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Fragmented multi-layered graphene films were directly synthesized via chemical vapor deposition (CVD) on dielectric substrates with a pre-deposited copper catalyst. We demonstrate that the thickness of the sacrificial copper film, process temperature, and growth time essentially influence the integrity, quality, and disorder of the synthesized graphene. Atomic force microscopy and Kelvin probe force microscopy measurements revealed the presence of nano-agglomerates and charge puddles. The potential gradients measured over the sample surface confirmed that the deposited graphene film possessed a multilayered structure, which was modelled as an ensemble of randomly oriented conductive prolate ellipsoids. THz time domain spectroscopy measurements gave the ac conductivity of the graphene flakes and homogenized graphitic films as being around 1200 S cm−1 and 1000 S cm−1, respectively. Our approach offers a scalable fabrication of graphene structures composed of graphene flakes, which have effective conductivity sufficient for a wide variety of THz applications.

Raman spectroscopy was used to determine mechanisms for neutrophil activation upon interaction with multi-walled carbon nanotubes (MWCNTs) possessing CO2H groups (MWCNTCO2H) and in complexes with DNA (MWCNT–CO2H/DNA). Short MWCNT–CO2H and MWCNT–CO2H/DNA (100–300 nm) were found to interact with neutrophils, causing cytoskeleton reorganization, namely, activation of G-actin to give F-actin in cells. Raman spectroscopy showed that these CNTs are not phagocytized by neutrophils. MWCNT–CO2H enhances the generation of reactive oxygen and chlorine species by neutrophils as indicated by an increase in the NADPH–oxidase activity in a cell suspension. The interaction of MWCNT–CO2H/DNA leads to inhibition of the oxygen-activating ability of phagocytes and formation of neutrophilic extracellular traps (NET) with the inclusion of carbon nanostructures. The free radical products generated by neutrophils disrupt the crystal structure of MWCNTs.

A systematic spectroscopic characterization of highly homogeneous water suspensions of ‘buckydiamonds’ comprising sp3 cubic nanodiamond (ND) core covered with disordered sp2 shell densely decorated with oxygen-containing groups demonstrates the excitation-wavelength-dependent photoluminescence (PL) given by at least four types of specific structures on the ND surface (hydroxyl, C=O containing ketones, carboxylic anhydrides, and carboxyl groups). PL properties of NDs suspensions possess concentration-dependent behavior revealing tendency of NDs to agglomerate. PL of NDs has been found to be strongly sensitive to pH of the environment in wide range of pH values, i.e. 2–11. We disclosed the mechanisms of pH sensitivity of the ‘buckydiamond’ and proved that it can serve as all-optical sensor of tiny pH variations suitable for further exploitation for pH sensing locally in the area where NDs have been delivered for any purpose, e.g. bioimaging or therapeutic needs.

In living organisms, redox reactions play a crucial role in the progression of disorders accompanied by the overproduction of reactive oxygen and reactive chlorine species, such as hydrogen peroxide and hypochlorous acid, respectively. We demonstrate that green fluorescence graphene quantum dots (GQDs) can be employed for revealing the presence of the hypochlorous acid in aqueous solutions and cellular systems. Hypochlorous acid modifies the oxygen-containing groups of the GQD, predominantly opens epoxide ring C–O–C, forms excessive C=O bonds and damages the carbonic core of GQDs. These changes, which depend on the concentration of the hypochlorous acid and exposure time, manifest themselves in the absorbance and fluorescence spectra of the GQD, and in the fluorescence lifetime. We also show that the GQD fluorescence is not affected by hydrogen peroxide. This finding makes GQDs a promising sensing agent for selective detecting reactive chlorine species produced by neutrophils. Neutrophils actively accumulate GQDs allowing to visualize cells and to examine the redox processes via GQDs fluorescence. At high concentrations GQDs induce neutrophil activation and myeloperoxidase release, leading to the disruption of GQD structure by the produced hypochlorous acid. This makes the GQDs a biodegradable material suitable for various biomedical applications.

We demonstrated that wide-field second harmonic generation (SHG) microscopy of lung tissue in combination with quantitative analysis of SHG images is a powerful tool for fast and label-free visualization of the fibrosis pathogenesis in pulmonary arterial hypertension (PAH). Statistical analysis of the SHG images revealed changes of the collagen content and morphology in the lung tissue during the monocrotaline-induced PAH progression in rats. First order statistics disclosed the dependence of the collagen overproduction on time, the second order statistics indicated tightening of collagen fiber network around blood vessels and their spreading into the alveolar region. Fourier analysis revealed that enhancement of the fiber orientation in the collagen network with PAH progression was followed with its subsequent reduction at the terminating phase of the disease. Proposed approach has potential for assessing pulmonary fibrosis in interstitial lung disease, after lung(s) transplantation, cancer, etc.

Single-walled carbon nanotubes (SWCNTs) demonstrate a strong potential as an optically activated theranostic nano-agent. However, using SWCNTs in theranostics still requires revealing mechanisms of the SWCNT-mediated effects on cellular functions. Even though rapid and delayed cellular responses can differ significantly and may lead to undesirable consequences, understanding of these mechanisms is still incomplete. We demonstrate that introducing short (150–250 nm) SWCNTs into C6 rat glioma cells leads to SWCNT-driven effects that show pronounced time dependence. Accumulation of SWCNTs is carried out due to endocytosis with modification of the actin cytoskeleton but not accompanied with autophagy. Its initial stage launches a rapid cellular response via significantly heightened mitochondrial membrane potential and superoxide anion radical production, satisfying the cell demand of energy for SWCNT transfer inside the cytoplasm. In the long term, SWCNTs agglomerate to micron-sized structures surrounded by highly active mitochondria having parameters return to control values. SWCNTs postponed effects are also manifested themselves in the suppression of the cell proliferative activity with further restoration after five passages. These results demonstrate relative cellular inertness and safety of SWCNTs eliminating possible side effects caused by optically activated theranostic applications.

Integration of living cells with nonbiological surfaces (substrates) of sensors, scaffolds, and implants implies severe restrictions on the interface quality and properties, which broadly cover all elements of the interaction between the living and artificial systems (materials, surface modifications, drug-eluting coatings, etc.). Substrate materials must support cellular viability, preserve sterility, and at the same time allow real-time analysis and control of cellular activity. We have compared new substrates based on graphene and pyrolytic carbon (PyC) for the cultivation of living cells. These are PyC films of nanometer thickness deposited on SiO2 and black silicon and graphene nanowall films composed of graphene flakes oriented perpendicular to the Si substrate. The structure, morphology, and interface properties of these substrates are analyzed in terms of their biocompatibility. The PyC demonstrates interface biocompatibility, promising for controlling cell proliferation and directional intercellular contact formation while as-grown graphene walls possess high hydrophobicity and poor biocompatibility. By performing experiments with C6 glioma cells we discovered that PyC is a cell-friendly coating that can be used without poly-l-lysine or other biopolymers for controlling cell adhesion. Thus, the opportunity to easily control the physical/chemical properties and nanotopography makes the PyC films a perfect candidate for the development of biosensors and 3D bioscaffolds.

Carbon nanotubes are a very promising class of nanomaterials for biological and medical applications. The work aims at studying the interaction of rat C6 glioma cells with single-walled carbon nanotubes in complex with DNA (DNA-SWCNTs) paying the special attention to the changes in the structural, mechanical, and physical properties of the cell surface layer and the parameters of the DNA-SWCNT accumulation in cells. The properties of cells incubated with DNA-SWCNTs for 1–24 h were studied by atomic force microscopy, patch-clamp method, and Raman spectroscopy. The surface of glioma cells exposed to DNA-SWCNTs for 1 h had invaginations of size similar to the length of nanotubes (100–250 nm). The parameters of sliding friction force measured between the AFM tip and cell surface and the resting membrane potential were significantly reduced during the initial phase of DNA-SWCNT uptake (1–2 h) by cells. With further increase in incubation time (18–24 h), when DNA-SWCNTs were accumulated in the cytoplasm, the friction force parameters increased and became higher than the corresponding parameters for control cells. Using the decision tree algorithm for classification, the difference in the spatial distribution of the cell surface mechanical properties for C6 glioma cells exposed to DNA-SWCNTs during 1 or 24 h was shown with high classification accuracy. Our results prove that endocytosis is the main mechanism of DNA-SWCNT uptake by C6 rat glioma cells. The reorganization of the cell surface layer accompanying nanotube endocytosis leads to changes in the mechanical and physical properties of the cell surface.

The objective of the study is to determine the patterns of regulation of single-walled carbon nanotube accumulation, distribution, and agglomeration in glioma cells exposed to an external electric field. C6 glioma cells were treated with 5 μg/ml DNA wrapped single-walled carbon nanotubes and exposed to bi-phasic electric pulses (6.6 V/m, 200 Hz, pulse duration 1 ms). Nanotube accumulation was determined by Raman microspectroscopy and their intracellular local concentration was evaluated using the G-band intensity in Raman spectra of single-walled carbon nanotubes. It was revealed that the low-frequency and low-strength electric field stimulation of glioma cells exposed to single-walled carbon nanotubes led to facilitation and, thus, to amplification of nanotube accumulation inside the cells. The number of nanotubes in intracellular agglomerates increased from (28.8 ± 13.1) un./agglom. and (84.0 ± 28.7) un./agglom. in control samples to (60.6 ± 21.4) un./agglom. and (184.2 ± 53.4) un./agglom. for 1 h and 2 h stimulation, respectively. Thus, the tumor exposure to an external electric field makes it possible to more effectively regulate the accumulation and distribution of carbon nanotubes inside glioma cells allowing to reduce the applied therapeutic doses of carbon nanomaterial delivered anticancer drugs.

Rinnakkaistallenne avautuu 28.10.2021 https://repository.kaust.edu.sa/handle/10754/665699 Black silicon (bSi) refers to an etched silicon surface comprising arrays of microcones that effectively suppress reflection from UV to near-infrared (NIR) while simultaneously enhancing the scattering and absorption of light. This makes bSi covered with a nmthin layer of plasmonic metal, i.e., gold, an attractive substrate material for sensing of bio-macromolecules and living cells using surface-enhanced Raman spectroscopy (SERS). The performed Raman measurements accompanied with finite element numerical simulation and density functional theory analysis revealed that at the 785 nm excitation wavelength, the SERS enhancement factor of the bSi/Au substrate is as high as 108 due to a combination of electromagnetic and chemical mechanisms. This finding makes the SERS-active bSi/Au substrate suitable for detecting trace amounts of organic molecules. We demonstrate the outstanding performance of this substrate by highly sensitive and specific detection of a small organic molecule of 4-mercaptobenzoic acid and living C6 rat glioma cell nucleic acids/proteins/lipids. Specifically, the bSi/Au SERS-active substrate offers a unique opportunity to investigate the living cells' malignant transformation using characteristic protein disulfide Raman bands as a marker. Our findings evidence that bSi/Au provides a pathway to the highly sensitive and selective, scalable, and low-cost substrate for lab-on-a-chip SERS biosensors that can be integrated into silicon-based photonics devices.

Interaction of neutrophils with carbon nanotubes (CNT), 100–400 nm in length, functionalized with -COOH groups and non-covalently functionalized with DNA molecules, were investigated by means of Raman spectroscopy, luminol- and lucigenin-enhanced chemiluminescence, and fluorescence microscopy. It was shown that multi-walled CNT with COOH-groups and conjugated with DNA do not accumulate in neutrophils but induce reorganization of cells actin filaments and lead to neutrophils activation. It was established that according to the type of CNT and the method of CNT functionalization they are able to regulate the redox activity of neutrophils. We found that the interaction of double-walled CNT/DNA complexes with cells leads to an enhancement of superoxide generation, but the total amount of free radicals generated by neutrophils remains unaffected. Under the action of multi-walled CNT functionalized with - COOH groups on non-adherent neutrophils, NADPH oxidase is activated as well as an enhancement of the reactive oxygen and chlorine species generation is observed. The interaction of these CNT with neutrophils, stimulated with fMLP and adhesion, leads to the superoxide yield decrease. Thus, CNT could regulate the activation and redox processes in neutrophils.

Distribution of single-walled carbon nanotubes (SWCNT) in C6 glioma cells was investigated by means of confocal Raman microscopy. The analysis was done using short-length (100–250 nm) SWCNT previously dispersed in DNA aqueous solutions. Cytotoxic concentrations of short-length SWCNT for rat C6 glioma cells were established. SWCNT-DNA in nontoxic concentrations were found to penetrate and accumulate in cells after 6–8 h of cells cultivation in the presence of the complexes. SWCNT-DNA complexes distribution was uneven and maintained in cells during 5 passages. Simultaneous combination of confocal Raman microscopy and fluorescent microscopy using JC-1 or MitoSOX made it possible to establish, that the complexes are located predominantly near actively functioning mitochondria, causing their shape modification and slight mitochondrial membrane potential increase, electron transfer and superoxide production in complexes I and III modification. Prolonged C6 cells cultivation in the presence of SWCNT-DNA complexes led to the decrease in cells proliferative activity during the first 3 passages followed by its further recovery, causing, therefore, activation of cell stress adaptation mechanisms.