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Micro and nanotechnologies for life sciences : single molecule combing, microfluidics for neurosciences and infrared
stimulation of action potential in neurons.
Benoît CHARLOT, Fabrice BARDIN
IES : Lambert PARIS, Patrice QUINTANA, Didier LAUX INM : Jean VALMIER, Patrick CAROLL, Pascale BOMONT
MMDN : Christelle LASBLEIZ, Jean Michel VERDIER IGMM : Etienne SCHWOB
GIN: Maxime CAZORLA, Frédéric SAUDOU
Labex NUMEV
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Outline 1. Lab overview 2. Single molecule : DNA combing 3. Microsystems for neurosciences
Neuron growth containment Microfluidics Optical stimulation Mechanical stimulation
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1 Lab overview TIMA (Grenoble) 1998-2005 Ph.D (MEMS testing) Fingerprint sensor Micro power sources
LIMMS (Tokyo) 2005-2007 Nanomechanical memories Field emission microresonator
IES (Montpellier) 2007-2015 …. DNA combing Microfluidics for Neurosciences
3 So I am not a neuroscientist….sorry
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CNRS / Univ. Montpellier 159 persons Faculty 64, CNRS 8, Post doc& Ph.D 58, technicians 29
3 research depts. - Photonics and Waves IR lasers & detectors, Terahertz - Energy, Reliability and Radiations Power electronics, Rad. Hard electronics - Sensors, Devices and Systems MEMS, microfluidics, Photovoltaïcs, acoustics … Microfabrication facilities NEW !! 400 m2 Clean room (Litho, Sputter, Evap, E-beam, RIE, PECVD, ….) Microscopy (SEM, TEM, AFM, EDX …)
1 Lab overview
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Dept. Sensors Devices and Systems
Research activities Thin film Materials Piezo, Pyro, Thermoresistances
Thermal MEMS accelerometers,thermoelectrics
Photovoltaïcs concentrated PV Flex substrates, paper, polymer, RFid
Acoustics, ultrasound, piezo
BioMEMS, Cell chips & microfluidics
1 Lab overview
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Part 2. Single molecule handling : DNA combing
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2 DNA Combing DNA organisation
Persistance length: 100 nm for dsDNA and 2 nm for ssDNA
In solution : Pellets, Coiling need of uncoiling techniques for observation
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Nanofluidic containment
D.Bensimon, ENS
A.Cerf, LAAS CNRS
J. Guan and L. J.Lee Ohio State University
F. Westerlund, Chalmers
Deposition by dewetting
D.E.Streng; North Carolina state Univ.
2 DNA Combing How to uncoil DNA ?
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Capillary force assembly : interactions between particles mediated by fluid interfaces.
Peter A. Kralchevsky
2 DNA Combing
Coffee ring
Blood droplet
Convection
Capillary force
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The standard DNA combing technique used everyday at IGMM
Human genomic DNA, YOYO tagged, 237 x 177 μm image
Substrate Silanization DNA ends anchoring at pH 5,4 Slow dewetting
2 DNA Combing
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Ordered deposition Forced dewetting on perturbations PDMS substrate No functionalization
2 DNA Combing
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composite Image (GFP Fluorescence + transmited light) of λ phage DNA (48kbp) tagged with YOYO intercalating dye. Dewetting 400µm/s. Red: uncoiled DNA pellets, 5µm below surface
B. Charlot, et al. “Elongated unique DNA strand deposition on microstructured substrate by receding meniscus assembly and capillary force”, Biomicrofluidics 8, 014103 (2014).
2 DNA Combing
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Combing on nanogratings : rectilinear conformation 800nm pitch nanogratings : Interference lithography
B.Charlot, R.Teissier, M.Drac, E.Schwob, “DNA on rails: Combing DNA fibers on nanogratings”, Applied Physics Letters, 105, 243701 (2014).
2 DNA Combing
Stretching ratio 150%
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2 DNA Combing Next steps :
Combination of perturbation dewetting (PDMS) + nanogratings
Long dsDNA fragments
Chromatin combing
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Part 3. Microsystems for Neuroscience
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Objectives : analysis of the effect of physical stimulations on neurons
- Optical stimulations (Lasers, LED, IR, visible) ! optogenetics - Mechanical / acoustic stimulations - Thermal / thermodynamics of the nervous influx
Through the use of MEMS, BioMEMS, microfluidics, nanostuff Micro sensors and actuators, piezos, electrodes
And several collaborations…
3 Microsystems for neuroscience
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3.1 Neuron Growth Engineering
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Protein stamping 2D containment
PEG-DMA microstructures 2,5D containment
PDMS microfluidics 3D containment
M.Cazorla
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3.1 PEG-DMA scaffolds
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2,5 D cell culture pattern Confinement Cell adhesion selectivity
Non-immunogenicity Non-antigenicity Protein rejection
Cortical DRG
ß tubulin DAPI PEG-DMA autofluorescence
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3.1 Neuron growth on nanogratings
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Regenerative medecine - Dorsal Root Ganglions neurons (axotomized) - « straight line » axonal growth - Interference lithography
B.Charlot, et al., “Axonal growth guidance by surface nano-topology for the regeneration of sensori motor neurons”, IEEE EMBS Neural engineering conference, Montpellier, France, (2015)
Control Nanograting Nanograting
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3.1 Neuron growth on nanogratings
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On Cortical neurons…. Less effect…
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3.2 Microfluidics for Neurosciences
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Microfluidic Compartiment cell culture chambers - Campenot cell design - Oriented neuronal networks / guided growth - Microchannels for Soma / Axon separation
- SU8 /PDMS replica molding
Taylor et al.
DRG
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3.2 Microfluidics for Neurosciences
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In-vitro Neuronal junctions reconstruction - Pathologic brain model (Alzheimer, Huntington..) - Cortico-striatal junctions - Electrophysiologie+ HR microscopy
- Drug perfusion in synaptic chambers - Axonal Transport (pre-synaptic vesicles) - + localised Physical stimulation
M.Cazorla, GIN
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3.2 Microfluidics for Neurosciences Microfluidic chamber + Micro Electrode Array - 64 channels (plugged on Multi Channel System) - Electrode network : Chrome/ Platinum - 5x5 cm glass chip, 170µm thick for microscopy - Branch design for axonal splitting
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3.2 Microfluidics for Neurosciences Extracel recording and stimulations - Impedance - Noise - Signal /noise ratio
Pt and Au electrode Impedance measurement
PEDOT:PSS electrodes
To improve S/N : lowering of the impedance - Black Platinum, black gold
- Nanostructured electrodes
- PEDOT:PSS conductive polymer
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3.3 Optical stimulation
25 with LED (5min) w/o LED Neuron growth rate increasing
645nm low level Light
Proliferation stimulation
Cellular differentiation
Tissue regeneration
L.Paris, et al. “Neurite growth acceleration of adult Dorsal Root Ganglion neurons illuminated by low-level Light Emitting Diode light at 645 nm”, Journal of Biophotonics 1–9 (2014).
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3.3 Optical stimulation Firing of action potentials and depolarizations by infrared (1875nm) millisecond laser pulses
On dorsal root ganglion, vestibular and retinal ganglion cells
Patch clamp recording with whole cell configuration of laser stimulation pulses on cultured retinal ganglion cells
It works….. Ok, But why? Energy threshold for AP generation 26
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- Infrared light absorption in water - High power density (200mW to 1W)
3.3 Optical stimulation
- Open Patch method : Laser burst on the pipette modification of its impedance and measured current as function of voltage steps
�Temperature induced phenomenon?
Temperature rise in the vicinity of neurons during Laser pulses
Temperature rise for AP triggering is equivalent for different laser conditions : 55°C- 60°C
�Temperature seems to be involved, but what is the link with neurons ? Ion channels? 27
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Temperature sensitive ion channel activation by IR laser TRPV (Transient Potential Receptor Vanilloid channels) (Albert et al., 2012; Bec et al. , 2012)
3.3 Optical stimulation
TRPV ionic channel (heat sensitive) inhibition
TRPV4 ionic channel inhibition
Laser pulse
Inhibition of TRPV channels stops the laser induced AP generation
Same patched cell
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Modification of membrane potential induced by laser (IR 1875nm) - The level of depolarization is function of the optical energy received
Action potential triggering
- Threshold (n=15) 10,5±1,23 mV
Two distinct mechanisms :
- Change in membrane potential with the temperature increase (capacitive effect)
- Action potential generation with TRPV Channels
3.3 Optical stimulation What happens below the threshold?
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3.3 Optical stimulation Any mechanical aspect? (thermo mechanical wave)
11ms 12ms 30
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3.4 Mechanical stimulation Goal : replace laser pulses by focused pulsed ultrasound
time-lapse confocal imaging of calcium transients in hippocampal slice cultures
low-intensity, low-frequency ultrasound 440kHz
William J. Tyler Arizona State University
AP generation on single cell?
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Acoustics and rheology - Viscoelasticity of drying blood - Transverse ultrasound analysis at blood / glass interface
Precision Balance
Transverse sensor
Droplet Glass blade
3.4 Mechanical stimulation
Mass and reflexion transverse coef. vs time
Ultrasound generator to be plugged below MEA? 32
Screen printed PZT piezo on glass
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3.4 Mechanical stimulation
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Mesenchymal Stem Cells (MSC) Differentiation by mechanical stimulation. STIMDIF project (PEPS CNRS)
A.Leroy, C.Bony, C.Pinese, B.Charlot, X.Garric, D.Noël, J.Coudane, “PLA-poloxamer/poloxamine copolymers for ligament tissue engineering: sound macromolecular design for degradable scaffolds and MSC differentiation”, Biomaterials Science, DOI: 10.1039/C4BM00433G, (2015)
Biodegradable copolymer scaffold Mechanical stimulation of MSC differentiation
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- DNA combing by capillary force: use of nanogratings
- Neurosciences :
- Axonal guidance by micro engineered substrates: PEG-DMA / nanogratings
- Microfluidic MEA platform for in-vitro reconstruction of neural junctions.
- Physical stimulations :
- Laser pulses : thermo mechanical effect to be analysed
- Mechanical Stim and Ultrasound pulses : to be applied to neurons
- Thermal : to be developped
4 Summary
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Thank you