Annotation of the results obtained in 2022
Samples of hybrid materials were obtained according to established and verified protocols for the formation of previous stages. All samples before and after thermal modifications were studied by optical and scanning electron microscopy. The integrity of the outer sides of cell membranes, cell structures as a whole, was confirmed at temperatures not exceeding the range from 25 to 200-250 C with short-term (no more than 20 minutes) temperature effects in the air atmosphere (or flow exposure to argon) and in-situ annealing in ultrahigh vacuum. In general, exceeding the specified annealing durations even at temperatures of 200-250 C, and with increasing temperatures, lead to a violation of the integrity of cell structures distributed over the surface of the substrates. Thermal exposure in the Ar-H medium leads almost immediately to partial, and then, after 30 minutes, to a noticeable destruction of cell integrity. An increase in temperature and exposure time leads to the destruction of the hybrid material structures. For cell cultures based on E.coli, the stability of the processes of integration of Fe ions at exposure temperatures up to 300 C and with an exposure duration of 40 minutes is shown. An increase in temperature and exposure time leads to the destruction of the structures of this hybrid material. Structures with porous Si nanoparticles integrated with mammalian cells undergo significant changes after thermal effects. The obtained optical and electron microscopy data indicate partial destruction of hybrid structures at temperatures up to 250 C and almost complete removal of structures at values over 350 C. Based on the data from the study of Fe L2,3 edges and partially O K and C K synchrotron XANES data on the atomic and electronic structure during in situ/ex situ thermal transformations of inorganic particles in hybrid nanomaterials based on E.coli cell cultures with ultra–small Fe-O nanoparticles, the following is shown. Short times (up to 15 minutes) and exposure temperatures up to 250 C in an atmosphere of air, argon or in ultrahigh vacuum during in-situ annealing do not lead to transformations of the local environment of iron atoms of nanoparticles in cells. In the "restoring" atmosphere of Ar-H, the changes are significant in the direction of increasing the contribution of FeO. An increase in the time (up to 40 minutes) and/or the temperature of impacts up to 350 C in the atmosphere of air, Ar or during in-situ annealing leads to a change in the protein "matrix", up to destruction. Fe recovery does not occur even in an Ar-H atmosphere. The greatest interest for the transformations of hybrid nanomaterials based on E.coli cell cultures with ultra-small iron oxide nanoparticles is the possibility of restoring nanoparticles to a metallic state and removing the carbon "matrix" (as a container for delivering nanoparticles). An increase in exposure time (up to 40 minutes), temperatures (up to 500 C) lead to a reduction of Fe up to 50% with ex-situ exposure in the reducing atmosphere Ar-H. The XPS method data generally confirmed the XANES data. To achieve comparable sounding depths, focused ion etching was used, in the mildest modes established at the previous stages. The analysis was carried out by the fine structure of Fe 2p lines, as well as O and C 1s, the overall ratio of the line intensities of the survey spectra. A possible explanation of the experimental results is the formation of cluster-type agglomerates with a balanced composition of FexOy(H), where stoichiometry will be determined by the size and nature of the particle itself (the molecule in whose cavity the cluster accumulates) and the probability of the formation of a core-shell structure within the particle. Thus, the cell itself can actually act as a factory producing spherical dps nanomolecules (about 10 nm) with an inorganic nanonuclear iron and its compounds in the inner cavity (no more than 6 nm). At the same time, thermal action can restore nanoparticles of the Fe – O composition to a metallic state. Based on data from the study of Si L2,3 edges and partially O K and C K edges of the XANES synchrotron method on the atomic and electronic structure of the structure during in situ/ex situ thermal transformations of inorganic particles in hybrid nanomaterials based on mammalian cell cultures decorated with Si – O nanoparticles, including at different incubation times of nanoparticles in cells, the following is shown. At temperatures not exceeding the range from room temperature to 200 C, with short-term (no more than 20 minutes) temperature effects in the air atmosphere, flowing Ar exposure and in-situ annealing in ultrahigh vacuum, no noticeable conformation violations are observed according to microscopy data. There are also no changes in the specifics of the local environment of Si atoms in the nanoparticles of the cell array, the electronic spectrum. The changes are fragmentary, temperatures and times are not enough to activate the processes of Si oxidation, including due to the destruction of the organic environment. An increase in temperature over 350 C and times over 20 minutes leads to a significant removal of cellular material and almost complete oxidation of Si nanoparticles. Heating at the same temperatures in a "reducing" atmosphere stimulates the oxidation of Si nanoparticles, probably due to the higher rate of destruction of the organic part of the biohybrid structure. Silicon recovery has not been established. The XPS method data generally confirmed the XANES data. To achieve comparable sounding depths, focused ion etching was used, in the mildest modes established at the previous stages. The analysis was carried out by the fine structure of Si 2p lines, as well as O and C 1s, the overall ratio of the line intensities of the survey spectra. The fine structure of Si 2p lines, including after ion etching, showed greater susceptibility to thermal effects of mammalian cells with internalized nanoparticles of porous Si than E.coli with Fe – O particles. Changes in the fine structure of the Si 2p, O 1s and C 1s spectra indicate the removal of the organic part of the structures at temperatures above 350 C, especially in the "reducing" atmosphere of Ar-H. The XPS method did not show Si reduction in nanoparticles. The processes of active and destructive interaction with the intracellular medium with respect to Si nanoparticles in the hybrid material formed in vitro have been confirmed, which in the future can be controlled and ruled out thermally, for example, by local heating. The results of computer simulation showed that the alpha phase of Fe under consideration corresponds to an ordered magnetic structure, and all Fe atoms in full accordance with the experiment have co-directional magnetic moments. The analysis of partial densities of states indicates that the main contribution to the formation of the energy structure is given by d-states. There is a good correspondence between the calculated and measured X-ray emission and absorption spectra. A number of features of the fine structure have not been resolved, which requires clarification. The result of the analysis of the whole project realization is to establish the possibility of effective and controlled integration, combining small-sized inorganic nanoparticles with cellular material of various origins. Systems of inorganic particles in hybrid nanomaterials based on E.coli cell cultures with ultra–small Fe -O nanoparticles, as well as inorganic particles in hybrid nanomaterials based on mammalian cell cultures decorated with nanoparticles of porous Si and its oxides have been studied. The possibility of controlling and fine-tuning the features of the local atomic structure (environment), the electron-energy spectrum of inorganic nanoparticles in hybrid cell materials formed in vitro is shown. It seems promising to continue research on the influence of low temperatures, up to cryogenic, on the atomic and electronic structures and properties of hybrid cellular materials, on the interaction of biohybrid material with real 3D surfaces.

 

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Annotation of the results obtained in 2019
Using a unique world-class infrastructure facility of the Kurchatov synchrotron radiation source, a series of experiments was carried out to record synchrotron data from X-ray Absorption Near Edge Structure (XANES) and X-ray Photoelectron Spectroscopy (XPS). A set of calibration structures of iron-oxygen and silicon-oxygen systems, a number of others, as the main ones is determined. The choice of calibration structures is due to their phase and structural stability during long-term storage in laboratory conditions and multiple full reproducibility. Based on the selected methodological conditions and their confirmation by measurements of calibration structures, the registration of synchrotron XANES and XPS data for all reference structures was carried out. The detailed analysis of the electronic structure and specificity of the local atomic surrounding of the iron and silicon atoms, and in some cases oxygen and other studied data on physico-chemical state of reference objects conclusions about the specificity of the oxidation of iron nanoparticles and its oxides (hydroxides) in the free state and in a variety of surrounding, including the interaction with water and the main working solutions used in the preparation of cell cultures and nanoparticles of silicon and its oxides. Changes in the composition, structure, specificity of the local atomic surrounding and electronic spectrum, as well as other properties (morphology, substructure, clusterisation, formation and restructuring of phase and grain boundaries, etc.) as a result of natural oxidation processes and thermally stimulated in different regimes are shown. Focused ion beam profiling modes with estimated profiling rate from 1 A to 1 nm per minute are established. Temporary calibration criteria levels allowing to stabilize physical and chemical properties of the profiled surface including connections of systems iron-oxygen or silicon-oxygen are established. The registered results of evaluation of calibration and reference structures are systematized and collected in subject-thematic sequences. The results obtained by Scanning Electron Microscopy (SEM, including microanalysis data), X-ray diffraction, and, of course, the data of XANES and XPS methods, including ones after computer modeling, obtained using synchrotron radiation of the Kurchatov Institute source are generalized and systematized. Optical methods (for example, in the control of optical density for E.coli cell culture) established and controlled the processes of quantitative accumulation of cells, showing the stages of growth and saturation necessary for the preparation of hybrid structures. The general morphological stability of cell culture arrays to radiation from a number of electromagnetic radiation sources is shown: mercury lamp (quantum energy ~ 5 eV), X-ray tube (1486.61 eV), high-intensity synchrotron radiation (80-1200 eV). The preserved organic framework is insensitive to irradiation by focused electron beams (samples were subjected to numerous investigations by scanning electron microscopy) with energies of 2-20 kV. This picture is observed even after stimulated vacuum drying, including objects containing integrated inorganic particles iron-oxygen or silicon-oxygen in the concentrations of the initial samples of hybrid materials. The method of photoemission electron microscopy, applied for the first time in world practice for E.coli cells (initial samples) in combination with XPS and SEM studies confirmed this fact. Only extremely partial and fragmentary destruction of the membrane (surface) of cells during integration with particles, as well as during their long-term irradiation, is shown. A comprehensive analysis of data from synchrotron XANES and XPS studies of initial hybrid nanomaterials based on E.coli cell cultures with ultra-small iron oxide nanoparticles was carried out. It is shown that E.coli cultures in the initial state do not contain noticeable traces of iron and its oxides obtained by unicellular from the natural environment of growth and location. This fact may be evidence that even under conditions of Dps protein overproduction by cells, the accumulation of inorganic particles occurs extremely fragmentary and not necessarily on the outer surface of the cell membrane. However, even a small addition of a source of iron ions in the form of dissolved Mohr salt leads to the appearance of traces of iron oxides on the surface of cultures, which was noted only when saturated layering, when according to SEM cells constitute a dense micron layer formed on the substrate. Moreover, according to the set of provisions 2p iron core level shows a wide range of different oxides and hydroxides that exist on the surface of the samples, including even residual traces of Mohr salt. However, the XPS data of the 2p position of the iron core level and the fine structure of the XANES L2,3 spectra of the iron, including the position of the main resonance, indicate the absence of metallic iron. In general, the efficiency of using the Mohr salt (FeSO4*(NH4)2SO4*6H2O) as a source of iron ions is shown. The results of XANES spectroscopy are generally confirmed by the detailed consideration of oxygen absorption edge spectroscopy and mathematical modeling of XPS data on O1s and C1s core levels: when combined with the cell surface, a wide range of iron atoms states with different local environment character by oxygen atoms is formed: from the traditional and expected Fe2O3 to the persisting FeO throughout the experiments. It is concluded that it is necessary to optimize the process of washing samples to remove residual salts, fractal structures covering the entire surface of the sample in the absence of washing. Complete removal of salts must be carried out in order to isolate iron compounds in the composition of hybrid nanoparticles, with the accumulation in the cell itself, performing the role of a factory producing spherical nanomolecules Dps (about 10 nm) with an inorganic nanonuclear iron and its compounds in the inner cavity (not more than 6 nm). Criteria conditions of preparation and layering of a hybrid nanomaterial based on E.coli cell cultures with ultra-small iron oxide nanoparticles on the types of substrates used and modes of reliable detection of X-ray spectral signals using synchrotron radiation are determined. A comprehensive detailed analysis of X-ray spectral data of high resolution synchrotron XANES and XPS for the initial silicon powders of various methods of formation, consisting of nanoparticles of the silicon-oxygen system, as well as the initial hybrid nanomaterials based on mammalian cell cultures, decorated with silicon nanoparticles and its oxides. The composition and structure of silicon nanoparticles are significantly dependent on the technology of formation, drying and storage of powders before their integration with cell culture. The composition of the initial powders unambiguously includes a coating, within 5 nm, of natural silicon oxide, but of a different nature of the packing density of silicon-oxygen tetrahedra. Freeze-drying results in a denser package with an oxide layer thickness of less than 3 nm. Standard air drying results in a less dense oxide, up to about 4 nm thick. Silicon atoms are in a disordered state in the surface nanolayers, however, a number of features of the XANES Si L2, 3 and O K spectra may indicate partial preservation of the local atomic order. The presence of a number of silicon suboxides distributed in the surface layer of powder particles not exceeding 5 nm at SiOx oxidation states with x values from 1 to 2 is noted. The thickness of the transition layer also depends significantly on the way the particles are prepared for integration with cell cultures. Combining particles with mammalian cell cultures 3T3NIH (mouse embryonic fibroblast line) changes the physicochemical state of the surface of silicon oxygen nanoparticles. Chemical shifts of 2p silicon core level and 1s oxygen core level confirm the transformation of the physico-chemical state of powder particles during integration with cells and the stability of their composition during prolonged storage in laboratory conditions and a partial increase in the thickness of the silicon oxide layer is shown. The slope and position of the main characteristic features of the fine structure of the Si L2,3 and O K XANES spectra also confirms a wide range of silicon oxides with a disturbed oxidation state and the local environment of silicon atoms with oxygen. Sufficient thickness of the assumed oxide shell is the reason for the stability of their composition when integrated into cells with marked partial oxidation. As in the case of hybrid nanomaterials based on E.coli cultures, a significant issue is the cleaning of the surface from the salts of working solutions, which is the cornerstone for obtaining the most reliable data. Criteria levels of silicon nanoparticles and its oxides concentrations during integration with cell culture, layering modes on the used substrate types and modes of reliable detection of X-ray spectral signals using synchrotron radiation of Unique Scientific Infrastructure Facility were determined. Preparation for the first-principle modeling of the main phases of the studied inorganic compounds was carried out. Calculations of zone structure, spectra of total and partial densities of states of stishovite - tetragonal superdense modification of SiO2 are performed. The influence of the choice of exchange-correlation potential on the obtained electronic structure of crystalline silicon is investigated. Three different approximations were used: LDA, GGA and mBJ (using the exchange-corellation potential mBJ as an amendment to GGA for silicon gave a result close to experimental). The principal possibility of combining inorganic nanoparticles based on iron oxides with E.coli cell cultures or inorganic nanoparticles based on silicon oxides with mammalian cell cultures (3T3NIH) is shown. The stability of the hybrid nanomaterials created, capable even in atmospheric conditions to act as carriers (containers) of nanoparticles preserving their properties for a long time, is established. The effect of combining natural cell cultures on the transformation of the local atomic and electronic structure and on the physical and chemical state of inorganic particles during their successful formation and / or integration is shown. For two types of hybrid nanomaterials, the relationship between the dimensional and structural properties of the developed surface and the specificity of the local environment of the atoms composing inorganic nanoparticles is shown. Iron nanoparticles are able to maintain a stable composition for a long time, different from that expected for the developed surface (the outer part of the cell membrane) of iron oxide Fe2O3. FexOy(H) materials persist for a long time, due to the formation of core-shell structures or nanocluster structures within the inorganic nuclei of Dps protein molecules formed by E.coli. At the same time, under the conditions of protein overproduction by the cell, according to optical studies, and at the stage of long-term storage, the membrane does not experience structural-phase transformation, aggregating at the same time iron-containing nanoparticles. Nanoparticles of silicon and its oxides when integrated with mammalian cells after successful combination are also stable and are able to maintain dimensional and structural properties for a long time, including the established silicon suboxides. However, the activity of cell culture 3T3NIH in relation to the composition of nanoparticles, which is revealed in their oxidation, is shown.

 

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Annotation of the results obtained in 2020
Using a unique world-class infrastructure facility, the Kurchatov synchrotron radiation source, a series of experiments was performed to record synchrotron data from X-ray Absorption Near Edge Structure (XANES) and X-ray Photoelectron Spectroscopy (XPS). Studies of the reconstruction of the atomic and electronic structure with stimulated growth of the content of inorganic particles in hybrid nanomaterials based on cell cultures: E.coli with ultra-small iron oxide nanoparticles; mammals decorated with silicon and its oxides nanoparticles. Computer simulations were performed, including ab-initio calculations. The properties of the prepared samples were studied by optical microscopy (confocal and fluorescent) with a stimulated increase in the content of inorganic particles in hybrid nanomaterials. It is shown that the applied modes of saturation of cell cultures with nanoparticles of iron, silicon and its oxides do not lead to the destruction of the integrity (conformation) of cell structures, especially the outer sides of cell membranes. High-resolution microanalysis combined with selective surface mapping has been used to determine the efficiency of integration processes of iron ions formed in solutions with E. coli-based cell material. Significant penetration of iron atoms (ions) deep into the layer of cellular material was found. Data from the scanning electron microscopy method showed a possible manifestation of cellular visicles when saturated with iron particles in Dps molecules during protein superproduction. In this case, individual disorders in the area of cell membranes are detected, in our opinion, with a possible mechanism for the formation and destruction of visicular formations, which is accompanied by the release of molecular hybrid material into the extracellular space. The number of particles introduced into the culture medium for the integration of Si nanoparticles with mammalian 3T3NIH and MCF-7 cells varied with the introduction and with the variation of incubation time. Microscopic studies have shown the effectiveness of integrating nanoparticles without losses under the selected growth, saturation, integration (incubation) and subsequent fixation modes. A separate additional series of experiments was performed to refine the choice of substrates for sample deposition/layering/incubation. It is shown that the use of titanium foils relieves from numerous methodological difficulties, makes the process of recording synchrotron spectra effective, samples formed on the surface are stable, and the results of synchrotron studies are reproducible. High-resolution synchrotron experimental data of the XANES method on the atomic and electronic structure with stimulated growth of the content of inorganic particles in hybrid nanomaterials based on E.coli cell cultures with ultra-small iron oxide nanoparticles showed the following, based on data from the study of L2,3 iron edges and partially K oxygen edges. An increase in the content of nanoparticles in general does not lead to noticeable changes in the XANES Fe L2,3 signal intensity. In our opinion, this is due to the predominant formation of nanoparticles in the entire volume of the cell (in thickness within 1 microns), while the process is stable on the outside of the membrane due to the presence of a certain amount of Dps protein, which can be assumed to be unchanged or slightly changing in comparison with variations in the number of iron 2+ions presumably involved in the process (on the membrane and inside the cell). There is a preservation of conformation in the cell structure. In general, the analysis of the fine structure of the L2,3 iron spectra showed that there is no complete transition to Fe2O3 oxide in the formed nanoparticles, even with their stimulated growth. This is due to the formation of cluster-type agglomerates with a balanced composition of FexOy(H), where the stoichiometry will be determined by the size and nature of the particle itself (the molecule in the cavity of which the cluster accumulates) and the probability of formation of a core-shell structure within a single particle and their ultra-small agglomerates, sizes not exceeding hundreds of nanometers. At the same time, the introduction of bonds with hydrogen atoms in the formation of hydroxide groups also leads to a detectable rearrangement of the Fe L2, 3 XANES spectra with characteristic changes in the distribution of their fine structure and the energy positions of the main features. The results of mathematical modeling of XANES Fe L2, 3 spectra based on reference data confirmed this. It is shown that these processes occur independently of the amount of iron ions introduced from outside and are determined by the specific functioning of cells and their constituent Dps proteins. For example, the Dps protein superproduction shows an increase in the relative signal intensity when registering synchrotron XANES spectra, but the fine structure of these spectra does not show any noticeable changes. Criteria for conducting experiments and compatibility of cell cultures with inorganic nanoparticles based on the iron-oxygen system are established. Synchrotron and laboratory experimental data of survey and high-resolution spectra of the XPS method (Fe 2p, O1s, C1s, etc. in general, the conclusions made based on the analysis of the results of the XANES synchrotron method confirm the atomic and electronic structure of the stimulated growth of the content of inorganic particles in hybrid nanomaterials based on E.coli cell cultures with ultra-small iron oxide nanoparticles. However, it should be noted that the depth of the informative layer for all XPS studies was 2-3 nm according to our data. This made it possible to study the real surfaces of cell membranes. Accordingly, the number of nanoparticles formed in such a probed layer is estimated by us as an order of magnitude smaller, taking into account the thickness (width) of the cell, electron microscopy data, etc. As a result, at a resolution of ~ 0.1 eV, the required time was more than ten hours for detailed recording of chemical shifts in the core levels. The spectra of the 2p 1/2 2p 3/2 fine structure of iron show a set of contributions of components of 2-and 3- valence iron, metallic iron, in a number of cases of protonation, confirming the composition of FexOy (H). Criteria conditions for conducting experiments are established. Synchrotron high-resolution experimental data of the XANES method (Si L2,3, O K, other if necessary) on the atomic and electronic structure with a stimulated increase in the content of inorganic particles in hybrid nanomaterials based on mammalian cell cultures decorated with silicon nanoparticles and its oxides showed the following, based on data from the study of the L2,3 edges of silicon and partially K edges of oxygen. In contrast to hybrid materials based on E.coli cells, in which nanoparticle formation occurs "from the inside" in the cavities of spherical Dps nanomolecules, 3T3 NIH mouse fibroblasts and MCF-7 human breast cancer cells were saturated "from the outside" directly by adding a suspension of silicon nanoparticles to the culture medium. Due to this, the quantitative factor certainly affected the integration with cell cultures, primarily during the degradation of nanoparticles over the incubation time in cells. With the stimulated increase in the content of silicon nanoparticles in the cell medium, a significant dependence of the relative contribution and composition of these nanoparticles on the incubation time from 7 to 72 hours and the initial amount of introduced nanoparticles of 50 - 500 micrograms/ml is shown. It is shown that at the initial stages of incubation with cell cultures, a partial removal of the natural oxide layer from the surface of nanoparticles occurs. This degradation is the result of the beginning of the dissolution processes of silicon nanoparticles during physical and chemical interaction with the intracellular environment. At the same time, over the incubation time, the signal from silicon atoms is generally attenuated: up to 4 times. The relative intensity of the oxide part of the spectrum with respect to silicon increases over the incubation time, indicating that the process of reforming the oxide coating of particles is accompanied by its gradual dissolution by the intracellular medium. Criteria for conducting experiments and compatibility of cell cultures with inorganic nanoparticles based on the silicon-oxygen system are established. Synchrotron experimental data, overview and high resolution of the XPS method (Si 2p, O1s, C1s, etc. the results of the XANES synchrotron method generally confirm the conclusions made by the results of the XANES synchrotron method on the atomic and electronic structure with stimulated growth of the content of inorganic particles in hybrid nanomaterials based on mammalian cell cultures decorated with silicon and its oxides. An increase in the relative intensity of the contribution of 2p states of silicon atoms accompanies an increase in the number of nanoparticles introduced proportionally. This indicates the effectiveness of the processes of absorption of Si nanoparticles by cell cultures. An increase in the incubation time shows a decrease in the contribution of silicon to the surface composition of hybrid materials, confirming the ongoing dissolution processes. The fine structure of Si 2p lines shows a dynamic increase in the contribution of oxides and a gradual disappearance of the contribution from elementary silicon with increasing integration time. Criteria for conducting experiments and compatibility of cell cultures with inorganic nanoparticles based on the silicon-oxygen system are established. In our opinion, in the case of all hybrid materials, measurements occur almost at the sensitivity limit of the XANES and XPS methods, despite the use of high-intensity synchrotron radiation. Computer models of the electronic structure of silicon nanofilms with a thickness of 1 to 10 unit cells are obtained. Analysis of the full transformation spectrum of the electron state density with increasing thickness of nanofilms showed that for films with thickness in 1 cell spectrum is fundamentally different from the bulk crystal with increasing film thickness, this difference gradually diminishes, and the spectra of films with thickness of 6 and more cells show the main features of the electronic structure of the bulk crystal. The method of decomposition of experimental XANES L2,3 spectra into L2 and L3 components is implemented. The proposed method is used to decompose the experimental XANES L2, 3-spectrum of silicon into L2 and L3 components. In this case all the spin-doublets have been separated and assigned to different spectra. The theoretical XANES L3 spectrum of silicon is calculated and the contributions to the calculated spectrum from the s-and d-symmetry states are obtained. Based on the calculation, the interpretation of the features of the experimental spectrum is given. It is shown that near the edge of the spectrum in the energy range ~2 eV wide, the s-and d-states make a comparable contribution to the spectrum, and at high XANES L3 transition energies, the silicon spectrum is formed mainly by d-symmetry states. All data from experimental measurements (obtained using synchrotron radiation from the Kurchatov Institute) and theoretical calculations and simulations for the XANES and XPS methods are generalized and collected in separate thematic assemblies by the type of measured spectra or by the type of objects studied. Under conditions of stimulated growth of nanoparticles of hybrid materials, the general morphological stability of cell culture arrays to the accumulated radiation dose of a number of electromagnetic radiation sources, including long - term exposure: X-ray tube (1486.61 eV), high-intensity synchrotron radiation (80-1200 eV). Also, the preserved organic framework is insensitive to irradiation with focused electron beams (the samples were subjected to numerous studies by scanning electron microscopy) with energies of 2-20 kV.

 

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Annotation of the results obtained in 2021
Prior to ion profiling, a series of repeated registration of XPS spectroscopy data (if necessary with XANES control) for calibration structures was carried out. The choice of calibration structures is due to their phase and structural stability during long-term storage in laboratory conditions and multiple complete reproducibility of the composition according to the fine structure of the recorded spectra. When registering XPS data, where it is possible, we have decided to use an "internal" calibration for 1s level of hydrocarbon contamination (285 eV). However, when working with materials of natural origin, this is not always possible. We carried out calibration for core levels Au 4f 7/2, Cu 2p 3/2, as well as a number of others. The XPS data of the initial reference structures of the main compounds planned for detection in functional inorganic nanoparticles, including taking into account ion profiling, were re-registered. Ion profiling was carried out for all calibration and reference materials. When understanding the order of the thicknesses of the layers of natural oxide, the known thicknesses of films, the level of carbon-containing contaminants, it was decided to use two basic values of accelerating voltages of 1 and 3 kV with etching times from 1 minute to half an hour, their stepwise combinations with registration of verification data, or a full XPS experiment cycle after each etching stage. Etching rates were experimentally tested. A voltage of 1 kV was chosen as the lowest stable accelerating voltage in terms of value, providing soft removal of ultra-thin layers of the surface. However, in order to minimize phase transformations with long profiling times, a single etching step was no more than 10 minutes. A voltage of 3 kV was used to understand and refine possible phase transformations with a significant increase in the profiling rate, which is significant for inorganic nanoparticles and their bio-environment. For fine profiling, which practically excludes interatomic mixing as a result of ion beam irradiation, the 1 kV mode is preferable, as well as step-by-step etching with the control of XPS survey spectra, for which a step of 1 eV is sufficient to register. The newly obtained samples of hybrid materials were double-checked by optical and scanning electron microscopy, including the variability of the content of inorganic particles in hybrid nanomaterials. The results of scanning electron microscopy control over pointed ion profiling showed the following. The morphology of cell cultures and hybrid nanostructures based on them undergoes significant changes caused by profiling in the local area exposed to argon ions. The geometry of the ion source location made it possible to subject all cells located in the etching (profiling) area to partial destruction. The removal rates are shown to be different. Thus, for hybrid nanomaterials based on cell cultures: E.coli with ultra-small iron oxide nanoparticles, a partial absence of part of the evacuated cells was shown after about 10 steps of sequential exposure to an ion beam at an energy of 1 kV and a step duration of 5 minutes (removal of the upper part of the membrane). Taking into account the fact that profiling takes place at an angle with respect to the total surface of the sample, the part of the cell wall opposite to the ion source partially retains its shape even after 10 profiling steps. Thus, the inner part of the cell under conditions of preservation of ultra-high vacuum becomes accessible to probing by the XPS method. The situation looks different with the surface of hybrid nanomaterials profiled by a focused ion beam based on mammalian cell cultures decorated with silicon and its oxides nanoparticles. At the selected concentrations of nanoparticles, changes in the shape of cells can occur in the membrane region (decoration) and when nanoparticles penetrate inside. Profiling, at selected values of accelerating stresses and times, primarily leads to the removal of the surface of silicon nanoparticles (or rather core-shell silicon - silicon oxide). The observed profile formation rates are significantly lower, especially at high concentrations of nanoparticles and short incubation times. Thus, in the case of hybrid nanomaterials based on mammalian cell cultures decorated with silicon nanoparticles and its oxides, it is first of all possible to obtain a profile of covering (decorating) silicon nanoparticles, and only with an increase in the number of scans to study the physicochemical state of those particles that penetrated deep into the cells during incubation. For a time of no more than 3 minutes, profiling is possible at an energy of 3 kV, which makes it more efficient to overcome the "external", decorating layer of nanoparticles. Synchrotron and laboratory experimental data of survey spectra and high-resolution spectra of the XPS method (Fe 2p, O1s, C1s, etc. on the atomic and electronic structure of hybrid nanomaterials based on E.coli cell cultures with ultra-small iron oxide nanoparticles, the following was shown. Nanoparticles of the iron-oxygen system are present in the region of the cell membrane. The minimum signal intensity at the first steps of profiling is associated with the predominant formation of nanoparticles throughout the cell volume. However, the data obtained from the outer surface of the membrane and at the first steps of profiling (partial separation of the membrane) showed the absence of a complete transition to Fe2O3 oxide in the nanoparticles formed. The partial removal of the cell volume with a further increase in the profiling steps shows a relative increase in the number of nanoparticles formed in the probed layer of the inner part of the cell. The fine structure of the spectra practically did not change, indicating the constancy of the composition and physico-chemical state of the nanoparticles inside the cells and on the membrane surface. The spectra of the fine structure of 2p 1/2 2p 3/2 iron show a set of contributions of the components of 2- and 3-valence iron, metallic iron, in some cases protonation, confirming the composition of FexOy(H). The observed contribution of spectral components generally confirms the formation of cluster-type agglomerates with a balanced composition of FexOy(H), where stoichiometry will be determined by the size and nature of the particle itself (the molecule in whose cavity the cluster accumulates) and the probability of formation of a core-shell structure within one particle and their ultra-small agglomerates, sizes not exceeding hundreds of nanometers. Criteria conditions for conducting experiments and compatibility of cell cultures with inorganic nanoparticles based on the iron-oxygen system have been established and clarified. The absence of a 2p line of iron states on the survey spectra can also be observed with point profiling by an ion beam, which does not prove the real absence of a signal from Fe atoms. For reliable confirmation of the presence of a signal from iron atoms and its registration, it is necessary to record the corresponding interval of the 2p region of iron states with large accumulations. A five-stage etching with sequential exposure to an ion beam at an energy of 1 kV and a step duration of 5 minutes is sufficient to remove a sufficient part of the membrane and increase the efficiency of accumulation of a weak signal from iron-oxygen nanoparticles. The successful formation of a hybrid structure, the accumulation of nanoparticles in the cell itself, acting as a factory producing dps nanomolecules with an inorganic iron nanocore and its compounds in the inner cavity (no more than 6 nm), is shown. Unlike hybrid materials based on E.coli cells, in which the formation of nanoparticles occurs "from the inside" in the cavities of spherical dps nanomolecules, 3T3 NIH mouse fibroblast cells and MCF-7 human breast cancer were "saturated from the outside" (decoration). According to XPS data (Si 2p, O1s, C1s, etc. degradation of nanoparticles was observed, including during profiling. Differences in the ratio of surface oxide covering nanoparticles and non-oxidized silicon depending on the method of formation and storage, including with point ion etching, are shown. The profiling confirmed that, in general, the structures were of the "core-shell" type for Si nanoparticles, and the thicknesses of natural silicon oxide SiO2 and the transition layer of silicon suboxides were primarily functions of the preparation mode. A significant increase in the degree of oxidation of those particles that decorate the cell, as opposed to the particles inside the cell, was noted. According to our data, the surface layer of the oxide of "external" decorating particles was about 10 nm. The degradation of the composition and structure of nanoparticles as a result of the dissolution of silicon during physicochemical interaction with the intracellular medium has been confirmed. An increase in the number of particles leads to a slowdown of these processes, but their course is confirmed by studying the "inner" part of the cells. Reliable detection of XPS data, including ion profiling, requires an increase in accumulation times, especially with a decrease in the number of silicon nanoparticles integrated with cell culture. Criteria conditions for conducting experiments and compatibility of cell cultures with inorganic nanoparticles based on the silicon-oxygen system have been established and clarified. The use of point ion profiling made it possible to study the composition of the particles decorating the cell, for which significant surface oxidation was shown as a result of interaction with the environment during formation and subsequent storage. Stepwise point etching, with a variation in the number of steps and an increase to 3 kV of accelerating voltages, allows us to obtain data from nanoparticles internal to the cell having a different composition, confirming the processes of active and destructive interaction with the intracellular medium with respect to silicon nanoparticles in the hybrid material formed in vitro. The method of decomposition of XANES L2,3-spectra into L2- and L3-components is implemented, the decomposition of experimental XANES L2,3-spectra of crystalline silicon, silicon dioxide and Si sample covered with a layer of natural oxide into L2- and L3-components is performed. Theoretical reference XANES L3 spectra of silicon and silicon dioxide have been calculated. The contribution of each of the reference phases in the resulting experimental XANES spectrum of a multiphase system and the determination of the contribution to the absorption of a layer of natural oxide is carried out. Contributions to the calculated XANES spectra from s- and d-symmetry states are obtained. It is shown that in crystalline silicon near the edge of the spectrum in the energy range with a width of ~2 eV, the s- and d-states make a comparable contribution to the spectrum, and at high XANES L3 transition energies, the silicon spectrum is formed mainly by d-symmetry states. In silicon dioxide, the absorption edges of the s- and d-symmetry states are separated in energy by ~2.5 eV, while the lowest energy maximum is formed almost entirely by s-states, and the wider main maximum of the spectrum, which is ~2.5 eV away from it in the direction of high absorption energies, is due mainly to absorption by d-symmetry states. Computer models of the band structure, total and local partial densities of electronic states, as well as XANES K-spectra of iron in iron monosilicide are obtained. Based on calculations, it is shown that the absorption edge is at an energy of ~7110 eV, followed by a local maximum at an energy of ~7124 eV. The main maximum is at an energy of ~7140 eV. At energies above ~7145 eV, another scattering mechanism, EXAFS (extended fine structure of X–ray absorption), makes a noticeable contribution to the absorption spectrum along with XANES. The study of the interrelationships of properties, primarily morphology, of hybrid materials subjected to controlled focused ion etching (profiling) with data on atomic and electronic structure showed significant differences during profiling for hybrid nanomaterials based on cell cultures: E.coli with ultra-small iron oxide nanoparticles; mammals decorated with silicon and its oxides nanoparticles. Soft profiling modes were found to be the most effective for studying E.coli cell cultures with ultra-small iron oxide nanoparticles. Decorating mammalian cell cultures with silicon oxide nanoparticles makes it necessary to increase the number of profiling steps.

 

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