nano and Microtechnologies of hybrid bioelectronic systems Ordered conducting grooves Rough conducting substrate Random nano-topography insulating substrate Ordered insulating grooves Perturbed ordered insulating grooves Nano topography Types of nano-topography

25. Nano and Microtechnologies of hybrid bioelectronic systems Ra Symmetry

26. Nano and Microtechnologies of hybrid bioelectronic systems Carbon nanotube based neuro- chips for engineering and recording of cultured neural networks

27. Nano and Microtechnologies of hybrid bioelectronic systems Recording from cultured neural networks Ben-Jacob, TAUFromherz, MPIBauman, URosGross, UNT

28. Nano and Microtechnologies of hybrid bioelectronic systems Multi electrode arrays E. Ben-Jacob 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

29. Nano and Microtechnologies of hybrid bioelectronic systems Multi electrode arrays

30. Nano and Microtechnologies of hybrid bioelectronic systems Outline How can we make better/new MEA How do we manipulate cells on substrates? Properties of our new MEAs

31. Nano and Microtechnologies of hybrid bioelectronic systems How can we make better/new MEA? Signal fidelity ~ Noise CeCe ReRe C sh R spread R met R seal C hd Soma CeCe ReRe C sh R spread R met R seal C hd Kovacs, 1994

32. Nano and Microtechnologies of hybrid bioelectronic systems Cell-substrate interactions Wong et al. Surface chemistry 2004

33. Nano and Microtechnologies of hybrid bioelectronic systems Nano-topography Hu et al. Mattson et al. J. Mol. Neurosci 2000 Craighead, Cornell

34. Nano and Microtechnologies of hybrid bioelectronic systems Electronic properties (CNTs) armchair zigzag

35. Nano and Microtechnologies of hybrid bioelectronic systems Carbon nanotubes  Biocompatible  Super capacitors  Compatibility with micro fabrication CNT electrodes  Self-cell-organization  Network engineering  Excellent recording Carbon nanotube multi-electrode arrays

36. Nano and Microtechnologies of hybrid bioelectronic systems CNT based MEA Mo electrodes SOG passivation RIE etch PDMS stencil CNTs

37. Nano and Microtechnologies of hybrid bioelectronic systems NN on CNT islands

38. Nano and Microtechnologies of hybrid bioelectronic systems Engineered Networks Tension competes with adhesion to surface

39. Nano and Microtechnologies of hybrid bioelectronic systems Neuronal tissue on CNT electrodes

40. Nano and Microtechnologies of hybrid bioelectronic systems 0.67 F/m 2 Platinum wire 3.4 F/m 2 Pt Black (Commercial MEA) 300 F/m 2 CNT super capacitor 2.45 F/m 2 CNT MEA <0.01 F/m 2 Mo DC Electrochemical Performances Comparable to Commercial MEA 137 mM NaCl, 2.7mM KCL, pH 7.4 at 25ºC Cyclic voltammetrySpecific capacitance Electrode Capacitance

41. Nano and Microtechnologies of hybrid bioelectronic systems Electrical activity (patch clamp) Stimulated electrical activity

42. Nano and Microtechnologies of hybrid bioelectronic systems Electrical activity (CNTs) Spontaneous electrical activity

43. Nano and Microtechnologies of hybrid bioelectronic systems Cell-surface interaction Mo electrode Craighead, Cornell

44. Nano and Microtechnologies of hybrid bioelectronic systems Summary CNT are excellent substrates for neuronal growth Self-organization of neurons Engineered networks Very good recording properties

45. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Topography Peter Fromherz, Max Planck Institute

46. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Topography Fromherz (http://www.biochem.mpg.de/mnphys/)

47. Nano and Microtechnologies of hybrid bioelectronic systems CNT FET Bio-sensors

48. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Topography

49. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (V) : Topography

50. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (VII) : Wells Pine, Caltech

51. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (VII) : Wells Neuro-wells / neuro-cages Space for neurite growth

52. Nano and Microtechnologies of hybrid bioelectronic systems Approaches (VI) : Wells

53. Nano and Microtechnologies of hybrid bioelectronic systems References Zeck G, Fromherz P, Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip, P NATL ACAD SCI USA 98 (18): 10457-10462 AUG 28 2001 St John PM, Davis R, Cady N, et al., Diffraction-based cell detection using a microcontact printed antibody grating, ANAL CHEM 70 (6): 1108-1111 MAR 15 1998 Craighead HG, James CD, Turner AMP, Chemical and topographical patterning for directed cell attachment, CURR OPIN SOLID ST M 5 (2-3): 177-184 APR-JUN 2001 Segev R, Benveniste M, Hulata E, et al., Long term Behavior of lithographically prepared in vitro neuronal networks, PHYS REV LETT 88 (11): Art. No. 118102 MAR 18 2002 Yousaf MN, Houseman BT, Mrksich M, Using electroactive substrates to pattern the attachment of two different cell populations, P NATL ACAD SCI USA 98 (11): 5992-5996 MAY 22 2001 Yeo WS, Hodneland CD, Mrksich M, Electroactive monolayer substrates that selectively release adherent cells, CHEMBIOCHEM 2 (7-8): 590-+ AUG 3 2001 Chen CS, Mrksich M, Huang S, et al., Geometric control of cell life and death, SCIENCE 276 (5317): 1425-1428 MAY 30 1997 Folch A, Toner M, Microengineering of cellular interactions, ANNU REV BIOMED ENG 2: 227-+ 2000