Planar Waveguides: How Nano Layers Enable to Detect Zepto Moles of Macro Molecules in Pico Liter Spots on Micro Arrays

Transcription

1 Planar Waveguides: How Nano Layers Enable to Detect Zepto Moles of Macro Molecules in Pico Liter Spots on Micro Arrays Dr. Markus Ehrat Zeptosens A Division of Bayer Schweiz AG SSOM Meeting March 16 /17 29 Engelberg What are Biochemical Microarrays? 1 to 1 spots per cm 2 = photo lithography arrays 1 to 1 spots per cm 2 = spotted arrays Each spot binds a specific analyte of the sample solution Detection signal: e.g. fluorescence labels Spots with selective recognition elements Glass or polymer support (e.g. microscope slide) 1

2 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 #Entries

3 Information Obtained from 2 µl of Sample 96 well plate Detection area (mm 2 ) 4 Information per cm 2* well plate 15 µm spot microarray 15 µm spot microarray 1.5 µm spot microarray x x x * Sample solution of 2 µl Microarrays - Small detection areas Nanoliters of sample volumes: Require high detection sensitivity 3

4 Thin Film Planar Waveguide (PWG) Chip Design Grating Period: 2-75 nm Depth: 5-2 nm Ta 2 O 5 waveguiding layer 1 2 nm glass substrate Light Intensity vs. Waveguide Thickness d = 1nm d = 15nm d = 2nm d = 25nm d = 3nm Parameters: n sub =1.52, n sup =1.335, n PWG =2.15, m=, λ=635nm 4

5 Planar Waveguide Principle - High Sensitivity Fluorescence Microarray Detection surface confined excitation of bound label CCD camera Advantages of Fluorescence Excitation on PWG Conventional excitation ZeptoREADER - Evanescent excitation sample solution support background problems Confocal excitation: ~ no background Evanescent excitation: Separation of excitation and detection directions Ultimate sensitivity Fast time to result Less sample preparation Direct measurement in blood or serum Focus depth ~ 2µm Depth ~ 1nm 5

6 ZeptoREADER PWG Inside Ultra sensitive: Planar wave guide technology based evanescent field fluorescence excitation Exceptionally fast: Over 12 data points in 6 hours Increase efficiency: Extended walk away time using 6 slides integrated autoloader Absolutely reliable: Swiss designed and manufactured for highest quality and precision possible ZeptoREADER Setup Plate Storage Electronics Optics Measurement Chamber 6

7 Optical Scheme of the ZeptoREADER TM 532 nm 635 nm 1. Laser 2. Shutter 3. Gray filter wheel 4. Coupling unit 5. ZeptoCHIP TM 6. Front lens unit 7. Emission filter wheel 8. Camera lens unit and CCD High Sensitivity of PWG Signal Detection Dilution series 1..1 pm Cy5-BSA fluorescence intensity x image saturation (16 bit) background Cy5-BSA [pm] LOD = 1 zeptomol (6 proteins) per spot 7

8 ZeptoMARK Assay Sensitivity Spiking series of recombinant receptor in 2 mg/ml lysate RFI (avg 3 arrays) Spotted and analyzed at 4 dilutions, duplicate spots Average and std dev of 3 arrays RFI () + 3 σ c (rec. receptor) / pm LOD of ~2 receptor protein molecules per spot mean RFI of 2 arrays mean RFI of 2 arrays Assay Reproducibility: Different Operators Different Days Different ZeptoREADERs operator 1 - ZeptoREADER 1 operator 2 - ZeptoREADER operator 1 - ZeptoREADER 1 operator 2 - ZeptoREADER P-Akt (Ser473) lysate samples P-marker A lysate sample 8

9 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 5 years Cancer therapies are still essentially one size fits all,... 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 drug development Science, Vol 312, May 26, pp 1157, 1165, 1166, (26) 9

10 Signatures Guide Drug Choice Julian Downward. 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 Nature 439, (19 January 26) Pathway branching PI3K/AKT pathway Proliferation Survival NF-κB IKK PDK Akt PI3K PIP2 PIP3 PTEN Src p27 p21 MDM2 Caspase BAD FOXO ASK GSK3β mtor p53 Bcl-2 FasL β-catenin p7 S6K 4EBP Cell Cycle Apoptosis Proliferation Protein Progression Resistance Migration Translation 1

11 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 1 or more elements Conclusion: an array-based solution will provide the required scalability and throughput Protein Microarrays Two Formats Forward Protein Arrays Reverse Protein Arrays Array of target-specific capture molecules (e.g. antibodies) One sample measured per array Array of samples on chip One analyte measured per array 11

12 Reverse Protein Microarrays Cell Lysate Arrays Array of samples Sample volume 4 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 ZeptoMARK Reverse Arrays From Cells to Protein Profiles Cellular systems Cell lysis Spotting Blocking I II I II III III IV IV V V V V Ab1 Ab1 Ab2 Ab2 Ab3 Ab3 Ab4 Ab4 Ab5 Ab5 Ab6 Ab6 Pathway Profiles Data evaluation Readout Assay 12

13 Zeptosens Reverse Arrays From Cells to Protein Profiles # 1...# n Cellular systems Drug treatment Cell lysis Spotting Sample ~1 5 cells Lysis 5 µl lysis Spotting 4 pl spotting ~ 1 mg tissue 2 mg total protein/ml Spotting: Reproducibility & Quality 4pL 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% 13

14 Chip and Array Layout 6 Arrays / Chip Ref Ref Ref Ref Zeptosens Buffer Standards (Ref) 32 Reference Sample dilutions (mg/ml) Reference reference spots containing constant fluorescence up to 32 samples (4 dilutions, duplicates) per array up to 192 samples (4 dilutions, duplicates) per chip Highly Parallel Monitoring of Signaling Events Treatment Raf Ab Erk2 Ab P-Erk2 Ab 6 identical Lysate Arrays P-Akt Ab Akt Ab Bcl-2 Ab Effect From Cell Signaling Technology 14

15 The System Response Profile Buffer Standards <RFI> Sample # Stimulation with Insulin Inhibition with Wortmannin Insulin.4.3 P-Akt (S473) InsR IGFR IRSs RFI Ctrl EGF(t) RFI.2.1. P-Akt (S473) W(t) W+EGF(t) W(c)+Ins Ctrl EGF(t) Ins(t) W(t) W+EGF(t) Cell growth W(c)+Ins mtor S6 kinase S6 Ins(t) PKB/Akt GSK3 PI3K (p11,p85) PIP 3 PIP 2 PTEN PDKs Glycogen synthase Wortmannin A431, starvation over night Stimulation with Insulin (15nM) 7 time points, -24min 15

16 Compound Profiling using Reverse Protein Arrays P-Akt (Ser473) IGF receptor 12 1 trec 5 = 171 nm PTEN PI3K (p11,p85) IRSs inhibitor % Inhibition PIP 2 PIP 3 PDKs 2. ctrl Log Concentration (nm) FHs (AFX1,FKHR) Transcription PKB/Akt GSK3 Glut4 Glucose transport Regulation of glycogen synthase % Inhibition P-GSK3beta trec 5 = 263 nm ctrl Log Concentration (nm) trec5" (transduced EC5) curves of an IGFR inhibitor at different pathway nodes Effects on EGFR signaling in A431 cells + EGF cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 percent of control percent of control percent of control percent of control percent of control compound conc (um) compound conc (um) compound conc (um) compound conc (um) compound conc (um) P-EGFR cpd 1.6 cpd cpd 3 >1 cpd 4 >1 cpd 5 Stimul. P-STAT3 P-PLCg >1 >1 >1 >1 Stimul. Stimul. P-ERK >1 >1 plateau 6-7% of ctrl P-AKT <.14 Stimul. inhibitor of: EGFR, EGFR PI3K PI3K MEK 16

17 Detection and Monitoring of Biomarkers Pathway Atlas Disease Model versus Wild-type GSK-3b +/- GSK-3b +/+ heart and pancreas heart and pancreas 3 GSK-3b 12 GSK-3a Heart WT Heart GSK3b +/- Pancreas WT Pancreas GSK3b +/- Heart WT Heart GSK3b +/- Pancreas WT Pancreas GSK3b +/- 6 Phospho-p44/42 MAPK 2 Phospho-PTEN Heart Heart Pancreas Pancreas Heart Heart Pancreas Pancreas WT GSK3b +/- WT GSK3b +/- WT GSK3b +/- WT GSK3b +/- In collaboration with Novartis 17

18 Liver Cirrhosis Biomarker Evaluation in Urine Comparison of 16 pairs of sex and age matched Controls (C) / Patients (P) 1 liver cirrhosis candidate markers were measured whereas results for one are illustrated here Protein concentrations between 2.5 to 1 µg/ml No concentration step required In collaboration with Fernando Corrales, CIMA Microarrays for Highly Efficient Reconnaissance of Complex Signaling Network Systems 18