Egor Ukraintsev

Micro-Pattern Guided Adhesion of Osteoblasts on Diamond Surfaces

Microscopic chemical patterning of diamond surfaces by hydrogen and oxygen surface atoms is used for self-assembly of human osteoblastic cells into micro-arrays. The cell adhesion and assembly is further controlled by concentration of cells (2,500-10,000 cells/cm2) and fetal bovine serum (0-15%). The cells are characterized by fluorescence microscopy of actin fibers and nuclei. The serum protein adsorption is studied by atomic force microscopy (AFM). The cells are arranged selectively on O-terminated patterns into 30-200 μm wide arrays. Higher cell concentrations allow colonization of unfavorable H-terminated regions due to mutual cell communication. There is no cell selectivity without the proteins in the medium. Based on the AFM, the proteins are present on both H- and O-terminated surfaces. Pronounced differences in their thickness, surface roughness, morphology, and phase images indicate different conformation of the proteins and explain the cell selectivity.

Catalyst-free site-specific surface modifications of nanocrystalline diamond films via microchannel cantilever spotting

The properties of nanocrystalline diamond (NCD) films offer great potential for the creation of various sensing and photonic devices. A great challenge in order to materialize such applications lies in achieving the micrometrically resolved functionalization of NCD surfaces. In the present work, we introduce a facile approach to meet this challenge employing the novel strain-promoted alkyne–azide cycloaddition “click” chemistry reaction, a catalyst-free ligation protocol compatible with biomolecules. The ability to achieve well-resolved multicomponent patterns with high reproducibility is demonstrated, paving the way for the fabrication of novel devices based on micropatterned NCD films.

Epithelial cell morphology and adhesion on diamond films deposited and chemically modified by plasma processes

The authors show that nanocrystalline diamond (NCD) thin films prepared by microwave plasma enhanced chemical vapor deposition apparatus with a linear antenna delivery system are well compatible with epithelial cells (5637 human bladder carcinoma) and significantly improve the cell adhesion compared to reference glass substrates. This is attributed to better adhesion of adsorbed layers to diamond as observed by atomic force microscopy (AFM) beneath the cells. Moreover, the cell morphology can be adjusted by appropriate surface treatment of diamond by using hydrogen and oxygen plasma. Cell bodies, cytoplasmic rims, and filopodia were characterized by Peakforce AFM. Oxidized NCD films perform better than other substrates under all conditions (96% of cells adhered well). A thin adsorbed layer formed from culture medium and supplemented with fetal bovine serum (FBS) covered the diamond surface and played an important role in the cell adhesion. Nevertheless, 50-100 nm large aggregates formed from the RPMI medium without FBS facilitated cell adhesion also on hydrophobic hydrogenated NCD (increase from 23% to 61%). The authors discuss applicability for biomedical uses.

bOptimizing atomic force microscopy for characterization of diamond-protein interfaces

Atomic force microscopy (AFM) in contact mode and tapping mode is employed for high resolution studies of soft organic molecules (fetal bovine serum proteins) on hard inorganic diamond substrates in solution and air. Various effects in morphology and phase measurements related to the cantilever spring constant, amplitude of tip oscillations, surface approach, tip shape and condition are demonstrated and discussed based on the proposed schematic models. We show that both diamond and proteins can be mechanically modified by Si AFM cantilever. We propose how to choose suitable cantilever type, optimize scanning parameters, recognize and minimize various artifacts, and obtain reliable AFM data both in solution and in air to reveal microscopic characteristics of protein-diamond interfaces. We also suggest that monocrystalline diamond is well defined substrate that can be applicable for fundamental studies of molecules on surfaces in general.

Nanostructured diamond layers enhance the infrared spectroscopy of biomolecules.

We report on the fabrication and practical use of high-quality optical elements based on Au mirrors coated with diamond layers with flat, nanocolumnar, and nanoporous morphologies. Diamond layers (100 nm thickness) are grown at low temperatures (about 300 °C) from a methane, carbon dioxide, and hydrogen gas mixture by a pulsed microwave plasma system with linear antennas. Using grazing angle reflectance (GAR) Fourier transform infrared spectroscopy with p-polarized light, we compare the IR spectra of fetal bovine serum proteins adsorbed on diamond layers with oxidized (hydrophilic) surfaces. We show that the nanoporous diamond layers provide IR spectra with a signal gain of about 600% and a significantly improved sensitivity limit. This is attributed to its enhanced internal surface area. The improved sensitivity enabled us to distinguish weak infrared absorption peaks of <10-nm-thick protein layers and thereby to analyze the intimate diamond-molecule interface.

Diamond as functional material for bioelectronics and biotechnology

Understanding the interaction between the biological environment (tissues, cells, proteins, electrolytes, etc.) and a solid surface is crucial for biomedical applications such as bio-sensors, bio-electronics, tissue engineering and the optimization of implant materials. Cells, the cornerstones of living tissue, perceive their surroundings and subsequently modify it by producing extracellular matrix (ECM), which serves as a basis to simplify their adhesion, spreading and differentiation (Shakenraad & Busscher, 1989). This process is considerably complex, flexible and strongly depends on the cell cultivation conditions including the type of the substrate. Surface roughness of the substrate plays an important role (Babchenko et al., 2009; Kalbacova et al., 2009; Kromka et al., 2009; Zhao et al., 2006), other influential factors include both the porosity (Tanaka et al., 2007) and the wettability of the substrate, the latter influencing protein conformation (Browne et al., 2004; Rezek, Ukraintsev, Michalikova, Kromka, Zemek & Kalbacova, 2009) as well as the adsorption and viability of cells (Grausova et al., 2009; Kalbacova, Kalbac, Dunsch, Kromka, Vanecek, Rezek, Hempel & Kmoch, 2007). Materials which are commonly employed as substrates for in vitro testing are polystyrene and glass. In this context, diamond as a technological material can provide a relatively unique combination of excellent semiconducting, mechanical, chemical as well as biological properties (Nebel et al., 2007). Diamond also meets the basic requirements for large-scale industrial application, most notably, it can be prepared synthetically. Diamond can be synthesized either as a bulk material under high-pressure and high-temperature conditions, or in the form of thin films by chemical vapor deposition of methane and hydrogen on various substrates including glass and metal (Kromka et al., 2008; Potocky et al., 2007). Moreover, the application of selective nucleation makes it possible to directly grow conductive diamond microstructures, which operate e.g. as transistors or pH sensors (Kozak et al., 2010). Nowadays, it is possible to deposit diamond even on large areas (600 cm2 or more) using linear antennas (Kromka et al., 2011; Tsugawa et al., 2010). The excellent compatibility of diamond with biological materials and environment (Bajaj et al., 2007; Grausova et al., 2009; Diamond as Functional Material for Bioelectronics and Biotechnology

Stochastic model explains formation of cell arrays on H/O-diamond patterns

Cell migration plays an important role in many biological systems. A relatively simple stochastic model is developed and used to describe cell behavior on chemically patterned substrates. The model is based on three parameters: the speed of cell movement (own and external), the probability of cell adhesion, and the probability of cell division on the substrate. The model is calibrated and validated by experimental data obtained on hydrogen- and oxygen-terminated patterns on diamond. Thereby, the simulations reveal that: (1) the difference in the cell movement speed on these surfaces (about 1.5×) is the key factor behind the formation of cell arrays on the patterns, (2) this difference is provided by the presence of fetal bovine serum (validated by experiments), and (3) the directional cell flow promotes the array formation. The model also predicts that the array formation requires mean distance of cell travel at least 10% of intended stripe width. The model is generally applicable for biosensors using diverse cells, materials, and structures.

Dose-Rate Effects in Breaking DNA Strands by Short Pulses of Extreme Ultraviolet Radiation

In this study, we examined dose-rate effects on strand break formation in plasmid DNA induced by pulsed extreme ultraviolet (XUV) radiation. Dose delivered to the target molecule was controlled by attenuating the incident photon flux using aluminum filters as well as by changing the DNA/buffer-salt ratio in the irradiated sample. Irradiated samples were examined using agarose gel electrophoresis. Yields of single- and double-strand breaks (SSBs and DSBs) were determined as a function of the incident photon fluence. In addition, electrophoresis also revealed DNA cross-linking. Damaged DNA was inspected by means of atomic force microscopy (AFM). Both SSB and DSB yields decreased with dose rate increase. Quantum yields of SSBs at the highest photon fluence were comparable to yields of DSBs found after synchrotron irradiation. The average SSB/DSB ratio decreased only slightly at elevated dose rates. In conclusion, complex and/or clustered damages other than cross-links do not appear to be induced under the radiation conditions applied in this study.

Diamond nanoparticles suppress lateral growth of bacterial colonies

Diamond nanoparticles (DNPs) of various types have been recently reported to possess antibacterial properties. Studies have shown a decrease of the colony forming ability on agar plates of the bacteria that had been previously co-incubated with DNPs in the suspension. Before plating, bacteria with DNPs were adequately diluted in order to obtain a suitable number of colony forming units. However, residual DNPs were still present on an agar plate, concentrated on the surface during the plating process; this introduces a potential artifact which might affect colony growth. The effect of DNPs remaining on the surface, alongside growing bacteria, has not been previously investigated. In this work, we present the experiments designed to investigate the effect of DNPs on bacterial survival and on the growth of the bacterial colony on a solid media. We employed Escherichia coli and Bacillus subtilis as models of Gram-negative and Gram-positive bacteria, respectively, and Proteus mirabilis as a model of bacterium exhibiting swarming motility on the surfaces. We analyzed the number, area, and weight of bacterial colonies grown on the agar surface covered with DNPs. We did not observe any bactericidal effect of such applied DNPs. However, in all bacterial species used in this work, we observed the appreciable reduction of colony area, which suggests that DNPs obstruct either bacterial growth or motility. The most obvious effect on colony growth was observed in the case of motile P. mirabilis. We show that DNPs act as the mechanical barrier blocking the lateral colony growth.

Is there any dose-rate effect in breaking DNA strands by short pulses of extreme ultraviolet and soft x-ray radiation? (Conference Presentation)

Possible dose-rate effects in a plasmid DNA exposed to pulsed extreme ultraviolet (XUV) and soft x-ray (SXR) water window radiation from two different table-top plasma-based sources was studied. Dose delivered to the target molecule was controlled by attenuating the incident photon flux with aluminum thin foils as well as varying the DNA/buffer-salt ratio in the irradiated sample. Irradiated samples were analyzed using the agarose gel electrophoresis. Some additional bands were identified in gel electrophoretograms as results of a DNA cross-linking. They were inspected by atomic force microscopy (AFM). Yields of single- and double-strand breaks (Gy-1 Da-1) were determined as a function of incident dose rate. Both yields decreased with a dose rate increasing. The ratio of single- and double-strand breaks exhibited only a slight increase at elevated dose rates. In conclusion, complex and/or clustered damages do not seem to be initiated under these irradiation conditions.