Nanotechnology Tools for Life Sciences

Nanotechnology Tools for Life Sciences

Harry Heinzelmann
VP Nanotechnology & Life Sciences

Neuchâtel, June 2009

v1.19

CSEM profile

Privately held Innovation Center, incorporated, not for profit
Privately held Innovation Center, incorporated, not for profit
since 1984, from watchmaking
since 1984, from watchmaking
about 70 shareholders (mostly private)
about 70 shareholders (mostly private)

2008:
2008:
>65 Mio. CHF annual turnover, 395 employees
>65 Mio. CHF annual turnover, 395 employees
30 start-ups created since 2000
30 start-ups created since 2000

Activities:
Activities:
Applied research (contract with Swiss Government)
Applied research (contract with Swiss Government)
Industrialization of technologies, product development
Industrialization of technologies, product development

Technologies:
Technologies:
Micro- and Nanotechnology, Information Technology,
Micro- and Nanotechnology, Information Technology,
and System Engineering
and System Engineering

Copyright 2009 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 1

CSEM profile

Bridge from Science to Innovation

Applied Product
Basic Research Res & Dev Development Marketing
PhD programs Industrialisation Sales
Teaching Customers

Science & Market
Education Success

• technologies
for innovations

• research partners:


CSEM profile

Bridge from Science to Innovation

• wide range of technologies, large experience and network innovative solutions
• highly qualified and experienced staff fast developments
• IP portfolio to support the customers’ application protected business

• shareholders include

• technologies for Green Solutions


CSEM profile

Technologies I (divisions in Neuchâtel)

Microelectronics
Circuit Design, RF, Information Processing

Nanotechnology & Life Sciences
Optical and Bio MNT, Self-assembly, Sensors

Systems Engineering
Mechatronics, Signal Processing, Communication

Time and Frequency
Atomic Clocks, Optical Advanced Systems

Microsystems
MEMS, Cleanroom Infrastucture, Microscopy & Analysis


CSEM profile

Technologies II (divisions outside Neuchâtel)

Photonics (Zurich)
Image Sensing, Optoelectronics

Robotics (Alpnach)
Lab Automation, Packaging, Assembly

Thin Film Optics (Basel)
Optoelectronics, Replicated Optics

Nano Medicine (Landquart)
Imaging, Medical Sensors

CSEM UAE

CSEM Brazil


Technologies and Applications

Nanostructuring

• extended experience in self-assembly of
polymer and nanoparticle systems
• block copolymer microphase separation
and copolymer lithography / MEMS
• molecular grafting chemistries, from and to
• controlled self-assembly of beads

• partnerships & projects:

• industrial collaborations:


Nanostructuring

Top-Down vs. Bottom-Up

10
classical (micro-) fabrication
1 mm

100 MEMS: Micro Electro Mechanical Systems
lithography:
10
VIS
1 µm
UV, X-ray, e – beam
100 FIB (serial)
10

1 nm
molecular self-assembly
1 Å “molecular nanotechnology” 250 nm
4 µm


Nanostructuring

Polymeric Self-Assembly I : Polymer Demixing
50% PMMA / 50% PS 90% PMMA / 10% PS

• demixing of immiscible polymer blends
• qualitative structures on the micron scale
• control over feature size and properties
• large variety of polymers available
80 µm 5 µm

• simple deposition technique
• selective solvent can remove one polymer type
• scalable to
large surfaces

large 5µm med 2µm small <1µm

inexpensive and flexible method to control surface properties on a micron scale


Nanostructuring – Polymer Demixing

Nanoporous Layers for Ink-jet Printer Paper

Polymer paper Nanoporous paper
Nanoporous layer
Polymer layer (Alumina film)
Cellulose Cellulose

stable images fast up-take, small spot size
slow ink uptake, big spot size image fading (light, gas,…)

polymer film
transferred
on paper

paper, 5 µm x 5 µm polymer on Si



Security Features for Anti-Counterfeiting Applications

• market size for counterfeit goods (2004): 500 Bill. US$
for art pieces: >10 Bill. US$ (Europe)

• nanoscale structures are
difficult to counterfeit,
and are mass-producible

• self-assembly structures are random and unique

*patent
pending

• security features can be mass produced at low cost, both for mass id and unique fingerprints



Topography Gradients for Surface Interaction Screening

PMMA / P2VP demixing on a pre-prepared surface chemistry gradient

• surface coatings with controlled properties,
varying over short length scales

• combinatorial studies of cell-substrate interactions: effect of surface
roughness on cell adhesion and proliferation, with gradients adapted
to typical distances travelled by cells study of cell locomotion

Blondiaux et al., submitted


Nanostructuring

Polymeric Self-Assembly II : Microphase Separation

• block copolymer A-b-B

-A-A-A- -B-B-B-
• Microphase Separation
• inexpensive & flexible method to generate
10 -100 nm
ordered structures on the molecular scale
• wide choice of functions and chemistries:
mechanical, chemical / catalytic,
optical, electrical, magnetic, …

high high
A-fraction B-fraction

Krishnamoorthy et al., materials today (September 2006)


Nanostructuring – Copolymer Microphase Separation

(Random) Nanostructures with Order and Function

Function: Order:
• PI-b-PFS poly(isoprene-b-ferrocenylsilane) • random and short range
• spincoating of 30nm thin film, plasma etch • can be improved by templating
• high density magnetic pattern: 4 1011 /cm2 • topographical, chemical, temp, fields, …
H
CH3 Fe
Si

CH3
n-Bu m n

PI-b-PFS

different FexOy stochiometries PS-PFS
from Korczagin, Vancso et al., Mesa+ from Stoykovich et al., Science (2005)



Copolymer Lithography for Nano-Pillars and Nano-Pores

etch mask from
inverted micelles etch mask
copolymer patterns
from polymer
constituents with
different etch rates

in some cases it is
necessary to provide
an “amplification” of RIE
the etch contrast

Krishnamoorthy et al., Nanotechnology (2008)



Non-Wetting Surfaces with Nanopillar Structures

• self-cleaning surfaces by functionalization
with perfluorosilane (wet or PVD)
transition from Wenzel to Cassie-Baxter wetting mode
for structure aspect ratio > 2:1
planar SiNx silanised
with perfluorosilane:
contact angle 111°

Nanopillars in SiNx, 90nm high, 100nm periodicity
silanised with perfluorosilane:
water contact angle 150˚, highly mobile drop
WCA adv 160° (compare to 110° on a flat surface)
Krishnamoorthy et al., Nanotechnology (2008) hysteresis 5°, rolling angle 6°, 10ml droplet


Nanostructuring – Nanoporous Membranes

Osmotic Biosensor based on Nanoporous Membranes

• nanoporous membranes from
copolymer lithography
• macro prototyping of osmotic sensor
• size selectivity supported specific
binding chemistry


Nanostructuring – Nanoporous Membranes

Wafer Scale Replication of Copolymer Lithography Patterns

• replication by polymer casting • replication by embossing into PC foil
master by Ni electroplating

wafer scale PDMS casting

• PMMA nanoporous membranes

small medium large

• nanostructured surfaces for cell studies
influence on:
• cell growth
• protein expression
• cytoskeleton organ.



BioMEMS for Nanotoxicity Tests

• experience in cell handling
dedicated infrastructure
• established knowledge in microfabrication
and replication technologies, in house fab
• nanotechnology / nanoparticle handling
• microfluidics design and prototyping

• partnerships & projects: InLiveTox

• industrial collaborations:


BioMEMS

Nanotoxicology – Risks of Nanoparticle Technology

• molecular nanotechnology “hype” • new class of nano-materials with “unknown”
• “grey goo” & “green goo” properties: carbon (CNT, buckyballs, …),
TiO2, SiO2, metallic (Au…), quantum dots
(CdS, CdSe, CdTe, etc.), polymeric…
gold

latex

Catalytic CO Oxidation by a Gold
CNTs Nanoparticle, N. Lopez and J.K.
Norskov, J.Am.Chem.Soc.(2002)

• … in widespread applications: catalysts,
sunscreens, fuel cells, solar panels, …


BioMEMS - Nanotoxicology

Translocation Measurement Device – EU IP Nanosafe2

• problem: unknown effects of nanoparticles on human organisms
• microfabricated chip for the in vitro study of model epithelia transport properties

nanoparticle suspension
coming in

confluent layer of
epithelial cells

porous Si3N4
electrodes for TEER membrane
measurements detection of nanoparticles
that cross the cell layer

detection of inorganic nanoparticles off-line using inductively
coupled plasma mass spectrometry (ICP-MS)



On-Chip Electrical Characterization of Cell Layers

• microfabricated chip with cell culture wells
• porous membranes at the basis of each well
to allow toxins or drugs to pass through
• TransEpithelial Electrical Resistance (TEER)
to determine the tightness of a cell layer
Calu-3 cells grown in one of five wells

• electrical contacts
• plastic holder
• glass support, to seal the
fluidic network
• PDMS fluidics
• SiN membrane
• PDMS in plastic holder,
electrical contacts at the bottom



Intestinal, Liver & Endothelial NP Toxicity – InLiveTox

• CSEM, 4 university partners, Helmholtz Zentrum Berlin, Kirkstall Ltd, Alma
• objectives:
• develop in vitro test system to reduce/replace animal tests of nanoparticle toxicity
• replace the “lab rat”
‘Gastro Intestinal tract’ ‘Intestinal epithelium’
by a setup of (co-culture of epithelial cells,
‘Bloodstream’
• microfluidics and monocytes and dendritic cells)

• cell cultures ‘Vascular endothelium’
of model organs (endothelial cells)
‘Liver’ (hepatocytes)

Nanoparticles Sampling ports

• 3Rs: Replace, Reduce and Refine animal tests
• REACH: Registration, Evaluation, Authorisation & Restriction of Chemical Substances


Nanotools

Probe Array Technology – PROBART

• speed up single probe operation
by parallel
imaging and
sensing

• PROBART for Life Science applications, for
nisenet.org - bioarrays
- cells

but: operation in liquids!


Nanotools – Probe Arrays

Force Spectroscopy on Cells

• information about adhesion proteins,
cell mechanics, kinetics, …
• cell-surface, cell-cantilever, cell-cell
• meaningful only with sufficient
statistics, which makes experiments
rather tedious
• at current rate of a few cells per day,
not useful for screening formats

• array format would improve statistics
and make high throughput screening
formats more accessible
source: JPK



PROBART for Parallel Imaging

VEE (- 6V)
Rlever
Rref (~ 20 kohm)

R ref Vout
R1 R2
R lever

probe
#6 4x4 array imaging in
buffer solution with
probe continuous zoom-in
#13

probe
#15



Cell Adhesion Forces

similar adhesion forces for cells in all
phases of the cell cycle (thus no need
for synchronization in future studies)

Human osteoblasts, growing on
hemispherical pits (a, diameter 27 µm) and
nanopillars (b, 45nm high, replicated in a non-
metallic bone implant material



Nanotools for Ultimate Pipetting

• vast experience in Scanning Probe Methods
• MEMS design and fabrication in house
• fluidics design and fabrication
• surface chemistry and characterization
• experience in handling biomaterials,
nanoparticles in solutions

• partnerships & projects:

• industrial collaborations: first contacts with instrument makers


Nanotools – Nanoscale Dispensing

Nanoscale Dispensing – NADIS

deposition of liquids
in ultrasmall volumes
from microscopic tips

• functional biomolecules for microarrays, such as
Molecules in solution proteins or DNA

• metallic nanoparticles to form connects, catalyst
Nanoparticle suspensions
particles, optical and chemical functions, …

• etch resist materials, sol-gel precursors, …

Materials for processing



NADIS with FIB Modified Probes

• apertures with Ø down to 200 nm
• flexibility in location (off-center, …)
• possible to keep sharp AFM tips

1 µm

sub-attoliter
volumes
Meister et al., App.Phys.Lett. (2004)



NADIS of Fluorophores in Liquid Environments

3 µm
1

Intensity [a.u.]
0.5

0
0 2 4 6 8

applied pressure ~ 2mbar



NADIS for Liquid Exchange with Living Cells

• injection after perforation
of the cell membrane
• extraction of cytoplasm for
remote analysis
• towards patch clamping

viable neuroblastoma cells
Cell TrackerTM green staining


Conclusions

Nanostructuring

• polymer demixing
for random but regular microstructures

• co-polymer microphase separation
for well-arranged functional nanostructures and lithography

THANK YOU !
• collaborators from CSEM: AM Popa, M Klein, W Li, F Montage, R Pugin, …
• cleanroom team from COMLAB and CMI EPF Lausanne
• partners from U Mulhouse, U Twente, EPFL, …


Conclusions

Nanotools

• probe array platform
for parallel force spectroscopy in biological environments

• nanoscale dispensing (NADIS)
for liquid arraying and cell manipulation

THANK YOU !
• collaborators from CSEM: J Przybylska, M Favre, J Polesel, A Meister, M Liley, …
• cleanroom team from COMLAB and CMI EPF Lausanne
• partners from IMT U Neuchâtel, U Lund, U Trento, ETHZ, EPFL, …


Nanotechnology Tools for Life Sciences

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