patlisci

Harry HeinzelmannVP Nanotechnology & Life Sciences

PATLiSci – Probe Array Technology for Life Sciences

Bern, May 2011

Copyright 2011 I Nanotechnology & Life Sciences I Harry Heinzelmann I page 2

“nano” -tera

PATLiSci – Probe Array Technology for Life Science Applications


Science Museum London“The Making of the Modern World”


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IBM

IBM


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?


Probe Array Technology


• cantilever arraysfor nanomechanical sensing

• measure the presence of minute concentrations of analytes (N channels)

• use for R&D, optimization, integration

• point probe arraysfor parallel force spectroscopy

• measure interaction forces and mechanical properties (N statistics)

• proof of principle, use for R&D


A Nose for Cancer Detection



Project Partners


H. Vogel EPFL

Membr protein immobilisation

H.P. Herzig EPFL-IMT

Optics

N. de Rooij, P. Vettiger, J. Brugger

EPFL-IMT, MEMS design & fab

A. Mariotti CePO, CHUV Melonoma progression F. Beermann

ISREC, EPFL Tumorigenesis

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


Cantilever Sensing – Technology

detection in liquids:

• biomarkers for cancer in DNA/cell samples

• measured by optical beam deflection

detection in the gas phase:

• volatile organic compounds (VOCs) in patient‘s breath – non-invasive early recognition of cancer

• measured with integrated piezoresistors


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


Cantilever Sensing – Results

Detection of mutant DNA (in liquid)

B-Raf oncogene, in 50-60% of all melanoma tumors

Detection of VOCs (in gas phase)


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

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-120

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DNA from normal cells DNA from melanoma cells

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


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 – Technology

• information about adhesion proteins, cell mechanics, kinetics, …

• statistics! parallel force spectroscopy novel cantilever deflection readout probe array microfabrication living melanoma cell array


source: JPK


Force Spectroscopy – Results


M. Favre et al., J. Mol. Recogn. 24 (2011) 446)

pore

cell

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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)



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


Thank you for your attention.


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

Probe Array Technology for Life Science Applications


Force Spectroscopy on Cells

• information about adhesion proteins, cell mechanics, kinetics, …

• cell-surface, cell-cantilever, cell-cell

• meaningful only with sufficient statistics, which makes experiments rather tedious

• at current rate of a few cells per day,not useful for screening formats

• array format and parallel operation will greatly improve statistics and allow high throughput screening formats


source: JPK


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


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



Safety Production

Food Quality

Environment

Diagnostics

Impact beyond the Scope of this Project


Research,Screening

NEMS / nano

ICT / tera


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

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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


Nanoscale Dispensing – NADIS

Nanotools – Nanoscale Dispensing

Nanoparticle suspensions

Materials for processing

Molecules in solution

• functional biomolecules for microarrays, such as

proteins or DNA

• metallic nanoparticles to form connects, catalyst

particles, optical and chemical functions, …

• etch resist materials, sol-gel precursors, …

deposition of liquidsin ultrasmall volumesfrom microscopic tips


Closed Channel NADIS Cantilevers

• single probes

• 1-dim arrays

• one and two channel design, on-chip reservoirs

• closed channels for- better control- operation in liquids

• new microfabrication process



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Inte

nsity [a

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3 μm

applied pressure ~ 2mbar

NADIS of Fluorophores in Liquid Environments


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

Technology

life science applicationsits

dufrnenature nanotechnology

cancer cells

science museum londonthe

cancer detectioncopyright

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cell mechanical properties

early stage