patlisci
TRANSCRIPT
Harry HeinzelmannVP Nanotechnology & Life Sciences
PATLiSci – Probe Array Technology for Life Sciences
Zurich, April 2012
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Probe Technology
PATLiSci – Probe Array Technology for Life Science Applications
IBM
for Cancer Research and Detection
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Project Partners
PATLiSci – Probe Array Technology for Life Science Applications
H. Vogel EPFL
Membrane prot. immobilisation
H.P. Herzig EPFL-IMT
Optics
A. Mariotti CePO, CHUV Melonoma progression
P. Romero LICR U Lausanne Head & neck
carcinoma
E. Meyer Ch. Gerber Uni Basel
Cantilever sensors
D. Rimoldi LICR U Lausanne
Melanoma
H. Heinzelmann CSEM (Coord) Probe array technologies
P. Renaud EPFL-IMT Fluidics
N. de Rooij, P. Vettiger, J. Brugger
EPFL-IMT, MEMS design & fab
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Probe Array Tech – high potential in Cancer Research
PATLiSci – Probe Array Technology for Life Science Applications
• cantilever arrays (without tips)for nanomechanical sensing
• measure the presence of minute concentrations of analytes (N channels)
• personalized healthcare & diagnostics
• probe arrays with tipsfor parallel force spectroscopy
• measure interaction forces and mechanical properties (N statistics)
• R&D, cell based screening
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Force Spectroscopy
• information about adhesion proteins, cell mechanics, kinetics, …
• statistics! parallel force spectroscopy novel cantilever deflection readout probe array microfabrication living melanoma cell array
PATLiSci – Probe Array Technology for Life Science Applications
source: JPK
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Motivation: metastatic cancer development
PATLiSci – Force Spectroscopy
Lee et al., Trends Biotechnol. 2007
all cells
tumor cells
normal cells
S.E. Cross et al., Nature Nanotech (2007)
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Probe Arrays for parallel force spectroscopy
PATLiSci – Force Spectroscopy
F. Loizeau et al., MicroNanoLetter 2012 (in press)
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SiN probe arrays with (active) actuation
PATLiSci – Force Spectroscopy
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Setup and readout
PATLiSci – Force Spectroscopy
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Elasticity of Melanoma cells
PATLiSci – Force Spectroscopy
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Elasticity, Mobility, Vesicle formation
PATLiSci – Force Spectroscopy
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Elasticity of different malignant cell types
• Probing melanoma cell elasticity by pulling and relaxing membrane nanotubes using optical tweezers
PATLiSci – Force Spectroscopy
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Parallel force spectroscopy on living cells
• Proof of Principle parallel force spectroscopy force on WM239 melanoma cells
PATLiSci – Force Spectroscopy
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Nanomechanical Sensing
detection in liquids :
• BRAF mutation in DNA samples
• capture of melanoma cells
detection in the gas phase :
• volatile organic compounds for early diagnosis
PATLiSci – Probe Array Technology for Life Science Applications
J. Fritz et al., Science 288, 316-318 (2000); D. Schmid et al., Eur. J. Nanomedicine 1, 44-47 (2008)
Cantilever is a Nanomechanical Sensorspecific adsorption/docking of molecules creates mechanical stress bending
melanoma naevus (National Cancer Inst.)
B-Raf oncogenein 50-60% of all melanoma tumors
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Principle of nanomechanical biosensing
• each cantilever is functionalized for molecular recognition (ex: oligonucleotides)
• Probe cantilevers coated with a specific layer for target capture
• Reference cantilevers coated with a non-specific layer
• Differential measurement reveals net signal and cancels thermal drift
PATLiSci – Nanomechanical Sensing
Injection Baseline
baseline
injection
di
ff. d
efle
ctio
n Dx
time
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Functionalization for BRAF V600E
PATLiSci – Nanomechanical Sensing
SH-GAGATTTCTCTGTAGCTA
SH-GAGATTTCTCTGTAGCTA
SH-GAGATTTCTCTGTAGCTA
SH-GAGATTTCTCTGTAGCTA
SH-ACACACACACACACACAC
SH-ACACACACACACACACAC
SH-ACACACACACACACACAC
SH-ACACACACACACACACAC
reference cantilever: unspecific oligonucleotide (#1-4) SH-ACACACACACACACACAC
sensing cantilever: probe oligonucleotide(#5-8) SH-GAGATTTCTCTGTAGCTA
Au / Ti layer (for thiol binding top side)
PEG-silane (for passivation) 1.
2.
3. thiol-oligonucleotide self-assembly
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Detection of single point BRAF mutation in DNA
PATLiSci – Nanomechanical Sensing
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Detection of single point BRAF mutation in RNA
PATLiSci – Nanomechanical Sensing
BRAF mutation in RNA detected within minutes !
0 20 40 60 80 100-100
-80
-60
-40
-20
0
20
40
*
diffe
rent
ial d
efle
ctio
n Dx
/nm
time /min
**
injection of RNA
injection of buffer
first personalized medical drug:• 60% of melanoma patients carry the
BRAF V600E mutation• RG7204 shows a significant survival
benefit in melanoma.
ZELBORAF available in Switzerland since Feb 2012
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Functionalization for melanoma cell capture
PATLiSci – Nanomechanical Sensing
PEG-silane (for passivation)
Au / Ti layer
antibodies covalently attached to Au Si
Melanoma cells expressing High Molecular Weight Melanoma-Associated Antigen (HMM-MAA)
antibodies:• sensing: anti-HMW-MAA highly specific to melanoma cells
anti-MHC-Class-I molecules less specific to melanoma cells• reference: anti-Hemagglutinin (HA) non-binding
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Specific capturing of melanoma cells
PATLiSci – Nanomechanical Sensing
N. Backmann et al., unpublished (2012)
specific Ab: -HLA-Class-I
non-specific Ab: -HA
specific Ab: -HMW-MAA
1
6
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Microfluidic system for preselection of cells
PATLiSci – Nanomechanical Sensing
• Separation of melanoma cells from single cell suspensions from biopsies
• goal: capture CTCs from blood samples
MINACEL extension, EPFL & UniBAScytometer chip (A) with electrodes (B)
• microfabricated flow cytometerbased on dielectrophoresis
• tests in progress
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Membrane Surface Stress Sensors & Electronics
PATLiSci – Nanomechanical Sensing
F. Loizeau, EPFL-IMT (2012)
A. Tonin, UniBAS
Inkjet spotting for MSS coatinge.g. polymers such as PSS, Dextran, CMC, PVP
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Membrane sensor response to solvent vapor
PATLiSci – Nanomechanical Sensing
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Principal Component Analysis (PCA)
PATLiSci – Nanomechanical Sensing
• measured signals from CL arraygive multi-dimensional data set
• PCA seeks „optimized“ projection matrix into 2 dimensionswith maximum information content
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Selectivity to breath and VOC samples
PATLiSci – Nanomechanical Sensing
test substance
clustering is robust againststorage for 48 h at 4°C and variations in injection cycles
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Next steps
• optimization of setup for parallel force spectroscopy
• demonstration experiment if possible on cell arrays, alternatively on vesicle arrays
• implementation of bioreactors and cell sorting with microfluidics (MINACEL)
• Clinical study with breath samples from head & neck cancer patientsObjective: identify cancer and track drug treatment efficacy
• approved by Ethics Committee April 2012, start of the study May 2012double-blind scheme with patients before and after treatment, and healthy people
• with Ludwig Center for Cancer Research LICR
PATLiSci – Probe Array Technology for Life Science Applications
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Thank you for your attention.
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Thank you for your attention.
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Literature – Force Spectroscopy on Cancer Cells
PATLiSci – Probe Array Technology for Life Science Applications
from S.E. Cross et al., Nanotechnology (2008)
from S.E. Cross et al., Nature Nanotech (2007)
all cells
Tumor cells
normal cells
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Safety Production
Food Quality
Environment
Diagnostics
Impact beyond the Scope of this Project
Probe Array Technology for Life Science Applications
Research,Screening
NEMS / nano
ICT / tera
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NADIS for Liquid Exchange with Living Cells
Nanotools – Nanoscale Dispensing
• injection after perforation of the cell membrane
• extraction of cytoplasm for remote analysis
• towards patch clamping
viable neuroblastoma cells Cell TrackerTM green staining
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Motivation: Metastatic cancer development
PATLiSci: Force spectroscopy
Adhesion properties of cells Elastic properties of cells
Characterization by force
spectroscopy
Parameters involved in metastatic proliferation
Weder et al. 2009Biointerph. 4:27
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Parallel force spectroscopy on living cells
• Harry, nous n’avons pas encore de telles courbes de force, mais nous en aurons une pour le meeting NanoTera
PATLiSci: Force spectroscopy
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Types of melanoma cell analyzed
• Primary melanocyte: non cancerous healthy cells used as reference
• Radial growth phase (RGP): Cells spread horizontally through the epidermis (cell line SBCL2)
• Vertical growth phase (VGP): Cells begin to grow deeper into the skin and invade the organism via the blood and lymphatic vessels (cell line WM115)
• Metastatic (M): Cells from metastasis in the organism (cell line WM239)
PATLiSci: Force spectroscopy
Radial growth phase Vertical growth phase
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it’s much much more than microscopy…
PATLiSci – Probe Array Technology for Life Science Applications
Müller and DufrêneNature Nanotechnology (2008)
U Pennsylvania
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Cancer is Relevant
PATLiSci – Probe Array Technology for Life Science Applications
bfs.admin.ch
• how do cancer cells differ in cell mechanical properties ?
• how do cancer cells adhere to substrates, or to other cells ?
• can we find better ways to detect cancer in an early stage ?
• can we bring a test device to POC?
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Cantilever Sensing – Outlook and Next Steps
in liquids
DNA, mRNA, and tumor cell detection
• melanoma associated antigens
• test of mutation/antigen and cell binding
• detection limits of the assays
• optimization of DNA and antigen binding
• optimization of cell capture
• implementation of a microfluidic system for an initial cell sorting step (PATLiSci extension MINACEL)
in gas phase
Breath analysis of from cancer patients
• feasibility EBS of head & neck cancer patients
• representative study on EBS of head & neck or lung cancer patients
• optimization of readout hard-/software
• functionality and reliability tests
• portable device prototype
• implementation of a micro bioreactorin combination with cantilever arrays (PATLiSci extension MINACEL)
PATLiSci – Probe Array Technology for Life Science Applications
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Force Spectroscopy – Outlook and Next Steps
• Measure cell elasticity at different growth phases
• Analysis of cell adhesion (cell-surface, cell-cell) in the presence of extra cellular matrix proteins
• Compact optical cantilever deflection read-out
• Individual cantilever actuation (force control)
• implementation of cell separation and sorting (PATLiSci extension MINACEL)
PATLiSci – Probe Array Technology for Life Science Applications
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Migration and invasion properties
• There is no clear relationship between cell stiffness and cell motility
PATLiSci – Force Spectroscopy
Sbcl2(in Tu) WM115 WM239A0
100
200
300
400
500Invasion
Prim Mel Sbcl2 WM115 WM239A0
100
200
300
400
500
600Migration
% m
igra
tion
com
pare
d to
WM
239A
cel
ls
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MINACEL: Micro- and Nanofluidics for Cell Handling
PATLiSci – Probe Array Technology for Life Science Applications
bring competence in fluidics to PATLiSci
• micro Bioreactor with tumor cells producing VOCs for gas phase analysis
• Cell Sorting device to isolate CTC and adherent cells
• Nanofluidics for single cell microinjection using NADIS technology
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backup slides
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Cantilever Sensing in Gaseous and in Liquid Environments
Non-Invasive Diagnostics for early detection of eg. lung, head & neck cancer • higher specificity and sensitivity to VOC with
coatings based on natural odorant receptors
• piezo-resistive cantilevers
• handheld device for POC applications
Detection of melanoma specific somatic mutations in blood samples• detection of dissolved tumor specific
markers with suitable anti-bodies, or direct binding of melanoma cells (CTC)
• no prior amplification or labeling
Probe Array Technology for Life Science Applications
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Project Goals
• develop point probe array system (microfabricated array and read-out system)
• demonstrate parallel measurement of cell mechanics
• demonstrate cell adhesion measurements with improved statistics
• assess potential in diagnostics and cell based screening
• improve performance of cantilever array sensors
• demonstrate detection of cancer via breath analysis
• improve sensitivity and demonstrate detection of disorders in patients’ blood samples via various biomarkers (library)
• integrate system into a handheld cantilever-based diagnostic device prototype
Probe Array Technology for Life Science Applications
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PROBART for Parallel Imaging
Nanotools – Probe Arrays
R lever
R ref
VEE (- 6V)Rlever
Rref
R1 R2
Vout
(~ 20 kohm)
4x4 array imaging inbuffer solution with continuous zoom-in
probe #6
probe #13
probe #15
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ArrayFM with Optical Read-out – First Results
Nanotools – Probe Arrays
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PROBART for Force Spectroscopy
Nanotools – Probe Arrays
600 pN/div
√
√√
√√
√√
in “expert reviews in molecular medicine”, http://www-ermm.cbcu.cam.ac.uk
Force resolution = 160 pN
sufficient for mostdonor/acceptor complexes
glass surface
PBS (0.01M)Polylysin (5mg/l)
18µm
6.4µm Mapping of the elastic response of a cell
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ArrayFM with Optical Read-out
Nanotools – Probe Arrays
where are we with this?
first demonstration in ambient conditions and on solid substrates
topography detail reproduced down to nm scale and nm sensitivity
what is still missing?
• improve sensitivity / noise equivalent force
• adapt optics to operation in liquids
• adapt optics to large arrays
• interface with software, data transfer
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ArrayFM with Optical Read-out – Some More Tricks
Nanotools – Probe Arrays
• solving phase ambiguity
• LabView based software interface
• Si and sol-gel replicated cantilevers
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Cell Adhesion Forces
Nanotools – Probe Arrays
what is still missing?
• work on arrays of cells(immobilized arrays)
• work on arrays of vesicles, and assess feasibility
• for cell-cell (vesicle-vesicle) studies, develop protocols on how to get these on the probe tip
• work on probes, tip geometry, functionalization
• work probe actuation
• work on probe array homogeneity, and alignment issues
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Thank you for your attention.