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1. Neuron Based Biosensors Definition: a biosensor that uses living neural cells to detect substance of interest
Why neuron based biosensors?
Key advantage: a single neuron-based sensor can potentially detect a vast number of chemical and biological agents
A healthy neuron generates voltage pulses (action potentals) spontaneously on the membrane of the axon.
Changes in environment (presence of chemicals or biological agents) modulate the neurons electrical activity.
Neuron exhibits a unique electrical response to particular agents
2. Review: Detection Favored method of detection is Microelectrode Arrays (MEAs).
Electrodes fabricated on surface of device
Monitor signal externally; doesnt damage cells
Neural signals typically in the range of 100s of uVpp
Many working neuron-based sensors utilize MEAs
Much research focused on improving control of neural growth on MEAs.
3. Analysis of Neural Response Time domain analysis
Characterize response for various substances
Duration of burst
Time interval between bursts
4. Analysis of Neural Response Frequency domain analysis
Example: Prasad et al. 2004: Examine and characterize frequency components of neural response for particular substances
5. Challenges Controlling interaction of living neuron to device.
Ideal of 1:1 association of neurons to electrodes is difficult to achieve
Affects signal-to-noise ratio
Affects reproducibility and repeatability of response
Long term maintenance of cells in vitro
Stability of device (corrosion, biofouling, etc)
6. Review: Cell Patterning Techniques
7. Cell Patterning Using SAMs SAMs form a single layer of molecules on a substrate.
Creates a biocompatible membrane like microenvironment
Supporting structure for growth
Relatively easy to create
Long term stability
Many Types of SAMs
Recent research has focused on using thiols on gold substrates
8. Early work Potember (1995)
Selective UV irradiation to remove/pattern SAM
Surface made bioactive using synthetic peptide, covalently attached using a cross-linker
5um line widths
Optical microscopy to evaluate neural attachment and growth
Cells remained restricted to pattern for over 15 days in culture
9. Thiol-based SAMs Structure:
Alkane chain, typically with 10-20 methylene units
Head group with a strong preferential adsorption to the substrate used. Eg: Thiol (-SH) head groups and Au(111) substrates
Tail group gives the SAM its functionality
10. Thiols on Au(111) Thiol head group bonds to the threefold hollow site on gold surface.
Van der Waals forces between alkane chain causes them to lie at 30 degree angle
11. Nam et al. 2004 Contribution: Coated microelectrode arrays with gold in order to use alkanthiol-based SAM techniques
Coat MEAs with 50-80A of gold
Immerse in MUA solution for 2 hours to create SAM
Expose SAM to other compounds to produce layer of NHS esters
Use uCP to apply poly-D-lysine. Stable PDL layer created by covalent linking to SAM layer
Unstamped areas covered with chemical that inhibits cell growth
12. Nam et al. 2004 Results:
Demonstrated cell viability on PDL linked gold surface
Good resolution stamped 100 x 100um grid pattern of 10um line width
Cells complied to pattern for > 2 weeks
Recording of spontaneous neural activity to verify cell activity.
Enhanced amplitudes up to 500uVpp (100-200uVpp typical)
Gold MEAs were not reusable
13. Nam et al. 2006 Updated process
different SAM 3-glycidoxypropyl trimethoxysilane (3-GPS)
uCP for protein pattern stamping
Neurons complied to patters for 2-3 weeks
Spontaneous neural activity recorded:
Note SAM increased impedance by factor of 2-3
Mean SNR of 6.5 at 2 weeks
mean amplitude of extracellular spikes was 25uVpp at 7 DIV and 50uVpp at 20-24 DIV.
Background noise 2.9uVpp
14. Palyvoda et al. 2007 Technique:
Create gold electrodes
Immerse in 11-AUT solution to create SAM
Studied effect of pad size on neural guidance
Contribution: used SAM to support and guide neural growth directly
No intermediate protien layer, e.g. polylysine (which is difficult to pattern, nonphysiological, toxic under some conditions)
15. Palyvoda et al. 2007 Results
Effects of pad size on cell counts:
350x350um: 57 +/-10
200x200um: 16 +/- 5
100x100um: 4.14 +/- 2
50x50um: 1 +/- 0.87
50x50um pad size comes close to single neuron immobilization, with error.
No measurement of electrical activity was performed
16. Other Characterization Studies Naka et al. (2002)
Investigated effects of different functional tail groups of thiol based SAMs on neuron growth: Amino groups (NH2), carboxyl (COOH), methyl (CH3).
Concludes 11-amino-1-undecanethiol is best
Neurons detach after about 2 weeks.
Slaughter et al. (2004)
Characterized protein attachment to two thiol SAMs on gold electrodes using florescence microscopy, atomic force microscopy, and ac impedance.
Faucheux et al. (2004)
Characterized SAM with various functional groups by examining wettability, layer thickness, and roughness
Examined protein adsorption to these SAMs
17. Other Recent Studies Romanova et al. (2006)
Studied how chemical modifications to SAM affected neuron growth and neurite extension.
Widge et al. (2007)
Studied how mixed SAMs of thiol and conductive polymers affected electrical impedance and phase of gold coated MEAs.
Adding conductive polymers reduces impedance by adding surface area (roughness)
Lin et al. (2008)
Characterized physical structure (thickness, bonding characteristics) of gold electrodes modified MUA-SAMs coated with poly-D-lysine using infared reflection and AFM.
Demonstrated that neurons adhere better to MUA-SAM modified gold electrodes better than to bare gold.
18. Conclusions Neuron-based biosensors appear poised to become an effective biosensor technology.
Stability of culture and micro device
Reproducibility of results
Maintaining health of cells over long term
19. Conclusions SAMs are attractive as a possible solution to these challenges
Relatively simple compared to other options
Increases stability of neuron-device interface
10-15 years or more of characterization studies of SAMs
No fully functional SAM-based biosensor for specific application to date
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Wikipedia Self-Assembled Monolayer. http://en.wikipedia.org/wiki/Self-assembled_monolayer. Accessed 5/15/0008.
Website: "Self Assembled Monolayers." Molecular Films and Surface Analysis group at the Laboratory of Applied Physics, Department of Physics, Linkping University in Sweden. http://www.ifm.liu.se/applphys/ftir/sams.html. Accessed 5/16/2008.