Smart Electrospun Nanofibers from Short Peptidomimetics Based on Pyrrolo-pyrazole Scaffold.

We prepared a small library of short peptidomimetics based on 3-pyrrolo-pyrazole carboxylate, a non-coded γ-amino acid, and glycine or alanine. The robust and eco-friendly synthetic approach adopted allows to obtain the dipeptides in two steps from commercial starting materials. This gives the possibility to shape these materials by electrospinning into micro- and nanofibers, in amounts required to be useful for coating surfaces of biomedical relevance. To promote high quality of electrospun fibers, different substitution patterns were evaluated, all for pure peptide fibers, free of any polymer or additive. The best candidate, which affords a homogeneous fibrous matrix, was prepared in larger amounts, and its biocompatibility was verified. This successful work is the first step to develop a new biomaterial able to produce pristine peptide-based nanofibers to be used as helpful component or stand-alone scaffolds for tissue engineering or for the surface modification of medical devices.

DNA data storage in electrospun and melt-electrowritten composite nucleic acid-polymer fibers.

Incorporating biomolecules as integral parts of computational systems represents a frontier challenge in bio- and nanotechnology. Using DNA to store digital data is an attractive alternative to conventional information technologies due to its high information density and long lifetime. However, developing an adequate DNA storage medium remains a significant challenge in permitting the safe archiving and retrieval of oligonucleotides. This work introduces composite nucleic acid-polymer fibers as matrix materials for digital information-bearing oligonucleotides. We devised a complete workflow for the stable storage of DNA in PEO, PVA, and PCL fibers by employing electrohydrodynamic processes to produce electrospun nanofibers with embedded oligonucleotides. The on-demand retrieval of messages is afforded by non-hazardous chemical treatment and subsequent PCR amplification and DNA sequencing. Finally, we develop a platform for melt-electrowriting of polymer-DNA composites to produce microfiber meshes of programmable patterns and geometries.

Pseudoheterodyne interferometry for multicolor near-field imaging.

We report the development and characterization of a detection technique for scattering-type scanning near-field optical microscopy (s-SNOM) that enables near-field amplitude and phase imaging at two or more wavelengths simultaneously. To this end, we introduce multispectral pseudoheterodyne (PSH) interferometry, where infrared lasers are combined to form a beam with a discrete spectrum of laser lines and a time-multiplexing scheme is employed to allow for the use of a single infrared detector. We first describe and validate the implementation of multispectral PSH into a commercial s-SNOM instrument. We then demonstrate its application for the real-time correction of the negative phase contrast (NPC), which provides reliable imaging of weak IR absorption at the nanoscale. We anticipate that multispectral PSH could improve data throughput, reduce effects of sample and interferometer drift, and help to establish multicolor s-SNOM imaging as a regular imaging modality, which could be particularly interesting as new infrared light sources become available.

Electron microscopy and calorimetry of proteins in supercooled water.

Some of the best nucleating agents in nature are ice-nucleating proteins, which boost ice growth better than any other material. They can induce immersion freezing of supercooled water only a few degrees below 0 °C. An open question is whether this ability also extends to the deposition mode, i.e., to water vapor. In this work, we used three proteins, apoferritin, InaZ (ice nucleation active protein Z), and myoglobin, of which the first two are classified as ice-nucleating proteins for the immersion freezing mode. We studied the ice nucleation ability of these proteins by differential scanning calorimetry (immersion freezing) and by environmental scanning electron microscopy (deposition freezing). Our data show that InaZ crystallizes water directly from the vapor phase, while apoferritin first condenses water in the supercooled state, and subsequently crystallizes it, just as myoglobin, which is unable to nucleate ice.

Tuning the magnetic properties of NiPS3 through organic-ion intercalation.

Atomically thin van der Waals magnetic crystals are characterized by tunable magnetic properties related to their low dimensionality. While electrostatic gating has been used to tailor their magnetic response, chemical approaches like intercalation remain largely unexplored. Here, we demonstrate the manipulation of the magnetism in the van der Waals antiferromagnet NiPS3 through the intercalation of different organic cations, inserted using an engineered two-step process. First, the electrochemical intercalation of tetrabutylammonium cations (TBA+) results in a ferrimagnetic hybrid compound displaying a transition temperature of 78 K, and characterized by a hysteretic behavior with finite remanence and coercivity. Then, TBA+ cations are replaced by cobaltocenium via an ion-exchange process, yielding a ferrimagnetic phase with higher transition temperature (98 K) and higher remanent magnetization. Importantly, we demonstrate that the intercalation and cation exchange processes can be carried out in bulk crystals and few-layer flakes, opening the way to the integration of intercalated magnetic materials in devices.

Influenza A virus hemagglutinin prevents extensive membrane damage upon dehydration.

While the molecular mechanisms of virus infectivity are rather well known, the detailed consequences of environmental factors on virus biophysical properties are poorly understood. Seasonal influenza outbreaks are usually connected to the low winter temperature, but also to the low relative air humidity. Indeed, transmission rates increase in cold regions during winter. While low temperature must slow degradation processes, the role of low humidity is not clear. We studied the effect of relative humidity on a model of Influenza A H1N1 virus envelope, a supported lipid bilayer containing the surface glycoprotein hemagglutinin (HA), which is present in the viral envelope in very high density. For complete cycles of hydration, dehydration and rehydration, we evaluate the membrane properties in terms of structure and dynamics, which we assess by combining confocal fluorescence microscopy, raster image correlation spectroscopy, line-scan fluorescence correlation spectroscopy and atomic force microscopy. Our findings indicate that the presence of HA prevents macroscopic membrane damage after dehydration. Without HA, fast membrane disruption is followed by irreversible loss of lipid and protein mobility. Although our model is principally limited by the membrane composition, the macroscopic effects of HA under dehydration stress reveal new insights on the stability of the virus at low relative humidity.

Nanoscale Wetting of Single Viruses.

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ~1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

Peptide-Based Electrospun Fibers: Current Status and Emerging Developments.

Electrospinning is a well-known, straightforward, and versatile technique, widely used for the preparation of fibers by electrifying a polymer solution. However, a high molecular weight is not essential for obtaining uniform electrospun fibers; in fact, the primary criterion to succeed is the presence of sufficient intermolecular interactions, which function similar to chain entanglements. Some small molecules able to self-assemble have been electrospun from solution into fibers and, among them, peptides containing both natural and non-natural amino acids are of particular relevance. Nowadays, the use of peptides for this purpose is at an early stage, but it is gaining more and more interest, and we are now witnessing the transition from basic research towards applications. Considering the novelty in the relevant processing, the aim of this review is to analyze the state of the art from the early 2000s on. Moreover, advantages and drawbacks in using peptides as the main or sole component for generating electrospun nanofibers will be discussed. Characterization techniques that are specifically targeted to the produced peptide fibers are presented.

Electrospinning of pyrazole-isothiazole derivatives: nanofibers from small molecules.

We investigate the electrospinning of small molecules, specifically designed peptide derivatives of the pyrazole-isothiazole scaffold. Such non-natural peptides enhance the spectrum of fundamental materials used for electrospinning. Unlike standard electrospun materials, our peptides are not polymeric, but able to aggregate in solution and especially during processing. They contain donor/acceptor groups that can form hydrogen bonds, and groups that are able to generate π-stacking interactions, which are known as important requirements for assembly processes. The pyrazole-isothiazole derivatives were synthesized by means of a 1,3-dipolar cycloaddition reaction, which is completely regioselective, affording only one isomer. We demonstrate that our compounds can be electrospun from fluoroalcohol solution into solid, quasi-endless micro- and nanofibers. The electrospinnability varies substantially, depending on the amino acids linked to the scaffold. Some compounds provide only short fibers, while Fmoc-glycyl-(N-benzyl)-pyrazole-isothiazole-tert-butyl carboxylate-1,1-dioxide forms continuous, homogenous, and bead-free fibers (droplet-like beads are a common problem in electrospinning). We analyzed the compounds and the fibers with various spectroscopic techniques (MS, IR and Raman). Electrospinning does not change chemical composition and configuration, suggesting the monomeric form of the compounds even in the fibers. Interestingly, we found that the stereochemistry of the scaffold can affect the ability of the peptide to be electrospun.

TMV-Templated Formation of Metal and Polymer Nanotubes.

Metals and polymers are probably the most important construction materials, but also have many more functions, e.g., for electronics. The interaction of metal ions with tobacco mosaic virus (TMV) was originally used for the preparation of heavy metal isomorphic replacement for structural analysis. Metal ions can also be the precursors for metal clusters, particles, and layers. Various strategies have been developed, which allow the creation of metal layers on the external surface of TMV. Such layers can be made as metal tubes, enveloping a complete virion. An alternative strategy is adsorption of metal nanoparticles. If a dense coating of TMV is achieved, again a tube results. Nanoscale tubes have various physical properties that depend on size, crystallinity, uniformity, but especially on the nature of the metal. Polymer coatings are as yet rarely investigated, though they can be prepared quite easily.Here, a series of exemplary protocols is provided, which covers all of these different concepts.

Catalysis of a 1,3-dipolar reaction by distorted DNA incorporating a heterobimetallic platinum(ii) and copper(ii) complex.

A novel catalytic system based on covalently modified DNA is described. This catalyst promotes 1,3-dipolar reactions between azomethine ylides and maleimides. The catalytic system is based on the distortion of the double helix of DNA by means of the formation of Pt(ii) adducts with guanine units. This distortion, similar to that generated in the interaction of DNA with platinum chemotherapeutic drugs, generates active sites that can accommodate N-metallated azomethine ylides. The proposed reaction mechanism, based on QM(DFT)/MM calculations, is compatible with thermally allowed concerted (but asynchronous) [π4s + π2s] mechanisms leading to the exclusive formation of racemic endo-cycloadducts.

Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies.

The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus-host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

Virus-Templated Near-Amorphous Iron Oxide Nanotubes.

We present a simple synthesis of iron oxide nanotubes, grown under very mild conditions from a solution containing Fe(II) and Fe(III), on rod-shaped tobacco mosaic virus templates. Their well-defined shape and surface chemistry suggest that these robust bionanoparticles are a versatile platform for synthesis of small, thin mineral tubes, which was achieved efficiently. Various characterization tools were used to explore the iron oxide in detail: Electron microscopy (SEM, TEM), magnetometry (SQUID-VSM), diffraction (XRD, TEM-SAED), electron spectroscopies (EELS, EDX, XPS), and X-ray absorption (XANES with EXAFS analysis). They allowed determination of the structure, crystallinity, magnetic properties, and composition of the tubes. The protein surface of the viral templates was crucial to nucleate iron oxide, exhibiting analogies to biomineralization in natural compartments such as ferritin cages.

Multifrequency Force Microscopy of Helical Protein Assembly on a Virus.

High-resolution microscopy techniques have been extensively used to investigate the structure of soft, biological matter at the nanoscale, from very thin membranes to small objects, like viruses. Electron microscopy techniques allow for obtaining extraordinary resolution by averaging signals from multiple identical structures. In contrast, atomic force microscopy (AFM) collects data from single entities. Here, it is possible to finely modulate the interaction with the samples, in order to be sensitive to their top surface, avoiding mechanical deformations. However, most biological surfaces are highly curved, such as fibers or tubes, and ultimate details of their surface are in the vicinity of steep height variations. This limits lateral resolution, even when sharp probes are used. We overcome this problem by using multifrequency force microscopy on a textbook example, the Tobacco Mosaic Virus (TMV). We achieved unprecedented resolution in local maps of amplitude and phase shift of the second excited mode, recorded together with sample topography. Our data, which combine multifrequency imaging and Fourier analysis, confirm the structure deduced from averaging techniques (XRD, cryoEM) for surface features of single virus particles, down to the helical pitch of the coat protein subunits, 2.3 nm. Remarkably, multifrequency AFM images do not require any image postprocessing.

Structural analysis and mapping of individual protein complexes by infrared nanospectroscopy.

Mid-infrared spectroscopy is a widely used tool for material identification and secondary structure analysis in chemistry, biology and biochemistry. However, the diffraction limit prevents nanoscale protein studies. Here we introduce mapping of protein structure with 30 nm lateral resolution and sensitivity to individual protein complexes by Fourier transform infrared nanospectroscopy (nano-FTIR). We present local broadband spectra of one virus, ferritin complexes, purple membranes and insulin aggregates, which can be interpreted in terms of their α-helical and/or ß-sheet structure. Applying nano-FTIR for studying insulin fibrils--a model system widely used in neurodegenerative disease research--we find clear evidence that 3-nm-thin amyloid-like fibrils contain a large amount of α-helical structure. This reveals the surprisingly high level of protein organization in the fibril's periphery, which might explain why fibrils associate. We envision a wide application potential of nano-FTIR, including cellular receptor in vitro mapping and analysis of proteins within quaternary structures.

Charge and spin transport in PEDOT:PSS nanoscale lateral devices.

The electrical transport of the highly conductive poly-(3,4-ethylenedioxythiophene):poly(styrenesulfonic acid) (PEDOT:PSS) is investigated with Ohmic and spin-polarized tunnel contacts at nanoscale lateral dimensions. Temperature-dependent charge transport measurements reveal that electrical conductivity scales non-linearly as a function of electrode spacing, which is attributed to the localization of carriers induced by the disorder introduced by the PSS polyelectrolyte. In addition, we demonstrate the integration of this conducting polymer in nanoscale lateral spin-valve devices by increasing the pH of the PEDOT:PSS solution. We present charge and magnetotransport measurement results of NiFe/AlOx/PEDOT:PSS/AlOx/NiFe lateral structures for various thicknesses of the alumina tunnel barriers. We discuss the absence of magnetoresistance of our spin valves within the framework of Valet-Fert theory, and estimate an upper limit for the spin lifetime of carriers in PEDOT:PSS to τsf ≤ 50 ns.

The condensation of water on adsorbed viruses.

The wetting and dewetting behavior of biological nanostructures and to a greater degree single molecules is not well-known even though their contact with water is the basis for all biology. Here, we show that environmental electron microscopy (EM) can be applied as a means of imaging the condensation of water onto viruses. We captured the formation of submicrometer water droplets and filaments on single viral particles by environmental EM and by environmental transmission EM. The condensate structures are compatible with capillary condensation between adsorbed virus particles and with known droplet shapes on patterned surfaces. Our results confirm that such droplets exist down to <50 nm. The viruses preserved their shape after a condensation/evaporation cycle as expected from their stability in air and water. Moreover we developed procedures that overcome problems of beam damage and of resolving structures with a low atomic number.

Nanoscale science and technology with plant viruses and bacteriophages.

Nanoscale science refers to the study and manipulation of matter at the atomic and molecular scales, including nanometer-sized single objects, while nanotechnology is used for the synthesis, characterization, and for technical applications of structures up to 100 nm size (and more). The broad nature of the fields encompasses disciplines such as solid-state physics, microfabrication, molecular biology, surface science, organic chemistry and also virology. Indeed, viruses and viral particles constitute nanometer-sized ordered architectures, with some of them even able to self-assemble outside cells. They possess remarkable physical, chemical and biological properties, their structure can be tailored by genetic engineering and by chemical means, and their production is commercially viable. As a consequence, viruses are becoming the basis of a new approach to the manufacture of nanoscale materials, made possible only by the development of imaging and manipulation techniques. Such techniques reach the scale of single molecules and nanoparticles. The most important ones are electron microscopy and scanning probe microscopy (both awarded with the Nobel Prize in Physics 1986 for the engineers and scientists who developed the respective instruments). With nanotechnology being based more on experimental than on theoretical investigations, it emerges that physical virology can be seen as an intrinsic part of it.

pH control of the electrostatic binding of gold and iron oxide nanoparticles to tobacco mosaic virus.

We report the binding of nanoparticles (NPs) to wild type (unmodified) tobacco mosaic virus (TMV). The viruses are simply mixed with citrate-coated, negatively charged gold and iron oxide nanoparticles (IONPs) in acidic solution. This results in TMV decorated along its whole length by the respective particles. Such a decoration usually requires chemical modification or mutation of TMV (e.g., cysteine residues), but here we simply reduce TMV's natural negative charge by protonation. The particles are protonated to a much smaller extent. This charge-based mechanism does not operate for neutral particles.

Integration of plant viruses in electron beam lithography nanostructures.

Tobacco mosaic virus (TMV) is the textbook example of a virus, and also of a self-assembling nanoscale structure. This tubular RNA/protein architecture has also found applications as biotemplate for the synthesis of nanomaterials such as wires, as tubes, or as nanoparticle assemblies. Although TMV is, being a biological structure, quite resilient to environmental conditions (temperature, chemicals), it cannot be processed in electron beam lithography (eBL) fabrication, which is the most important and most versatile method of nanoscale structuring. Here we present adjusted eBL-compatible processes that allow the incorporation of TMV in nanostructures made of positive and negative tone eBL resists. The key steps are covering TMV by polymer resists, which are only heated to 50 °C, and development (selective dissolution) in carefully selected organic solvents. We demonstrate the post-lithography biochemical functionality of TMV by selective immunocoating of the viral particles, and the use of immobilized TMV as direct immunosensor. Our modified eBL process should be applicable to incorporate a wide range of sensitive materials in nanofabrication schemes.