Pt-Black-Modified (Hemi)spherical AFM Sensors: In Situ Imaging of Light-Driven Hydrogen Peroxide Evolution.

In this work, we present (hemi)spherical atomic force microscopy (AFM) sensors for the detection of hydrogen peroxide. Platinum-black (Pt-B) was electrodeposited onto conductive colloidal AFM probes or directly at recessed microelectrodes located at the end of a tipless cantilever, resulting in electrocatalytically active cantilever-based sensors that have a small geometric area but, due to the porosity of the films, exhibit a large electroactive surface area. Focused ion beam-scanning electron microscopy tomography revealed the porous 3D structure of the deposited Pt-B. Given the accurate positioning capability of AFM, these probes are suitable for local in situ sensing of hydrogen peroxide and at the same time can be used for (electrochemical) force spectroscopy measurements. Detection limits for hydrogen peroxide in the nanomolar range (LOD = 68 ± 7 nM) were obtained. Stability test and first in situ proof-of-principle experiments to achieve the electrochemical imaging of hydrogen peroxide generated at a microelectrode and at photocatalytically active structured poly(heptazine imide) films are demonstrated. Force spectroscopic data of the photocatalyst films were recorded in ambient conditions, in solution, and by applying a potential, which demonstrates the versatility of these novel Pt-B-modified spherical AFM probes.

Multimodal Analysis of Light-Driven Water Oxidation in Nanoporous Block Copolymer Membranes.

Heterogeneous light-driven catalysis is a cornerstone of sustainable energy conversion. Most catalytic studies focus on bulk analyses of the hydrogen and oxygen evolved, which impede the correlation of matrix heterogeneities, molecular features, and bulk reactivity. Here, we report studies of a heterogenized catalyst/photosensitizer system using a polyoxometalate water oxidation catalyst and a model, molecular photosensitizer that were co-immobilized within a nanoporous block copolymer membrane. Via operando scanning electrochemical microscopy (SECM), light-induced oxygen evolution was determined using sodium peroxodisulfate (Na2 S2 O8 ) as sacrificial electron acceptor. Ex situ element analyses provided spatially resolved information on the local concentration and distribution of the molecular components. Infrared attenuated total reflection (IR-ATR) studies of the modified membranes showed no degradation of the water oxidation catalyst under the reported light-driven conditions.

Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution.

High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

Investigation of modified nanopore arrays using FIB/SEM tomography.

The investigation of electrochemical processes at the interface of two immiscible electrolyte solutions (ITIES) is of great interest for sensing applications, and serves as a surrogate to the study of biological transport phenomena, e.g. ion channels. Alongside e-beam lithography, focused ion beam (FIB) milling is an attractive method to prototype and fabricate nanopore arrays that support nanoITIES. Within this contribution, we explore the capability of FIB/scanning electron microscopy (SEM) tomography to visualize the actual pore structure and interfaces at silica-modified nanoporous membranes. The nanopores were also characterized by atomic force microscopy (AFM) using ultra-sharp AFM probes to determine the pore diameter, and using scanning transmission electron microscopy (STEM) and energy dispersive X-ray (EDX) spectroscopy, providing additional information on the elemental composition of deposits within the pores. Si-rich particles could be identified within the pores as well as at the orifice that had faced the organic electrolyte solution during electrochemical deposition. The prospects of the used techniques for investigating the interface at or within FIB-milled nanopores will be discussed.

Achievement of Diffusional Independence at Nanoscale Liquid-Liquid Interfaces within Arrays.

In this work, independent radial diffusion at arrayed nanointerfaces between two immiscible electrolyte solutions (nanoITIES) was achieved. The arrays were formed at nanopores fabricated by focused ion beam milling of silicon nitride (SiN) membranes, enabling the reproducible and systematic design of five arrays with different ratios of pore center-to-center distance (rc) to pore radius (ra). Voltammetry across water-1,6-dichlorohexane nanoITIES formed at these arrays was examined by the interfacial transfer of tetrapropylammonium ions. The diffusion-limited ion-transfer current increased with the ratio rc/ra, reaching a plateau for rc/ra ≥ 56, which was equivalent to the theoretical current for radial diffusion to an array of independent nanoITIES. As a result, mass transport to the nanoITIES arrays was greatly enhanced due to the decreased overlap of diffusion zones at adjacent nanoITIES, allowing each interface in the array to behave independently. When the rc/ra ratio increased from 13 to 56, the analytical performance parameters of sensitivity and limit of detection were improved from 0.50 (±0.02) A M(-1) to 0.76 (±0.02) A M(-1) and from 0.101 (±0.003) µM to 0.072 (±0.002) µM, respectively. These results provide an experimental basis for the design of arrayed nanointerfaces for electrochemical sensing.

Antibacterial Action of Zn2+ Ions Driven by the In Vivo Formed ZnO Nanoparticles.

Antibacterial formulations based on zinc oxide nanoparticles (ZnO NPs) are widely used for antibiotic replacement in veterinary medicine and animal nutrition. However, the undesired environmental impact of ZnO NPs triggers a search for alternative, environmentally safer solutions. Here, we show that Zn2+ in its ionic form is a more eco-friendly antibacterial, and its biocidal action rivals that of ZnO NPs (<100 nm size), with a minimal biocidal concentration being 41(82) µg mL-1 vs 5 µg mL-1 of ZnO NPs, as determined for 103(106) CFU mL-1 E. coli. We demonstrate that the biocidal activity of Zn2+ ions is primarily associated with their uptake by E. coli and spontaneous in vivo transformation into insoluble ZnO nanocomposites at an internal bacterial pH of 7.7. Formed in vivo nanocomposite then damages E. coli membrane and intracellular components from the inside, by forming insoluble biocomposites, whose formation can also trigger ZnO characteristic reactions damaging the cells (e.g., by generation of high-potential reactive oxygen species). Our study defines a special route in which Zn2+ metal ions induce the death of bacterial cells, which might be common to other metal ions capable of forming semiconductor oxides and insoluble hydroxides at a slightly alkaline intracellular pH of some bacteria.

Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies.

PML nuclear bodies (PML-NBs) are dynamic interchromosomal macromolecular complexes implicated in epigenetic regulation as well as antiviral defense. During herpesvirus infection, PML-NBs induce epigenetic silencing of viral genomes, however, this defense is antagonized by viral regulatory proteins such as IE1 of human cytomegalovirus (HCMV). Here, we show that PML-NBs undergo a drastic rearrangement into highly enlarged PML cages upon infection with IE1-deficient HCMV. Importantly, our results demonstrate that dual signaling by interferon and DNA damage response is required to elicit giant PML-NBs. DNA labeling revealed that invading HCMV genomes are entrapped inside PML-NBs and remain stably associated with PML cages in a transcriptionally repressed state. Intriguingly, by correlative light and transmission electron microscopy (EM), we observed that PML cages also entrap newly assembled viral capsids demonstrating a second defense layer in cells with incomplete first-line response. Further characterization by 3D EM showed that hundreds of viral capsids are tightly packed into several layers of fibrous PML. Overall, our data indicate that giant PML-NBs arise via combined interferon and DNA damage signaling which triggers entrapment of both nucleic acids and proteinaceous components. This represents a multilayered defense strategy to act in a cytoprotective manner and to combat viral infections.

Silica nanoparticle aggregation in calcite replacement reactions.

Natural nanoparticles are fundamental building blocks of Earth's bio- and geosphere. Amorphous silica nanoparticles are ubiquitous in nature, but fundamental knowledge of their interaction mechanisms and role in mineral replacement reactions is limited. Here we show how silica nanoparticles replace Cretaceous calcite bivalve shells in a volume- and texture-preserving process. Electron tomography reveals that mineral replacement transfers calcite crystallographic orientations to twinned photonic crystals composed of face-centered cubic silica sphere stacks. During the face-specific replacement process, silica nanoparticles continuously nucleate, aggregate, and form a lattice of uniform spheres parallel to calcite low-energy facets. We explain the replacement process with a new model that unifies recently proposed, probably universal mechanisms of interface-coupled dissolution-precipitation and aggregation-based crystallization; both key mechanisms in geological processes and nanomaterials design and synthesis.

3D Analysis of HCMV Induced-Nuclear Membrane Structures by FIB/SEM Tomography: Insight into an Unprecedented Membrane Morphology.

We show that focused ion beam/scanning electron microscopy (FIB/SEM) tomography is an excellent method to analyze the three-dimensional structure of a fibroblast nucleus infected with human cytomegalovirus (HCMV). We found that the previously described infoldings of the inner nuclear membrane, which are unique among its kind, form an extremely complex network of membrane structures not predictable by previous two-dimensional studies. In all cases they contained further invaginations (2nd and 3rd order infoldings). Quantification revealed 5498HCMV capsids within two nuclear segments, allowing an estimate of 15,000 to 30,000 capsids in the entire nucleus five days post infection. Only 0.8% proved to be enveloped capsids which were exclusively detected in 1st order infoldings (perinuclear space). Distribution of the capsids between 1st, 2nd and 3rd order infoldings is in complete agreement with the envelopment/de-envelopment model for egress of HCMV capsids from the nucleus and we confirm that capsid budding does occur at the large infoldings. Based on our results we propose the pushing membrane model: HCMV infection induces local disruption of the nuclear lamina and synthesis of new membrane material which is pushed into the nucleoplasm, forming complex membrane infoldings in a highly abundant manner, which then may be also used by nucleocapsids for budding.