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x : Experimental values * : Calibration Values NER: NANO-GRATING FORCE SENSOR FOR MEASUREMENT OF NEURON MEMBRANE CHARACTERISTICS UNDER GROWTH AND CELLULAR DIFFERENTIATION

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slide1

x: Experimental values

*: Calibration Values

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

Results

Fabrication Sequence

  • Cell experiences stress and strain
    • Physical/Chemical cues (growth)
    • Environmental perturbation (robustness)
  • 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 mechano-transduction[4]
    • Size-dependant mechanical properties have not been characterized
  • PC12-a cell line derived from rat pheochromocytoma
    • Serve as good models of neuron cells
    • Easily be induced to extend neurites with NGF
  • Significance
    • guided nerve regeneration
    • wound healing
    • cell based drug delivery

Cell Structure [1]

  • Probing causes motion of grating
  • Change in diffraction spot
  • Determines the amount of force applied. (F=kx)

Suspension Stiffness

  • Two symmetric nanogratings provide mechanical balance and maximal optical surface area.
  • Nanogratings consist of flexure folding beams suspended between two parallel cantilevers of known stiffness[7].

Flexure Stiffness

PC12 Cells [5]

SEM Images

Simulation Results

  • 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

Basic structure of neuron [6]

Principle of Operation

  • Probe displacement, therefore the force, can be measured through the spatial frequency of far-field diffraction pattern.

Conclusions

  • 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

m is diffraction order,λ is wavelength, p is grating pitch, αis illumination angle, and θis diffraction angle.

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

Reference:

[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.php

[6] See: http://www.med.osaka-u.ac.jp/pub/molonc/www/Esignal.html

[7] Stephen D. Sentura “ Microsystem Design”,2001

  • 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.

Eigen modes under mechanical vibration were simulated using CoventorWare™ .

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)