Patlisci - Probe Array Technology For Life Sciences

PATLiSci Probe Array Technology for Life Sciences
Harry Heinzelmann VP Nanotechnology & Life Sciences

Zurich, April 2012
PATLiSci Probe Array Technology for Life Science Applications
Probe Technology for Cancer Research and Detection
IBM
Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 1
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 A. Mariotti CePO, CHUV Melonoma progression
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
Probe Array Tech high potential in 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)
R&D, cell based screening
personalized healthcare & diagnostics
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
PATLiSci Force Spectroscopy
Motivation: metastatic cancer development
all cells
normal cells
tumor cells
Lee et al., Trends Biotechnol. 2007
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
Cantilever is a Nanomechanical Sensor

specific adsorption/docking of molecules creates mechanical stress bending
B-Raf oncogene in 50-60% of all melanoma tumors
melanoma naevus (National Cancer Inst.)
J. Fritz et al., Science 288, 316-318 (2000); D. Schmid et al., Eur. J. Nanomedicine 1, 44-47 (2008)
PATLiSci Nanomechanical Sensing
Principle of nanomechanical biosensing
diff. deflection Dx
baseline
Baseline Injection
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
Functionalization for BRAF V600E

1. 2. 3. PEG-silane (for passivation) Au / Ti layer (for thiol binding top side) thiol-oligonucleotide self-assembly
SH-ACACACACACACACACAC SH-ACACACACACACACACAC SH-ACACACACACACACACAC SH-ACACACACACACACACAC
reference cantilever: unspecific oligonucleotide (#1-4) SH-ACACACACACACACACAC
SH-GAGATTTCTCTGTAGCTA SH-GAGATTTCTCTGTAGCTA SH-GAGATTTCTCTGTAGCTA
sensing cantilever: (#5-8)
probe oligonucleotide SH-GAGATTTCTCTGTAGCTA
SH-GAGATTTCTCTGTAGCTA
Detection of single point BRAF mutation in DNA
Detection of single point BRAF mutation in RNA

40
differential deflection Dx /nm
20 0 -20 -40 -60 -80 -100 0 20 40
injection of RNA *
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
injection of buffer **
60 80 100
time /min
BRAF mutation in RNA detected within minutes !
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: reference: anti-HMW-MAA anti-MHC-Class-I molecules anti-Hemagglutinin (HA) highly specific to melanoma cells less specific to melanoma cells non-binding
Specific capturing of melanoma cells

1
non-specific Ab: -HA

specific Ab: -HMW-MAA
specific Ab: -HLA-Class-I
N. Backmann et al., unpublished (2012)

Microfluidic system for preselection of cells

Separation of melanoma cells from single cell suspensions from biopsies goal: capture CTCs from blood samples
microfabricated flow cytometer based on dielectrophoresis

tests in progress
cytometer chip (A) with electrodes (B) MINACEL extension, EPFL & UniBAS
Membrane Surface Stress Sensors & Electronics
A. Tonin, UniBAS
F. Loizeau, EPFL-IMT (2012)
Inkjet spotting for MSS coating e.g. polymers such as PSS, Dextran, CMC, PVP
Membrane sensor response to solvent vapor
Principal Component Analysis (PCA)

measured signals from CL array give multi-dimensional data set
PCA seeks optimized projection matrix into 2 dimensions with maximum information content
Selectivity to breath and VOC samples

test substance
clustering is robust against storage for 48 h at 4C 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 patients Objective: identify cancer and track drug treatment efficacy approved by Ethics Committee April 2012, start of the study May 2012 double-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

all cells
Tumor cells
normal cells
from S.E. Cross et al., Nanotechnology (2008)
from S.E. Cross et al., Nature Nanotech (2007)
Probe Array Technology for Life Science Applications
Impact beyond the Scope of this Project
Safety
Production
NEMS / nano
Research, Screening Diagnostics
Environment
Food Quality
ICT / tera
Nanotools Nanoscale Dispensing
NADIS for Liquid Exchange with Living Cells

injection after perforation of the cell membrane extraction of cytoplasm for remote analysis towards patch clamping
viable neuroblastoma cells Cell TrackerTM green staining
PATLiSci: Force spectroscopy
Motivation: Metastatic cancer development

Parameters involved in metastatic proliferation Adhesion properties of cells Elastic properties of cells
Characterization by force spectroscopy
Weder et al. 2009 Biointerph. 4:27

Parallel force spectroscopy on living cells

Harry, nous navons 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
its much much more than microscopy
U Pennsylvania
Mller and Dufrne Nature Nanotechnology (2008)

Cancer is Relevant
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?

bfs.admin.ch
Cantilever Sensing Outlook and Next Steps

in liquids in gas phase
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)
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 bioreactor in 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
Migration
% migration compared to WM239A cells
Invasion
500
700 600 500 400 300 200 100 0
400
300
200
100
Prim Mel
Sbcl2
WM115
WM239A
Sbcl2(in Tu)
WM115
WM239A
There is no clear relationship between cell stiffness and cell motility
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 melanoma specific somatic detection of eg. lung, head & neck cancer mutations in blood samples
higher specificity and sensitivity to VOC with detection of dissolved tumor specific coatings based on natural odorant receptors markers with suitable anti-bodies, or direct binding of melanoma cells (CTC) piezo-resistive cantilevers handheld device for POC applications 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
Nanotools Probe Arrays
PROBART for Parallel Imaging
VEE (- 6V)
Rlever
(~ 20 kohm)
Rref
R ref
R1
Vout R2
R lever
probe #6 probe #13 probe #15
4x4 array imaging in buffer solution with continuous zoom-in
ArrayFM with Optical Read-out First Results
PROBART for Force Spectroscopy

600 pN/div
Polylysin (5mg/l)
PBS (0.01M) glass surface
Force resolution = 160 pN
sufficient for most donor/acceptor complexes

in expert reviews in molecular medicine, http://www-ermm.cbcu.cam.ac.uk
18m
6.4m
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

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PATLiSci Probe Array Technology for Life Sciences

Harry Heinzelmann VP Nanotechnology & Life Sciences

PATLiSci Probe Array Technology for Life Science Applications

Probe Technology for Cancer Research and Detection

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 1

PATLiSci Probe Array Technology for Life Science Applications

D. Rimoldi LICR U Lausanne Melanoma

P. Renaud EPFL-IMT Fluidics

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

PATLiSci Probe Array Technology for Life Science Applications

Probe Array Tech high potential in Cancer Research

R&D, cell based screening

personalized healthcare & diagnostics

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 3

PATLiSci Probe Array Technology for Life Science Applications

PATLiSci Force Spectroscopy

Motivation: metastatic cancer development

Lee et al., Trends Biotechnol. 2007

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

PATLiSci Force Spectroscopy

Probe Arrays for parallel force spectroscopy

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

PATLiSci Force Spectroscopy

SiN probe arrays with (active) actuation

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 7

PATLiSci Force Spectroscopy

Setup and readout

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 8

PATLiSci Force Spectroscopy

Elasticity of Melanoma cells

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 9

PATLiSci Force Spectroscopy

Elasticity, Mobility, Vesicle formation

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 10

PATLiSci Force Spectroscopy

Elasticity of different malignant cell types

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 12

PATLiSci Force Spectroscopy

Parallel force spectroscopy on living cells

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 13

PATLiSci Probe Array Technology for Life Science Applications

Cantilever is a Nanomechanical Sensor

PATLiSci Nanomechanical Sensing

Principle of nanomechanical biosensing

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 15

PATLiSci Nanomechanical Sensing

Functionalization for BRAF V600E

reference cantilever: unspecific oligonucleotide (#1-4) SH-ACACACACACACACACAC

SH-GAGATTTCTCTGTAGCTA SH-GAGATTTCTCTGTAGCTA SH-GAGATTTCTCTGTAGCTA

sensing cantilever: (#5-8)

probe oligonucleotide SH-GAGATTTCTCTGTAGCTA

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 16

PATLiSci Nanomechanical Sensing

Detection of single point BRAF mutation in DNA

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 17

PATLiSci Nanomechanical Sensing

Detection of single point BRAF mutation in RNA

differential deflection Dx /nm

20 0 -20 -40 -60 -80 -100 0 20 40

BRAF mutation in RNA detected within minutes !

Copyright 2012 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 18

PATLiSci Nanomechanical Sensing