PATLiSci

Harry HeinzelmannVP Nanotechnology & Life Sciences

PATLiSci – Probe Array Technology for Life Sciences

Zurich, April 2012

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 2

Probe Technology

PATLiSci – Probe Array Technology for Life Science Applications

IBM

for Cancer Research and Detection


Project Partners


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


Probe Array Tech – high potential in Cancer Research


• 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


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


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)


Probe Arrays for parallel force spectroscopy


F. Loizeau et al., MicroNanoLetter 2012 (in press)


SiN probe arrays with (active) actuation



Setup and readout



Elasticity of Melanoma cells



Elasticity, Mobility, Vesicle formation



Elasticity of different malignant cell types

• Probing melanoma cell elasticity by pulling and relaxing membrane nanotubes using optical tweezers



Parallel force spectroscopy on living cells

• Proof of Principle parallel force spectroscopy force on WM239 melanoma cells



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


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

http://upload.wikimedia.org/wikipedia/commons/6/6c/Melanoma.jpg

http://upload.wikimedia.org/wikipedia/commons/6/6c/Melanoma.jpg


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


Functionalization for BRAF V600E


SH-GAGATTTCTCTGTAGCTA




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


Detection of single point BRAF mutation in DNA



Detection of single point BRAF mutation in RNA


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


Functionalization for melanoma cell capture


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


Specific capturing of melanoma cells


N. Backmann et al., unpublished (2012)

specific Ab: -HLA-Class-I

non-specific Ab: -HA

specific Ab: -HMW-MAA

1

6


Microfluidic system for preselection of cells


• 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


Membrane Surface Stress Sensors & Electronics


F. Loizeau, EPFL-IMT (2012)

A. Tonin, UniBAS

Inkjet spotting for MSS coatinge.g. polymers such as PSS, Dextran, CMC, PVP


Membrane sensor response to solvent vapor



Principal Component Analysis (PCA)


• measured signals from CL arraygive multi-dimensional data set

• PCA seeks „optimized“ projection matrix into 2 dimensionswith maximum information content


Selectivity to breath and VOC samples


test substance

clustering is robust againststorage for 48 h at 4°C and variations in injection cycles


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



Thank you for your attention.




Literature – Force Spectroscopy on Cancer Cells


from S.E. Cross et al., Nanotechnology (2008)

from S.E. Cross et al., Nature Nanotech (2007)

all cells

Tumor cells

normal cells


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


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


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


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



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)


Radial growth phase Vertical growth phase


it’s much much more than microscopy…


Müller and DufrêneNature Nanotechnology (2008)

U Pennsylvania


Cancer is Relevant


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?


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)



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)



Migration and invasion properties

• There is no clear relationship between cell stiffness and cell motility


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


MINACEL: Micro- and Nanofluidics for Cell Handling


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


backup slides


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



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



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


ArrayFM with Optical Read-out – First Results



PROBART for Force Spectroscopy


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


ArrayFM with Optical Read-out


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


ArrayFM with Optical Read-out – Some More Tricks


• solving phase ambiguity

• LabView based software interface

• Si and sol-gel replicated cantilevers


Cell Adhesion Forces


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



PATLiSci

Documents

parallel force

patlisci nanomechanical

patlisci force

probe array

nanotools

life science

patlisci extension

force spectroscopy