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IMTEK powerpoint template 2008: Version 2 of the first slide

Biochip-Technologies

T. Brandstetter

T. Brandstetter/ 09.05.2014 / slide 2 www.imtek.de/cpi

• 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)

Content


Our profile

Research and teaching

• 22 faculties

• 300 researchers and technicians

• highly interdisciplinary world of

microsystem technology

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


Biochip-technologies http://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 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.



• 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.



3

8

13

HPV 6

HPV_3D_Katrin_N_30s_Cy5

substrat

dot

microarray

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


Manufacturing of biochips – in general(1)

3. Immobilisation

2. Microarray printing

1. Untreated slide

mixed analyte solution


step1:

print polymer mixed with DNA

step 2:

photocrosslinking

via UV-irradiation

step 3:

hybridisation

and

readout

C OH

Manufacturing of biochips – in general(2)


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

• 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


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


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.


2D chips using SAMs (self assembled monolayers)

typical DNA-chip design:

+ sensitivity

adapted from: E. Southern, K. Mir, M. Shchepinov, Nature Gen., 27 (1999) 5

+ surface properties

+ reproducibility (why is acceptance of microarrays below expectations in non-research areas?

sequence of the probe

polyT(thymine) tailer

Weakness:


A „skyscraper“-approach

“polymer layer” – approach allows to

improve the sensitivity

adjust properties of the surface

(hydrophilicity, reactivity)

polymer brushes

attachment of

oligonucleotide probes

2D

3D

polymer networks

3D


chemisorption of polymers

grafting of polymers on

plasma modified

surfaces

photochemical attachment

of polymers

blockcopolymers

via macroinitators

growth of polymers

on surfaces

surface-attached

polymer networks „grafting in between“

Functional polymer monolayers


C O C O C

CCH

350 nmOH

CC

OH

hydrogen

abstraction

recombination

= 100 µ s

Photochemistry of benzophenone

265 nm

triplet formation upon n,* excitation

biradical reacts with C,H bonds

Toomey R., Freidank D. and

Rühe J.. Swelling Behavior of

Thin, Surface-Attached

Polymer Networks.

Macromolecules, Vol. 37, 2004,

882-887.


Me

O O

O

ON

Me

Me

swelling in

water (2h)

polymeric substrate

(e.g. polyurethane)

photocrosslinkableovercoat

ca. 20 µm

~ 1 mm

simultaneous crosslinking

and surface attachment

via pendant benzophenone

units

Polymer networks attached to polymeric substrates


Microstructuring in biochip technologies, two procedures

I. Contact printing

http://www.anopoli.com/ http://www.anopoli.com/

Print pins Printhead


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 pin

Solid pin

•Spot diameters : 75µm to 215 µm

•Uptake volumes : 0.25µl to 0.64 µl

•Spot diameters : 75µm to 450 µm

htt

p:/

/ww

w.a

nopoli.

com

/


PDMAA(Polydimethylmetacrylate) PS (Polystyrene)


Printing with different, not aqueous, solutions is possible.

printing medium: ethanol printing medium: toluene

200 µm



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-co-

2,5%VPA



scale lining

PDMAA layer

PMMA (5 mg/ml) lining

Printing medium toluene

exposure after

photocrosslinkage


1. copolymers 2. buffer 3. PT-6000 tungsten

2D

3D

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

2D_16_04_07_P2Dsp.2a 2D_16_04_07_N2Dsp.4b

3D_12_04_07_P3Dsp.11a

a. a.

3D_12_04_07_N3Dsp.2a

a. a. b.

b.

2D_04_04_07_N2Ds.4a

3D_03_04_07_N3Ds.4a

b.

2D_04_04_07_P2Ds.1a

3D_03_04_07_P3Ds.11

b.


3D


1. copolymers 2. buffer 3. PT-6000 tungsten

2D

3D

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

2D_16_04_07_P2Dsp.2a

3D_12_04_07_P3Dsp.11a

a. a.

3D_12_04_07_N3Dsp.2a

a. a. b.

b.

2D_xx_04_07_N2Ds.x

3D_xx_04_07_N3Ds.x

b.

2D_xx_04_07_P2Ds.x

3D_xx_04_07_P3Ds.x

b.

2D_16_04_07_N2Dsp.4b


2D

3D


Microstructuring in biochip technologies, contactless printing

II. Contactless printing/Piezo Electric Dispenser

http://www.scienion.de



II. Piezo Electric dispenser

Piezo 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%


Photos after print


2D

3D

3D

2D = printing with PBS without polymer

3D = printing with PBS 1 mg/ml PDMAA-co-

5%MABP-co-2,5%VPA



Droplet stacking

1mg/ml polymer in distilled water

PSS = Polystyrenesulfanit

PMMA = Polymethylmetacrylate

Small droplet with 10x

Large droplets with 20x

Photo after print

PSS PMMA



“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

AFM

Fluorescence microscope

Raster electron microscope



Printing with additives, avoiding

“donut”-morphology

1mg/ml PDMAA-co-5%MABP-

co-2,5%VPA in PBS

Additive Glycerol

Photo after print

0 2.5 5 10 25%(v/v)



Printing with/withoutTrehalose

1mg/ml PDMAA-co-5%MABP

-co-2,5%VPA in PBS

125 mg/ml Trehalose (T) in PBS

“Donut”-structure without

Trehalose

Homogeneity in the dot

morphology, using Trehalose

-T

-T

+T

+T

α-Ｄ-glucopyranosyl α-Ｄ-

glucopyranoside(α,α‐Trehalose)

http://en.wikipedia.org/wiki/Trehalose

Exposure with a fluorescence microscope


Printing on microstructured surfaces

500 µm


Microstructuring in biochip technologies

Micronas Biochip technlogy



80% humidity, RT

droplet volume 390 pl,

photodiode diameter 180 µm

printing on structured surfaces






printing directly on a photodiode

180 µm





printing directly on a photodiode

pattern matching using a software

no

t p

rin

ted

pri

nte

d


Piezo Electric dispenser (Scienion AG®)


Droplet volume control

Droplet diameter tunable (>100µm)

Printing only with aqueous solutions

1mg/ml polymer


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%

Microstructuring in biochip technologies, summary


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.

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.

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