IMTEK powerpoint template 2008: Version 2 of the first

IMTEK powerpoint template 2008: Version 2 of the first slide

Biochip-Technologies (6)

T. Brandstetter

T. Brandstetter/ 19.07.2013 / slide 2 www.imtek.de/cpi

• 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 (05.07.13)

• Other applications + Summary(19.07.13)

Content


Where can I find the lecture stuff/script?


Other applications


miRNA


Breast cancer is an extremely heterogenous disease

• “There is no one single breast tumor. Each tumor is different."

Diagnosis is based on a sub-classification of the malignancy

Problem: Factors insufficient for an accurate sub-classification

• often wrong treatment is given

Breast Cancer

Lymph node Status Tumor Size Histology Hormone Receptor Status HER2

sub-groups 1-5


To improve diagnosis a more precise sub-classification is needed based on molecular markers

For molecular classification several regulatory levels of gene expression are monitored

• Genomic level

• Transcriptional level

• Protein level

Breast Cancer

Molecular signatures: Gene Expression Profiling MicroRNA Profiling Mutation Analysis Proteine Profiling sub-groups 1-x


different biological assays are needed

Problems

uneconomic

limited amount of samples

Solution

combination of different biological assays in a single test

Breast Cancer

Vorführender

Präsentationsnotizen

Fine needle aspiration biopsy: minimal invasive 1µg RNA 1 cell 10 pg RNA? 1-5 % mRNA


Aim of the study

x

Extraction of RNA

Amplification

Detection

Tumor sample Microarray production • Printing of Probe (DNA) and Polymer • Immobilization via UV-light Process described in Rendl, Boenisch. et al. 2011

Lehr et al, 2003; (1) Slide, (2) Microarray, (3) Flow cell, (4) Laser, (5) Cooling element, (6) Peltier (7) CCD Camera, (8) Filter

TIRF based read-out

© Andreas Mader

Vorführender


Lehr, H. P., M. Reimann, et al. (2003). "Real-time detection of nucleic acid interactions by total internal reflection fluorescence." Anal Chem 75(10): 2414-20.


MicroRNAs (miRNAs)

are small

encoded in the genomes of plants and animals (Figure 1)

highly conserved

~21-mer RNA

regulate the expression of genes by binding to the 3'-untranslated regions

(3'-UTR) of specific mRNAs.

miRNA , what are these biomolecules?

Figure 1. Transcription of miRNAs. Approximately 60% of miRNAs are expressed independently, 15% of miRNAs are expressed in clusters, and 25% are in introns. [http://www.ambion.com/techlib/resources/miRNA/mirna_intro.html]


miRNA Amplifikation by stem-loop RT-NASBA

Theory

5' miRNA

Stem-Loop-Primer

AMV-RT

3'

5' P1_T7

AMV-RT, RNaseH, T7 RNA polymerase

5‘ Sense RNA

Reverse Primer

3‘ cDNA

Cycle

Step 1 Reverse Transcription

Step 2 NASBA

Figure 1. Schematic of stem-loop RT NASBA for isothermal amplification of miRNAs. First, the stem-loop RT primer is anneals to the 3’ end of the miRNA and is extended by the reverse transcriptase enzyme to make a cDNA complementary to the miRNA. Second, the cDNA is added to a NASBA reaction containing an oligonucleotide primer pair plus the enzymes AMV-RT, RNaseH and the T7 RNA polymerase. The miRNA specific forward primer (P1_T7) is carrying a T7 RNA polymerase promoter sequence where the T7 RNA polymerase binds to produce copies of sense RNA that can either hybridize to specific probes or used as templates for the reverse primer to generate cDNA that is again template for the T7 promotor carrying forward primer.


mRNA Compton et al, 1991

mRNA (+)

RNA (-)

Primer Enzymes

1. Reverse Transcription

2. NASBA

mRNA and miRNA

mRNA (+) miRNA (+)

tag sequences

Universal Primer Enzymes

miRNA (+)

miRNA

RNA (+)

cDNA

Primer Enzymes

3. Detection Northern Blot, DNA Microarray

NASBA (nucleic acid sequence based amplification)-Concept

Vorführender


Compton, J. (1991). "Nucleic acid sequence-based amplification." Nature 350: 91-92. Cox, A. (2007). "A common coding variant in CASP8 is associated with breast cancer risk." Nature Genetics 39(3): 352-688. Siva, A. C., L. J. Nelson, et al. (2009). "Molecular assays for the detection of microRNAs in prostate cancer." Mol Cancer 8: 17.


• 5-plex NASBA (Mader, Riehle et al. 2010)

• 5-plex NASBA with RNA extracted from FFPE tumor samples

Multiplex NASBA assays-mRNA

CC S18 206 210 125b 125a 21 16 10b CC

DC S18 206 210 125b 125a 21 16 10b NC

DC S18 206 210 125b 125a 21 16 10b CC

DC F4 PGR S2 C8 YB1 M11 HER ER CC

NC F4 PGR S2 C8 YB1 M11 HER ER NC

CC F4 PGR S2 C8 YB1 M11 HER ER CC

R² = 0,7907

0

2

4

6

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0 2 4 6 8 10

Sign

al In

tens

ity R

eplic

ate

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Signal Intensity Replicate 1

Chip layout CC: Coupling control, DC: Detection control, NC: Negative control, S18-ER: DNA Probes

T1

T3

T2

RNA Preparation 2 RNA Preparation 1

Tum

or sa

mpl

es

Replicates


0

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Multiplex NASBA assays - miRNA

C 4-plex D 7-plex

A 1-plex CC 210 206 125b 125a 21 16 10b CC

NC 210 206 125b 125a 21 16 10b NC

NC 210 206 125b 125a 21 16 10b CC

DC 210 206 125b 125a 21 16 10b NC

DC 210 206 125b 125a 21 16 10b CC

DC 210 206 125b 125a 21 16 10b NC

DC 210 206 125b 125a 21 16 10b CC

DC 210 206 125b 125a 21 16 10b NC

NC 210 206 125b 125a 21 16 10b CC

NC 210 206 125b 125a 21 16 10b NC

CC 210 206 125b 125a 21 16 10b CC

2-plex

4-plex 7-plex

1-plex

Fluo

resc

ence

sig

nal

Fluo

resc

ence

sig

nal

0 1 2 3 4 5

0 0,5

1 1,5

2 2,5

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Chip layout CC: Coupling control DC: Detection control NC: Negative control 10b-210: miRNA Probes

B 2-plex


Multiplex NASBA assays – miRNA (RNA extracted from FFPE samples)

0 0,5

1 1,5

2 2,5

3

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0 0,2 0,4 0,6 0,8

1 1,2

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Fluo

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Fluo

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sig

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CC 210 206 125b 125a 21 16 10b CC

NC 210 206 125b 125a 21 16 10b NC

NC 210 206 125b 125a 21 16 10b CC

DC 210 206 125b 125a 21 16 10b NC

DC 210 206 125b 125a 21 16 10b CC

DC 210 206 125b 125a 21 16 10b NC

DC 210 206 125b 125a 21 16 10b CC

DC 210 206 125b 125a 21 16 10b NC

NC 210 206 125b 125a 21 16 10b CC

NC 210 206 125b 125a 21 16 10b NC

CC 210 206 125b 125a 21 16 10b CC

Chip layout CC: Coupling control DC: Detection control NC: Negative control 10b-210: miRNA Probes

C 4-plex D 7-plex

A 1-plex B 2-plex 2-plex

4-plex 7-plex

1-plex


Aim of the study

x

Extraction of RNA

Amplification

Detection

Tumor sample

•Method: NASBA •Genetic Parameters •Primers •Conditions

Works for 5 parameters in parallel


Method works on Tumor samples

Vorführender




• Reverse Transcription: multiple primer pairs

• NASBA: 1 primer pair

• combined mRNA and miRNA analysis

Universal NASBA

mRNA and miRNA

mRNA (+) miRNA (+)

cDNA

tag sequences

Primer pair Enzymes

1. Reverse Transcription

2. NASBA

Reverse Transcription: (mRNA + miRNA)

Amplification

Literature Universal NASBA


0 0,5

1 1,5

2 2,5

3 3,5

4 4,5

Fluo

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nten

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t

NASBA 9plex

NASBA -RT

NASBA NTC

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miR

-10b

miR

-16

miR

-21

miR

-125

a

miR

-125

b

miR

-206

miR

-210

RPS1

8

ESR1

HER2

MM

P11

YBX1

CASP

8

SOD2

PGR

FGFR

4

NASBA 9plex

NASBA -RT

NASBA NTC

Multiplex universal NASBA

9-plex NASBA

• miRNA (3)

• mRNA (6)

0 2 4 6 8

10 12 14 16 18

Fluo

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nten

sitä

t

NASBA 9plex

NASBA -RT

NASBA NTC

miRNA mRNA


Aim of the study

x

Extraction of RNA

Amplification

Detection

Tumor sample

•Method: NASBA •Genetic Parameters •Primers •Conditions



Method works on Tumor samples

9 parameters in parallel Combined mRNA and miRNA

analysis

Vorführender




polysaccharides


Polysaccharides are

polymeric carbohydrate structures

formed of repeating units (either mono- or di-saccharides) joined together by glycosidic bonds

often linear, but may contain various degrees of branching

often quite heterogeneous, containing slight modifications of the repeating unit

Definition of polysaccharides

3D structure of cellulose, a beta glucan poly-saccharide [http://en.wikipedia.org/wiki/Polysaccharides]


Polysaccharide

• Biosensors for in-vitro diagnostics Global market of point-of-care devices (espicom study)

Examples: Products on the market or still in research

• Applications of biosensors in glycomics

• Own strategy

Baader_Diss2010


Importance of glycomics

• As a model assay the Micronas Chip is functionalized with capsular polysaccharides of the bacteria Streptococcus pneumoniae. Protein-carbohydrate interactions can be detected

• What is the importance of monitoring protein-carbohydrate interactions?

• Glycoproteins on the cell surface play a critical role in bacterial and viral recognition

• Glycans are involved in cellular signaling pathways

• There are many glycan-specific diseases

E.g. eertain glycan structures are cancer markers


R & D, challenges in the assay process development

Protein

biocompatibility

nativity of the analytes

incubation time

quantification Polysaccharides

Clin Chem Lab Med 2005;43(12):1291–1302 2005 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2005.223


R & D, microarray-based processes

Protein

Immunoassay

competitive and non- competitve ELISA

Polysaccharides

Clin Chem Lab Med 2005;43(12):1291–1302 2005 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2005.223


R & D, microarray-based processes


POC: Commercial products

Triage System (Biosite)

• Fluorescence-based detection (laser incorporated)

• Sample is filtered by “microcapilariy fluidic”

• Results available in approximately 15 minutes

• Normally six analytes measured in parallel

Nanoplex (Oxonica)

• Silicon-coated surface and enhanced Raman

scattering (SERS)-active metallic nanoparticles

(colloidal gold core).

• Multiplexed detection capabilities

• Benchtop reader


Strategy

• A silicon semiconductor chip with an area of ca. (7x7) mm2 integrates 32 photodiodes each 190μm in diameter and at 500μm spacing.

• Each photodiode is individually addressable by integrated electronics and can be used for independent analysis: each photodiode can monitor a different receptor ligand reaction.


Micronas Chip: sensor principle

reset

out

negfb

sample

adressdec pix(0-31) gainadjust

pix(0-31)

Luminol ox. + h*ν

H2O2 H2O + OHRP

+ Luminol

428 nmPhotodiode

• The active pixel design of the biochip specific silicon layer with integrated

photodiodes (red symbol in drawing) and

integrated amplifiers (white symbol in drawing).

• Voltage change is immediately amplified and serves as output signal for each individual pixel.

• Due to the direct positioning of the receptor ligand complex on the sensor surface, nearly 50% of the emitted detection light is captured.


Microarrays in glycomics: Applications

P. Seeberger. Chem. Soc. Rev., 2008, 37, 1414–1422

Overview:


Principle of polysaccharide microarray detection

Baader J., Klapproth H., Bednar S., Brandstetter T., Rühe J. und Freund I.. Polysaccharide microarrays with a CMOS based signal detection unit. Biosens Bioelectron. 2010 Jan 28. [Epub ahead of print] [PMID: 20181471]

http://www.ncbi.nlm.nih.gov/pubmed/20181471�

http://www.ncbi.nlm.nih.gov/pubmed/20181471�


µarrays in glycomics: enzymatic activity

Glycosilation monitoring

• In organism carbohydrates and polysaccharides are synthesized by enzymes

• Microarrays can be used to assay for glycosyltransferase activity

• Time-dependent glycolsylation of alpha- and beta-linked carbohydrates was monitored

S. Park, I. Shin. Org. Lett., Vol. 9, No. 9, 2007


µarrays in glycomics: binding analysis

J. Stevens, Nat. Rev. Microbiol., 2006, 4, 857ff

Virus mutation monitoring

• Binding preferences of hemagglutinin proteins (HG) were assessed

• HG contributes to generate the host species barrier of influenza viruses

• Fast test to analyze for dangerous mutations of avian flu virus


µarrays in glycomics: cell interactions

M.D. Disney, Chem. Biol., 2004, 11, 1701.

Monitoring cell-sugar interactions

• Carbohydrate ligands at the surface mimic cell-interfaces

• Binding inhibitors can be found and there IC50-values determined

• Diagnostic test systems to detect for harmful pathogens (adherent vs. non-adherent cells)


µarrays in glycomics: antibody screening

J.W. Pickering et al., Am.J.Clin.Pathol., 2007, 128, 23ff

New ELISA formats

• Chemoluminescent microarray combined with ELISA plate format

• A 22-plex assay for anti-PnPS IgG was developed by the group of Pickering

• Vaccination response could be successfully determined

• Comparison to standard ELISA: Correlation factor r² = 0.91-0.97

• Drawbacks:

Low sensitivity

Difficult printing process

Detection unit not integrated


µarrays in glycomics: The Micronas Chip

• Highly sensitive and specific antibody detection by using the CMOS Micronas polysaccharide chip Improved lower detection limit of

approx. one decade

• No pretreatment of receptor molecules is needed as pneumococcal polysaccharides are well bound to the silicon dioxide sensor interface

• Test time is about three hours for multi-analyte measurements

test duration 16 h*status quo ~4.5 h,

with flow cell ~1.5 hsera volumes 30 µl* ~1 µlrequired antigen volume 100 µl/well 2.4 nl/photodiodelower detection limit 10-50 ng/ml ~15 pg/ml

material effortplate photometer, incubator, tumbler pump

* test for 10 different serotypes, value scales with amount of analyzed antigens







* test for 10 different serotypes, value scales with amount of analyzed antigens


The Micronas Chip: validation

0 4000 8000 12000 16000

0

4000

8000

12000

16000

chip_768 hospital_768

mea

sure

d co

ncen

tratio

n /

ng/m

l

concentration of int. reference Ko768 / ng/ml

Modell Line

Gleichung y = A + B*x

Chi-Quadr Reduziert

157157.18145

Kor. R-Qua 0.99438Wert Standardfeh

F A 0 0F B 1.1600 0.02557

Modell Line

Gleichung y = A + B*x

Chi-Quadr Reduziert

933662.76948

Kor. R-Quad 0.94567Wert Standardfehl

C A 0 0

C B 0.9087 0.06233

Correlation with the gold standard

• Internat. reference serum Ko768 analyzed by both biochip and ELISA (University Hospital Freiburg)

• Both methods in good agreement to averaged values of internat. laboratories


Cell biochips


Cell analysis with biochips

http://www.google.de/imgres?imgurl=http://www.nanoink.net/i/NanoStemAboutImg1.jpg&imgrefurl=http://www.nanoink.net/NanoStem_about.htm&usg=__bNdjUS4kXsVo8JWFd4lnCgC36ss=&h=431&w=600&sz=67&hl=de&start=312&itbs=1&tbnid=nPtJjWzVApi6OM:&tbnh=97&tbnw=135&prev=/images%3Fq%3Dcell%2Bbiochips%26start%3D300%26hl%3Dde%26sa%3DN%26gbv%3D2%26ndsp%3D20%26tbs%3Disch:1


Tissue arrays

Tissue Array (or Tissue MicroArray) is a method of relocating multiple tissues from conventional histologic paraffin blocks so that tissues from multiple patients can be seen on a same slide.

http://www.tissue-array.com/ver3/index.php

Identifying/colouring different cell components

In situ hybridization, RNA or DNA

Fluorescent in situ hybridization (FISH)

In situ PCR, RNA or DNA


miRNA analysis ca. 21mer Stem loop detection

polysaccharides glucane Glycosilation monitoring Virus mutation monitoring Cell-sugar interaction

Cell analysis Cell differentiation Tissue arrays

Other microarray based biochip techniques, summary


Thank you for your attention!

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


Literature

• Rendl M., Bönisch A., Mader A., Schuh K., Prucker O., Brandstetter T. and Rühe J.. A Simple One-Step Procedure for Immobilization of Biomolecules on Plastic Surfaces using Surface- Attached Polymer Networks. Langmuir, 2011, 27 (10), pp 6116–6123

• Lehr, H.-P., Reimann, M., Brandenburg, A., Sulz, G., Klapproth, H., 2003. Real-Time Detection of Nucleic Acid Interactions by Total Internal Reflection Fluorescence. Anal. Chem., 75, pp. 2414-2420.

• Mader A., Riehle U., Brandstetter T., Stickeler E., zur Hausen A. and Rühe J.. Microarray-based Amplification and Detection of RNA by Nucleic Acid Sequence-Based Amplification (NASBA). Anal Bioanal Chem. 2010 Aug;397(8):3533-41. Epub 2010 Jul 2.

• Clinical Chemistry 53, No. 11, 2007

• P. Seeberger. Chem. Soc. Rev., 2008, 37, 1414–1422

• Baader J., Klapproth H., Bednar S., Brandstetter T., Rühe J. and Freund I.. Polysaccharide microarrays with a CMOS based signal detection unit. Biosens Bioelectron. 2010 Jan 28. [Epub ahead of print]

• Park S.-J. et al. (2002), Science , 295, 1503-1506

• J. Stevens, Nat. Rev. Microbiol., 2006, 4, 857ff

• M.D. Disney, Chem. Biol., 2004, 11, 1701

• J.W. Pickering et al., Am.J.Clin.Pathol., 2007, 128, 23ff


Biochip-technologies http://portal.uni-freiburg.de/cpi/biochip-group-dr-brandstetter

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IMTEK powerpoint template 2008: Version 2 of the first