Transcript Slide 1

NER: NANO-GRATING FORCE SENSOR FOR MEASUREMENT OF NEURON MEMBRANE CHARACTERISTICS
UNDER GROWTH AND CELLULAR DIFFERENTIATION
Ashwini Gopal1, Zhiquan Luo2, Karthik Kumar1, Jae Young Lee3, Kazunori Hoshino1, Bin Li2, Christine Schmidt1, 3, Paul S. Ho2, and Xiaojing Zhang1
Departments of Biomedical Engineering1, Mechanical Engineering2, Chemical Engineering3The University of Texas at Austin,Texas, USA
Motivation
Device Design
• Cell experiences stress and strain
Physical/Chemical cues (growth)
Environmental perturbation (robustness)
Cell Structure [1]
• Neuronal shape depend on both progressive and
regressive processes such as axonal elongation and
axonal elimination [2-3]
• Mechanical tension is a direct stimulus for neurite
initiation and elongation from peripheral neurons
Classical concepts fail to explain mechanotransduction[4]
Suspension Stiffness
• Two symmetric nanogratings
Size-dependant mechanical properties have not been
3
provide mechanical balance and
2 Ew t
characterized
maximal optical surface area.
k
s 
3
• PC12-a cell line derived from rat
Ls
• Nanogratings consist of flexure
Flexure Stiffness
pheochromocytoma
folding beams suspended between
[5]
PC12
Cells
•Serve as good models of neuron cells
3
two parallel cantilevers of known
8
Ew
t
•Easily be induced to extend neurites with NGF
k
f 
[7]
3
stiffness .
Lf
• Significance
guided nerve regeneration
Simulation Results
wound healing
cell based drug delivery
Basic structure of neuron [6]
Fabrication Sequence
x: Experimental values
*: Calibration Values
•Probing causes motion of grating
•Change in diffraction spot
•Determines the amount of force applied. (F=kx)
SEM Images
• Experiment was conducted on 10-15 cells
• Time for testing cells-30mins
• An average force of 22+8μN caused the neurite length to
contract up to 6μm
Principle of Operation
• Probe displacement, therefore the force, can be measured
through the spatial frequency of far-field diffraction
pattern.
m  psin   sin  
Conclusions
m is diffraction order,λ is wavelength, p is grating pitch,
α is illumination angle, and θ is diffraction angle.


p
m
 m 

p 1  
 p 
2
2
Results
Absence of stress across the gratings and stress concentrated
on the flexure beams
• Sensitivity of the device can be greatly improved by
reducing the critical feature size, p, of the grating to the
nanometer regime to match the illumination wavelength.
Experimental Setup
Opto-mechanical sensing interface to study cell mechanics
• Design, simulation, and fabrication of nanograting
displacement-force sensor
• Displacement range of 10µm & force sensitivity 8N/m
• Eigen modes vs. optical detection
• Localized, quantitative interactions in microenvironment
Neuron(PC12) mechanics characterization
• Neurite contraction under mechanical stimulation
• Elastic modulus of neuron membrane 425±30 Pa
Platform to study mechano-transduction in other cell lines
• Hippocampal cells, spinal cord motor neuron
Reference:
Eigen modes under mechanical vibration were simulated
using CoventorWare™ .
• Nanograting sensor is attached to a piezoelectric actuator
• Placed in a liquid media (serum containing F12K media)
to probe the PC12 cells (Neurons)
• Illuminated with a 635 nm He-Ne laser diode
• Spot size covering the grating area of 120μm x 120μm.
[1] See: http://www.uic.edu/classes/bios/bios100/lecturesf04am/cytoskeleton.jpg
[2] Cowan, W.M, Fawcett, J. W, O'Leary, D. D. M, and Stanfield, B. B “Regressive
events in neumgenesis.” Science. 225, p1258-1265, 1984.
[3] Purves, D, and Lichtman, J. W, “Elimination of synapses in the developing nervous
system.”, Science, 210, p153-157, 1980.
[4] McKintosh F C, Kas J and Janmey P A 1995 Phys. Rev. Lett. 75 4425
[5] See:
http://scienceblogs.com/purepedantry/2006/07/background_to_the_20_year_coma.ph
p
[6] See: http://www.med.osaka-u.ac.jp/pub/molonc/www/Esignal.html
[7] Stephen D. Sentura “ Microsystem Design”,2001
Acknowledgement: NSF Nanoscale Exploratory Research Award (NER), ECS-0609413, the Welch Foundation and the Strategic Partnership for Research in Nanotechnology (SPRING), and NSF NNIN Facilities at UT Austin and Stanford Center
for Integrated Systems (Grant # 0335765 and 9731293 respectively)