Nano and Microtechnologies of hybrid bioelectronic systems
Nano and Microtechnologies of hybrid bioelectronic
systems
(Lecture 2- nano-topography)
Dr. Yael Hanein
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
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Nanotopography
• Ancient methods• Micro-methods
– Silicon pillars– Silicon grass
• Nano-methods– Carbon nanotubes
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
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Nanotopography
http://www.hgc.cornell.edu/neupostr/lrie.htm
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Nanotopography
http://www.wadsworth.org/divisions/nervous/nanobio/DG06.htm
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Nanotopography
Craighead
RIE: Cl2,CF4,O2
Photoresist
Wet etching: HF, nitric acid, H2O
Resist removal, Cleaning
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Nanotopography
LRM55 Astroglial cells – prefer smooth surfacesCortical astrocytes – Preferred rough surface
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 )!?
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
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
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
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
Nano and Microtechnologies of hybrid bioelectronic systems
Nickel die fabrication100 nm thick resist with nanopits
Sputter coating of a 50nm Ni-V laye
Electroplating of Ni to a thickness of 300 m
Nickel Die
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
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 no of adherent cells and measuring their orientation
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
Nano and Microtechnologies of hybrid bioelectronic systems
Many cells possess surface nanometric features
Filopodia and microspikes may be the organelle whosemajor 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
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
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???
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
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?
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
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
Nano and Microtechnologies of hybrid bioelectronic systems
Ra
Symmetry
Nano and Microtechnologies of hybrid bioelectronic systems
Carbon nanotube based neuro-chips for engineering and recording of cultured
neural networks
Nano and Microtechnologies of hybrid bioelectronic systems
Recording from cultured neural networks
Ben-Jacob, TAU Fromherz, MPIBauman, URosGross, UNT
Nano and Microtechnologies of hybrid bioelectronic systems
Multi electrode arrays
E. Ben-Jacob
Large electrically active networks, Long term (weeks), Relevant biological activity
BUTLarge electrodes, Poor sealing, Average (many neurons) signal, Poor electrode-cell coupling, Random networks
Nano and Microtechnologies of hybrid bioelectronic systems
Multi electrode arrays
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
Nano and Microtechnologies of hybrid bioelectronic systems
How can we make better/new MEA?
• Signal fidelity ~• Noise•
Ce
Re
Csh
RspreadRmet
Rseal
Chd
Soma
Ce
ReCsh
Rspread
Rmet
Rseal
Chd
Kovacs, 1994
fkTRV Nrmsnoise 4
sealR
ARn
1~
Nano and Microtechnologies of hybrid bioelectronic systems
Cell-substrate interactions
Wong et al. Surface chemistry 2004
Nano and Microtechnologies of hybrid bioelectronic systems
Nano-topography
Hu et al.Mattson et al.J. Mol. Neurosci 2000
Craighead, Cornell
Nano and Microtechnologies of hybrid bioelectronic systems
Electronic properties (CNTs)
armchair
zigzag
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
Nano and Microtechnologies of hybrid bioelectronic systems
CNT based MEA
Mo electrodes
SOG passivation
RIE etchPDMS stencil
CNTs
Nano and Microtechnologies of hybrid bioelectronic systems
NN on CNT islands
Nano and Microtechnologies of hybrid bioelectronic systems
Engineered Networks
Tension competes with adhesion to surface
Nano and Microtechnologies of hybrid bioelectronic systems
Neuronal tissue on CNT electrodes
Nano and Microtechnologies of hybrid bioelectronic systems
Platinum wire
0.67 F/m2
Pt Black(Commerci
al MEA)3.4 F/m2
CNT super capacitor
300 F/m2
CNT MEA 2.45 F/m2
Mo <0.01 F/m2
DC Electrochemical Performances Comparable to Commercial MEA
137 mM NaCl, 2.7mM KCL, pH 7.4 at 25ºC
Cyclic voltammetry Specific capacitance
Electrode Capacitance
Nano and Microtechnologies of hybrid bioelectronic systems
Electrical activity (patch clamp)
Stimulated electrical activity
Nano and Microtechnologies of hybrid bioelectronic systems
Electrical activity (CNTs)
Spontaneous electrical activity
Nano and Microtechnologies of hybrid bioelectronic systems
Cell-surface interaction
Mo electrode
Craighead, Cornell
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
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Topography
Peter Fromherz, Max Planck Institute
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Topography
Fromherz (http://www.biochem.mpg.de/mnphys/)
Nano and Microtechnologies of hybrid bioelectronic systems
CNT FET Bio-sensors
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Topography
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (V) : Topography
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (VII) : Wells
Pine, Caltech
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (VII) : Wells
Neuro-wells / neuro-cages
Space for neurite growth
Nano and Microtechnologies of hybrid bioelectronic systems
Approaches (VI) : Wells
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