Diamond-based Platforms for Long-term Growth and Investigations of Neurons
Synthesis and Properties of Functional Materials
Final Report Abstract
The main focus of this project was not only to study in detail the individual technological steps towards the fabrication of multi-electrode arrays (MEAs) based on ultrananocrystalline diamond (UNCD) films prepared by microwave plasma chemical vapor deposition but also on their general application in biotechnology, especially as substrates for in vitro cell growth using the advantageous bio-properties of UNCD to expand the possibilities for research in neurosciences. For anchorage-dependent cells, such as neurons, the adhesion to a substrate is a crucial step. Thus, the topography and the surface chemistry are playing the most important roles. The UNCD films are composed of diamond nanocrystallites with sizes of up to 10 nm surrounded by amorphous carbon representing a nanocomposite material. Furthermore they develop a specific nanotopography determined by the structure with a typical surface roughness in the order of 9-14 nm. The films after deposition are H-terminated and exhibited contact angles of 70-80° against water. The UV/O3 treatment, O2 and NH3 plasma modifications resulted in exchange of the termination bringing different O-containing or/and amine functional groups to the surface. Both modification processes rendered the UNCD surface strongly hydrophilic with contact angles below 10° without affecting the topography. The UNCD films investigated in the project provided a favorable combination of surface parameters (topography, wettability, surface chemical groups, etc.) allowing fast and strong attachment of neurons without application of adhesion proteins. This advanced attachment allowed for considerable improvement of primary cell culture techniques, for example, the exclusion of the centrifugation step during which most of the cell extensions are torn apart. In addition to the fast and strong attachment of the neurons on the UNCD surfaces, no adverse effects were observed on cell viability, expressed by their spontaneous activity and their responses to the neurotransmitter acetylcholine in Ca2+ imaging experiments. Thus, the results confirmed the biocompatibility of the as-grown and modified UNCD films established before with other cell lines. Another advantage of the enhanced neuron adhesion was the reduction of the cell losses during immunocytochemical characterization of cell cultures by more than one order of magnitude. Furthermore, the integration of a Ti grid structure beneath the UNCD allowed for the identification of individual cells while switching between the experimental setups. The surface patterning of a biomaterial, i.e. placing areas with different surface chemistries next to each other is an important step for various biomedical and biotechnological applications. In the project we also investigated the patterning of the surface termination of UNCD films. In order to achieve a high wettability contrast in the neighboring areas O- and F-terminations were selected. The sequence of the modifications and the feasibility of different masking techniques were tested in advance. Initially the whole UNCD surface was modified by UV/O3 treatment rendering the surface O-terminated and strongly hydrophilic. The partial masking of this surface before the second modification was achieved with a gold hard mask photolithographically structured and wet etched in form of a grid. After the second modification with CF3 plasma which provided F-termination and hydrophobic character of the surface (contact angle > 110°), the gold layer was removed. The results of the patterning of the surface termination were visualized by scanning electron microscopy revealing different contrasts of the areas with different terminations. Human SH-SY5Y neuroblastoma cells were plated on the patterned UNCD surfaces and they exhibited preferential attachment and growth on the hydrophilic grid.
Publications
- Bio-properties and applications of ultrananocrystalline diamond films, Proceedings of the International Workshop „Progressive Biomedical Materials and Technologies 2015“, October 9-10, 2015, Kladno, Czech Republic, ISBN: 978-80-01-05818-3, pp. 11-18
C. Popov
- Plasma surface fluorination of ultrananocrystalline diamond films, Surface and Coatings Technology 302 (2016) 448-453
W. Kulisch, A. Voss, D. Merker, J.P. Reithmaier, R. Merz, M. Kopnarski, C. Popov
(See online at https://doi.org/10.1016/j.surfcoat.2016.06.029) - Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces, Materials Science and Engineering C 64 (2016) 278-285
A. Voss, H. Wei, Y. Zhang, S. Turner, G. Ceccone, J.P. Reithmaier, M. Stengl, C. Popov
(See online at https://doi.org/10.1016/j.msec.2016.03.092)
