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Planar Waveguides: How Nano Layers Enable to Detect
Zepto Moles of Macro Molecules inPico Liter Spots on
Micro Arrays
Dr. Markus EhratZeptosens – A Division of Bayer Schweiz AG
SSOM Meeting March 16 /17 2009
Engelberg
What are Biochemical Microarrays?
Glass or polymer support (e.g. microscope slide)
Spots with selective recognition elements
Each spot binds a specific analyte ofthe sample solution Detection signal: e.g. fluorescence labels
100 to 10’000 spots per cm2 = spotted arrays
10’000 to 100’000 spots per cm2 = “photo lithography” arrays
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Microarrays -A powerful technology to measure
thousands of samples and thousands of analytes- genes or proteins -,
in a short period of time
Entries in PubMed Database, Search Term „Microarray“
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Information Obtained from 200 µL of Sample
45’000’0001.7 x 10-61.5 µm spot microarray
450’0001.7 x 10-415 µm spot microarray
45001.7 x 10-2150 µm spot microarray
162.51536 well plate
14096 well plate
Information per cm2*Detection area(mm2)
* Sample solution of 200 µL
Microarrays -Small detection areas
Nanoliters of sample volumes:
Require high detection sensitivity
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Thin Film Planar Waveguide (PWG) Chip Design
Ta2O5 waveguiding layer100 – 200 nm
glass substrate
GratingPeriod: 200 - 750 nmDepth: 5 - 20 nm
Light Intensity vs. Waveguide Thickness
Parameters:nsub=1.52, nsup=1.335, nPWG=2.15, m=0, λ=635nm
d = 100nm d = 150nm d = 200nm
d = 250nm d = 300nm
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Planar Waveguide Principle - High Sensitivity Fluorescence Microarray Detection
surface confined excitation of bound label
CCD camera
Advantages of Fluorescence Excitation on PWG
samplesolution
support
Conventional excitation
background problems ~ no background
ZeptoREADER™ - Evanescent excitation
• Separation of excitation and detection directions
• Ultimate sensitivity
• Fast time to result
• Less sample preparation
• Direct measurement in blood or serumConfocal excitation:
Focus depth ~ 2µm
Evanescent excitation:
Depth ~ 100nm
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ZeptoREADER – PWG Inside
Ultra sensitive:Planar wave guide technology based evanescent field fluorescence excitation
Exceptionally fast:Over 120’000 data points in 6 hours
Increase efficiency:Extended walk away time using 60 slides integrated autoloader
Absolutely reliable:Swiss designed and manufactured for highest quality and precision possible
ZeptoREADER Setup
Plate StorageElectronicsOpticsMeasurementChamber
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Optical Scheme of the ZeptoREADERTM
1. Laser
2. Shutter
3. Gray filter wheel
4. Coupling unit
5. ZeptoCHIPTM
6. Front lens unit
7. Emission filter wheel
8. Camera lens unit and
CCD
635 nm
532 nm
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fluor
esce
nce
inte
nsity
x 1
0-3
Cy5-BSA [pM]
High Sensitivity of PWG Signal Detection
image saturation (16 bit)
background
Dilution series
1..1
0000
pM
Cy5
-BSA
LOD = 1 zeptomol (600 proteins) per spot
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0 .0 0 .1 1 1 0 1 00 1 00 00 .0 0
0 .0 2
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0 .0 6
0 .0 8
0 .1 0
R F I (0 ) + 3 σ
RFI
(avg
3 a
rray
s)
c (re c . re ce p to r) / p M
ZeptoMARK Assay Sensitivity
Spiking series of recombinant receptor in 2 mg/mL lysate
Spotted and analyzed at 4 dilutions, duplicate spots
Average and std dev of 3 arrays10 100 1000
0.01
0.1
1
LOD of ~2000 receptor protein molecules per spot
Assay Reproducibility: Different Operators – Different Days –Different ZeptoREADERs
P -A k t (S e r4 7 3 )
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mea
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FI o
f 2 a
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o p e ra to r 1 - Ze p to R E A D E R 1o p e ra to r 2 - Ze p to R E A D E R 2
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o p e ra to r 1 - Z e p to R E A D E R 1o p e ra to r 2 - Z e p to R E A D E R 2
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High detection sensitivity
What is the value in the real world?
The Systems Approach
“…the rate of mortality from cancer has changed very little over the past 50 years“ “Cancer therapies are still essentially “one size fits all”,…..”.
Science, Vol 312, May 26, pp 1157, 1165, 1166, (2006)
“Targeting systems, rather than single molecules, will likely result in more durable responses in cancers considered non-responsive to treatment.” “Thus the ”omic” technology is promising an approach both
- to evaluate the heterogeneity of cancer patients- and as a means of identifying biosystems as target for new drugdevelopment”
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Signatures Guide Drug Choice Julian Downward
Nature 439, 274-275 (19 January 2006)
….“Cancer drugs are increasingly designed to target specific cell-signalling pathways…”
….“Pathways can be activated at different points [ ]. If a factor at the top of a signalling cascade is unaffected, for instance, one cannot assume that the pathway is not involved, as a factor further downstream might have been activated”…
PIP2
PIP3
PTENPI3K
PDK
Akt
Src
p27 p21
IKKNF-κB
FOXOBAD
β-cateninp70 S6K
4EBP
MDM2
p53
ASK GSK3β mTORCaspase
Cell Cycle
Progression
Apoptosis
Resistance
Protein
Translation
Proliferation
Migration
Proliferation
Survival
Bcl-2 FasL
PI3K/AKT pathwayPathway branching
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Requirements for Pathway Proteomics Approaches
Signaling is highly dynamic– protein phosphorylations in seconds/minutes – protein synthesis/degradation in hours/days – extensive sampling required to obtain time resolution
Many signals are post-translational modifications– issue for classical MS-based methods
Highly parallel measurements of analytes is important – a thorough pathway profile can easily comprise 100 or more elements
Conclusion: an array-based solution will provide the required scalability and throughput
Protein Microarrays – Two FormatsReverse Protein Arrays
• Array of samples on chip• One analyte measured
per array
Forward Protein Arrays
• Array of target-specific capture molecules (e.g. antibodies)
• One sample measured per array
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Reverse Protein Microarrays
• Array of samples• Sample volume 400 pL• Specific detection with
target-specifc antibodies• One Ab per target /array• High flexibility in study
design – minimum assay development effort
• Sample volume is never a bottle neck to measure multiple analytes
Cell Lysate Arrays
Cellular systems Cell lysis
Data evaluation ReadoutPathway Profiles
III
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Ab1
Ab2
Ab3
Ab5
Ab4
Ab6
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Ab1
Ab2
Ab3
Ab5
Ab4
Ab6
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Ab1
Ab2
Ab3
Ab5
Ab4
Ab6
III
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Ab1
Ab2
Ab3
Ab5
Ab4
Ab6
ZeptoMARK Reverse Arrays – From Cells to Protein Profiles
Blocking
Assay
Spotting
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Zeptosens Reverse Arrays – From Cells to Protein Profiles
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Cellular systems SpottingCell lysisDrug treatment
# 1
...# n
Cellular systems SpottingCell lysisDrug treatment
Sample~105 cells
~ 1 mg tissue
Lysis50 µl lysis
2 mg total protein/mL
Spotting400 pL spotting
400pL sample volume
Non-contact ink-jet spotting technology
Up to 256 lysates/array or 1536 lysates/chip
Reproducibilities of mean spot signals: CV‘s ≤ 2%
Spotting: Reproducibility & Quality
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Chip and Array Layout
Zept
osen
s
6 Arrays / Chip Ref Ref Ref Ref
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BufferStandards
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(Ref) reference spots containing constant fluorescenceup to 32 samples (4 dilutions, duplicates) per arrayup to 192 samples (4 dilutions, duplicates) per chip
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Ref
eren
ce
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Ref
eren
ce
Sample dilutions (mg/ml)
Highly Parallel Monitoring of Signaling Events
Raf Ab
P-Akt Ab
Akt Ab
Erk2 Ab
Bcl-2 Ab
P-Erk2 Ab
6 identical Lysate Arrays
From Cell Signaling Technology
Treatment
Effect From Cell Signaling Technology
TreatmentTreatment
Effect
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The System Response Profile
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Sample #
<RFI
>
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Sample #
<RFI
>
Stimulation with Insulin – Inhibition with Wortmannin
InsRIGFR IRSs
PI3K(p110,p85)
PKB/Akt
GSK3
PDKs
PIP3 PIP2
PTEN
Glycogen synthase
mTOR
S6 kinase
S6
Insulin
Ctrl
EG
F(t)
W(t)
W+E
GF(
t)
W(c
)+In
s
Ins(
t)
P-Akt (S473)
0.0
0.1
0.2
0.3
0.4
RFI
Cellgrowth
A431, starvation over nightStimulation with Insulin(150nM) 7 time points,
0-240min
Wortmannin
Ctrl
EG
F(t)
W(t)
W+E
GF(
t)
W(c
)+In
s
Ins(
t)
P-Akt (S473)
0.0
0.1
0.2
0.3
0.4
RFI
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Compound Profiling using Reverse Protein Arrays
IGF receptor
IRSsPI3K(p110,p85)
PKB/Akt
GSK3
PDKs
inhibitor
Glut4
PIP2PIP3
PTEN
Regulation of glycogen synthase
Glucose transport
FHs(AFX1,FKHR)
Transcription
1 2 3 4 5-20
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P-GSK3beta
Log Concentration (nM)
% In
hibi
tion
trEC50 = 263 nM
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1.0
1.5
2.0
ctrl 0
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100 30
1 2 3 4 50
20
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60
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P-Akt (Ser473)
Log Concentration (nM)
% In
hibi
tion
0.00
0.25
0.50
ctrl 0
1000
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100 30
trEC50 = 171 nM
“trEC50" (transduced EC50) curves of an IGFR inhibitor at different pathway nodes
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0.001 0.01 0.1 1
compound conc (uM)
perc
ent o
f con
trol
-25
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0.01 0.1 1 10
compound conc (uM)
perc
ent o
f con
trol
0255075
100125150175200225250275300325350375400425
0.01 0.1 1 10
compound conc (uM)
perc
ent o
f con
trol
-25
0
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0.01 0.1 1 10
compound conc (uM)
perc
ent o
f con
trol
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0.01 0.1 1 10
compound conc (uM)
perc
ent o
f con
trol
cpd 1 cpd 2 cpd 3 cpd 4 cpd 5
Stimul.
plateau 60-70% of ctrl
Stimul. Stimul.
Stimul.cpd 5
<0.0140.4931.3490.012P-AKT
>10>104.2430.129P-ERK
>10>101.0480.012P-PLCg>10>100.4750.006P-STAT3
>10>101.0380.006P-EGFRcpd 4cpd 3cpd 2cpd 1
Effects on EGFR signaling in A431 cells + EGF
EGFR, EGFR PI3K MEKPI3Kinhibitor of:
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Detection and Monitoring of Biomarkers
Pathway Atlas Disease Model versus Wild-type
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HeartWT
HeartGSK3b +/-
PancreasWT
PancreasGSK3b +/-
GSK-3b
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GSK3b +/-
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Phospho-p44/42 MAPK
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WT
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GSK3b +/-
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GSK3b +/-
Phospho-PTEN
GSK-3b +/- GSK-3b +/+heart and pancreasheart and pancreas
In collaboration with Novartis
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In collaboration with Fernando Corrales, CIMA
Comparison of 16 pairs ofsex and age matchedControls (C) / Patients (P)
10 liver cirrhosiscandidate markers weremeasured whereasresults for one areillustrated here
Protein concentrations between 2.5 to 10 µg/mlNo concentration step required
Liver Cirrhosis Biomarker Evaluation in Urine
Microarrays for Highly Efficient Reconnaissance of Complex Signaling Network Systems
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