Post on 29-Jan-2017
IMTEK powerpoint template 2008:Version 2 of the first slide
Biochip TechnologiesBiochip-Technologies
T. Brandstetter
Content
• Materials and surface modifications (26.04.13)
• Manufacturing of Biochips (14.06.13)
• Biochip technologies – Between research and routine diagnostics (state of the art, 21.06.13)
• Nucleic acid based techniques (28.06.13)
• Biochips for protein analytics (05.06.13)
• Other applications (12.07.13)
S (19 07 13)
T. Brandstetter/ 26.04.2013 / slide 2www.imtek.de/cpi
• Summary (19.07.13)
Our profile
R h d t hiResearch and teaching
• 22 faculties
• 300 researchers and technicians• 300 researchers and technicians
• highly interdisciplinary world of microsystem technology
IMTEK and industry
• Many industrial cooperations• Many industrial cooperations
• MSTBw
Core competences of CPI
• Preparation of surfaces with tailor-made propertiesproperties
• Topological and chemical micro structuring of surfaces
T. Brandstetter/ 26.04.2013 / slide 3www.imtek.de/cpi
• AFM
• Biochip-technologies
Biochip‐technologieshttp://portal.uni-freiburg.de/cpi/biochip-group-dr-brandstetter
T. Brandstetter/ 26.04.2013 / slide 4www.imtek.de/cpi
Biochips – what are they?(1)
• devices that can contain anywhere from tens to tens of millions of individualsensor elements (or biosensors)( )
• The sensors are packed together into a package typically the size of ap g p g yp ymicroscope slide. Because so many sensors can be put into such a smallarea, a huge number of distinct tests can be done very rapidly.
• Biochips are often made using the same microfabrication technology usedto make microchips Unlike microchips however biochips are generally notto make microchips. Unlike microchips, however, biochips are generally notelectronic (although they can be).
• The key premise behind biochips is, that they can do chemistry on a smallscale. Each biosensor can be thought of as a "microreactor“, which does
T. Brandstetter/ 26.04.2013 / slide 5www.imtek.de/cpi
chemistry designed to sense a specific analyte.
Biochips – what are they?(2)
• Biosensors can be made to sense a wide variety of analytes, including DNA,protein, antibodies, and small biological molecules.p , , g
• Fluorescence is often used to indicate a sensing event. Automatedgmicroscopy systems can be used to "read" the chip, i.e. determine whichsensors are fluorescing
• Most biochips are 2D arrays of sensors placed carefully in a gridarrangement The position of the sensor on the chip determines its functionarrangement. The position of the sensor on the chip determines its function.
• To place the sensors in precise coordinates sophisticated and expensive• To place the sensors in precise coordinates, sophisticated and expensivemicrodeposition techniques are used. The sensors are essentially placed oneat a time, or serially, on the chip.
T. Brandstetter/ 26.04.2013 / slide 6www.imtek.de/cpi
Biochips – what are they?(3)
HPV_3D_Katrin_N_30s_Cy5 substrat
HPV 6
dot
38
HPV 6
813 microarray
http://en.wikipedia.org/wiki/Biochip#History
T. Brandstetter/ 26.04.2013 / slide 7www.imtek.de/cpi
Manufacturing of biochips – in general(1)
1. Untreated slidemixed analyte solution
2. Microarray printing
3 I bili ti3. Immobilisation
T. Brandstetter/ 26.04.2013 / slide 8www.imtek.de/cpi
Manufacturing of biochips – in general(2)
step1:print polymer mixed with DNA
step 3:hybridisation andreadoutreadout
step 2:step 2:photocrosslinkingvia UV-irradiation
T. Brandstetter/ 26.04.2013 / slide 9www.imtek.de/cpiC OH
Materials and surface modifications
T. Brandstetter/ 26.04.2013 / slide 10www.imtek.de/cpi
Biochip materials (1)
Microscope slide of glass Commercial microscope glass slides
• Silica (SiO2) + vitreous silica
• Sodium carbonate (Na2CO3) + soda-lime-silicate glass• Sodium carbonate (Na2CO3) + soda lime silicate glass
• Limestone (CaCO3) + borosilicate glass-pyrex
• Magnesium Carbonate (MgCO3) + aluminosilicate glass
+ borosilicate glass
Detailled informationFrontiers in biochip technologyby Wan-Li Xing, Jing ChengEdition: illustrated
T. Brandstetter/ 26.04.2013 / slide 11www.imtek.de/cpi
Edition: illustratedPublished by Birkhäuser, 2006ISBN 0387255680, 9780387255682357 pages
Biochip materials (2)
Microscope slide of plastic Commercial plastic slides
• PMMA (polymethymethacrylate) + PMMA
• Polystyrene + Polystyrene• Polystyrene + Polystyrene
• COC (cyclic olefin copolymer) + TOPAS
• Polycarbonate + Polycarbonate
• Polypropyrene + Polypropyrene
T. Brandstetter/ 26.04.2013 / slide 12www.imtek.de/cpi
Lab Chip, 2007, 7, 856 - 862, DOI: 10.1039/b700322f
Biochip coatings
directly chemically modified surfaces
I it th i l ti t d l b l b dii id• In situ synthesis on glass + activated glass by poly-carbodiimide, aminosilane, aldehyde
Sil i t d b difi d l ft ti l ili ( l )• Silanizated probes on unmodified glass + graft coating polymers on silicon (glass)
• Photocrosslinking on unmodified plastic + plastic-based DNA microarrays using b dii id h i tcarbodiimide 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.
T. Brandstetter/ 26.04.2013 / slide 13www.imtek.de/cpi
, , , g, , , ,• 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.
2D chips using SAMs (self assembled monolayers)
typical DNA-chip design:
sequence of the probepolyT(thymine) tailer
adapted from: E. Southern, K. Mir, M. Shchepinov, Nature Gen., 27 (1999) 5
+ reproducibility (why is acceptance of microarrays below expectations in non-research areas?
Weakness:
+ sensitivity
+ surface properties
T. Brandstetter/ 26.04.2013 / slide 14www.imtek.de/cpi
A „skyscraper“‐approach
2D
attachment of li l tid b
2D
oligonucleotide probes3D
polymer brushes
“polymer layer” – approach allows to improve the sensitivity adjust properties of the surface
(hydrophilicity, reactivity)3D
T. Brandstetter/ 26.04.2013 / slide 15www.imtek.de/cpi
polymer networks
Functional polymer monolayers
chemisorption of polymers growth of polymers chemisorption of polymerson surfaces
blockcopolymers
grafting of polymers on plasma modified
surfaces
p yvia macroinitators
T. Brandstetter/ 26.04.2013 / slide 16www.imtek.de/cpi
photochemical attachment of polymers
surface-attached polymer networks „grafting in between“
Photochemistry of benzophenone
triplet formation upon n,* excitation
biradical reacts with C,H bonds
C O C O C
CCH350 nm
O
H265 nm
hydrogenabstraction
= 100 µ s
CCOH
recombinationToomey R., Freidank D. and Rühe J.. Swelling Behavior of Thi S f Att h d
T. Brandstetter/ 26.04.2013 / slide 17www.imtek.de/cpi
Thin, Surface-Attached Polymer Networks. Macromolecules, Vol. 37, 2004,882-887.
Polymer networks attached to polymeric substrates
photocrosslinkableovercoat
Me
O OONMesimultaneous crosslinkingand surface attachment
O
Mepolymeric substrate
(e.g. polyurethane)
and surface attachmentvia pendant benzophenoneunits
O
swelling in water (2h)water (2h)
ca. 20 µm~ 1 mm
T. Brandstetter/ 26.04.2013 / slide 18www.imtek.de/cpi
µ
Microstructuring in biochip technologies, two procedures
I. Contact printing
Print pins PrintheadPrint pins Printhead
T. Brandstetter/ 26.04.2013 / slide 19www.imtek.de/cpi
http://www.anopoli.com/http://www.anopoli.com/
Microstructuring in biochip technologies, contact printing
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/ 26.04.2013 / slide 20www.imtek.de/cpi
Microstructuring in biochip technologies, contact printing
Pin heads make the difference.
Split pinp p•Spot diameters : 75µm to 215 µm
•Uptake volumes : 0.25µl to 0.64 µl
li.co
m/
http
://w
ww
.ano
pol
Solid pin
•Spot diameters : 75µm to 450 µm
T. Brandstetter/ 26.04.2013 / slide 21www.imtek.de/cpi
Microstructuring in biochip technologies, contact printing
Printing with different, not aqueous, solutions is possible.
PDMAA(Polydimethylmetacrylate) PS (Polystyrene)
200 µm µ
T. Brandstetter/ 26.04.2013 / slide 22www.imtek.de/cpi
printing medium: ethanol printing medium: toluene
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 400mM Napi and 1mg/ml PDMAA-co-5%MABP-co-2,5%VPA
T. Brandstetter/ 26.04.2013 / slide 23www.imtek.de/cpi
Microstructuring in biochip technologies, contact printing
l li i scale lining PDMAA layer
PMMA (5 mg/ml) lining( g ) g
Printing medium toluene
exposure after photocrosslinkagephotocrosslinkage
T. Brandstetter/ 26.04.2013 / slide 24www.imtek.de/cpi
Microstructuring in biochip technologies, contact printing
1. copolymers 2. buffer 3. PT-6000 tungstenPDMAA-co-5%MABP-co-2,5%VPA (a) 400 mM Napi
plastic/PMMA glass/Epoxy
PDMAA co 5%MABP co 2,5%VPA (a) 400 mM Napi(b) 200 mM Napi/3xSSC/0.75 M betaine
a a bb
2D
a. a. b.b.
2D_16_04_07_P2Dsp.2a 2D_16_04_07_N2Dsp.4b
a. a. b.2D_04_04_07_N2Ds.4a2D_04_04_07_P2Ds.1a
b.
3D3D
T. Brandstetter/ 26.04.2013 / slide 25www.imtek.de/cpi
3D_12_04_07_P3Dsp.11a 3D_12_04_07_N3Dsp.2a 3D_03_04_07_N3Ds.4a3D_03_04_07_P3Ds.11
Microstructuring in biochip technologies, contact printing
1. copolymers 2. buffer 3. PT-6000 tungstenPDMAA-co-5%MABP-co-2,5%VPA (a) 400 mM Napi
plastic/PMMA glass/Epoxy
(b) 200 mM Napi/3xSSC/0,75 M betaine
a a bb
2D
a. a. b.b.
2D2D
2D_16_04_07_P2Dsp.2a
a. a. b.2D_xx_04_07_N2Ds.x2D_xx_04_07_P2Ds.x
b.2D_16_04_07_N2Dsp.4b
3D3D
T. Brandstetter/ 26.04.2013 / slide 26www.imtek.de/cpi
3D_12_04_07_P3Dsp.11a 3D_12_04_07_N3Dsp.2a 3D_xx_04_07_N3Ds.x3D_xx_04_07_P3Ds.x
Microstructuring in biochip technologies, contactless printing
II. Contactless printing/Piezo Electric Dispenser
http://www.scienion.de
T. Brandstetter/ 26.04.2013 / slide 27www.imtek.de/cpi
Microstructuring in biochip technologies, contactless printing
II. Piezo Electric dispenserPiezo Electric dispenser(Scienion AG®)
Contactless printing procedure
65% h idit RT 65% humidity, RT
droplet volume 410 pl,
droplet diameter 175 µm droplet diameter 175 µm
droplet volume and diameter is adjustable
Process variance < 10%
T. Brandstetter/ 26.04.2013 / slide 28www.imtek.de/cpi
Microstructuring in biochip technologies, contactless printing
Photos after print
2D 3D
3D2D = printing with PBS without polymer
3D = printing with PBS 1 mg/ml PDMAA-co-5%MABP-co-2,5%VPA
T. Brandstetter/ 26.04.2013 / slide 29www.imtek.de/cpi
Microstructuring in biochip technologies, contactless printing
D l t t ki Droplet stacking
1mg/ml polymer in distilled water
PSS = Polystyrenesulfanit PSS Polystyrenesulfanit
PMMA = Polymethylmetacrylate
Small droplet with 10x p
Large droplets with 20x
Photo after print
T. Brandstetter/ 26.04.2013 / slide 30www.imtek.de/cpi
PSS PMMA
Microstructuring in biochip technologies, contactless printing
“don t” str ct ring “donut”-structuring
1mg/ml PDMAA-co-5%MABP-co-2,5%VPA in
PBS
Exposure after wash with PBS and 0.1% (v/v) Tween)( ) )
T. Brandstetter/ 26.04.2013 / slide 31www.imtek.de/cpi
Dot morphology, how to analyze?
Dot morphology, depending on
surface properties
print solution contact angle
analyte concentration
Dot morphology analyzed by Dot morphology, analyzed by
AFM
Fluorescence microscope Fluorescence microscope
Raster electron microscope
T. Brandstetter/ 26.04.2013 / slide 32www.imtek.de/cpi
Microstructuring in biochip technologies, contactless printing
Printing ith additi es a oiding Printing with additives, avoiding “donut”-morphology
1mg/ml PDMAA-co-5%MABP-co-2,5%VPA in PBS
Additive Glycerol
Photo after print Photo after print
0 2.5 5 10 25%(v/v)
T. Brandstetter/ 26.04.2013 / slide 33www.imtek.de/cpi
Microstructuring in biochip technologies, contactless printing
P i ti ith/ ith tT h l Printing with/withoutTrehalose
1mg/ml PDMAA-co-5%MABP -co-2,5%VPA in PBS
-T+T
125 mg/ml Trehalose (T) in PBS
“Donut”-structure without Trehalose +TTrehalose
Homogeneity in the dot morphology, using Trehalose
-T+T
α-D-glucopyranosyl α-D-
http://en.wikipedia.org/wiki/Trehalose
T. Brandstetter/ 26.04.2013 / slide 34www.imtek.de/cpi
α-D-glucopyranosyl α-D-glucopyranoside(α,α‐Trehalose) Exposure with a fluorescence microscope
Printing on microstructured surfaces
500 µm
T. Brandstetter/ 26.04.2013 / slide 35www.imtek.de/cpi
MicrostructuringMicrostructuring in in biochipbiochip technologiestechnologies
MicronasMicronas BiochipBiochip technlogytechnlogy
Piezo Electric dispenser(Scienion AG®)
Contactless printing procedure
80% humidity, RT
droplet volume 390 pl,
photodiode diameter 180 µm
i ti t t d f printing on structured surfaces
Process variance < 10%
T. Brandstetter/ 26.04.2013 / slide 36www.imtek.de/cpi
Microstructuring in biochip technologies
Micronas Biochip technlogy
Piezo Electric dispenser(Scienion AG®)
printing directly on a photodiode
180 µm
T. Brandstetter/ 26.04.2013 / slide 37www.imtek.de/cpi
Microstructuring in biochip technologies
Micronas Biochip technlogy
Piezo Electric dispenser(Scienion AG®)
printing directly on a photodiode
pattern matching using a software
ted
not p
rint
dpr
inte
T. Brandstetter/ 26.04.2013 / slide 38www.imtek.de/cpi
MicrostructuringMicrostructuring in biochip technologies, summaryin biochip technologies, summary
Piezo Electric dispenser (Scienion AG®) Contactless printing procedure Contactless printing procedure Droplet volume control Droplet diameter tunable (>100µm)
P i i l i h l i Printing only with aqueous solutions 1mg/ml polymer Process variance < 10%
Omnigrid from GeneMachine®
Contact printing procedure Contact printing procedure Steel or tungsten needle with reservoir droplet volume 400 – 600 pl droplet diameter approx 200 µm droplet diameter approx. 200 µm Printing of different solutions > 1mg/ml polymer possible
T. Brandstetter/ 26.04.2013 / slide 39www.imtek.de/cpi
Process variance > 10%
Thank you for your attention!
http://www.bilder-welten.net/de/produkt_detail.php?id=23019&catid=1623
T. Brandstetter/ 26.04.2013 / slide 40www.imtek.de/cpi
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• 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.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 5838Ehrat 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.
T. Brandstetter/ 26.04.2013 / slide 41www.imtek.de/cpi
, , ,• Toomey R., Freidank D. and Rühe J.. Swelling Behavior of Thin, Surface-Attached Polymer
Networks. Macromolecules, Vol. 37, 2004, 882-887.