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Nanofair Gesim
1. µ-Contact Printing
Method to create µm and nm Patterns on Cell Based Bio-Chips
Steffen Howitz, Frank Baudisch, Felix Franz, GeSiM mbH Großerkmannsdorf
Michael Gepp and Heiko Zimmermann, Fraunhofer IBMT St.Ingbert
Stefan Fiedler and Michael Zwanzig, Fraunhofer IZM-Berlin
www.gesim.de
2. Content
• motivation
• comparison of print technologies
• new PDMS based stamp concept
• µCP2.1 for printing and imprinting
• first results
• conclusion
www.gesim.de
3. 1. motivation
• rough overview of surface functionalization methods
• research platform µCP2.1 for nano surface
programming and imprinting
• explain new PDMS-stamp concept
• first results
www.gesim.de
4. 2. print technologies for surface functionalization
how we can bring surface relevant factors to biochips?
soluble
differentiation using ++ signal
factors
immobilised
Cell Programming by
Nanoscaled Devices
www.gesim.de
5. 2. comparison of print technologies
Feature Piezo-Printing Pin-Tool Printer µ-Contact-Printing
NP2.1 NP2.1 µCP2.1
Foot Print of a 60...150µm 50…120µm < 50nm
single spot
Array Density ~ 5x5 spots ~ 5x5 spots ~ 5000 x 5000 spots
per mm²
Surface Contact non contact contact contact
Print Mode serial parallel parallel
Sample Access programmable Well-Plate correlated one sample
per Ink Step
programmable Pin-Tool head Stamp correlated
correlated
Array Desing
www.gesim.de
6. 2. piezo-non-contact-printing at NP2.1
Ref.: IBMT- R. Strelow
System Features 1. Piezo-Pipette non-contact printing of DNA and Proteins
2. Integrated Imaging System
3. Windows Software NPC16
4. Customized Work-Plate
5. Coolable Plate-Holder and Slidetray
6. Humidifyer , 40....80% rel. Humidity
www.gesim.de
7. 2. pin-tool-contact-printing at NP2.1
System Features
• Silicon-Pin Tools from Parallel Synthesis Technologies
• Integrated Imaging System
• Windows Software NPC16
• Customized Work-Plate
• Coolable Plate-Holder and Slidetray
• Humidifyer , 40....80% rel. Humidity
www.gesim.de
8. 2. µ-contact-printing at µCP2.1
c
b
a
• (a) inking station with 4 ink pads
• (b) drying nozzles, two per stamp
• (c) stamping unit
µCP- print process
• inking for 30…60sec
• drying at 1bar/N2 and 60sec
• stamping at 0,2bar and 60sec
1 print cycle 3 minutes
www.gesim.de
9. 3. new stamp design for system µCP2.1
compressed air
Stamp Chamber Stampholder Stamp Chamber
Stampframe
PDMS-Membran
nm/µm Patterns
a) Basic Mode: PDMS-membran planar b) Print-Mode: PDMS-membran deflected
SEM picture Si-Master Master in Casting Staion Casting of PDMS-Stamp PDMS-Stamp µCP-Stamping Unit
www.gesim.de
11. 3. stamp gets in contact to a glass slide
www.gesim.de
12. 4. µ-contact-printing at µCP2.1
Stamping Unit
Slide Tray
Microscope
www.gesim.de
13. 4. 3d-imprinting at µCP2.1
µCP2.1 with UV-light source
www.gesim.de
14. 5. first results of surface functionalization
soluble
differentiation using ++ signal
factors
immobilised
Cell Programming by
Nanoscaled Devices
www.gesim.de
15. 5. µ-contact-printing with µm-scaled stamps
Fig.1: first µ-Contact-Print of CY3- Fig.2: second print without new inking Fig.3: third print without new inking
labeled fibronectin on hydrophilic glass
slide, stamp area 1x1cm²
method:
a) inking of stamp 1min,
b) drying of stamps with 25x25µm²
compressed air 2 bar/30sec, Quadrate
c) contact print applying 0,25bar
stamp pressure for 60sec.
2µm Stege
www.gesim.de
16. 5. µ-contact-printing with a defined parallel shift
Pattern: A=100
Ø A
A
Ø
2xA
A
A
2xA
Fig.1: µ-contact-print of CY3-labeled fibronectin Fig.2: µ-contact-print like figure 1
on glass slide, stamp area 1x1cm² with larger patterns, stamp area 1x1cm²
Method:
• first print after inking the stamp
• second print realised with a parallel shift and without new inking
www.gesim.de
17. 5. Cell Adhesion Assay with L929 Mouse Fibroblasts
ECM Stamping on hydrophilic Glass Slides
Patter : A=100
n
ØA
ØA
2x A
A
A
2xA
1) 1..5 h µCP-pattern related cell adhesion visible,
2) 5…20 h a general cell adhesion occurs, patterns are then invisble
Next Experiments: ECM µ-ContactPrints on cell repulsive surfaces like Teflon!
www.gesim.de
18. 5. Cell surface interaction with nano-structured surfaces
L929 fibroblast on silicon substrate
50 – 100 nm wide fibronectin lines were printed
Images courtesy AMO and FhG-IBMT
Cell Programming by
Nanoscaled Devices
www.gesim.de
19. 5. µ-contact-printing to transfer nano-particles
25x25µm²
Quadrat e
2µm Stege
3µm 8µm
µ-contact-printing of spherical gold nano-particles (Ø=37nm, diluted in water) on
a hydrophilic glass slide
www.gesim.de
20. 5. imprinting of spin-coated photo restist films
50µm 50µm
Fig.1: Imprinted photo resist on glass Fig.2: Imprinted photo resist on glass slides,
slides, thickness 8µm thickness 500nm
IMPRINTING – A fast way to fabricate simple fluidics on glass slides!
www.gesim.de
21. 5. MagnaLab – Non-Contact and magnetic handling of glass
based cell-carriers in micro fluidic channels
Cell Programming by
Nanoscaled Devices
www.gesim.de
22. 5. Demands for Cell-Carrier,functional layers
biology
cell repulsive
NanoScapeTM
glass basis
technology
magnetic
shielding
sliding
Cell Programming by
Nanoscaled Devices
www.gesim.de
23. 5. cell carrier with NanoScapes
Cell-carrier chip processed
completely
Cell Programming by
Nanoscaled Devices
www.gesim.de
24. 6. conclusion
• µCP technology close the gap from µm to nm
for surface programming for printing and imprinting
• status quo of µCP2.1 - semi automatically research tool
• future oportunities enlarge print area and increase automation
• µCP reasonable tool for 3D-surface patterning
• potental application
basic material research
bio-sensor technology
cell-bacteria-virus research
substrates for cell storing e.g. in cryo-biotechnology
disposable micro-fluidic systems
combination with MEA-technolgy e.g. for neuronal networks combined
with fluidics …..
www.gesim.de