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Nanoscale extracellular vesicle (EVs) have been found to play a key role in intercellular communication, offering opportunities for both diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for their physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The investigation was performed with small EVs (sEVs) isolated from a non-small cell lung cancer (NSCLC) cell line H1975 as well as from a human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter >70 nm), the microscopy based approach showed a better capacity for analyses on smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.

Nanosized extracellular vesicles (EVs) have been found to play a key role in intercellular communication, offering opportunities for both disease diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The study was performed with small EVs (sEVs) isolated from a non-small-cell lung cancer (NSCLC) cell line as well as from human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter > 70 nm), the microscopy based approach showed a better capacity for analyses of smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.

Molecularly imprinted polymers (MIPs) are gaining great interest as tailor-made recognition materials for the development of biomimetic sensors. Various approaches have been adopted to interface MIPs with different transducers, including the use of pre-made imprinted particles and the in situ preparation of thin polymer layers directly on transducer surfaces. In this work we functionalized quartz crystal microbalance (QCM) sensor crystals by coating the sensing surfaces with pre-made molecularly imprinted nanoparticles. The nanoparticles were immobilized on the QCM transducers by physical entrapment in a thin poly(ethylene terephthalate) (PET) layer that was spin-coated on the transducer surface. By controlling the deposition conditions, it was possible to gain a high nanoparticle loading in a stable PET layer, allowing the recognition sites in nanoparticles to be easily accessed by the test analytes. In this work, different sensor surfaces were studied by micro-profilometry and atomic force microscopy and the functionality was evaluated using quartz crystal microbalance with dissipation (QCM-D). The molecular recognition capability of the sensors were also confirmed using radioligand binding analysis by testing their response to the presence of the test compounds, (R)- and (S)-propranolol in aqueous buffer.

We have used magnetic particles to study specific binding of prostate specific antigen (PSA) to the surfaces of the bioparticles. The used particles have a mean diameter of about 130 nm and are placed in phosphate buffer saline (PBS). Each particle is composed of clusters of magnetic single domains of magnetite, which are covered with dextran. Changes in surface chemistry of the particles give rise to a change in the hydrodynamic volume of the particles. The later is mirrored by the changed frequency response of the complex magnetic susceptibility of a fluid containing these particles. Using ordinary induction coils and the lock-in amplifier technique it is possible to measure the complex magnetic susceptibility of the particle solution in a frequency range from about 10 Hz up to 10 kHz. From the measurement of the complex susceptibility versus the excitation frequency (both at the excitation frequency as well as at higher harmonics) we have shown that it is possible to quantitatively study the binding of PSA to the surfaces of the magnetic particles and thus to determine the PSA concentration in solution containing known concentration of nanoparticles functionalised with a monoclonal PSA antibody. Our method allows to perform an immunoassay in a single step and is much faster and cheaper compared to conventional ELISA procedures.

The adsorption of glycine deposited at 95 K on the graphite basal (0001) plane was studied from submonolayer to multilayer coverages by thermal desorption spectroscopy (TDS) and high- resolution electron loss spectroscopy (HREELS). At all coverages glycine adsorbs in three-dimensional (3D) clusters (no distinct monolayer formation was observed). Glycine molecules desorb around 300 K in a single desorption peak (α peak) with a binding energy of 0.85 eV, representing the cohesive energy of glycine. HREELS analysis of these overlayers is severely hampered by charging of the 3D clusters; only one vibrational feature at 26 meV was observed reproducibly. The glycine adlayers are very sensitive to electron irradiation. This effect was studied in some detail by TDS. Electrons of ∼50 eV energy cause: (i) conversion of glycine molecules from the α state to (surprisingly) a more weakly bound β state (desorption temperature ∼250 K), interpreted as 2D adsorbates, and (ii) dissociation of glycine into fragments that ei...

Iron in amphibole asbestos is implicated in the pathogenicity of inhaled fibers. Evidence includes the observation that iron chelators can suppress fiber-induced tissue damage. This is believed to occur via the diminished production of fiber-associated reactive oxygen species. The purpose of this study was to explore possible mechanisms for the reduction of fiber toxicity by iron chelator treatments. We studied changes in the amount and the oxidation states of bulk and surface iron in crocidolite and amosite asbestos that were treated with iron-chelating desferrioxamine, ferrozine, sodium ascorbate, and phosphate buffer solutions. The results have been compared with the ability of the fibers to produce free radicals and decompose hydrogen peroxide in a cell-free system in vitro. We found that chelators can affect the amount of iron at the surface of the asbestos fibers and its valence, and that they can modify the chemical reactivity of these surfaces. However, we found no obvious or direct correlations between fiber reactivity and the amount of iron removed, the amount of iron at the fiber surface, or the oxidation state of surface iron. Our results suggest that surface Fe3+ ions may play a role in fiber-related carboxylate radical formation, and that desferrioxamine and phosphate groups detected at treated fiber surfaces may play a role in diminishing and enhancing, respectively, fiber redox activity. It is proposed that iron mobility in the silicate structure may play a larger role in the chemical reactivity of asbestos than previously assumed.

The influence of oxygen on the hydrogen uptake by Pd-coated Mg films was investigated, by exposing the Mg films to various amounts of oxygen, after deposition of the Mg film but before deposition of the Pd layer. The hydrogen uptake rate of these PdMgOxMg samples shows a peculiar dependence on the thickness of the MgOx layer. For thin oxide layers (small added amounts of oxygen) the hydrogen rate uptake increases, compared with pure PdMg samples. Thicker oxide films (larger amounts of added oxygen) cause a monotonic decline of the hydrogen uptake rate. However, even very large oxygen exposures/uptakes cannot prevent hydrogen uptake completely. MgOx is thus only a partial but not a perfect diffusion barrier for hydrogen. Repeated hydriding-dehydriding cycles increase the hydrogen uptake rate by PdMgOxMg samples, compared with the rate during the first cycle. The results are discussed in terms of a combined effect of the MgOx, layer, affecting both hydrogen transport and nucleation of the hydride phase.

Resistivity measurements were performed during hydriding—decomposition cycles of UHV-prepared Mg films. The hydrogen exposures were made in situ at pH2 = 0.6 torr and 350 K. To obtain measurable uptake rates at these conditions a thin layer of Pd (≈ 7 nm) was deposited on top of the typically 380 nm thick Mg films. Mg and Pd film thicknesses and hydrogen uptakes were determined by the frequency shift of a quartz crystal microbalance (QCM). Both the QCM and the quartz substrate onto which the Mg resistor film was deposited were mounted on the same sample holder. The p, T conditions were chosen so that hydrogen was distributed nearly homogeneously through the sample. The total hydrogen uptakes were in most cases kept small (

The adsorption on a gold surface of 5–50-nm silica particles in solution has been studied by the piezoelectric quartz crystal microbalance technique. The parameters of the solution where a reproducible layer by layer deposition is possible have been characterized. The uptake depends strongly on the ionic strength of the solution, but only weakly on the particle size or concentration. The strong dependence on ionic strength is attributed to electrostatic screening.

Titanium implants have been used widely and successfully for various types of bone-anchored reconstructions. It is believed that properties of oxide films covering titanium implant surfaces are of crucial importance for a successful osseointegration, in particular at compromized bone sites. The aim of the present study is to investigate the surface properties of anodic oxides formed on commercially pure (c.p.) titanium screw implants as well as to study 'native' oxides on turned c.p. titanium implants. Anodic oxides were prepared by galvanostatic mode in CH3COOH up to the high forming voltage of dielectric breakdown and spark formation. The oxide thicknesses, measured with Auger electron spectroscopy (AES), were in the range of about 200-1000 nm. Barrier and porous structures dominated the surface morphology of the anodic film. Quantitative morphometric analyses of the micropore structures were performed using an image analysis system on scanning electron microscopy (SEM) negatives. The pore sizes wereor = 8 microm in diameter and had 1.27-2.1 microm2 opening area. The porosity was in the range of 12.7-24.4%. The surface roughness was in the range of 0.96-1.03 microm (Sa), measured with TopScan 3D. The crystal structures of the titanium oxide were amorphous, anatase, and a mixtures of anatase and rutile type, as analyzed with thin-film X-ray diffractometry (TF-XRD) and Raman spectroscopy. The chemical compositions consisted mainly of TiO2, characterized with X-ray photoelectron spectroscopy (XPS). The native (thermal) oxide on turned implants was 17.4 nm (+/- 6.2) thick and amorphous. Its chemical composition was TiO2. The surface roughness had an average height deviation of 0.83 microm (Sa). The present results are needed to elucidate the influence of the oxide properties on the biological reaction. The results of animal studies using the presently characterized surface oxides on titanium implants will be published separately.