Towards High-Temperature MEMS: Two-Step Annealing Suppressed Recrystallization in Thin Multilayer Pt-Rh/Zr Films.

Platinum-based thin films are widely used to create microelectronic devices operating at temperatures above 500 °C. One of the most effective ways to increase the high-temperature stability of platinum-based films involves incorporating refractory metal oxides (e.g., ZrO2, HfO2). In such structures, refractory oxide is located along the metal grain boundaries and hinders the mobility of Pt atoms. However, the effect of annealing conditions on the morphology and functional properties of such multiphase systems is rarely studied. Here, we show that the two-step annealing of 250-nm-thick Pt-Rh/Zr multilayer films instead of the widely used isothermal annealing leads to a more uniform film morphology without voids and hillocks. The composition and morphology of as-deposited and annealed films were investigated using X-ray diffraction and scanning electron microscopy, combined with energy-dispersive X-ray spectroscopy. At the first annealing step at 450 °C, zirconium oxidation was observed. The second high-temperature annealing at 800-1000 °C resulted in the recrystallization of the Pt-Rh alloy. In comparison to the one-step annealing of Pt-Rh and Pt-Rh/Zr films, after two-step annealing, the metal phase in the Pt-Rh/Zr films has a smaller grain size and a less pronounced texture in the <111> direction, manifesting enhanced high-temperature stability. After two-step annealing at 450/900 °C, the Pt-Rh/Zr thin film possessed a grain size of 60 ± 27 nm and a resistivity of 17 × 10-6 Ω·m. The proposed annealing protocol can be used to create thin-film MEMS devices for operation at elevated temperatures, e.g., microheater-based gas sensors.

Nanoisland SERS-Substrates for Specific Detection and Quantification of Influenza A Virus.

Surface-enhanced Raman spectroscopy (SERS)-based aptasensors for virus determination have attracted a lot of interest recently. This approach provides both specificity due to an aptamer component and a low limit of detection due to signal enhancement by a SERS substrate. The most successful SERS-based aptasensors have a limit of detection (LoD) of 10-100 viral particles per mL (VP/mL) that is advantageous compared to polymerase chain reactions. These characteristics of the sensors require the use of complex substrates. Previously, we described silver nanoisland SERS-substrate with a reproducible and uniform surface, demonstrating high potency for industrial production and a suboptimal LoD of 4 × 105 VP/mL of influenza A virus. Here we describe a study of the sensor morphology, revealing an unexpected mechanism of signal enhancement through the distortion of the nanoisland layer. A novel modification of the aptasensor was proposed with chromium-enhanced adhesion of silver nanoparticles to the surface as well as elimination of the buffer-dependent distortion-triggering steps. As a result, the LoD of the Influenza A virus was decreased to 190 VP/mL, placing the nanoisland SERS-based aptasensors in the rank of the most powerful sensors for viral detection.

Lithographic SERS Aptasensor for Ultrasensitive Detection of SARS-CoV-2 in Biological Fluids.

In this paper, we propose a technology for the rapid and sensitive detection of the whole viral particles of SARS-CoV-2 using double-labeled DNA aptamers as recognition elements together with the SERS method for detecting the optical response. We report on the development of a SERS-aptasensor based on a reproducible lithographic SERS substrate, featuring the combination of high speed, specificity, and ultrasensitive quantitative detection of SARS-CoV-2 virions. The sensor makes it possible to identify SARS-CoV-2 in very low concentrations (the limit of detection was 100 copies/mL), demonstrating a sensitivity level comparable to the existing diagnostic golden standard-the reverse transcription polymerase chain reaction.

A Combination of Membrane Filtration and Raman-Active DNA Ligand Greatly Enhances Sensitivity of SERS-Based Aptasensors for Influenza A Virus.

Biosensors combining the ultrahigh sensitivity of surface-enhanced Raman scattering (SERS) and the specificity of nucleic acid aptamers have recently drawn attention in the detection of respiratory viruses. The most sensitive SERS-based aptasensors allow determining as low as 104 virus particles per mL that is 100-fold lower than any antibody-based lateral flow tests but 10-100-times higher than a routine polymerase chain reaction with reversed transcription (RT-PCR). Sensitivity of RT-PCR has not been achieved in SERS-based aptasensors despite the usage of sophisticated SERS-active substrates. Here, we proposed a novel design of a SERS-based aptasensor with the limit of detection of just 103 particles per ml of the influenza A virus that approaches closely to RT-PCR sensitivity. The sensor utilizes silver nanoparticles with the simplest preparation instead of sophisticated SERS-active surfaces. The analytical signal is provided by a unique Raman-active dye that competes with the virus for the binding to the G-quadruplex core of the aptamer. The aptasensor functions even with aliquots of the biological fluids due to separation of the off-target molecules by pre-filtration through a polymeric membrane. The aptasensor detects influenza viruses in the range of 1·103-5·1010 virus particles per ml.

Microlens-assisted microscopy for biology and medicine.

The addition of dielectric transparent microlens in the optical scheme is an effective and at the same time simple and inexpensive way to increase the resolution of a light microscope. For these purposes, spherical and cylindrical microlenses with a diameter of 1-100 µm are usually used. The microlens focuses the light into a narrow beam called a photonic nanojet. An enlarged virtual image is formed, which is captured by the objective of the light microscope. In addition to microscopy, the microlenses are successfully applied to amplify optical signals, increase the trapping force of optical tweezers and are used in microsurgery. This review considers the design and principle of microlens-assisted microscopes. Taking into account the advantages of the super-resolution optical methods for research in life science, the examples of the use of the microlenses in biomedical practice are discussed in detail.

High resolution imaging of viruses: Scanning probe microscopy and related techniques.

Scanning probe microscopy is a group of measurements that provides 3D visualization of viruses in different environmental conditions including liquids and air. Besides 3D topography it is possible to measure the properties like mechanical rigidity and stability, adhesion, tendency to crystallization, surface charge, etc. Choosing the right substrate and scanning parameters makes it much easier to obtain reliable data. Rational interpretation of experimental results should take into account possible artifacts, proper filtering and data presentation using specially designed software packages. Animal and human virus characterization is in the focus of many intensive studies because of their potential harm to higher organisms. The article focuses on high-resolution visualization of plant viruses. Tobacco mosaic virus, potato viruses X and B and others are not dangerous for the human being and are widely used in different applications such as vaccine preparation, construction of building units in nanotechnology and material science applications, nanoparticle production and delivery, and even metrology. The methods of virus's deposition, visualization, and consequent image processing and interpretation are described in details. Specific examples of viruses imaging are illustrated using the FemtoScan Online software, which has typical and all the necessary built-in functions for constructing three-dimensional images, their processing and analysis. Despite visible progress in visualizing the viruses using probe microscopy, many unresolved problems still remain. At present time the probe microscopy data on viruses is not systemized. There is no descriptive atlas of the images and morphology as revealed by this type of high resolution microscopy. It is worth emphasizing that new virus investigation methods will appear due to the progress of science.

Label-free sensitive detection of influenza virus using PZT discs with a synthetic sialylglycopolymer receptor layer.

We describe rapid, label-free detection of Influenza A viruses using the first radial mode of oscillations of lead zirconate titanate (PZT) piezoelectric discs with a 2 mm radius and 100 µm thickness fabricated from a piezoelectric membrane. The discs are modified with a synthetic sialylglycopolymer receptor layer, and the coated discs are inserted in a flowing virus suspension. Label-free detection of the virus is achieved by monitoring the disc radial mode resonance frequency shift. Piezo transducers with sialylglycopolymer sensor layers exhibited a long lifetime, a high sensitivity and the possibility of regeneration. We demonstrate positive, label-free detection of Influenza A viruses at concentrations below 105 virus particles per millilitre. We show that label-free, selective, sensitive detection of influenza viruses by home appliances is possible in principle.

Virus-Like Particle Facilitated Deposition of Hydroxyapatite Bone Mineral on Nanocellulose after Exposure to Phosphate and Calcium Precursors.

We produced and isolated tobacco mosaic virus-like particles (TMV VLPs) from bacteria, which are devoid of infectious genomes, and found that they have a net negative charge and can bind calcium ions. Moreover, we showed that the TMV VLPs could associate strongly with nanocellulose slurry after a simple mixing step. We sequentially exposed nanocellulose alone or slurries mixed with the TMV VLPs to calcium and phosphate salts and utilized physicochemical approaches to demonstrate that bone mineral (hydroxyapatite) was deposited only in nanocellulose mixed with the TMV VLPs. The TMV VLPs confer mineralization properties to the nanocellulose for the generation of new composite materials.

Highly sensitive detection of influenza virus with SERS aptasensor.

Highly sensitive and rapid technology of surface enhanced Raman scattering (SERS) was applied to create aptasensors for influenza virus detection. SERS achieves 106-109 times signal amplification, yielding excellent sensitivity, whereas aptamers to hemagglutinin provide a specific recognition of the influenza virus. Aptamer RHA0385 was demonstrated to have essentially broad strain-specificity toward both recombinant hemagglutinins and the whole viruses. To achieve high sensitivity, a sandwich of primary aptamers, influenza virus and secondary aptamers was assembled. Primary aptamers were attached to metal particles of a SERS substrate, and influenza viruses were captured and bound with secondary aptamers labelled with Raman-active molecules. The signal was affected by the concentration of both primary and secondary aptamers. The limit of detection was as low as 1 · 10-4 hemagglutination units per probe as tested for the H3N2 virus (A/England/42/72). Aptamer-based sensors provided recognition of various influenza viral strains, including H1, H3, and H5 hemagglutinin subtypes. Therefore, the aptasensors could be applied for fast and low-cost strain-independent determination of influenza viruses.

Blister formation during graphite surface oxidation by Hummers' method.

Graphite oxide has a complex structure that can be modified in many ways to obtain materials for a wide range of applications. It is known that the graphite precursor has an important role in the synthesis of graphite oxide. In the present study, the basal-plane surface of highly annealed pyrolythic graphite (HAPG) was oxidized by Hummers' method and investigated by Raman spectroscopy and atomic force microscopy. HAPG was used as a graphite precursor because its surface after cleavage contains well-ordered millimeter-sized regions. The treatment resulted in graphite intercalation by sulfuric acid and blister formation all over the surface. Surprisingly, the destruction of the sp2-lattice was not detected in the ordered regions. We suggest that the reagent diffusion under the basal plane surface occurred through the cleavage steps and dislocations with the Burgers vector parallel to the c-axis in graphite.

Direct Observation of Changes in Focal Conic Domains of Cholesteric Films Induced by Ultraviolet Irradiation.

The helical supramolecular structure of cholesteric liquid crystalline (LC) films predetermines their outstanding optical properties and the unique nanostructure of their surface. The introduction of photochromic dopants in these films opens up an interesting possibility for creation of smart cholesteric materials with photocontrollable optical and photovariable surface properties. Using atomic force microscopy (AFM), we performed in situ measurements of the surface topography of cyclosiloxane LC cholesteric oligomer films during the cholesteric helix twisting caused by their preliminary ultraviolet (UV) irradiation. A chiral-photochromic isosorbide-based dopant was introduced in the films to control the cholesteric helix pitch by UV-irradiation. The initial films are characterized by planar texture with the presence of focal conic domains having the double-spiral relief on their surface. UV-irradiation of these films leads to the cholesteric helix twisting resulting in a decrease in the surface relief period, and the enlargement of defect areas between the domains. The detailed mechanisms of the rearrangement of the film surface structure due to the cholesteric helix twisting are suggested. They include the rotation and displacement of cholesteric layers in the bulk, and the nucleation of new ones at the surface in defect regions.

Effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters.

BACKGROUND: Over the past years there are increasing evidences that the interplay between two molecules of RNA polymerases, initiating transcription from promoters, oriented in opposite (convergent) directions, can serve as a regulatory factor of gene expression. The data concerning the molecular mechanisms of this so-called transcriptional interference (TI) are not well understood. METHODS: The interaction of RNA polymerase with circular DNA templates, containing the convergent promoters, was investigated in a series of in vitro transcription assays and atomic force microscopy (AFM). RESULTS: In this work, to study the mechanisms of transcription interference a series of plasmids with oppositely oriented closely spaced artificial promoters, recognized by Escherichia coli RNA polymerase, was constructed. The constructs differ in promoter structure and distance between the transcription start sites. We have demonstrated that the transcripts ratio (RNA-R/RNA-L) and morphology of convergent open promoter complexes (OPC) are highly dependent on the interpromoter distance. CONCLUSIONS: The obtained results allowed us to suggest the novel model of TI, which assumes the DNA bending upon binding of RNA polymerase with promoters and explains the phenomenon of complete inactivation of weaker promoter by the stronger one. GENERAL SIGNIFICANCE: The results show that the conformational transitions in DNA helix, associated with DNA bending upon binding of RNA polymerase with promoters, play crucial role in OPC formation in the systems with convergent promoters.

A Genetically Modified Tobacco Mosaic Virus that can Produce Gold Nanoparticles from a Metal Salt Precursor.

We genetically modified tobacco mosaic virus (TMV) to surface display a characterized peptide with potent metal ion binding and reducing capacity (MBP TMV), and demonstrate that unlike wild type TMV, this construct can lead to the formation of discrete 10-40 nm gold nanoparticles when mixed with 3 mM potassium tetrachloroaurate. Using a variety of analytical physicochemical approaches it was found that these nanoparticles were crystalline in nature and stable. Given that the MBP TMV can produce metal nanomaterials in the absence of chemical reductants, it may have utility in the green production of metal nanomaterials.

Surface Relief Changes in Cholesteric Cyclosiloxane Oligomer Films at Different Temperatures.

The development of new approaches for the surface topography control is an important topic as the relief significantly affects physical and chemical properties of surfaces. We studied cholesteric cyclosiloxane oligomeric films on which surface focal conic domains with double-helix pattern were observed by means of AFM. In situ investigation of the dependence of the films topography on temperature showed that the surface relief formation can be effectively managed by varying conditions of thermal treatment. Obtained structures can be frozen by cooling the films below glass-transition temperature.

Biosynthesis of stable iron oxide nanoparticles in aqueous extracts of Hordeum vulgare and Rumex acetosa plants.

We report the synthesis and characterization of amorphous iron oxide nanoparticles from iron salts in aqueous extracts of monocotyledonous (Hordeum vulgare) and dicotyledonous (Rumex acetosa) plants. The nanoparticles were characterized by TEM, absorbance spectroscopy, SAED, EELS, XPS, and DLS methods and were shown to contain mainly iron oxide and iron oxohydroxide. H. vulgare extracts produced amorphous iron oxide nanoparticles with diameters of up to 30 nm. These iron nanoparticles are intrinsically unstable and prone to aggregation; however, we rendered them stable in the long term by addition of 40 mM citrate buffer pH 3.0. In contrast, amorphous iron oxide nanoparticles (diameters of 10-40 nm) produced using R. acetosa extracts are highly stable. The total protein content and antioxidant capacity are similar for both extracts, but pH values differ (H. vulgare pH 5.8 vs R. acetosa pH 3.7). We suggest that the presence of organic acids (such oxalic or citric acids) plays an important role in the stabilization of iron nanoparticles, and that plants containing such constituents may be more efficacious for the green synthesis of iron nanoparticles.

The use of tobacco mosaic virus and cowpea mosaic virus for the production of novel metal nanomaterials.

Due to the nanoscale size and the strictly controlled and consistent morphologies of viruses, there has been a recent interest in utilizing them in nanotechnology. The structure, surface chemistries and physical properties of many viruses have been well elucidated, which have allowed identification of regions of their capsids which can be modified either chemically or genetically for nanotechnological uses. In this review we focus on the use of such modifications for the functionalization and production of viruses and empty viral capsids that can be readily decorated with metals in a highly tuned manner. In particular, we discuss the use of two plant viruses (Cowpea mosaic virus and Tobacco mosaic virus) which have been extensively used for production of novel metal nanoparticles (<100nm), composites and building blocks for 2D and 3D materials, and illustrate their applications.

Electrochemical nanoprobes for single-cell analysis.

The measurement of key molecules in individual cells with minimal disruption to the biological milieu is the next frontier in single-cell analyses. Nanoscale devices are ideal analytical tools because of their small size and their potential for high spatial and temporal resolution recordings. Here, we report the fabrication of disk-shaped carbon nanoelectrodes whose radius can be precisely tuned within the range 5-200 nm. The functionalization of the nanoelectrode with platinum allowed the monitoring of oxygen consumption outside and inside a brain slice. Furthermore, we show that nanoelectrodes of this type can be used to impale individual cells to perform electrochemical measurements within the cell with minimal disruption to cell function. These nanoelectrodes can be fabricated combined with scanning ion conductance microscopy probes, which should allow high resolution electrochemical mapping of species on or in living cells.

The role of the 5'-cap structure in viral ribonucleoproteins assembly from potato virus X coat protein and RNAs.

The potato virus X (PVX) virion can be reconstituted in vitro from the virus coat protein (CP) and RNA; heterologous RNAs may be used as well. In our recent study, structure and properties of cognate and heterologous viral ribonucleoproteins (vRNPs) were demonstrated to be similar to those of native virions. The assembly was found to be initiated at the 5' terminus of an RNA and was not dependent on RNA sequence. The aim of the present study was to search for a signal or an essential structural element that directs packaging of viral genetic material into vRNPs. vRNPs were formed by incubation of the PVX CP with heterologous capped RNAs, their functional fragments lacking the cap structure, as well as the capped and uncapped transcripts corresponding to the 5'-terminal region of the genomic PVX RNA. Experimental data show that the presence of the cap structure at the 5' end of a nucleic acid is an important condition for vRNP assembly from RNA and CP. Presumably, the 5'-cap affects conformational state of the RNA region responsible for the efficient interaction with CP and creates conformational encapsidation signal for vRNP assembly.

Atomic force microscopy study of surface topography of films of cholesteric oligomer- and polymer-based mixtures with photovariable helix pitch.

The surface topography of glass-forming polymers and oligomer cholesteric systems with a phototunable helix pitch was studied. For this purpose several mixtures based on nematic polyacrylate and cholesteric cyclosiloxanes doped with chiral-photochromic dopant were prepared and investigated. The molecules of chiral-photochromic dopant consist of isosorbide chiral moiety and cinnamic C=C double bonds capable of E-Z photoisomerizing. UV irradiation of the planarly oriented films of mixtures leads to dopant photoisomerization and changes of its helical twisting power. During this process irreversible changes of helix pitch values take place, which allows one to obtain the same cholesteric systems with different helix pitch values. The films of the annealed mixtures were studied by atomic force microscopy and transmission electron microscopy. The correlations between the features of surface topography and helix pitch of cholesteric supramolecular structure were found and discussed.

The model of amyloid aggregation of Escherichia coli RNA polymerase σ70 subunit based on AFM data and in vitro assays.

To propose a model for recently described amyloid aggregation of E.coli RNA polymerase σ(70) subunit, we have investigated the role of its N-terminal region. For this purpose, three mutant variants of protein with deletions Δ1-73, Δ1-100 and Δ74-100 were constructed and studied in a series of in vitro assays and using atomic force microscopy (AFM). Specifically, all RNA polymerase holoenzymes, reconstituted with the use of mutant σ subunits, have shown reduced affinity for promoter-containing DNA and reduced activity in run-off transcription experiments (compared to that of WT species), thus substantiating the modern concept on the modulatory role of N-terminus in formation of open complex and transcription initiation. The ability of mutant proteins to form amyloid-like structures has been investigated using AFM, which revealed the increased propensity of mutant proteins to form rodlike aggregates with the effect being more pronounced for the mutant with the deletion Δ1-73 (10 fold increase). σ(70) subunit aggregation ability has shown complex dependence on the ionic surrounding, which we explain by Debye screening effect and the change of the internal state of the protein. Basing on the obtained data, we propose the model of amyloid fibril formation by σ(70) subunit, implying the involvement of N-terminal region according to the domain swapping mechanism.