IMTEK powerpoint template 2008: Version 2 of the first ...
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IMTEK powerpoint template 2008: Version 2 of the first slide
Biochip-Technologies (6)
T. Brandstetter
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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
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T. Brandstetter/ 19.07.2013 / slide 3 www.imtek.de/cpi
Where can I find the lecture stuff/script?
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T. Brandstetter/ 19.07.2013 / slide 4 www.imtek.de/cpi
Other applications
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T. Brandstetter/ 19.07.2013 / slide 5 www.imtek.de/cpi
miRNA
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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
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T. Brandstetter/ 19.07.2013 / slide 7 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 8 www.imtek.de/cpi
different biological assays are needed
Problems
uneconomic
limited amount of samples
Solution
combination of different biological assays in a single test
Breast Cancer
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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
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T. Brandstetter/ 19.07.2013 / slide 10 www.imtek.de/cpi
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]
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T. Brandstetter/ 19.07.2013 / slide 11 www.imtek.de/cpi
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.
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T. Brandstetter/ 19.07.2013 / slide 12 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 13 www.imtek.de/cpi
• 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
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Sign
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Signal Intensity Replicate 1
Chip layout CC: Coupling control, DC: Detection control, NC: Negative control, S18-ER: DNA Probes
T1
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T2
RNA Preparation 2 RNA Preparation 1
Tum
or sa
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Replicates
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T. Brandstetter/ 19.07.2013 / slide 14 www.imtek.de/cpi
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
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sig
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Fluo
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Chip layout CC: Coupling control DC: Detection control NC: Negative control 10b-210: miRNA Probes
B 2-plex
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T. Brandstetter/ 19.07.2013 / slide 15 www.imtek.de/cpi
Multiplex NASBA assays – miRNA (RNA extracted from FFPE samples)
0 0,5
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Fluo
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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
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T. Brandstetter/ 19.07.2013 / slide 16 www.imtek.de/cpi
Aim of the study
x
Extraction of RNA
Amplification
Detection
Tumor sample
•Method: NASBA •Genetic Parameters •Primers •Conditions
Works for 5 parameters in parallel
Works for 7 parameters in parallel
Method works on Tumor samples
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T. Brandstetter/ 19.07.2013 / slide 17 www.imtek.de/cpi
• 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
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0 0,5
1 1,5
2 2,5
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NASBA 9plex
NASBA -RT
NASBA NTC
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miR
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RPS1
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ESR1
HER2
MM
P11
YBX1
CASP
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SOD2
PGR
FGFR
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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
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NASBA 9plex
NASBA -RT
NASBA NTC
miRNA mRNA
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T. Brandstetter/ 19.07.2013 / slide 19 www.imtek.de/cpi
Aim of the study
x
Extraction of RNA
Amplification
Detection
Tumor sample
•Method: NASBA •Genetic Parameters •Primers •Conditions
Works for 5 parameters in parallel
Works for 7 parameters in parallel
Method works on Tumor samples
9 parameters in parallel Combined mRNA and miRNA
analysis
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T. Brandstetter/ 19.07.2013 / slide 20 www.imtek.de/cpi
polysaccharides
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T. Brandstetter/ 19.07.2013 / slide 21 www.imtek.de/cpi
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]
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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
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T. Brandstetter/ 19.07.2013 / slide 23 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 24 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 25 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 26 www.imtek.de/cpi
R & D, microarray-based processes
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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
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T. Brandstetter/ 19.07.2013 / slide 28 www.imtek.de/cpi
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.
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T. Brandstetter/ 19.07.2013 / slide 29 www.imtek.de/cpi
Micronas Chip: sensor principle
reset
out
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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.
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Microarrays in glycomics: Applications
P. Seeberger. Chem. Soc. Rev., 2008, 37, 1414–1422
Overview:
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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]
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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
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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
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T. Brandstetter/ 19.07.2013 / slide 34 www.imtek.de/cpi
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)
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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
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T. Brandstetter/ 19.07.2013 / slide 36 www.imtek.de/cpi
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 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 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
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T. Brandstetter/ 19.07.2013 / slide 37 www.imtek.de/cpi
The Micronas Chip: validation
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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
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T. Brandstetter/ 19.07.2013 / slide 38 www.imtek.de/cpi
Cell biochips
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T. Brandstetter/ 19.07.2013 / slide 39 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 40 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 41 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 42 www.imtek.de/cpi
Thank you for your attention!
http://www.bilder-welten.net/de/produkt_detail.php?id=23019&catid=1623
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T. Brandstetter/ 19.07.2013 / slide 43 www.imtek.de/cpi
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
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T. Brandstetter/ 19.07.2013 / slide 44 www.imtek.de/cpi
Biochip-technologies http://portal.uni-freiburg.de/cpi/biochip-group-dr-brandstetter