Nanotechnology Tools for Life Sciences

Harry Heinzelmann

VP Nanotechnology & Life Sciences

Nanotechnology Tools for Life Sciences

Neuchâtel, June 2009

v1.19

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

CSEM profile

Privately held Innovation Center, incorporated, not for profit

since 1984, from watchmaking

about 70 shareholders (mostly private)

Privately held Innovation Center, incorporated, not for profit

since 1984, from watchmaking

about 70 shareholders (mostly private)

2008:

>65 Mio. CHF annual turnover, 395 employees

30 start-ups created since 2000

2008:

>65 Mio. CHF annual turnover, 395 employees

30 start-ups created since 2000

Activities:

Applied research (contract with Swiss Government)

Industrialization of technologies, product development

Activities:

Applied research (contract with Swiss Government)

Industrialization of technologies, product development

Technologies:

Micro- and Nanotechnology, Information Technology,

and System Engineering

Technologies:

Micro- and Nanotechnology, Information Technology,

and System Engineering


Bridge from Science to Innovation

CSEM profile

• research partners:

Applied

Res & Dev

Science &

Education

Basic Research

PhD programs

Teaching

Product

Development

Market

Success

Marketing

Sales

Customers

Industrialisation

• technologies

for innovations


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

CSEM profile

• technologies for Green Solutions

• shareholders include


Technologies I (divisions in Neuchâtel)

CSEM profile

Microelectronics

Circuit Design, RF, Information Processing

Nanotechnology & Life Sciences

Optical and Bio MNT, Self-assembly, Sensors

Systems Engineering

Mechatronics, Signal Processing, Communication

Microsystems

MEMS, Cleanroom Infrastucture, Microscopy & Analysis

Time and Frequency

Atomic Clocks, Optical Advanced Systems


Technologies II (divisions outside Neuchâtel)

CSEM profile

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


• 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

Technologies and Applications


Top-Down vs. Bottom-Up

Nanostructuring

classical (micro-) fabrication

MEMS: Micro Electro Mechanical Systems

lithography:

VIS

UV, X-ray, e – beam

FIB (serial)

10

1 mm

1 µm

10

100

100

10

1 nm

1 Å

molecular self-assembly

“molecular nanotechnology” 4 µµµµm250 nm


Polymeric Self-Assembly I : Polymer Demixing

80 µm

50% PMMA / 50% PS 90% PMMA / 10% PS

5 µm

• demixing of immiscible polymer blends

• qualitative structures on the micron scale

• control over feature size and properties

• large variety of polymers available

Nanostructuring

• simple deposition technique

• selective solvent can remove one polymer type

• scalable to

large surfaces

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

large 5µm med 2µm small <1µm


Nanoporous Layers for Ink-jet Printer Paper

Nanostructuring – Polymer Demixing

Polymer paper

stable images

slow ink uptake, big spot size

Polymer layer

Cellulose

Nanoporous layer

(Alumina film)

Cellulose

Nanoporous paper

fast up-take, small spot size

image fading (light, gas,…)

paper, 5 µm x 5 µm

polymer film

transferred

on paper

polymer on Si


*patentpending

• nanoscale structures are

difficult to counterfeit,

and are mass-producible

Security Features for Anti-Counterfeiting Applications


• market size for counterfeit goods (2004): 500 Bill. US$

for art pieces: >10 Bill. US$ (Europe)

• self-assembly structures are random and unique

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


�

Topography Gradients for Surface Interaction Screening

Blondiaux et al., submitted

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


�

Polymeric Self-Assembly II : Microphase Separation

Nanostructuring

Krishnamoorthy et al., materials today (September 2006)

• Microphase Separation

• inexpensive & flexible method to generate

ordered structures on the molecular scale

• wide choice of functions and chemistries:

mechanical, chemical / catalytic,

optical, electrical, magnetic, …

10 -100 nm

-A-A-A- -B-B-B-

high

A-fraction

high

B-fraction

• block copolymer A-b-B


(Random) Nanostructures with Order and Function

Function:

• PI-b-PFS poly(isoprene-b-ferrocenylsilane)

• spincoating of 30nm thin film, plasma etch

• high density magnetic pattern: 4 1011 /cm2

Order:

• random and short range

• can be improved by templating

• topographical, chemical, temp, fields, …

Nanostructuring – Copolymer Microphase Separation

Fe

Si

CH3

CH3

H

n-Bu nm

PI-b-PFS

different FexOy stochiometries

from Korczagin, Vancso et al., Mesa+

PS-PFS

from Stoykovich et al., Science (2005)


�

Copolymer Lithography for Nano-Pillars and Nano-Pores


Krishnamoorthy et al., Nanotechnology (2008)

etch mask from

copolymer patterns

from polymer

constituents with

different etch rates

in some cases it is

necessary to provide

an “amplification” of

the etch contrast

inverted micelles etch mask

RIE


�

Non-Wetting Surfaces with Nanopillar Structures


Krishnamoorthy et al., Nanotechnology (2008)

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

• self-cleaning surfaces by functionalization

with perfluorosilane (wet or PVD)

transition from Wenzel to Cassie-Baxter wetting mode

for structure aspect ratio > 2:1

WCA adv 160° (compare to 110° on a flat surface)

hysteresis 5°, rolling angle 6°, 10ml droplet


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

Nanostructuring – Nanoporous Membranes

• replication by polymer casting

wafer scale PDMS casting

influence on:

• cell growth

• protein expression

• cytoskeleton organ.

• nanostructured surfaces for cell studies

• PMMA nanoporous membranes

• replication by embossing into PC foil

master by Ni electroplating

small medium large


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:



Nanotoxicology – Risks of Nanoparticle Technology

• molecular nanotechnology “hype”

• “grey goo” & “green goo”

• new class of nano-materials with “unknown”

properties: carbon (CNT, buckyballs, …),

TiO2, SiO2, metallic (Au…), quantum dots

(CdS, CdSe, CdTe, etc.), polymeric…

BioMEMS

gold

latex

CNTs Catalytic CO Oxidation by a Gold

Nanoparticle, N. Lopez and J.K.

Norskov, J.Am.Chem.Soc.(2002)

• … in widespread applications: catalysts,

sunscreens, fuel cells, solar panels, …


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

BioMEMS - Nanotoxicology

detection of nanoparticles

that cross the cell layer

porous Si3N4

membrane

confluent layer of

epithelial cells

electrodes for TEER

measurements

nanoparticle suspension

coming in

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


• electrical contacts

• plastic holder

• glass support, to seal the

fluidic network

• PDMS fluidics

• SiN membrane

• PDMS in plastic holder,

electrical contacts at the bottom

Calu-3 cells grown in one of five wells


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”

by a setup of

• microfluidics and

• cell cultures

of model organs

• 3Rs: Replace, Reduce and Refine animal tests

• REACH: Registration, Evaluation, Authorisation & Restriction of Chemical Substances

‘Bloodstream’

Nanoparticles Sampling ports

‘Gastro Intestinal tract’ ‘Intestinal epithelium’

(co-culture of epithelial cells,

monocytes and dendritic cells)

‘Vascular endothelium’

(endothelial cells)

‘Liver’ (hepatocytes)


Probe Array Technology – PROBART

• speed up single probe operation

by parallel

imaging and

sensing

Nanotools

nisenet.org

• PROBART for Life Science applications, for

- bioarrays

- cells

but: operation in liquids!


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

Nanotools – Probe Arrays

source: JPK


PROBART for Parallel Imaging


R lever

R ref

VEE (- 6V)Rlever

Rref

R1 R2

Vout

(~ 20 kohm)

4x4 array imaging in

buffer solution with

continuous zoom-in

probe

#6

probe

#13

probe

#15


Cell Adhesion Forces


Human osteoblasts, growing on

hemispherical pits (a, diameter 27 µm) and

nanopillars (b, 45nm high, replicated in a non-

metallic bone implant material

similar adhesion forces for cells in all

phases of the cell cycle (thus no need

for synchronization in future studies)


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



Nanoscale Dispensing – NADIS

Nanotools – Nanoscale Dispensing

Nanoparticle suspensions

Materials for processing

Molecules in solution

• functional biomolecules for microarrays, such as

proteins or DNA

• metallic nanoparticles to form connects, catalyst

particles, optical and chemical functions, …

• etch resist materials, sol-gel precursors, …

deposition of liquidsin ultrasmall volumesfrom microscopic tips


�

NADIS with FIB Modified Probes


Meister et al., App.Phys.Lett. (2004)

1 µm

sub-attoliter volumes

• apertures with Ø down to 200 nm

• flexibility in location (off-center, …)

• possible to keep sharp AFM tips



0

0.5

1

0 2 4 6 8

Inte

nsit

y [a

.u.]

3 µm

applied pressure ~ 2mbar

NADIS of Fluorophores in Liquid Environments


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


Nanostructuring

Conclusions

• 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, …


Nanotools

Conclusions

• 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, …

Thank you for your attention.

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