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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

Image source: http://www.cic-caracas.org/departments/science/Topic11.php

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.

Photo Source: Nam, et al., Gold-coated microelectrode array with thiol linked self-assembled monolayers for engineering neuronal cultures. IEEE Transactions on Biomedical Engineering, 51:1 (2004) 158-165.

Analysis of Neural Response

Time domain analysis
Characterize response for various substances
Amplitude Duration of burst Time interval between bursts
MEA recordings of neural activity a) Spontaneous activity b) Cyclothiazide c) MK-801 d) NBQX

Source: Chiappalone et a. Networks of neurons coupled to microelectrode arrays: a neuronal sensory system for pharmacological applications Biosensor and Bioelectronics, 18:5-6 (2003), 627-634

Analysis of Neural Response

Frequency domain analysis
Example: Prasad et al. 2004: Examine and characterize frequency components of neural response for particular substances
Neural response to hydrogen peroxide

Neural response to ethanol

Source: Prasad et al. Neurons as sensors: individual and cascaded chemical sensing Biosensor and Bioelectronics, 19:12 (2004), 1599-1610

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)

Review: Cell Patterning Techniques

Goal is to enhance detectibility of action potentials by patterning neurons over electrodes
Topographical Patterning Dielectrophoresis

Source: Craighead, et al., 1998.

Source: Prasad, et al., 2003.

Physical Immobilization

Chemical Patterning

Source: Tooker, et al., 2004.

Source: James, et al., 2004.

Cell Patterning Using SAMs

SAMs form a single layer of molecules on a substrate. Advantages:
Creates a biocompatible membrane like microenvironment
Supporting structure for growth Directs growth

Relatively easy to create Long term stability Customizable

Many Types of SAMs Recent research has focused on using thiols on gold substrates

Early work
Potember (1995)
SAM: n-octa-decyltrichlorosilane 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
Source: Potember et al. Conducting networks from cultured cells on selfassembled monolayers Synthetic Metals, 71(1995) 1997-1999.

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

Source: Self Assembled Monolayers http://www.ifm.liu.se/applphys/ftir/sams.html

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

Source: Self Assembled Monolayers http://www.ifm.liu.se/applphys/ftir/sams.html

Commonly used SAMs: MUA: 11-mercaptoundecanoic acid 11-AUT: 11-amino-1-undecanethiol

Nam et al. 2004

Contribution: Coated microelectrode arrays with gold in order to use alkanthiol-based SAM techniques Techninque:
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

Source: Nam, et al., Gold-coated microelectrode array with thiol linked self-assembled monolayers for engineering neuronal cultures. IEEE Transactions on Biomedical Engineering, 51:1 (2004) 158-165.

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

Source: Nam, et al., Gold-coated microelectrode array with thiol linked self-assembled monolayers for engineering neuronal cultures. IEEE Transactions on Biomedical Engineering, 51:1 (2004) 158-165.

Nam et al. 2006

Updated process
different SAM 3-glycidoxypropyl trimethoxysilane (3-GPS) uCP for protein pattern stamping

Results
Neurons complied to patters for 2-3 weeks Spontaneous neural activity recorded:
400ms

Source: Nam et al. Epoxy-silane linking of biomolecules is simple and effective for patterning neuronal cultures. Biosensors and Bioelectronics 22 (2006) 589597

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

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)

Source: Palyvoda, et. al., Culturing neuron cells on electrode with self-assembly monolayer Biosensor and Bioelectronics, 22 (2007) 2346-2350.

Image of neurons on 50x50um SAM coated gold electrode

Palyvoda et al. 2007

Results
Effects of pad size on cell counts:

Source: Palyvoda, et. al., Culturing neuron cells on electrode with self-assembly monolayer Biosensor and Bioelectronics, 22 (2007) 2346-2350.

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

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

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.

Conclusions
Neuron-based biosensors appear poised to become an effective biosensor technology. Challenges:
Stability of culture and micro device Reproducibility of results Maintaining health of cells over long term

Conclusions
SAMs are attractive as a possible solution to these challenges
Relatively simple compared to other options Highly biocompatible Customizable Increases stability of neuron-device interface

But
10-15 years or more of characterization studies of SAMs No fully functional SAM-based biosensor for specific application to date

Questions?

References
Chiappalone, M., Vato, A., Tedesco, M.B., Marcoli, M., Davide, F., Martinoia, S., 2003. Networks of neurons coupled to microelectrode arrays: a neuronal sensory system for pharmacological applications Biosensor and Bioelectronics 18(5-6), 627-634. Chun, C., Hickman, J.J., Wang, W., Gregory, C. Narayanan, S. Poeta, M. "Neuronal cell patterning on covalently bound protein patterns by microcontact printing techniques and the functioning of proteins bound on silane monolayers." Conference Paper. Craighead, H.G., Turner, S.W., Davis, R.C., James, C., Perez, A.M., John, P.M., Isaacson, M.S., Kam, L. Shain, W., Turner, J.N, Banker, G., 1998. Chemical and Topographical Surface Modifcation for Control of Central Nervous System Cell Adhesion. Biomedical Microdevices 1, 49-64. Faucheux, N., Schweiss, R., Lutzow, K., Werner, C., Groth, T., 2004. "Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies." Biomaterials 25 2721-2730. Griscom, L., Degenaar, P. LePioufle, B., Tamiya, E., Fujita, H., 2002. Techniques for patterning and guidance of primary culture neurons on microelectrode arrays. Sensors and Actuators B 83, 15-21. James, C.D., Spence, A.J.H., Dowell-Mesfin, N.M., Hussain, R.J., Smith, K.L., Craighead, H.G., Isaacson, M.S., Shain, W., Turner, J.N., 2004. Extracellular recordings from patterned neuronal networks using planar microelectrode arrays. IEEE Transactions on Biomedical Engineering 51(9), 1640-1648. Lin, S., Chen, J.J., Liaao, J., Tzeng, S., 2008. "Characterization of surface modification on microelectrode arrays for in vitro cell culture." Biomedical Microdevices 10, 99-111. Maher, M.P., Pine, J., Wright, J., Tai, Y., 1999. "The Neurochip: a new multielectrode device for stimulating and recording from cultured neurons." Journal of Neuroscience Methods 87, 45-56. Naka, Y., Eda, A., Takei, H. Shimizu, N. 2002. "Neurite outgrowths of neurons on patterned self-assembled monolayers." Journal of Bioscience and Bioengineering 94(5), 434-439. Nam, Y., Chang, J.C., Wheeler, B.C., Brewer, G.J., 2004. Gold-coated microelectrode array with thiol linked self-assembled monolayers for engineering neuronal cultures. IEEE Transactions on Biomedical Engineering 51(1), 158-165.

References
Nam, Y., Branch, D.W., Wheeler, B.C., 2006. Epoxy-silane linking of biomolecules is simple and effective for patterning neuronal cultures. Biosensors and Bioelectronics 22, 589597. Palyvoda, O., Chen, C., Auner, G.W., 2007. Culturing neuron cells on electrode with self-assembly monolayer Biosensor and Bioelectronics 22, 2346-2350. Potember, R.S., Matsuzawa, M., Liesi, P., 1995. Conducting networks from cultured cells on self-assembled monolayers Synthetic Metals 71, 19971999. Prasad, S., Yang, M., Zhang, X., Ozkan, C.S., Ozkan, 2003, Electric field assisted patterning of neuronal networks for the study of brain functions. Biomedical Microdevices 5(2), 125-137. Prasad, S., Zhang, X., Yang, M., Ozkan, C.S., Ozkan, M., 2004. Neurons as sensors: individual and cascaded chemical sensing. Biosensor and Bioelectronics 19(12), 1599-1610 Romanova, E.V., Oxley, S.P., Rubakhin, S.S., Bohn., P.W., Sweedler, J.V., "Self-assembled monolayers of alkanethiols on gold modulate electrophysiological parameters and cellular mophology of cultured neurons." Biomaterials 27, 1665-1669 Slaughter, G.E., Bieberich, E., Wnek, G.E., Wynne, K.J., Guiseppi-Elie, A., 2004 "Improving Nueron-to-electrode surface attachment via alkanthiol self-assembly: an alternating current impedance study." Laungmuir 20, 7189-7200. Tooker, A., Meng, E., Erickson, J., Tai, Y., Pine, J., 2004. Development of Biocompatible Parylene Neurocages. Proceedings, IEEE EMBS, San Francisco, September 2004, 2542-2545 Widge, A.S., Jeffries-El, M. Cui, X., Lagenaur, C.F., Matsuoka, Y., 2007. "Aelf-assembled monolayers of polythiophene conductive polymers improve biocompatibility and electrical impedance of neural electrodes." Biosensors and Bioelectronics 22, 1723-1732. 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.