Patlisci, Nano-Tera 2013

7/28/2019 Patlisci, Nano-Tera 2013

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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.jpg


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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-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



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


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


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.



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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.



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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

Documents

Patlisci, Nano-Tera 2013