(Nanowerk Spotlight) One area of nanobiotechnology that will have a massive impact on improving the live of disabled people lies at the interface (literally) between artificial functional materials and living neuronal tissues. Neuroprosthetics is an area of neuroscience that uses artificial microdevices to replace the function of impaired nervous systems or sensory organs (read more: "Nanotechnology coming to a brain near you"). Different biomedical devices implanted in the central nervous system, so-called neural interfaces, already have been developed to control motor disorders or to translate willful brain processes into specific actions by the control of external devices.
There is no lack of challenges for researchers, though. One are that has been quite difficult is the communication between biological tissues and artificial sensors – something that is key in building artificial retinas, for instance.
Researchers in Italy have now reported the functional interfacing of an organic semiconductor
with a network of cultured primary neurons. Their novel approach represents a new tool for neural active interfacing, which is a simpler alternative to the existing and widely used neuron optogenetic photostimulation techniques, and avoids gene transfer, which is potentially hazardous.
"We have successfully demonstrated the physiological stimulation of neuronal cells in a network by shaping visible light pulses at the polymer/electrolyte interface," Guglielmo Lanzani tells Nanowerk. "This new approach to the optical stimulation of neurons may stimulate further work towards the development of an artificial retina based on organic materials."
Lanzani is director of the Center for Nano Science and Technology at the Italian Institute of Technology (IIT) in Genova. Together with a team of neuroscientists from the Department of Neuroscience and Brain Technologies at ITT, lead by Fabio Benfenati, he published a paper in the January 17, 2011 online edition of Nature Communications ("A hybrid bioorganic interface for neuronal photoactivation") that describes their work of interfacing a network of primary neurons with an organic blend.
This is the first functional interfacing of an organic semiconductor with a network of cultured primary neurons.
Optical stimulation of neurons cultured onto an ITO/rr-P3HT:PCBM device. (a) Schematic representation of the optical stimulation paradigm including the localization of the stimulus in a region surrounding the patched neuron (scale bar, 10 µm). (b) Scheme of the photosensing interface, with the neuronal network grown on top of the polymer active layer during patch-clamp recordings. (c) Online monitoring of pH of the extracellular solution during the experiments in the presence (black, n = 4) or absence of photostimulation (red, n = 4). Data are presented as means ± s.e.m. (d) Action-potential generation in response to a photostimulation pulse (50 ms). (e) Example of spike train generated with 20 ms pulses repeated at 1 Hz (upper panel). Peristimulus time histogram (PSTH) count was computed and normalized by considering spike trains in all recorded neurons (bottom panel; n = 10, bins 20 ms). The right plot shows (means ± s.e.m.) the latency to the spike peak with respect to the light onset computed by averaging all spikes in the train obtained from all recorded neurons and the jitter calculated as the s.d. of spike latencies measured across all recorded neurons (n = 10). (Reprinted with permission from Nature Publishing Group)
Lanzani explains that the active layer of their interface is a prototype material commonly used for organic photovoltaic applications (rr-P3HT:PCBM). "In the bulk heterojunction structure, rr-P3HT works as an electron donor material, whereas PCBM is the electron acceptor, thus ensuring very high external quantum efficiency in the charge generation process."
The team realized their device through a multi-stage process: the active polymer film was spin-coated onto a glass substrate pre-coated with indium-tin oxide (ITO), which works as the anode of the photo-detector, and then the organic blend was annealed at 120 °C for 2 hours.
"The thermal treatment had a double role" according to Lanzani: "it improved the morphology of the polymeric film, enhancing the efficiency of charge photogeneration, and it prepared the film for subsequent cell culture by removing all residual traces of organic solvents – for example, acetone, methyl alcohol, chlorobenzene, which are highly toxic for the biological systems – and by sterilizing the substrate."
The polymer layer was then covered by poly-L-lysine (PLL) to improve adhesion, and primary rat embryonic hippocampal neurons were finally seeded and grown on it.
The researchers tested the performance of the bioorganic interface structure as an intercommunication device by evaluating the efficacy of light excitation to trigger the activity of whole-cell patched neurons. They found a deterministic correspondence between photostimulation of the organic semiconductor close to the cell body and neuronal activation was found. Even more, they demonstrated that by using organic semiconductors one can closely reproduce the color response functions of the human retina.
"Spike trains could be elicited with trains of light pulses (20 ms at 1 Hz) with a negligible percentage of failures" says Lanzani. We found that the single spikes were rapidly and precisely evoked by the light pulses."
What is interesting in this approach is that, in contrast to metal or silicon interfaces, the proposed interface of the Italian team works without any externally applied electric field and with minimal heat dissipation, favorably addressing the thermal issues, which are extremely relevant in an efficient biological interface. Since the mechanism is capacitive, i.e. based on space charge distribution, there is negligible electrical current during stimulation. Little current means low dissipation and no thermal stress for the cell.
"Our work suggests that π-conjugated materials can be taken into consideration for a new generation of biomimetic artificial retinal prostheses," says Lanzani. "Organic technology is characterized by simple and cheap fabrication techniques; existing deposition methods such as ink-jet printing, allow the realization of a variety of geometrical patterns with various active areas, up to few square micrometers, thus offering the possibility to specifically target selected groups of cells."
This work is a step further towards the realization of an organic artificial retina for color vision. Lanzani points out that, in principle, the organic film could be patterned with a retina-like chromophore distribution (for instance by ink-jet printing of active spots with different spectral response and space resolution). The simple soft-contact with the neuron system would allow transducing the optical system, without any mechanical intrusion.
Reference: Ghezzi, D., Antognazza, M., Dal Maschio, M., Lanzarini, E., Benfenati, F., & Lanzani, G. (2011). A hybrid bioorganic interface for neuronal photoactivation Nature Communications, 2 DOI: 10.1038/ncomms1164