The complex cell metabolism and its link to oncogenic signaling pathways have received huge interest within the last few years. But the lack of advanced analytical tools for the investigation of ...living cell metabolism is still a challenge to be faced. Therefore, we designed and fabricated a novel miniaturized microslot NMR detector with on-board heater integrated with a microfluidic device as NMR sample holder. For the first time, a tumor spheroid of 500 μm diameter and consisting of 9000 cells has been studied noninvasively and online for 24 h. The dynamic processes of production and degradation of 23 intra- and extracellular metabolites were monitored. Remarkably high concentrations of lactate and alanine were observed, being an indicator for a shift from oxidative to glycolytic metabolism. In summary, this methodical development has proven to be a successful analytical tool for the elucidation of cellular functions and their corresponding biochemical pathways. Additionally, the planar geometry of the microslot NMR detector allows the hyphenation with versatile lab-on-a chip (LOC) technology. This opens a new window for metabolomics studies on living cells and can be implemented into new application fields in biotechnology and life sciences.
Three-dimensional cell cultures are of growing importance in biochemical research as they represent tissue features more accurately than standard two-dimensional systems, but to investigate these ...challenging new models an adaptation of established analytical techniques is required. Spatially resolved data for living organoids are needed to gain insight into transport processes and biochemical characteristics of domains with different nutrient supply and waste product removal. Within this work, we present an NMR-based approach to obtain dynamically radial metabolite profiles for cell spheroids, one of the most frequently used 3D models. Our approach combines an easy to reproduce custom-made measurement design, maintaining physiological conditions without inhibition of the NMR experiment, with spatially selective NMR pulse sequences. To overcome the inherently low sensitivity of NMR spectroscopy we excited slices instead of smaller cube-like voxels in combination with an efficient interleaved measurement approach and employed a commercially available cryogenic NMR probe. Finally, radial metabolite profiles could be obtained via double Abel inversion of the measured one-dimensional intensity profiles. Applying this method to Ty82 cancer cell spheroids demonstrates the achieved spatial resolution, for instance confirming exceedingly high lactic acid and strongly decreased glucose concentrations in the oxygen-depleted core of the spheroid. Furthermore, our approach can be employed to investigate fast and slow metabolic changes in single spheroids simultaneously, which is shown as an example of a spheroid degrading over several days after stopping the nutrient supply.
Nanoparticle Tracking Analysis (NTA) allows for the simultaneous determination of both size and concentration of nanoparticles in a sample. This study investigates the accuracy of particle size and ...concentration measurements performed on an LM10 device. For experiments, standard nanoparticles of different sizes composed of two materials with different refractive indices were used. Particle size measurements were found to have a decent degree of accuracy. This fact was verified by the manufacturer-reported particle size—determined by transmission electron microscopy (TEM)—as well as by performed scanning electron microscopy (SEM) measurements. On the other hand, concentration measurements resulted in overestimation of the particle concentration in majority of cases. Thus, our findings confirmed the accuracy of nanoparticle sizing performed by the LM10 instrument and highlighted the overestimation of particle concentration made by this device. In addition, an approach of swift correction of the results of concentration measurements received for samples is suggested in the presented study.
The synthesis of grafted PMMA homopolymer films is reported using a surface-initiated reversible addition–fragmentation chain transfer (SI-RAFT) polymerization from a RAFT-agent immobilized on a ...silanized stainless steel surface. Therefore, stainless steel surfaces were hydroxylated with piranha solution followed by silanization with 3-aminopropylsilane (APS). The pendant primary amino groups of the cross-linked polysiloxane layer were reacted with 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid N-hydroxysuccinimide ester to produce a surface with covalently immobilized RAFT agents. PMMA homopolymers of different molecular weights between 13 060 and 45 000 g/mol were then prepared by a surface-initiated RAFT polymerization. Molecular weight (MW) and polydispersity index (PDI) were determined from sacrificial polymerization in solution. The different steps of stainless steel surface modification and the ultrathin films were investigated using atomic force microscopy (AFM), static, X-ray photoelectron spectroscopy (XPS), attenuated total reflectance infrared spectroscopy (ATR-IR), and ellipsometry.
Abstract
This study introduces a low-field NMR spectrometer (LF-NMR) featuring a multilayer Halbach magnet supported by a combined mechanical and electrical shimming system. This setup offers ...improved field homogeneity and sensitivity compared to spectrometers relying on typical Halbach and dipole magnets. The multilayer Halbach magnet was designed and assembled using three nested cylindrical magnets, with an additional inner Halbach layer that can be rotated for mechanical shimming. The coils and shim-kernel of the electrical shimming system were constructed and coated with layers of zirconia, thermal epoxy, and silver-paste resin to facilitate passive heat dissipation and ensure mechanical and thermal stability. Furthermore, the 7-channel shim coils were divided into two parts connected in parallel, resulting in a reduction of joule heating temperatures from 96.2 to 32.6 °C. Without the shimming system, the Halbach magnet exhibits a field inhomogeneity of approximately 140 ppm over the sample volume. The probehead was designed to incorporate a solenoidal mini coil, integrated into a single planar board. This design choice aimed to enhance sensitivity, minimize
$${B}_{1}$$
B
1
inhomogeneity, and reduce impedance discrepancies, transmission loss, and signal reflections. Consequently, the resulting linewidth of water within a 3 mm length and 2.4 mm inner diameter sample volume was 4.5 Hz. To demonstrate the effectiveness of spectral editing in LF-NMR applications at 29.934 MHz, we selectively excited hydroxyl and/or methyl protons in neat acetic acid using optimal control pulses calculated through the Krotov algorithm.
Polyethylene oxide (PEO) complexed with molecular iodine (
I
2
) forming PEO/
I
2
complex composites stand‐free films were investigated using dielectric relaxation, X-ray photoelectron spectroscopy ...(XPS), UV–Vis spectrophotometry, structural and morphological techniques. Scanning electron microscopy was used to monitor the variation in the surface morphology and the related roughness. 2D Energy-dispersive X-ray spectroscopy (EDX) measurements enabled to observe the distribution of iodine on the film surface. High resolution XPS measurements were used to define the iodine anion types and the metallic iodine existence, as well as the relevant concentrations based on the binding energies. The dielectric relaxation measurements were carried out over the frequency range from 0.1 to 10
7
Hz and temperature range from 155 to 330 K. Dielectric loss (ε′′) curves were fitted to the Havriliak–Negami (HN) model for one and/or two relaxation peaks (α and β), with and without the electrical conductivity contribution term, in order to deduce the relaxation time (τ) and the dielectric strengths (Δ
ε
), in addition to the electrical conductivities (
σ
). The temperature-dependent data of β- and σ- relaxations follow the law of Arrhenius thermal activation indicating the presence of typical glass-forming polymers. Δ
ε
of α-relaxation obeys the curvature pattern of Vogel-Tammann-Fulcher (VTF) thermal activation law. The electrical conductivity of the system increases 6000 folds by doping PEO with 5 wt% of iodine at the same temperature (293 K).
The recently developed surface plasmon microscopy of single nanoparticles offers a powerful tool for monitoring particle transfer from bulk solution to the adsorbing surface. In the present work, the ...possibility of absolute analysis of suspension, based on particle transfer observation, was examined. A model based on particle diffusion in a laminar flow, which allows analytical solution of mass transport equation, was considered. It was demonstrated that direct determination of particle concentration, based on counting the particles that bind to the sensor surface, is possible. The model contains only independent, easy to determine parameters: flow cell geometry, flow rate, particle size, and liquid viscosity. For applicability of the model, particles should be irreversibly bound when touching the sensor surface. It was demonstrated that the required “perfect binding” is provided in certain electrolyte concentration range because even particles oppositely charged to the surface do not bind in pure water. If the binding probability is less than 100%, “absoluteness” of the method is not violated. It will require the extension of the transport model with the sticking coefficient, which can be also measured by the surface plasmon microscopy directly. The present work shows the feasibility of the absolute analysis. It demonstrates the great potential of the method toward the comprehensive nanoparticle analytics and investigation of binding processes.
Near ambient pressure X-ray photoelectron spectroscopy (NAP-XPS) is a promising method to close the “pressure gap”, and thus, study the surface composition during heterogeneous reactions in situ. The ...specialized spectrometers necessary for this analytical technique have recently been adapted to operate with a conventional X-ray source, making it available for routine quantitative analysis in the laboratory. This is shown in the present in situ study of the partial oxidation of 2-propanol catalyzed with PdO nanoparticles supported on TiO2, which was investigated under reaction conditions as a function of gas composition (alcohol-to-oxygen ratio) and temperature. Exposure of the nanoparticles to 2-propanol at 30 °C leads to immediate partial reduction of the PdO, followed by a continuous reduction of the remaining PdO during heating. However, gaseous oxygen inhibits the reduction of PdO below 90 °C, and the oxidation of 2-propanol to carboxylates only occurs in the presence of oxygen above 90 °C. These results support the theory that metallic palladium is the active catalyst material, and they show that environmental conditions affect the nanoparticles and the reaction process significantly. The study also revealed challenges and limitations of this analytical method. Specifically, the intensity and fixed photon energy of a conventional X-ray source limit the spectral resolution and surface sensitivity of lab-based NAP-XPS, which affect precision and accuracy of the quantitative analysis.
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•Rational design of catalyst by means of a CO reduction treatment.•Very high Ce(III) fraction values obtained by CO reduction treatment.•Surface hydrophobicity plays an important role ...in the CeO2-x nanoparticles reduction.•Low reduction temperatures values found for CeO2-x nanoparticles.
Oxygen vacancies are one of the most active defects existing at the surface of metal oxides. Engineering of defect chemistry, especially oxygen vacancies, represents the possibility of controlling the catalytic properties of nanoparticles. This work was dedicated to study the reduction properties of CeO2-x nanoparticles prepared using different synthesis parameters when exposed to a CO reducing atmosphere. Near Ambient Pressure X-ray Photoelectron Spectroscopy (NAP-XPS) and in situ time-resolved X-Ray Absorption Near Edge Spectroscopy (XANES) techniques were used to probe in situ the electronic properties of these nanoparticles exposed to the CO atmosphere. Compared to typical results found for CeO2-x nanoparticles, an improved reduction tendency is observed for these synthesized nanoparticles. It was possible to tune the oxygen vacancy population and the reduction temperature (TR) by means of the combination of synthesis and CO reducing atmosphere. The TR and Ce(III) fraction values reached can be as low as 108°C and as high as 0.92, respectively. The influence of structural and electronic properties of the nanoparticles on the reduction kinetics of the CeO2-x reduction is elucidated. The results provide a valuable route for the rational design of CeO2-x nanoparticles with the desired oxygen vacancy population, and then preparing them in the best conditions for applications.
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