biochip technologies biochip-technologies

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


• AFM

• Biochip-technologies

Biochip‐technologieshttp://portal.uni-freiburg.de/cpi/biochip-group-dr-brandstetter


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


chemistry designed to sense a specific analyte.


• 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.



HPV_3D_Katrin_N_30s_Cy5 substrat

HPV 6

dot

38

HPV 6

813 microarray

http://en.wikipedia.org/wiki/Biochip#History


Manufacturing of biochips – in general(1)

1. Untreated slidemixed analyte solution

2. Microarray printing

3 I bili ti3. Immobilisation


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


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


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


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.


, , , 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


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


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


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


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


µ

Microstructuring in biochip technologies, two procedures

I. Contact printing

Print pins PrintheadPrint pins Printhead


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%



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



Printing with different, not aqueous, solutions is possible.

PDMAA(Polydimethylmetacrylate) PS (Polystyrene)

200 µm µ


printing medium: ethanol printing medium: toluene


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



l li i scale lining PDMAA layer

PMMA (5 mg/ml) lining( g ) g

Printing medium toluene

exposure after photocrosslinkagephotocrosslinkage



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


3D_12_04_07_P3Dsp.11a 3D_12_04_07_N3Dsp.2a 3D_03_04_07_N3Ds.4a3D_03_04_07_P3Ds.11


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


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



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%



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



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


PSS PMMA


“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)( ) )


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



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)



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


α-Ｄ-glucopyranosyl α-Ｄ-glucopyranoside(α,α‐Trehalose) Exposure with a fluorescence microscope

Printing on microstructured surfaces

500 µm


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%


Microstructuring in biochip technologies

Micronas Biochip technlogy


printing directly on a photodiode

180 µm


Microstructuring in biochip technologies

Micronas Biochip technlogy


printing directly on a photodiode

pattern matching using a software

ted

not p

rint

dpr

inte


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


Process variance > 10%

Thank you for your attention!

http://www.bilder-welten.net/de/produkt_detail.php?id=23019&catid=1623


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.


, , ,• Toomey R., Freidank D. and Rühe J.. Swelling Behavior of Thin, Surface-Attached Polymer

Networks. Macromolecules, Vol. 37, 2004, 882-887.

biochip technologies biochip-technologies

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