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PATLiSci – Probe Array Technology for Life Sciences

Ernst Meyer

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PATLiSci – Probe Array Technology for Life Science Applications

Project Partners
E. Meyer Ch. Gerber Uni Basel Cantilever sensors H. Heinzelmann CSEM (Coord) Probe array technologies

H.P. Herzig EPFL-IMT Optics H. Vogel EPFL Membrane prot. immobilisation

N. de Rooij, P. Vettiger, J. Brugger EPFL-IMT, MEMS design & fab P. Romero LICR U Lausanne Head & neck carcinoma

D. Rimoldi LICR U Lausanne Melanoma

P. Renaud EPFL-IMT Fluidics

A. Mariotti CePO, CHUV Melonoma progression

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Probe Array Tech – 2 promising approaches to Cancer Research

• probe arrays with tips for parallel force spectroscopy • measure interaction forces and mechanical properties (N statistics)

• cantilever arrays (without tips) for nanomechanical sensing • measure the presence of minute concentrations of analytes (N channels) • personalized healthcare & diagnostics (PHC)
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• R&D, cell based screening

Metastatic malignant melanoma at the heart

WikiMedia Commons

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Nanomechanical sensing of Human Melanoma
(collaboration with University Hospital of Lausanne)

Melanoma on a patient's skin (source : National Cancer Institute).

M. Volkenandt: Maligne Melanome. In: Dermatologie und Venerologie. O. Braun-Falco u. a. (Ed.), Verlag Springer, 2005, S. 1313–1324
development of lifetime risk for melanoma in the last 50 years

rate of incidence

Different growth phases: Radial, vertical, circulating tumor cells
Radial growth phase Vertical growth phase

0.010

0.008

0.006

0.004

0.002

0.000

1960

2006

year

F. Huber et al, Nature Nanotechnology 8, 125-129 (Feb. 2013)

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Principle of Nanomechanical Biosensors
diff. deflection Dx

Baseline Injection
baseline injection

time

• 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
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Functionalization for BRAF V600E
B-Raf oncogene (Rapidly accelerating fibrosarcoma B) => Functionalization of cantilevers with specific RNAsequence to detect BRAF V600E
: Single point mutation

Sensor : SH-GAGATTTC CTGTAGCTA Reference : SH-ACACACACACACACACAC 1. 2. PEG-silane (Passivation lower side) Au/Ti layer for thiol binding

SH-ACACACACACACACACAC SH-ACACACACACACACACAC SH-ACACACACACACACACAC SH-ACACACACACACACACAC SH-GAGATTTCTCTGTAGCTA

3.

Thiol-oligonucleotide self-assembly

Sensing cantilever: Probe oligonucleotide

SH-GAGATTTCTCTGTAGCTA
SH-GAGATTTCTCTGTAGCTA SH-GAGATTTCTCTGTAGCTA

Reference cantilever: unspecific oligonucleotide

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Detection of melanoma specific somatic mutations in B-Raf
• Concentration dependence B-Raf oncogene (Rapidly accelerating fibrosarcoma B)

40 20
wild type wild type:V600E = 50:1 wild type:V600E = 20:1 wild type:V600E = 10:1 wild type:V600E = 3:1 wild type:V600E = 1:1 V600E buffer flow 10 ng/µl DNA injection

differential deflection /nm

0 -20 -40 -60 -80 -100 -120 60 80 100 120 140 160 180 200

Wt / mut SNP
wt = wild type, not mutated

F. Huber et al, Nature Nanotechnology 8, 125-129 (Feb. 2013)

time /min

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BRAF concentration dependence - Langmuir isotherm

R2 = 0.97 indicates a reliable fit to the data.

F. Huber et al, Nature Nanotechnology 8, 125-129 (Feb. 2013)

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Braf V600E mutant response in total RNA background
Concentration dependence: 100 ng/μl & 20 ng/μl, no PCR required !
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100ng/µl wild type total RNA 100ng/µl V600E total RNA buffer RNA injection

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differential deflection /nm

differential deflection /nm

0

0

20ng/µl wild type total RNA 20ng/µl V600E total RNA buffer RNA injection

-20

-20

-40

-40

-60

-60

-80 0 10 20 30 40 50 60 70 80

-80 10 20 30 40 50 60 70 80

time /min BRafmtV600E _minus: SH-GAGATTTCTCTGTAGCTA 18-mer Unsp (reference): SH-ACACACACACACACACAC 18-mer Wt_long (reference): SH-TAGCTACAGTGAAATCTC 18-mer

time /min

Sensing cantilever: SH-BRafmt Reference cantilever: Unsp or Wt_long

F. Huber et al, Nature Nanotechnology 8, 125-129 (Feb. 2013)

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20 10 0

differential deflection (nm)

-10 -20 -30 -40 -50 -60 -70 -80 25 35 45 55 65 75 85 95 105 115 125

BRAF V600E cells

time (min)

20 10 0

differential deflection (nm)

-10 -20 -30 -40 -50 -60 -70 -80 25 35 45 55 65 75 85 95 105 115 125

SK-Mel37 V600E total RNA Me275 V600E total RNA Me246.M1 V600E total RNA T618A wild type total RNA T1405B wild type total RNA Buffer 100 ng/µl total RNA

BRAF wild type cells

time (min)

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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

Zelboraf First personalized medical drug

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Capture of Circulating Tumor Cells (CTC) using antibodies covalently attached to Au-coated cantilever surfaces
Functionalization protocol
Melanoma cells expressing The number of CTCs is High Molecular Weight MelanomaAssociated Antigen (HMW-MAA) low, about 1 to 10 in a billion cells per ml blood, Antibodies covalently attached to Au but more than 90% of all Au/Ti (20/2nm) cells are erythrocytes. PEGsilane

Si

Antibodies:
Sensing cantilevers: Reference cantilevers: anti-HMW-MAA anti-MHC-Class-I molecules anti-Hemagglutinin (HA) binding, highly specific to melanoma cells binding, less specific to melanoma cells non-binding

J. Zhang, N. Backmann et al. 2013

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Real-time capturing of live melanoma cells 4.2*105 cells/ml in RPMI/Hepes/Pen-strep

8.0 7.0 6.0

differential deflection m

Injection of cells

5.0 4.0 3.0 2.0 1.0 0.0 -1.0 -2.0 -3.0 -4.0 0 2 4

cl2345/4-cl1ref cl2345/4-cl6ref cl2345/4-cl7ref cl2345/4-cl8ref cl7ref-cl8ref cl6ref-cl8ref cl1ref-cl7ref

After washing 10 cells remain adhered
6 8 10 12 14 16 18 20

t hr

J. Zhang et al. 2013

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Experimental set-up for cell sorting

Rodrigo Martinez Duarte, Philippe Renaud EPFL PATLiSci Extension MINACEL
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Fraction concentration 2.5Mhz//10µl/min
2.5E+05

FIELD OFF, Cell release
2.0E+05

viable non-viable

cells per mililiter

1.5E+05

Trapping viable
1.0E+05

Retrieval viable

5.0E+04

0.0E+00

Control

1

2

3

4

5

6

7

8

15 ul fraction

%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 µm) from non viable ones and leukocytes (7-12 µm)

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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

source: JPK 19

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
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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. Röspel, et al. (1998). Human Reproduction 13(11): 32113219.

• 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

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Higher Tips Were Added in the Corners to Align the Chip with the Surface

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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) actuation
Actuator working principle

Metal

Structural layer, SixNy 𝜃

= 0
Metal

ΔT

• •

Thermal bimorph actuation. Stress inducing metallic layers are «balanced».


Δz 𝜃
= 0

Endpoint angle is kept constant during deflection.

Published in IEEE Sensors Journal, Special edition, ©2013 IEEE. 28

SiN probe arrays with (active) actuation
SEM images of actuated devices

Meandered bimorph actuator

Cantilever

• •

Top and bottom metal layer function both as resistors and as stress-inducing layers (left). Arrays to parallelize measurements (top).

Published in IEEE Sensors Journal, Special edition, ©2013 IEEE.
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30

550 µm

SiN probe arrays with (active) actuation
Displacement versus temperature in air

• • • •

17 µm displacement achieved in air. Linear relation between temperature and displacement. Thermomechanical sensitivity of 77.6 nm/K. Devices can be passivated for in liquid operation.

Published in IEEE Sensors Journal, Special edition, ©2013 IEEE.

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SiN probe arrays with (active) actuation
Comsol simulations

• Comsol simulations made to know the temperature profile during actuation. • A substantial temperature increase can be 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. 32

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. 33

Experimental platform for operating 2D probe arrays

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PATLiSci – Force Spectroscopy

PCM and interferometric images of an array and cells
Phase contrast microscope image Interferometric microscope image

cells

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Parallel force spectroscopy on SBCL2 cells

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Examples of analyzed curves

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Melanoma cells

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)

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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 on different substrates with different incubation times

Primary NHEM RGP SBCL2

VGP WM115 Metastatic WM239 PS 24h FN 24h FN 2h FN spot 2h PLL 2h
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Plasticity of melanoma cells stiffness increases with malignant progression
1400 1200 PS 24 h

Young's modulus (Pa)

1000 800

malignant progression

FN 24 h

FN 2 h 600 400 200
A B C D E A B C D E A B C D E A B C D E

FN-spots 2 h

PLL 2 h

0 NHEM normal melanocytes SBCl2 RGP WM115 VGP WM239A 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

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Head & Neck Cancer

http://diseasespictures.com/neck-cancer/
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Piezoresistive Membrane Surface Stress Sensor (PMSS) Results

H.P. Lang et al. 2013

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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
Cantilevers Membrane-type sensor

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Piezoresistive Membrane Surface Stress Sensors (PMSS)
G. Yoshikawa et al., Nano. Lett. 11, 1044 (2011) Diameter: 500 μm, thickness: 2 μm

F. Loizeau, T. Akiyama, S. Gautsch, IMT EPFL H.P. Lang et al., Univ. Basel 2012

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Functionalizsation of Membrane Sensors by Inkjet Technology

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Towards non-invasive diagnostics based on breath analysis
Principal Component Analysis (PCA) scores
PCA case scores
1.2

Nitrogen Acetone (Diabetes) Dimethylamine (Uramia)

1.0

0.7

0.5

Axis 2

0.2

-1.0

-0.7

-0.5

-0.2 -0.2

0.2

0.5

0.7

1.0

1.2

-0.5

Breath sample of a healthy patient

-0.7

-1.0 Axis 1

D. Schmid, P. Hunziker, Eur. J. Nanomedicine 1, 44-47 (2008)

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Principal Component Analysis (PCA) of Volatile Organic Compounds

H.P. Lang et al. 2011

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Ash tray in front of CHUV hospital

• •

Head and neck cancer patients and healthy persons are smokers Double blind study

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Diagnostics of Head & Neck Cancer

HP Lang et al (Univ. Basel), 2012, J.P. Rivals, University Hospital Lausanne 55

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cancer

cured bifurcation healthy cured healthy

HP Lang et al (Univ. Basel), 2013, J.P. Rivals et al., University Hospital Lausanne
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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

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Thank you for your attention.
Uni Basel: Andreas Tonin Heinz Breitenstein Sascha Martin Hans Peter Lang François Huber Jiayun Zhang Natalija Backmann Christoph Gerber Ernst Meyer CSEM: Gilles Weder Mélanie Favre Réal Ischer Joanna Bitterli Rita Smajda Marta Giazzon Martha Liley André Meister Harry Heinzelmann
EPFL-SAMLAB: Frédéric Loizeau Terunobu Akiyama Sebastian Gautsch Peter 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 Romero Donata Rimoldi Marielle Hendriks Agnese Mariotti

PATLiSci team
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Back up Slides

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Representative lung cancer biomarkers

P. Romero, J.P. Rivals, D. Rimoldi, CHUV / Ludwig Institute for Cancer Research, Lausanne H.P. Lang et al., 2012 Probe Array Technologies for Life Science Applications
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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.

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Results
200m P=10m
#3 𝑁𝑝

=10

200 𝜇𝑚 200 𝜇𝑚

#9

20°

200 𝜇𝑚

200 𝜇𝑚

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Principal Component Analysis (PCA)
explain the axis of the PCA plot, and how the analysis works SEE TEXT
Projection of multidimensional data in a two-dimensional plot Among the different possibilities for projection, PCA reveals maximized information content

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