This project is about further developing probe array techniques for life science applications, notably in the context of cancer research. The consortium shows the balance between experts in sensing technology as well as oncology.
Strategies for Landing an Oracle DBA Job as a Fresher
Patlisci
1. PATLiSci –
Probe Array Technology for Life Sciences
Harry Heinzelmann
VP Nanotechnology & Life Sciences
Bern, May 2011
2. PATLiSci – Probe Array Technology for Life Science Applications
“nano” -tera
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5. Science Museum London
“The Making of the Modern World”
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6. IBM
Drexel U, Philadelphia
IBM
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7. PATLiSci – Probe Array Technology for Life Science Applications
it’s much much more than microscopy…
U Pennsylvania
Müller and Dufrêne
Nature Nanotechnology (2008)
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8. PATLiSci – Probe Array Technology for Life Science Applications
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
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9. PATLiSci – Probe Array Technology for Life Science Applications
Probe Array Technology
• cantilever arrays • point probe arrays
for nanomechanical sensing for parallel force spectroscopy
• measure the presence of minute • measure interaction forces and
concentrations of analytes (N channels) mechanical properties (N statistics)
• use for R&D, optimization, integration • proof of principle, use for R&D
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10. PATLiSci – Probe Array Technology for Life Science Applications
A Nose for Cancer Detection
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11. PATLiSci – Probe Array Technology for Life Science Applications
Project Partners
E. Meyer H. Heinzelmann
Ch. Gerber CSEM (Coord)
Uni Basel Probe array
Cantilever sensors technologies H.P. Herzig
EPFL-IMT
Optics
H. Vogel
EPFL
N. de Rooij, P. Vettiger, J. Brugger Membr protein
EPFL-IMT, MEMS design & fab immobilisation
A. Mariotti
P. Romero
CePO, CHUV
LICR U Lausanne
Melonoma
Head & neck
D. Rimoldi F. Beermann progression
carcinoma
LICR U Lausanne ISREC, EPFL
Melanoma Tumorigenesis
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12. PATLiSci – Probe Array Technology for Life Science Applications
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
Cantilever is a Nanomechanical Sensor
specific adsorption/docking of molecules
creates mechanical stress bending
J. Fritz et al., Science 288, 316-318 (2000); D. Schmid et al., Eur. J. Nanomedicine 1, 44-47 (2008)
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13. PATLiSci – Probe Array Technology for Life Science Applications
Cantilever Sensing – Results
Detection of mutant DNA (in liquid) Detection of VOCs (in gas phase)
(National Cancer Inst.)
B-Raf oncogene, in 50-60%
of all melanoma tumors
DNA from normal cells
DNA from melanoma cells
20
0
-20
differential deflection /nm
-40
injection of DNA
-60
-80
-100
-120
-140
-160
-180
-200
20 40 60 80 100 120 140 160 180 200
time /min
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14. PATLiSci – Probe Array Technology for Life Science Applications
Cantilever Sensing – Outlook and Next Steps
in liquids in gas phase
DNA, mRNA, and tumor cell detection Breath analysis of from cancer patients
• melanoma associated antigens • feasibility EBS of head & neck cancer patients
• test of mutation/antigen and cell binding • representative study on
EBS of head & neck or lung cancer patients
• detection limits of the assays
• optimization of DNA and antigen binding • optimization of readout hard-/software
• optimization of cell capture • functionality and reliability tests
• portable device prototype
• implementation of a microfluidic system
for an initial cell sorting step • implementation of a micro bioreactor
(PATLiSci extension MINACEL) in combination with cantilever arrays
(PATLiSci extension MINACEL)
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15. PATLiSci – Probe Array Technology for Life Science Applications
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
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16. PATLiSci – Probe Array Technology for Life Science Applications
Force Spectroscopy – Results
A por B
e
C cell D
E F
M. Favre et al., J. Mol. Recogn. 24 (2011) 446)
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17. PATLiSci – Probe Array Technology for Life Science Applications
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)
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18. PATLiSci – Probe Array Technology for Life Science Applications
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
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19. Thank you for your attention.
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20. backup slides
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21. Probe Array Technology for Life Science Applications
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
• piezo-resistive cantilevers binding of melanoma cells (CTC)
• handheld device for POC applications • no prior amplification or labeling
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22. 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
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23. Probe Array Technology for Life Science Applications
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)
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24. Probe Array Technology for Life Science Applications
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
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25. Probe Array Technology for Life Science Applications
Impact beyond the Scope of this Project
Safety Production
NEMS / nano
Research,
Screening
Diagnostics
Environment
ICT / tera
Food
Quality
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26. Nanotools – Probe Arrays
PROBART for Parallel Imaging
VEE (- 6V)
Rlever
Rref (~ 20 kohm)
R ref Vout
R1 R2
R lever
probe
#6 4x4 array imaging in
buffer solution with
probe continuous zoom-in
#13
probe
#15
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27. Nanotools – Probe Arrays
ArrayFM with Optical Read-out – First Results
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28. Nanotools – Probe Arrays
PROBART for Force Spectroscopy
600 pN/div
√
√
Force resolution √
= 160 pN √
Polylysin PBS (0.01M)
(5mg/l)
glass surface √
sufficient for most
√
donor/acceptor complexes
√
in “expert reviews in molecular medicine”,
18µm http://www-ermm.cbcu.cam.ac.uk
6.4µm Mapping of the elastic
response of a cell
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29. Nanotools – Probe Arrays
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
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30. Nanotools – Probe Arrays
ArrayFM with Optical Read-out – Some More Tricks
• solving phase ambiguity
• LabView based
software interface
• Si and sol-gel replicated
cantilevers
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31. Nanotools – Probe Arrays
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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32. Nanotools – Nanoscale Dispensing
Nanoscale Dispensing – NADIS
deposition of liquids
in ultrasmall volumes
from microscopic tips
• functional biomolecules for microarrays, such as
proteins or DNA
Molecules in solution
• metallic nanoparticles to form connects, catalyst
particles, optical and chemical functions, …
Nanoparticle suspensions
• etch resist materials, sol-gel precursors, …
Materials for processing
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33. Closed Channel NADIS Cantilevers
• closed channels for
- better control
- operation in liquids
• new microfabrication process
• single probes
• 1-dim arrays
• one and two
channel
design, on-chip
reservoirs
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34. Nanotools – Nanoscale Dispensing
NADIS of Fluorophores in Liquid Environments
3 μm
1
Intensity [a.u.]
0.5
0
0 2 4 6 8
applied pressure ~ 2mbar
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35. 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
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Hinweis der Redaktion
nano-tera has both “nano” and “tera” in its namegoogle search for “nano” brings mostly hits like this:(I let you find out what a google search for “tera” brings up…)so people tend to forget the origins of nanotechnology…nano - STM and AFM by many considered the beginning of nanotechnologynice coincidence: the AFM has its 25th anniversary, first paper out in March 1986is Chris at the meeting ?
first STM 1981 by Binnig, Rohrer, Gerber, and Weibel
first AFM 1986 by Binnig, Quate, and Gerber, Phys. Rev. Lett. 56, 930-933 (1986)
original AFM from 1986 paperdisplayednext to the Apollo 10 command module from May 1969 (prepared the July 1969 Apollo 11 Lunar Landing mission)also witness the Sunbeam Ironmaster Model X21 electric dry iron (1955)French barber’s shaving bowl from the 18th century
IBM AlmadenIBMRüschlikonDrexel University, Philadelphia - http://einstein.drexel.edu/~wking/unfolding-disasters/posts/Force_spectroscopy/
basics to understandwhatthisprojectis aboutcl array: complements NMR, endoscopy, and blood test (PSA) tests, on single celllevel: biomarkers, RNA mutationspoint probe array: biophysicalproperties of cancer cells, and how biochemistry influences it
NOSE arrays from Basel benefit from piezolever development (today mostly “simple, commercial” cantilevers from IBM)NE could use some more surface functionalizationopticalread-out of CSEM mightbeinteresting for NOSE arrays as well
transduction of chemical recognition
B-Raf or proto-oncogene B-Raf is a protein that in humans is encoded by the BRAF gene. The B-Raf protein is involved in sending signals in cells and in cell growth. The BRAF gene may be mutated and as a consequence, the normal functioning of the B-Raf protein may be altered. Certain inherited BRAF mutations cause birth defects. Alternatively, other acquired mutations (oncogene) in adults may cause cancer. Drugs that treat those cancers by inhibiting B-Raf are being studied, and one, PLX4032, has started phase III clinical trials. cf. also Roche RG7204melanoma on skinPCA: principle component analysisPrOH = propylalcohol, « propanol »CMC = Carboxymethylcellulose, PSS = poly (sodium 4-styrenesulfonate)
melanoma associated antigen (HMW-MAA), expressed by melanoma tumorsEBS: exhaled breath samplesreadout hardwareincladaptation to further sensor chip generationsMINACEL in collaboration with P. Renaud, STI-LMIS EPFL
Numerous adhesion receptors of the selectin, integrin, or immunoglobulin family promote inflammatory cell recruitmentSingle Cell Force Measurements and Cell Adhesion Dr Torsten Müller & Dr Tanja Neumann, JPK Instruments This short review describes the relevance of cell adhesion in cell biology. Starting with a short overview of the force range of adhesion related biological events and the current biophysical techniques for investigating these events, it will conclude with a description of the use of single cell force spectroscopy for quantifying mechanical properties such as stiffness, surface tension, and bond disruption forces.MotivationThe past decade has seen the development of molecular cell biology and related biomedical/ pharmacological research. At the cell-level, the fundamental processes of genetics, metabolism and cell communication are under investigation. Looking ahead, processes in the life sciences will be redefined in the new concept of systems biology.Adhesion is one of the crucial mechanisms of interaction between living cells and their environment. Cell adhesion is a complex process that involves non-specific and specific binding of glycocalyx and plasma membrane surface molecules (e.g. integrins, selectins or cadherins) to the extracellular matrix proteins (ECM, mainly fibronectin, collagen) or to other cells respectively, down stream signalling that adhesion has occurred and a possible cellular response to this binding (e.g. cell shape, migration, proliferation). It is a dynamic and complex process which consists of physical interaction, biochemical response and physiological adaptation. Understanding this complexity and identifying the key triggers in specific biological responses of cell adhesion is of fundamental importance in a wide range of fields including cancer and stem cell research, developmental and infection biology, immunology and allergology, tissue engineering and implant research.There are many well-established techniques for studying cell adhesion; from fluorescence microscopy to biochemistry and molecular biology (e.g. gene and protein expressing assays or aggregation and migration assays). Based on the finding that the elasticity of extracellular matrix varies with the lineage of stem cells [1], new thoughts are discussed that not only (bio)chemical properties of the ECM/microenvironment but also their mechanical properties and forces can influence the connection to the plasma membrane and the cytoskeleton via adherence junction and focal adhesion as mechano-sensors [2,3].Current techniques to measure forces on single cellsThe forces involved in single molecules and single cells range widely from less than 1pN up to 1µN (Fig.1). Single protein-protein bonds are in the range of 1-20 pN, whereas single molecule unfolding requires up to 100pN. To remove a single cell from a substrate demands at least 1nN of force and sometimes more.Figure 1 - Overview of force range of adhesion relevant biological events and biophysical techniques used to investigate forces and mechanical properties of molecules and cells (at an approximate drawing speed of 500nm/s), for reviews see [3,4,5]. PFM – photonic force microscopy – is an OT variant.Optical and magnetic tweezers are best suited to measuring weaker forces on single molecules or local viscoelastic properties of cells. Force sensor arrays (FSA) and soft gel embedded particle tracking (GPT), and micro-plates cell pulling (MP) are mainly used to investigate cell responses to topographic patterns, and to measure traction and migration forces respectively. With a micro-plates cell puller, viscoelastic properties of adhered cells can be determined.Flow chamber, bioforce membrane probe technique (BFP) and atomic force microscopy (AFM) are characterized by a very wide force range from pN up to µN, and allow both measurements of unbinding forces of whole cells from ECM decorated substrates and of various pairs of receptor/ligand interactions.All these methods have their limitations. The AFM based single cell force spectroscopy (SCFS) is a versatile tool that uniquely fulfils many needs. These include (i) large vertical movement for cell/cell interaction measurements of up to 100µm, (ii) providing reproducible quantitative results for single cells with precision down to the single molecule level, (iii) combining with fluorescence microscopy or even confocal techniques, (iv) supporting flat as well topographical surfaces, cell monolayers and aggregates, and (v) dedicated professional software.Atomic force microscope-based force measurementsAFM uses a laser to measure the deflection of a flexible cantilever (Fig. 2) during probing. To run a typical cell adhesion experiment, the basic steps are as follows: An inverted optical microscope is used to facilitate the chemical binding of a cell to the cantilever.The cell is brought into contact with the target surface using a defined force. This will cause deformation of the cell.After a user-controlled contact time, the cell is withdrawn from the substrate by retracting the cantilever. The cell initially resists this process (the cantilever will bend in proportion to the unbinding force). Cell unbinding often requires effective pulling lengths of up to 100 µm [6] due to extrusion of membrane tethers during cell separation.After complete separation, the cell can be used again to address a new target surface.The main results extracted are: (i)-maximum unbinding force, (ii)-number and size of jumps, (iii)-number and length of tethers, (iv)- work (W) of separation until bonds start to break and (v)- slope of indentation for calculating contact stiffness and the elasticity cell.Example for SCFS in Developmental BiologyHow do zebra fish stem cells form the three germ layers during gastrulation? Very recently, biologists were able to explain this cell sorting and organization process using a CellHesion® system from JPK Instruments combined with a fluorescence microscope (Fig. 3). They quantified both adhesive forces and cell-cortex tension of individual endo-, meso-, and ectoderm cells [6] as basis for modelling simulations.In the first part of this work, the authors have investigated the homotypic adhesion between all 3 cell types using the workflow illustrated in Figure 2. They found that mesoderm and endoderm progenitor cells are more cohesive than ectoderm cells (maximum adhesion force at 30 s contact time is 6 nN, and 3 nN, respectively).To measure the actomyosin related cell-cortex tension of all 3 different germ-layer progenitor cells, the authors used a setup schematically drawn in figure 4.They calculated the tension from the force-indentation curves using the liquid droplet model. The authors found a rising value for cell-cortex tension from endoderm cells to mesoderm and ectoderm progenitor cells (60µN/m to 45 and 35 µN/m respectively). Additionally, blebbistatin reduced cell tension to same level in all 3 cell types. Furthermore, they found strong evidences for modulation of tension by Nodal/TGGβ-related signalling based on single cell force spectroscopy data.References[1] Engler, A. et al.: Cell 126, 677-689 (2006)[2] Toh, Y.C. et al.: Nanotoday 1/3, 34-43 (2006)[3] Girard, P. et al.: Soft Matter 3, 307-326 (2007)[4] van Fliet, KJ. et al.: ActaMaterialia 51, 5881-5905 (2003)[5] Robert, P. et al.: J. Mol. Recognit. 20, 432-447 (2007) [6] Krieg, M. et al.: Nature Cell Biology 10, 429-436 (2008)
to study the correlation of cell mechanics malignant progression by measuring elasticity of different cells corresponding to different invasive behavior. ???In the development of melanoma cancer, they are 3 development phase, radial growth phase (RGP), vertical growth phase (VGP), and metastatic (forming metastases at other locations inside the body). see blue sketchMalignancy refers to (i) cancerous cells that proliferate in a uncontrolled manner, and also to (ii) cancerous cells that forms metastases. For melanomas, all three types (RGP, VGP and metastatic) are malignant, wherefrom the 3rd type is of course the most dangerous . Meaning? We try to see if there is a link between the malignant progression (RGP -> VGP -> metastatic) and the cell elasticity
Microbioreactor / Cell Sorting / NADISbioreactor: detection of trace chemicals emitted by cancer cells in a microbioreactor, with CL connected to a microbioreactor, volatile by-products of individual cells can be detectedcell sorting: reduction of number of cells from blood samples to manageable levels,remove red blood cells and focus on cells with a nucleus because of a different dielectric response; and to selectively remove adherent cells that were characterized by force spectroscopy
in gas: Field study on 30 patients with pathologically confirmed head & neck cancer and 30 healthy personsDieseFallstudiewollenwirjaimRahmen des NanoTera Projektsdurchführen ... Daher Status: warten auf Bewilligung des ProjektsDie StudiesollzweiGruppen von Patientenumfassen:1. 30 patients with pathologically confirmed head & neck cancer (d.h. Patientenwelchenachweislich Krebs haben)2. 30 healthy personsin liquids: somaticcells = body cells (two copies of each chromosomes) <> gameteswhich are sperm or eggcells (one copy of each chromosome)Evaluate specificity and sensitivity using a cell line with BRAF V600E mutationReliable and robust lab resultsearlydetection of CTCsallows to understandbetter cancer formation and progression, and might help to identify cancer stem cellbiomarkersCTC = circulatingtumorcellsVOC = volatile organic compound
why force spectroscopy on cellsisinteresting, and whyweneeditparallel
only show if needvisual support to what I sayCL arrays: improve piezo read-out, improve surface functionalization for cancer markers .3ppm reachedtoday, dogscandetect parts per trillion pptoften exhale patterns of patterns of biochemical markers (frommetabolicwaste), thereforewemightneed a library
