PPT Final 1

IMTEK powerpoint template 2008:
Version 2 of the first slide
Biochip-Technologies
T. Brandstetter
Content
• Materials and surface modifications (09.05.14)
• Manufacturing of Biochips (23.05.14)
• Biochip technologies – Between research and routine diagnostics
(state of the art, 06.06.14)
• Nucleic acid based techniques (27.06.14)
• Biochips for protein analytics (04.07.14)
• Other applications (11.07.14)
• Summary (18.07.14)
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 2
Our profile
Research and teaching
•
•
22 faculties
•
highly interdisciplinary world of
microsystem technology
300 researchers and technicians
IMTEK and industry
•
•
Many industrial cooperations
MSTBw
Core competences of CPI
•
Preparation of surfaces with tailor-made
properties
•
Topological and chemical micro
structuring of surfaces
•
•
AFM
Biochip-technologies
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 3
Biochip-technologies
http://portal.uni-freiburg.de/cpi/biochip-group-dr-brandstetter
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 4
Biochips – what are they?(1)
• devices that can contain anywhere from tens to tens of millions of individual
sensor elements (or biosensors)
• The sensors are packed together into a package typically the size of a
microscope slide. Because so many sensors can be put into such a small
area, a huge number of distinct tests can be done very rapidly.
• Biochips are often made using the same microfabrication technology used
to make microchips. Unlike microchips, however, biochips are generally not
electronic (although they can be).
• The key premise behind biochips is, that they can do chemistry on a small
scale. Each biosensor can be thought of as a "microreactor“, which does
chemistry designed to sense a specific analyte.
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 5
Biochips – what are they?(2)
• Biosensors can be made to sense a wide variety of analytes, including DNA,
protein, antibodies, and small biological molecules.
• Fluorescence is often used to indicate a sensing event. Automated
microscopy systems can be used to "read" the chip, i.e. determine which
sensors are fluorescing
• Most biochips are 2D arrays of sensors placed carefully in a grid
arrangement. The position of the sensor on the chip determines its function.
• To place the sensors in precise coordinates, sophisticated and expensive
microdeposition techniques are used. The sensors are essentially placed one
at a time, or serially, on the chip.
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 6
Biochips – what are they?(3)
HPV_3D_Katrin_N_30s_Cy5
 substrat
 dot
HPV 6
3
8
13 microarray
www.imtek.de/cpi
http://en.wikipedia.org/wiki/Biochip#History
T. Brandstetter/ 09.05.2014 / slide 7
Manufacturing of biochips – in general(1)
1. Untreated slide
mixed analyte solution
2. Microarray printing
3. Immobilisation
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T. Brandstetter/ 09.05.2014 / slide 8
Manufacturing of biochips – in general(2)
step1:
print polymer mixed with DNA
step 3:
hybridisation
and
readout
step 2:
photocrosslinking
via UV-irradiation
C OH
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T. Brandstetter/ 09.05.2014 / slide 9
Materials and surface modifications
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T. Brandstetter/ 09.05.2014 / slide 10
Biochip materials (1)
Microscope slide of glass
Commercial microscope glass slides
• Silica (SiO2)
+
vitreous silica
• Sodium carbonate (Na2CO3)
+
soda-lime-silicate glass
• Limestone (CaCO3)
+
borosilicate glass-pyrex
• Magnesium Carbonate (MgCO3)
+
aluminosilicate glass
+
borosilicate glass
Detailled information
Frontiers in biochip technology
by Wan-Li Xing, Jing Cheng
Edition: illustrated
Published by Birkhäuser, 2006
ISBN 0387255680, 9780387255682
357 pages
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 11
Biochip materials (2)
Microscope slide of plastic
Commercial plastic slides
• PMMA (polymethymethacrylate)
+
PMMA
• Polystyrene
+
Polystyrene
• COC (cyclic olefin copolymer)
+
TOPAS
• Polycarbonate
+
Polycarbonate
• Polypropyrene
+
Polypropyrene
Lab Chip, 2007, 7, 856 - 862, DOI: 10.1039/b700322f
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 12
Biochip coatings
directly
chemically modified surfaces
• In situ synthesis on glass
+
activated glass by poly-carbodiimide,
aminosilane, aldehyde
• Silanizated probes on unmodified glass
+
graft coating polymers on silicon (glass)
• Photocrosslinking on unmodified plastic
+
plastic-based DNA microarrays using
carbodiimide chemistry
+
amine-modified PMMA substrates
+
activated polystyrene, polypropyrene,
polycarbonate (PC)
• S.A. Fodor, R. Rava, X.C. Huang, A.C. Pease, C.P. Holmes and C.L. Adams. Science 251 (1991) 767–773.
• M.J. Moorcroft, W.R. Meuleman, S.G. Latham, T.J. Nicholls, R.D. Egeland and E.M. Southern. NAR, 2005, Vol. 33, e75.
• N. Kimura, R. Oda, Y. Inaki and O. Suzuki. Nucleic Acids Research, 2004, Vol. 32, e68.
• H.-Y. Wang,R.L. Malek,A.E. Kwitek,A.S. Greene,T.V. Luu,B. Behbahani,B. Frank,J. Quackenbush, N.H. Lee, Genome Biol. 4 (2003), R5.
• M. Dufva, S. Petronis, L.B. Jensen, C. Krag and C.B. Christensen. Biotechniques 37 (2004) 286–292, 294, 296.
• A. Kumar, O. Larsson, D. Parodi, Z. Liang, Nucleic Acids Research, 2000, Vol. 28, e98.
• M. Schena, D. Shalon, R.W. Davis, P.O. Brown, Science 270 (1995), 467–470.
• De Paul S. M., Falconnet D., Pasche S., Textor M., Abel A. P., Kauffmann E., Liedtke R. and Ehrat M.. Anal. Chem. 2005, 77, 5831-5838.
• Johnson P. A., Gaspar M. A. and Levicky R. J. Am. Chem. Soc., 2004, 126, 9910-9911.
• N. Kimura, T. Nagasaka, J. Murakami, H. Sasamoto, M. Murakami, N. Tanaka and N. Matsubara. Nucleic Acids Research, 2005, Vol. 33, e46.
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 13
2D chips using SAMs (self assembled monolayers)
typical DNA-chip design:
sequence of the probe
polyT(thymine) tailer
adapted from: E. Southern, K. Mir, M. Shchepinov, Nature Gen., 27 (1999) 5
Weakness:
+ reproducibility (why is acceptance of microarrays below expectations in non-research areas?
+ sensitivity
+ surface properties
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T. Brandstetter/ 09.05.2014 / slide 14
A „skyscraper“-approach
2D
attachment of
oligonucleotide probes
3D
polymer brushes
“polymer layer” – approach allows to
 improve the sensitivity
 adjust properties of the surface
(hydrophilicity, reactivity)
3D
polymer networks
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T. Brandstetter/ 09.05.2014 / slide 15
Functional polymer monolayers
growth of polymers
on surfaces
chemisorption of polymers
blockcopolymers
via macroinitators
grafting of polymers on
plasma modified
surfaces
photochemical attachment
of polymers
www.imtek.de/cpi
surface-attached
polymer networks
T. Brandstetter/ 09.05.2014 / slide 16
„grafting in between“
Photochemistry of benzophenone

triplet formation upon n,* excitation

biradical reacts with C,H bonds
H C
C
350 nm
nm
265
C O
C O
C OH
 = 100 µ s
hydrogen
abstraction
C
www.imtek.de/cpi
C
OH
recombination
T. Brandstetter/ 09.05.2014 / slide 17
Toomey R., Freidank D. and
Rühe J.. Swelling Behavior of
Thin, Surface-Attached
Polymer Networks.
Macromolecules, Vol. 37, 2004,
882-887.
Polymer networks attached to polymeric substrates
photocrosslinkable
overcoat
Me
simultaneous crosslinking Me
N O
and surface attachment
Me
via pendant benzophenone
units
O
O
polymeric substrate
(e.g. polyurethane)
O
swelling in
water (2h)
~ 1 mm
ca. 20 µm
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T. Brandstetter/ 09.05.2014 / slide 18
Microstructuring in biochip technologies, two procedures
I.
Contact printing
Print pins
http://www.anopoli.com/
http://www.anopoli.com/
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T. Brandstetter/ 09.05.2014 / slide 19
Printhead
Microstructuring in biochip technologies, contact printing

www.imtek.de/cpi
Omnigrid from GeneMachine®

Contact printing procedure

65% humidity, RT

Steel or tungsten needle with reservoir

droplet volume 400 – 600 pl

droplet diameter 140 – 200 µm

Process variance > 10%
T. Brandstetter/ 09.05.2014 / slide 20
Microstructuring in biochip technologies, contact printing
Pin heads make the difference.
Split pin
•Uptake volumes : 0.25µl to 0.64 µl
Solid pin
•Spot diameters : 75µm to 450 µm
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 21
http://www.anopoli.com/
•Spot diameters : 75µm to 215 µm
Microstructuring in biochip technologies, contact printing
Printing with different, not aqueous, solutions is possible.
PDMAA(Polydimethylmetacrylate)

200 µm

printing medium: ethanol
www.imtek.de/cpi
PS (Polystyrene)
printing medium: toluene
T. Brandstetter/ 09.05.2014 / slide 22
Microstructuring in biochip technologies, contact printing
Spot diameter is not really controllable.
Split pin
Solid pin
Printing of 0.25 µm Cy5-labelled oligo-DNA in 400
mM Napi and 1mg/ml PDMAA-co-5%MABP-co2,5%VPA
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T. Brandstetter/ 09.05.2014 / slide 23
Microstructuring in biochip technologies, contact printing

scale lining

PDMAA layer

PMMA (5 mg/ml) lining

Printing medium toluene

exposure after
photocrosslinkage
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T. Brandstetter/ 09.05.2014 / slide 24
Microstructuring in biochip technologies, contact printing
1.
copolymers
2. buffer
PDMAA-co-5%MABP-co-2,5%VPA
(a) 400 mM Napi
(b) 200 mM Napi/3xSSC/0.75 M betaine
plastic/PMMA
a.
3. PT-6000 tungsten
glass/Epoxy
b.
a.
b.
2D
2D_04_04_07_P2Ds.1a
2D_16_04_07_P2Dsp.2a
a.
3D
b.
a.
b.
3D
3D_12_04_07_P3Dsp.11a
www.imtek.de/cpi
2D_04_04_07_N2Ds.4a
2D_16_04_07_N2Dsp.4b
3D_03_04_07_P3Ds.11
3D_12_04_07_N3Dsp.2a
T. Brandstetter/ 09.05.2014 / slide 25
3D_03_04_07_N3Ds.4a
Microstructuring in biochip technologies, contact printing
1. copolymers
2. buffer
PDMAA-co-5%MABP-co-2,5%VPA
(b) 200 mM Napi/3xSSC/0,75 M betaine
glass/Epoxy
b.
a.
b.
2D
2D
2D_xx_04_07_P2Ds.x
2D_16_04_07_P2Dsp.2a
a.
3D
PT-6000 tungsten
(a) 400 mM Napi
plastic/PMMA
a.
3.
b.
2D_xx_04_07_N2Ds.x
2D_16_04_07_N2Dsp.4b
a.
b.
3D
3D_12_04_07_P3Dsp.11a
www.imtek.de/cpi
3D_xx_04_07_P3Ds.x
3D_12_04_07_N3Dsp.2a
T. Brandstetter/ 09.05.2014 / slide 26
3D_xx_04_07_N3Ds.x
Microstructuring in biochip technologies, contactless printing
II.
Contactless printing/Piezo Electric Dispenser
http://www.scienion.de
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 27
Microstructuring in biochip technologies, contactless printing
II.
Piezo Electric dispenser
Piezo
www.imtek.de/cpi
Electric dispenser(Scienion AG®)

Contactless printing procedure

65% humidity, RT

droplet volume 410 pl,

droplet diameter 175 µm

droplet volume and diameter is
adjustable

Process variance < 10%
T. Brandstetter/ 09.05.2014 / slide 28
Microstructuring in biochip technologies, contactless printing
Photos after print
2D
3D
2D =
printing with PBS without polymer
3D =
printing with PBS 1 mg/ml PDMAA-co5%MABP-co-2,5%VPA
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 29
3D
Microstructuring in biochip technologies, contactless printing

Droplet stacking

1mg/ml polymer in distilled water

PSS = Polystyrenesulfanit

PMMA = Polymethylmetacrylate

Small droplet with 10x

Large droplets with 20x

Photo after print
PSS
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 30
PMMA
Microstructuring in biochip technologies, contactless printing

“donut”-structuring

1mg/ml PDMAA-co5%MABP-co-2,5%VPA in
PBS

Exposure after wash with
PBS and 0.1% (v/v) Tween)
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 31
Dot morphology, how to analyze?


Dot morphology, depending on

surface properties

print solution  contact angle

analyte concentration
Dot morphology, analyzed by

AFM

Fluorescence microscope

Raster electron microscope
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 32
Microstructuring in biochip technologies, contactless printing

Printing with additives, avoiding
“donut”-morphology

1mg/ml PDMAA-co-5%MABPco-2,5%VPA in PBS

Additive Glycerol

Photo after print
0
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 33
2.5
5
10
25%(v/v)
Microstructuring in biochip technologies, contactless printing

Printing with/withoutTrehalose

1mg/ml PDMAA-co-5%MABP
-co-2,5%VPA in PBS

125 mg/ml Trehalose (T) in PBS


+T
-T
“Donut”-structure without
Trehalose
+T
-T
Homogeneity in the dot
morphology, using Trehalose
http://en.wikipedia.org/wiki/Trehalose
α-Ｄ-glucopyranosyl α-Ｄglucopyranoside(α,α‐Trehalose)
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Exposure with a fluorescence microscope
T. Brandstetter/ 09.05.2014 / slide 34
Printing on microstructured surfaces

500 µm

www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 35
Microstructuring in biochip technologies
Micronas Biochip technlogy
Piezo
www.imtek.de/cpi
Electric dispenser(Scienion AG®)

Contactless printing procedure

80% humidity, RT

droplet volume 390 pl,

photodiode diameter 180 µm

printing on structured surfaces

Process variance < 10%
T. Brandstetter/ 09.05.2014 / slide 36
Microstructuring in biochip technologies
Micronas Biochip technlogy
Piezo

Electric dispenser(Scienion AG®)
printing directly on a photodiode
 180 µm 
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 37
Microstructuring in biochip technologies
Micronas Biochip technlogy
Piezo
Electric dispenser(Scienion AG®)
printing directly on a photodiode

pattern matching using a software
printed
not printed

www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 38
Microstructuring in biochip technologies, summary

Piezo Electric dispenser (Scienion AG®)
 Contactless printing procedure
 Droplet volume control
 Droplet diameter tunable (>100µm)
 Printing only with aqueous solutions
 1mg/ml polymer
 Process variance < 10%

Omnigrid from GeneMachine®
 Contact printing procedure
 Steel or tungsten needle with reservoir
 droplet volume 400 – 600 pl
 droplet diameter approx. 200 µm
 Printing of different solutions
 > 1mg/ml polymer possible
 Process variance > 10%
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 39
Thank you for your attention!
http://www.bilder-welten.net/de/produkt_detail.php?id=23019&catid=1623
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 40
Literature
•
•
•
•
•
•
•
•
•
•
•
•
•
•
E. Southern, K. Mir, M. Shchepinov, Nature Gen., 27 (1999) 5
Frontiers in biochip technology, by Wan-Li Xing, Jing Cheng, Edition: illustrated, published by
Birkhäuser, 2006, ISBN 0387255680, 9780387255682, 357 pages
Lab Chip, 2007, 7, 856 - 862, DOI: 10.1039/b700322f
S.A. Fodor, R. Rava, X.C. Huang, A.C. Pease, C.P. Holmes and C.L. Adams. Science 251
(1991) 767–773.
M.J. Moorcroft, W.R. Meuleman, S.G. Latham, T.J. Nicholls, R.D. Egeland and E.M. Southern.
NAR, 2005, Vol. 33, e75.
N. Kimura, R. Oda, Y. Inaki and O. Suzuki. Nucleic Acids Research, 2004, Vol. 32, e68.
H.-Y. Wang,R.L. Malek,A.E. Kwitek,A.S. Greene,T.V. Luu,B. Behbahani,B. Frank,J.
Quackenbush, N.H. Lee, Genome Biol. 4 (2003), R5.
M. Dufva, S. Petronis, L.B. Jensen, C. Krag and C.B. Christensen. Biotechniques 37 (2004)
286–292, 294, 296.
A. Kumar, O. Larsson, D. Parodi, Z. Liang, Nucleic Acids Research, 2000, Vol. 28, e98.
M. Schena, D. Shalon, R.W. Davis, P.O. Brown, Science 270 (1995), 467–470.
De Paul S. M., Falconnet D., Pasche S., Textor M., Abel A. P., Kauffmann E., Liedtke R. and
Ehrat M.. Anal. Chem. 2005, 77, 5831-5838.
Johnson P. A., Gaspar M. A. and Levicky R. J. Am. Chem. Soc., 2004, 126, 9910-9911.
N. Kimura, T. Nagasaka, J. Murakami, H. Sasamoto, M. Murakami, N. Tanaka and N.
Matsubara. Nucleic Acids Research, 2005, Vol. 33, e46.
Toomey R., Freidank D. and Rühe J.. Swelling Behavior of Thin, Surface-Attached Polymer
Networks. Macromolecules, Vol. 37, 2004, 882-887.
www.imtek.de/cpi
T. Brandstetter/ 09.05.2014 / slide 41

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