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

Nano and Microtechnologies of hybrid bioelectronic systems

Nano and Microtechnologies of hybrid bioelectronic

systems

(Lecture 2- nano-topography)

Dr. Yael Hanein


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


Approaches (V) : Nanotopography

• Ancient methods• Micro-methods

– Silicon pillars– Silicon grass

• Nano-methods– Carbon nanotubes



Thermally grown SiO2

Resist

Exposure, development

RIE, CHF3: oxide etch

Photoresist removal

RIE: Cl2, BCl3 Si etch

HF: Oxide removal



http://www.hgc.cornell.edu/neupostr/lrie.htm



http://www.wadsworth.org/divisions/nervous/nanobio/DG06.htm



Craighead

RIE: Cl2,CF4,O2

Photoresist

Wet etching: HF, nitric acid, H2O

Resist removal, Cleaning



LRM55 Astroglial cells – prefer smooth surfacesCortical astrocytes – Preferred rough surface


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 )!?


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


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


Methods

Arrays of nano-pits were prepared in a three-step process:

• Electron Beam Lithography

• Nickel die fabrication

• Hot embossing into polymers


Electron Beam Lithography

Positive resist ZEP 520A coating on silicon

EBL of pits, with diameter of

35, 75, 120 nm

Development


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


Hot embossing into polymers

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


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


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


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


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


• 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???


• 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


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?


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


Ordered conducting grooves

Rough conducting substrate

Random nano-topography insulating substrate

Ordered insulating grooves

Perturbed ordered insulating grooves

Nano topography

Types of nano-topography


Ra

Symmetry


Carbon nanotube based neuro-chips for engineering and recording of cultured

neural networks


Recording from cultured neural networks

Ben-Jacob, TAU Fromherz, MPIBauman, URosGross, UNT


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


Multi electrode arrays


Outline

• How can we make better/new MEA

• How do we manipulate cells on substrates?

• Properties of our new MEAs


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~


Cell-substrate interactions

Wong et al. Surface chemistry 2004


Nano-topography

Hu et al.Mattson et al.J. Mol. Neurosci 2000

Craighead, Cornell


Electronic properties (CNTs)

armchair

zigzag


Carbon nanotubes Biocompatible Super capacitors Compatibility with micro fabrication

CNT electrodes Self-cell-organization Network engineering Excellent recording

Carbon nanotube multi-electrode arrays


CNT based MEA

Mo electrodes

SOG passivation

RIE etchPDMS stencil

CNTs


NN on CNT islands


Engineered Networks

Tension competes with adhesion to surface


Neuronal tissue on CNT electrodes


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


Electrical activity (patch clamp)

Stimulated electrical activity


Electrical activity (CNTs)

Spontaneous electrical activity


Cell-surface interaction

Mo electrode

Craighead, Cornell


Summary

• CNT are excellent substrates for neuronal growth

• Self-organization of neurons• Engineered networks• Very good recording properties


Approaches (V) : Topography

Peter Fromherz, Max Planck Institute



Fromherz (http://www.biochem.mpg.de/mnphys/)


CNT FET Bio-sensors


Approaches (VII) : Wells

Pine, Caltech


Approaches (VII) : Wells

Neuro-wells / neuro-cages

Space for neurite growth


Approaches (VI) : Wells


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

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

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