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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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Cy5-BSA [pM]

High Sensitivity of PWG Signal Detection

image saturation (16 bit)

background

Dilution series

1..1

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pM

Cy5

-BSA

LOD = 1 zeptomol (600 proteins) per spot

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rray

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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

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LOD of ~2000 receptor protein molecules per spot

Assay Reproducibility: Different Operators – Different Days –Different ZeptoREADERs

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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

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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

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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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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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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

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W(t)

W+E

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t)

W(c

)+In

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Ins(

t)

P-Akt (S473)

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Cellgrowth

A431, starvation over nightStimulation with Insulin(150nM) 7 time points,

0-240min

Wortmannin

Ctrl

EG

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W(t)

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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

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P-GSK3beta

Log Concentration (nM)

% In

hibi

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trEC50 = 263 nM

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Log Concentration (nM)

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trEC50 = 171 nM

“trEC50" (transduced EC50) curves of an IGFR inhibitor at different pathway nodes

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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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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