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PATLiSciProbe Array Technology for
Life Sciences
Ernst Meyer
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Project PartnersPATLiSci 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 2 promising approaches to
Cancer Research
cantilever arrays (without tips)for nanomechanical sensing
measure the presence of minute
concentrations of analytes (N channels)
personalized healthcare & diagnostics (PHC)
probe arrays with tipsfor parallel force spectroscopy
measure interaction forces and
mechanical properties (N statistics)
R&D, cell based screening
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Metastatic malignant melanoma at the heart
WikiMedia Commons
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Nanomechanical sensing of Human Melanoma
M. Volkenandt: Maligne Melanome. In:
Dermatologie und Venerologie.
O. Braun-Falco u. a. (Ed.), Verlag Springer,2005, S. 13131324
Melanoma on a patient's skin
(source : National CancerInstitute).
1960 20060.000
0.002
0.004
0.006
0.008
0.010
rateofincidence
year
development of lifetime risk for
melanoma in the last 50 years
(collaboration with University Hospital of Lausanne)
Different growth phases:
Radial, vertical, circulating tumor cells
Radial growthphase
Verticalgrowth phase
F. Huber et al,
Nature Nanotechnology
8, 125-129 (Feb. 2013)
http://upload.wikimedia.org/wikipedia/commons/6/6c/Melanoma.jpg7/28/2019 Patlisci, Nano-Tera 2013
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Principle of Nanomechanical
Biosensors
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
InjectionBaseline
baseline
injection
diff.deflectionDx
time
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Functionalization for BRAF V600E
Au/Ti layer for thiol binding
PEG-silane (Passivation lower side)1.
2.
3. Thiol-oligonucleotide self-assembly
SH-GAGATTTCTCTGTAGCTA
SH-GAGATTTCTCTGTAGCTA
SH-GAGATTTCTCTGTAGCTA
SH-GAGATTTCTCTGTAGCTA
SH-ACACACACACACACACAC
SH-ACACACACACACACACAC
SH-ACACACACACACACACAC
SH-ACACACACACACACACAC
Sensing cantilever: Probe oligonucleotide
Reference cantilever: unspecific oligonucleotide
Sensor : SH-GAGATTTC CTGTAGCTA
Reference : SH-ACACACACACACACACAC
: Single point mutation
B-Raf oncogene (Rapidly accelerating fibrosarcoma B)
=> Functionalization of cantilevers with specific RNA-
sequence to detect BRAF V600E
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60 80 100 120 140 160 180 200-120
-100
-80
-60
-40
-20
0
20
40
differentialdeflection/nm
time /min
wild type
wild type:V600E = 50:1wild type:V600E = 20:1
wild type:V600E = 10:1
wild type:V600E = 3:1
wild type:V600E = 1:1
V600E
buffer flow
10 ng/l DNA injection
Detection of melanoma specific
somatic mutations in B-Raf Concentration dependence
B-Raf oncogene (Rapidly accelerating fibrosarcoma B)
wt =
wild type,
not mutated
Wt / mut
SNP
F. Huber et al,
Nature Nanotechnology
8, 125-129 (Feb. 2013)
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F. Huber et al,
Nature Nanotechnology8, 125-129 (Feb. 2013)
BRAF concentration dependence - Langmuir isotherm
R2 = 0.97
indicates a
reliable fit to
the data.
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Braf V600E mutant response in total RNA background
BRafmtV600E _minus:
SH-GAGATTTCTCTGTAGCTA 18-mer
Unsp (reference):
SH-ACACACACACACACACAC 18-mer
Wt_long (reference):
SH-TAGCTACAGTGAAATCTC 18-mer
Sensing cantilever:
SH-BRafmt
Reference cantilever:
Unsp or Wt_long
10 20 30 40 50 60 70 80-80
-60
-40
-20
0
2020ng/l wild type total RNA
20ng/l V600E total RNA
buffer
RNA injection
differentiald
eflection/nm
time /min
0 10 20 30 40 50 60 70 80-80
-60
-40
-20
0
20
differentia
ldeflection/nm
time /min
100ng/l wild type total RNA
100ng/l V600E total RNA
buffer
RNA injection
Concentration dependence: 100 ng/l & 20 ng/l, no PCR required !
F. Huber et al,
Nature Nanotechnology8, 125-129 (Feb. 2013)
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25 35 45 55 65 75 85 95 105 115 125-80
-70
-60
-50
-40
-30
-20
-10
0
10
20
differentialdeflection(nm
)
time (min)
25 35 45 55 65 75 85 95 105 115 125-80
-70
-60
-50
-40
-30
-20
-10
0
10
20
differential
deflection(nm)
time (min)
BRAF V600E cells
BRAF wild type cells
SK-Mel37 V600E total RNA
Me275 V600E total RNA
Me246.M1 V600E total RNAT618A wild type total RNA
T1405B wild type total RNA
Buffer
100 ng/l total RNA
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50% of melanoma patients carry the BRAF V600E mutation:RG7204 shows a significant survival benefit in melanoma.
FDA-Early approval for blockbuster meloma drug Venurafenib
ZelborafFirst personalized medical drug
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Capture of Circulating Tumor Cells (CTC) using antibodies
covalently attached to Au-coated cantilever surfaces
Antibodies:
Sensing cantilevers: anti-HMW-MAA binding, highly specific to melanoma cells
anti-MHC-Class-I molecules binding, less specific to melanoma cells
Reference cantilevers: anti-Hemagglutinin (HA) non-binding
PEG-
silane
Au/Ti (20/2nm)
Antibodies covalently attached to Au
Functionalization protocol
Si
Melanoma cells expressing
High Molecular Weight Melanoma-
Associated Antigen (HMW-MAA)
J. Zhang, N. Backmann et al. 2013
The number of CTCs is
low, about 1 to 10 in a
billion cells per ml blood,
but more than 90% of all
cells are erythrocytes.
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Real-time capturing of live melanoma cells
4.2*105 cells/ml in RPMI/Hepes/Pen-strep
Injection of cells
0 2 4 6 8 10 12 14 16 18 20
-4.0
-3.0
-2.0
-1.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
cl2345/4-cl1ref
cl2345/4-cl6ref
cl2345/4-cl7ref
cl2345/4-cl8ref
cl7ref-cl8ref
cl6ref-cl8ref
cl1ref-cl7ref
d
ifferentialdeflectionm
t hr
J. Zhang et al. 2013
After washing
10 cells remain
adhered
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Experimental set-up for cell sorting
Rodrigo Martinez Duarte, Philippe Renaud EPFL
PATLiSci Extension MINACEL
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0.0E+00
5.0E+04
1.0E+05
1.5E+05
2.0E+05
2.5E+05
Control 1 2 3 4 5 6 7 8
cellspermililiter
15 ul fraction
Fraction concentration2.5Mhz//10l/min
viable
non-viable
FIELD OFF,
Cell release
Trapping viable Retrieval
viable
%viable 95 84 95 94 88 84 83 97 100
Rodrigo Martinez Duarte, Philippe Renaud EPFL
PATLiSci Extention MINACEL
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MINACEL
User friendly platform for viability assay
Relatively fast (~0.5 hour assay) for 15 ul
samples
Efficient at purifying viable population
Separating viable melanoma cells (~20 m) from
non viable ones and leukocytes (7-12 m)
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Force Spectroscopy
information about adhesion proteins,
cell mechanics, kinetics,
statistics! parallelforce spectroscopy
novel cantilever deflection
readout
probe array microfabrication
living melanoma cell array
source: JPK
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Microfabrication Summary
Development and fabrication of 3x8 probe arrays
Complex 2-wafers process
Molded SiN cantilevers
200 m
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Cantilevers with Various
Spring Constants Were Needed
V-grooves along the cantilevers allow to define their stiffness without
changing their footprint
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Cantilevers with Various
Spring Constants Were Needed
V-grooves along the cantilevers allow to define their stiffness without
changing their footprint
50 m
Li i C ll M i l ti R i
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Living Cells Manipulation Requires
a Tip with a Large Radius of Curvature
Silica or polystyrene bead glued on tip Repeatable and large radius of
curvature
Postprocess technique
Serial process
Thie, M., R. Rspel, et al. (1998). Human Reproduction 13(11): 3211-3219.
Oxide removal in a pyramidal mold Wafer scale process
Small radius of curvature ( R < 500
nm )
G.M. Kim, A. K., J. Holleman, J. Brugger (2002).
Journal of Nanoscience and Nanotechnology 2:
55-59.
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Spherical tips have been developed
with an advanced molding process
10 m
Hi h Ti W Add d i th
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Higher Tips Were Added in the
Corners to Align the Chip with the Surface
The Alignment Tips are 15 m
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The Alignment Tips are 15 m
Higher than the Spherical Tips
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Flat Tips Can also Be Created by DRIE
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SiN probe arrays with (active) actuationActuator working principle
Metal
Metal
Structural layer, SixNy
= 0
= 0
T
z
Published in IEEE Sensors Journal, Special edition, 2013 IEEE.
Thermal bimorph actuation.
Stress inducing metalliclayers are balanced.
Endpoint angle is keptconstant during deflection.
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SiN probe arrays with (active) actuationSEM images of actuated devices
Published in IEEE Sensors Journal, Special edition, 2013 IEEE.
Meandered bimorph
actuator
Cantilever
Top and bottom metal layerfunction both as resistors and as
stress-inducing layers (left).
Arrays to parallelizemeasurements (top).
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550m
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SiN probe arrays with (active) actuationDisplacement versus temperature in air
Published in IEEE Sensors Journal, Special edition, 2013 IEEE.
17 m displacement achieved in air.
Linear relation betweentemperature and displacement.
Thermomechanical sensitivity of77.6 nm/K.
Devices can be passivated for inliquid operation.
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SiN probe arrays with (active) actuationComsol simulations
Comsol simulations made to know the
temperature profile during actuation.
A substantial temperature increase canbe achieved locally at the electrode.
Temperature at the water-to-cell
substrate interface is dominated by the
temperature of the substrate.
Published in IEEE Sensors Journal, Special edition, 2013 IEEE.
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SiN probe arrays with (active) actuation
A novel actuator system for parallelized
cantilever arrays was created.
This part of the project is ongoing.
Fabrication-related challenges are
resolved. The system is to be tested for
its real application.
Published in IEEE Sensors Journal, Special edition, 2013 IEEE.
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Experimental platform for operating 2D
probe arrays
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Parallel force spectroscopy on SBCL2 cells
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Examples of analyzed curves
M l ll
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Elastic analysis of melanoma cells
(primary and cancerous)
The different phases of melanoma cancer
development
For each phase, one cell line
Radial growth phase: SBCL2
Vertical growth phase: WM115
Metastatic: WM239
Two types of analysis
AFM-based force indentation curves
Probing cell elasticity with optical tweezers
(OT-pulled nanotube relaxation time)
Melanoma cells
Nanotube relaxation kinetics allows to
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Nanotube relaxation kinetics allows to
distinguish between different melanoma cell
lines
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Influence of the substrate on the cell elasticity (by AFM)
The cells (primary melanocytes, RGP, VGP, metastatic) were grown ondifferent substrates with different incubation times
Primary
NHEM
RGP
SBCL2
VGP
WM115
Metastatic
WM239
PS 24h FN 24h FN 2h FN spot 2h PLL 2h
Pl ti it f l ll tiff i ith li t i
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0
200
400
600
800
1000
1200
1400
NHEM SBCl2 WM115 WM239A
Young'smodulus(P
a)
PS 24 h
FN 24 h
FN 2 h
FN-spots 2 h
PLL 2 hA B C D E A B C D E A B C D E A B C D E
malignant progression
normal melanocytes RGP VGP Met
Cell stiffness decreases during malignant progression from NHEM to RGP and VGP cells
(conform to literature, cancerous cells are softer as healthy cells)
But metastatic cells are stiffer than the 3 less malignant types of cell.
Our explanation:
Plasticity of melanoma cells stiffness, i.e. their ability to vary their stiffness in response
to external stimuli, increases with progression to VGP and metastatic phase
Plasticity of melanoma cells stiffness increases with malignant progression
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42http://diseasespictures.com/neck-cancer/
Head & Neck Cancer
Pi i ti
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43H.P. Lang et al. 2013
Piezoresistive
Membrane Surface
Stress Sensor
(PMSS) Results
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From Simulation
Single clamped cantilever (PROBART-like) Optimized single clamped cantilever
Membrane-type sensor
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To Actual Sensors
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500 Micrometers in Diameter
and 2.5 Micrometers in Thickness
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Membranes are sensitive, have a fast
response and are easy to use
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They also have a linear response over the entire
measurement range during humidity characterization
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Membrane-type sensors show higher sensitivity
(6x) and better reproducibility over optimized
cantilevers
Membrane-type sensorCantilevers
Pi i ti M b S f St S (PMSS)
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Piezoresistive Membrane Surface Stress Sensors (PMSS)
F. Loizeau, T. Akiyama, S. Gautsch, IMT EPFL
H.P. Lang et al., Univ. Basel 2012
G. Yoshikawa et al., Nano. Lett. 11, 1044 (2011) Diameter: 500 m, thickness: 2 m
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Functionalizsation of Membrane Sensors by Inkjet Technology
Towards non-invasive diagnostics based on breath analysis
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PCA case scores
Axis2
Axis 1
-0.2
-0.5
-0.7
-1.0
0.2
0.5
0.7
1.0
1.2
-0.2-0.5-0.7-1.0 0.2 0.5 0.7 1.0 1.2
Nitrogen
Breath sample of a
healthy patient
Acetone (Diabetes)
Dimethylamine
(Uramia)
Principal Component Analysis (PCA) scores
D. Schmid, P. Hunziker, Eur. J. Nanomedicine 1, 44-47 (2008)
Towards non-invasive diagnostics based on breath analysis
Principal Component Analysis (PCA) of Volatile Organic Compounds
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Principal Component Analysis (PCA) of Volatile Organic Compounds
H.P. Lang et al. 2011
A h i f f CHUV h i l
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Ash tray in front of CHUV hospital
Head and neck cancer patients and healthy persons are smokers
Double blind study
Di ti f H d & N k C
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Diagnostics of Head & Neck Cancer
HP Lang et al (Univ. Basel), 2012, J.P. Rivals, University Hospital Lausanne
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cured
healthy
cancer
HP Lang et al (Univ. Basel), 2013, J.P. Rivals et al.,
University Hospital Lausanne
cured
healthybifurcation
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S d O tl k
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Summary and Outlook
Arrays with self sensing cantilevers and suitable probing
tips were developed
Cantilever Sensing was used to detect BRAF V600E in
complete RNA background and different cell lines
Circulating Tumor Cells were catched by suitably sensed
cantilevers
Elasticity of melanoma cells at different stages were
analyzed by parallelized force spectroscopy
Head and neck cancer was detected by breath analysis
with cantilever arrays
First experiments with cell lung cancer seem promising
Thank you for your attention
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PATLiSci team
Uni Basel:
Andreas Tonin
Heinz Breitenstein
Sascha MartinHans Peter Lang
Franois Huber
Jiayun Zhang
Natalija Backmann
Christoph Gerber
Ernst Meyer
CSEM:
Gilles Weder
Mlanie Favre
Ral Ischer
Joanna Bitterli
Rita Smajda
Marta Giazzon
Martha Liley
Andr Meister
Harry Heinzelmann
Thank you for your attention.
EPFL-SAMLAB:
Frdric Loizeau
Terunobu Akiyama
Sebastian GautschPeter Vettiger
Nico de Rooij
EPFL-LMIS1:
Jonas Henriksson
Maurizio Gullo
Juergen Brugger EPFL-OPT
Laura Chantadasantodomingo
Eric Logan
Hans Peter Herzig
EPFL-LCPPM:Horst Pick
Horst Vogel
EPFL-LMIS4:
Rodrigo Martinez Duarte
Philippe Renaud
CHUV-Ludwig-Institute for Cancer Research
(LICR):
Jean-Paul Rivals
Agnes Hiou
Pedro RomeroDonata RimoldiMarielle Hendriks
Agnese Mariotti
B k Slid
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Back up Slides
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Representative lung cancer biomarkers
Probe Array Technologies
for Life Science Applications
H.P. Lang et al., 2012
P. Romero, J.P. Rivals, D. Rimoldi,
CHUV / Ludwig Institute for Cancer Research, Lausanne
Working principle of the compact system
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Working principle of the compact system
In order to measured the depletion of
multiplexed cantilevers, the deflected beams
have to be smartly distributed in the CCD.
A grating deposited on the cantilevers tip will
result in a diffraction spot line. The cantilever tilt
results in a shift of the spots line which
corresponds to a phase change in the Fourier
domain.
Cantilevers can be multiplexed in different directions and their displacements analyse in
the Fourier domain.
Results
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Results
200m
P=10m =10
200
200
#3
20
#9
200 200
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Principal Component Analysis (PCA)
explain the axis of the PCA plot, and how the analysis works SEE TEXT
Projection of multi-
dimensional data in
a two-dimensional plot
Among the different
possibilities for projection,
PCA reveals maximized
information content