Flexible Physical Unclonable Functions Based on Non-deterministically Distributed Dye-Doped Fibers and Droplets.

The development of new anticounterfeiting solutions is a constant challenge and involves several research fields. Much interest is currently devoted to systems that are impossible to clone, based on the physical unclonable function (PUF) paradigm. In this work, a new strategy based on electrospinning and electrospraying of dye-doped polymeric materials is presented for the manufacturing of flexible free-standing films that embed simultaneously different PUF keys. The proposed films can be used to fabricate novel anticounterfeiting labels having three encryption levels: (i) a map of fluorescent polymer droplets, with random positions on a dense yarn of polymer nanofibers, (ii) a characteristic fluorescence spectrum for each label, and (iii) the unique speckle patterns that every label produces when illuminated with coherent laser light shaped in different wavefronts. The intrinsic uniqueness introduced by the manufacturing process encodes enough complexity into the optical anticounterfeiting tag to generate thousands of cryptographic keys. The simple and cheap fabrication process as well as multilevel authentication makes such colored polymeric unclonable tags a practical solution in the secure protection of goods in our daily life.

Macroscopic Biaxial Order in Multilayer Films of Bent-Core Liquid Crystals Deposited by Combined Langmuir-Blodgett/Langmuir-Schaefer Technique.

Bent-core liquid crystals, a class of mesogenic compounds with non-linear molecular structures, are well known for their unconventional mesophases, characterized by complex molecular (and supramolecular) ordering and often featuring biaxial and polar properties. In the nematic phase, their unique behavior is manifested in the formation of nano-sized biaxial clusters of layered molecules (cybotactic groups). While this prompted their consideration in the quest for nematic biaxiality, experimental evidence indicates that the cybotactic order is only short-ranged and that the nematic phase is macroscopically uniaxial. By combining atomic force microscopy, neutron reflectivity and wide-angle grazing-incidence X-ray scattering, here, we demonstrate that multilayer films of a bent-core nematic, deposited on silicon by a combined Langmuir-Blodgett and Langmuir-Schaefer approach, exhibit macroscopic in-plane ordering, with the long molecular axis tilted with respect to the sample surface and the short molecular axis (i.e., the apex bisector) aligned along the film compression direction. We thus propose the use of Langmuir films as an effective way to study and control the complex anchoring properties of bent-core liquid crystals.

Sunset Yellow Confined in Curved Geometry: A Microfluidic Approach.

The behavior of lyotropic chromonic liquid crystals (LCLCs) in confined environments is an interesting research field that still awaits exploration, with multiple key variables to be uncovered and understood. Microfluidics is a highly versatile technique that allows us to confine LCLCs in micrometric spheres. As microscale networks offer distinct interplays between the surface effects, geometric confinement, and viscosity parameters, rich and unique interactions emerging at the LCLC-microfluidic channel interfaces are expected. Here, we report on the behavior of pure and chiral doped nematic Sunset Yellow (SSY) chromonic microdroplets produced through a microfluidic flow-focusing device. The continuous production of SSY microdroplets with controllable size gives the possibility to systematically study their topological textures as the function of their diameters. Indeed, doped SSY microdroplets produced via microfluidics, show topologies that are typical of common chiral thermotropic liquid crystals. Furthermore, few droplets exhibit a peculiar texture never observed for chiral chromonic liquid crystals. Finally, the achieved precise control of the produced LCLC microdroplets is a crucial step for technological applications in biosensing and anticounterfeiting.

Nanoscale Structure of Langmuir-Blodgett Film of Bent-Core Molecules.

Bent-core mesogens (BCMs) are a class of thermotropic liquid crystals featuring several unconventional properties. However, the interpretation and technological exploitation of their unique behavior have been hampered by the difficulty of controlling their anchoring at surfaces. To tackle this issue, we report the nanoscale structural characterization of BCM films prepared using the Langmuir-Blodgett technique. Even though BCMs are quite different from typical amphiphilic molecules, we demonstrate that stable molecular films form over water, which can then be transferred onto silicon substrates. The combination of Brewster angle microscopy, atomic force microscopy, and X-ray reflectivity measurements shows that the molecules, once transferred onto a solid substrate, form a bilayer structure with a bottom layer of flat molecules and an upper layer of upright molecules. These results suggest that Langmuir-Blodgett films of BCMs can provide a useful means to control the alignment of this class of liquid crystals.

Low Cost and Easy Validation Anticounterfeiting Plasmonic Tags Based on Thin Films of Metal and Dielectric.

Multilevel anticounterfeiting Physical Unclonable Function (PUF) tags based on thin film of silver (Ag), Zinc Oxide (ZnO) and PolyVinylPyrrolidone (PVP), are experimentally demonstrated and validated. We exploit the low adhesion of silver to glass and consequent degradation during ZnO deposition to induce morphological randomness. Several photographs of the tag surfaces have been collected with different illumination conditions and using two smartphones of diverse brand. The photos were analyzed using an image recognition algorithm revealing low common minutiae for different tags. Moreover, the optical response reveals peculiar spectra due to labels of plasmonic nature. The proposed systems can be easily fabricated on large areas and represent a cost-effective solution for practical protection of objects.

Spherical Confinement of Chromonics: Effects of a Chiral Aminoacid.

Induced or spontaneous chirality in natural systems is an intriguing issue. In recent years, a lot of attention has been focused on chirality of chromonic liquid crystals, a class of materials that is able to self-assemble in columnar structures. However, the mechanism involved in the arising of chirality in these materials, that starts at the molecular level and controls the supramolecular structure, is poorly understood; however, it is certainly affected by ionic strength. In this work we present the results obtained doping Cromolyn, a chromonic material, with a strong helical-twisting-power peptide, and confining it in a spherical geometry. We demonstrate, by means of optical polarized microscopy and structural analysis, that both the geometrical constraint and the presence of the chiral dopant enhance the chiral effect; we also demonstrate that they favor the rise of a highly ordered helical superstructure, that may be optimized upon adding an ionic dye to the system. Finally, we report a procedure for the preparation of free-standing polymeric films, embedding and preserving the microspheres, and paving the way for the creation of biocompatible and eco-friendly optical devices to be used in the sensor and anticounterfeiting fields.

A novel phage display based platform for exosome diversity characterization.

We present an innovative approach allowing the identification, isolation, and molecular characterization of disease-related exosomes based on their different antigenic reactivities. The designed strategy could be immediately translated into any disease in which exosomes are involved. The identification of specific markers and their subsequent association with exosome subtypes, together with the possibility to engineer target-guided exosome-like particles, could represent the key for the effective adoption of exosomes in clinical practice.

Hybrid Plasmonic/Photonic Nanoscale Strategy for Multilevel Anticounterfeit Labels.

Innovative goods authentication strategies are of fundamental importance considering the increasing counterfeiting levels. Such a task has been effectively addressed with the so-called physical unclonable functions (PUFs), being physical properties of a system that characterize it univocally. PUFs are commonly implemented by exploiting naturally occurring non-idealities in clean-room fabrication processes. The broad availability of classic paradigm PUFs, however, makes them vulnerable. Here, we propose a hybrid plasmonic/photonic multilayered structure working as a three-level strong PUF. Our approach leverages on the combination of a functional nanostructured surface, a resonant response, and a unique chromatic signature all together in one single device. The structure consists of a resonant cavity, where the top mirror is replaced with a layer of plasmonic Ag nanoislands. The naturally random spatial distribution of clusters and nanoparticles formed by this deposition technique constitutes the manufacturer-resistant nanoscale morphological fingerprint of the proposed PUF. The presence of Ag nanoislands allows us to tailor the interplay between the photonic and plasmonic modes to achieve two additional security levels. The first one is constituted by the chromatic response and broad iridescence of our structures, while the second by their rich spectral response, accessible even through a common smartphone light-emitting diode. We demonstrate that the proposed architectures could also be used as an irreversible and quantitative temperature exposure label. The proposed PUFs are inexpensive, chip-to-wafer-size scalable, and can be deposited over a variety of substrates. They also hold a great promise as an encryption framework envisioning morpho-cryptography applications.

Nano- and Micro-Porous Chitosan Membranes for Human Epidermal Stratification and Differentiation.

The creation of partial or complete human epidermis represents a critical aspect and the major challenge of skin tissue engineering. This work was aimed at investigating the effect of nano- and micro-structured CHT membranes on human keratinocyte stratification and differentiation. To this end, nanoporous and microporous membranes of chitosan (CHT) were prepared by phase inversion technique tailoring the operational parameters in order to obtain nano- and micro-structured flat membranes with specific surface properties. Microporous structures with different mean pore diameters were created by adding and dissolving, in the polymeric solution, polyethylene glycol (PEG Mw 10,000 Da) as porogen, with a different CHT/PEG ratio. The developed membranes were characterized and assessed for epidermal construction by culturing human keratinocytes on them for up to 21 days. The overall results demonstrated that the membrane surface properties strongly affect the stratification and terminal differentiation of human keratinocytes. In particular, human keratinocytes adhered on nanoporous CHT membranes, developing the structure of the corneum epidermal top layer, characterized by low thickness and low cell proliferation. On the microporous CHT membrane, keratinocytes formed an epidermal basal lamina, with high proliferating cells that stratified and differentiated over time, migrating along the z axis and forming a multilayered epidermis. This strategy represents an attractive tissue engineering approach for the creation of specific human epidermal strata for testing the effects and toxicity of drugs, cosmetics and pollutants.

Spicy Bitumen: Curcumin Effects on the Rheological and Adhesion Properties of Asphalt.

Over the years, the need for the synthesis of biodegradable materials has facilitated the drift of the asphalt industry towards eco-sustainable and cost-effective production of road pavements. The principal additives in the asphalt industry to improve the performance of road pavements and increase its lifespan are majorly rheological modifiers, adhesion promoters and anti-oxidant agents. Rheological modifiers increase physico-chemical properties such as transition temperature of asphalt binder (bitumen), adhesion promoters increase the affinity between binder and stone aggregates while anti-oxidant agents reduce the effects of oxidation caused by exposure to air, water and other natural elements during the production of asphalt pavements. In this study, we tested the effectiveness of a food grade bio-additive on these three aforementioned properties. We also sought to hypothesize the mechanisms by which the additive confers these desired features on bitumen. We present this study to evaluate the effects of turmeric, a food-based additive, on bitumen. The study was conducted through dynamic shear rheology (DSR), atomic force microscopy, scanning electron microscopy (SEM) and boiling test analysis.

Sperm DNA Methylation at Metabolism-Related Genes in Vegan Subjects.

Objective: To investigate if epigenome of sperm cells could be dynamically affected by nutrition. Design and Methods: We assessed 40 healthy volunteers with different dietary habits and collected their demographic characteristics, as well as clinical and anthropometric parameters. We compared methylation profiles in sperm quantified by bisulfite pyrosequencing, at promoter-associated CpG sites of genes involved in metabolism including fat mass and obesity-associated (FTO) and melanocortin-4 receptor (MC4R) from six vegans and 34 omnivores. In addition, the FTO rs9939609 (T>A) was genotyped. Results: Higher DNA methylation levels were detected in the sperm of vegan at FTO gene CpG1 (p=0.02), CpG2 (p=0.001), CpG3 (p=0.004), and CpG4 (p=0.003) sites and at MC4R-CpG2 site [p=0.016] as compared to sperm of omnivores. This association was not related to FTO genotype. Conclusions: Although limited by the small number of investigated cases, our data provide insight into the role of diet on sperm DNA methylation in genes involved in metabolism.

Preparation and Characterization of Silver(I) Ethylcellulose Thin Films as Potential Food Packaging Materials.

Ag(I)-containing ethylcellulose (EC) films suitable as antbacterial packaging materials have been prepared and fully characterized. Different preparation methods, including the use of green casting solvents, are proposed. The Ag(I) acylpyrazolonato complexes, [Ag(Qpy,CF3 )(L)], L=benzylimidazole (Bzim) and L=ethylimidazole (EtimH), used as active additives, display different modes of interactions with EC, depending on their structural features. A thorough investigation of the EC liquid-crystalline lyotropic phase and its changes with the introduction of silver additives, has been conducted, revealing either the inclusion of complex molecules into the inner structure of the EC matrix or their dispersion on its surface. Moreover, the bactericidal activity of the prepared Ag(I) films seems to be related to the interaction between silver additives and the polymeric EC matrix. Indeed, the EC-2b films show a particularly good performance even with a low silver content, with a relative bacterial killing of about 100 %. Tests for Ag(I) migration have been performed by using three food stimulants under two assay conditions. Low values of silver release are recorded, particularly at low concentration of silver content, in the case of all new prepared Ag(I) films.

Adhesive Properties of Adsorbed Layers of Two Recombinant Mussel Foot Proteins with Different Levels of DOPA and Tyrosine.

Using a surface forces apparatus and an atomic force microscope, we characterized the adhesive properties of adsorbed layers of two recombinant variants of Perna viridis foot protein 5 (PVFP-5), the main surface-binding protein in the adhesive plaque of the Asian green mussel. In one variant, all tyrosine residues were modified into 3,4-dihydroxy-l-phenylalanine (DOPA) during expression using a residue-specific incorporation strategy. DOPA is a key molecular moiety underlying underwater mussel adhesion. In the other variant, all tyrosine residues were preserved. The layer was adsorbed on a mica substrate and pressed against an uncoated surface. While DOPA produced a stronger adhesion than tyrosine in contact with the nanoscopic Si3N4 probe of the atomic force microscope, the two variants produced comparable adhesion on the curved macroscopic mica surfaces of the surface forces apparatus. These findings show that the presence of DOPA is not a sufficient condition to generate strong underwater adhesion. Surface chemistry and contact geometry affect the strength and abundance of protein-surface bonds created during adsorption and surface contact. Importantly, the adsorbed protein layer has a random and dynamic polymer-network structure that should be optimized to transmit the tensile stress generated during surface separation to DOPA surface bonds rather than other weaker bonds.

Gi Protein Modulation of the Potassium Channel TASK-2 Mediates Vesicle Osmotic Swelling to Facilitate the Fusion of Aquaporin-2 Water Channel Containing Vesicles.

Vesicle fusion is a fundamental cell biological process similar from yeasts to humans. For secretory vesicles, swelling is considered a step required for the expulsion of intravesicular content. Here this concept is revisited providing evidence that it may instead represent a general mechanism. We report the first example that non-secretory vesicles, committed to insert the Aquaporin-2 water channel into the plasma membrane, swell and this phenomenon is required for fusion to plasma membrane. Through an interdisciplinary approach, using atomic force microscope (AFM), a fluorescence-based assay of vesicle volume changes and NMR spectroscopy to measure water self-diffusion coefficient, we provide evidence that Gi protein modulation of potassium channel TASK-2 localized in AQP2 vesicles, is required for vesicle swelling. Estimated intravesicular K⁺ concentration in AQP2 vesicles, as measured by inductively coupled plasma mass spectrometry, was 5.3 mM, demonstrating the existence of an inwardly K⁺ chemical gradient likely generating an osmotic gradient causing vesicle swelling upon TASK-2 gating. Of note, abrogation of K⁺ gradient significantly impaired fusion between vesicles and plasma membrane. We conclude that vesicle swelling is a potentially important prerequisite for vesicle fusion to the plasma membrane and may be required also for other non-secretory vesicles, depicting a general mechanism for vesicle fusion.

Temperature-tunable lasing from dye-doped chiral microdroplets encapsulated in a thin polymeric film.

In the last decade, much interest has grown around the possibility to use liquid-crystal droplets as optical microcavities and lasers. In particular, 3D laser emission from dye-doped cholesteric liquid crystals confined inside microdroplets paves the way for many applications in the field of sensors or tunable photonics. Several techniques can be used to obtain small microresonators as, for example, dispersing a liquid crystal inside an immiscible isotropic fluid to create an emulsion. Recently, the possibility to obtain a thin free-standing film starting from an emulsion having a mixture of water and polyvinyl alcohol as isotropic matrix has been reported. After the water evaporation, a polymeric film in which the microdroplets are encapsulated has been obtained. Bragg-type laser emission has been recorded from the emulsion as well as from the thin film. Here, we report on the possibility to tune the laser emission as a function of temperature. Using a chiral dopant with temperature dependent solubility, the emitted laser wavelength can be tuned in a range of 40 nm by a temperature variation of 18 °C. The proposed device can have applications in the field of sensors and for the development of anti-counterfeiting labels.

Non-invasive optical method for real-time assessment of intracorneal riboflavin concentration and efficacy of corneal cross-linking.

Keratoconus is the primary cause of corneal transplantation in young adults worldwide. Riboflavin/UV-A corneal cross-linking may effectively halt the progression of keratoconus if an adequate amount of riboflavin enriches the corneal stroma and is photo-oxidated by UV-A light for generating additional cross-linking bonds between stromal proteins and strengthening the biomechanics of the weakened cornea. Here we reported an UV-A theranostic prototype device for performing corneal cross-linking with the ability to assess corneal intrastromal concentration of riboflavin and to estimate treatment efficacy in real time. Seventeen human donor corneas were treated according to the conventional riboflavin/UV-A corneal cross-linking protocol. Ten of these tissues were probed with atomic force microscopy in order to correlate the intrastromal riboflavin concentration recorded during treatment with the increase in elastic modulus of the anterior corneal stroma. The intrastromal riboflavin concentration and its consumption during UV-A irradiation of the cornea were highly significantly correlated (R = 0.79; P = .03) with the treatment-induced stromal stiffening effect. The present study showed an ophthalmic device that provided an innovative, non-invasive, real-time monitoring solution for estimating corneal cross-linking treatment efficacy on a personalized basis.

Mixed emulsion of liquid crystal microresonators: towards white laser systems.

Microdroplet systems have attracted great interest because of their wide range of applications, easiness in processing and handling, feasibility in developing miniaturized devices with high performances and large flexibility. In this study, a stable emulsion based on different dye-doped chiral liquid crystal droplets has been engineered in order to achieve simultaneous omnidirectional lasing at different wavelengths. To obtain the mixed emulsion of dye doped Bragg onion-type microresonators the twofold action, as a surfactant and a droplet stabilizer, of the polyvinyl alcohol dissolved in water has been exploited. Multiple wavelengths lasing in all directions around the mixed emulsion is demonstrated. By water evaporation, a plastic sheet including different types of chiral droplets is also obtained, retaining all the emission characteristic of the precursor emulsion. A relevant feature is the large flexibility of the preparation method that enables an easy and full control of the lasing spectrum addressing white laser systems. However, the simplicity of the procedure based on a single-step process as well as the high stability of the mixed emulsion is a relevant result, envisaging strong potentiality for developing easy and friendly technologies useful in the field of identification, sensing, imaging, coating and lab-on-a-chip architectures.

Biomechanical Strengthening of the Human Cornea Induced by Nanoplatform-Based Transepithelial Riboflavin/UV-A Corneal Cross-Linking.

Purpose: The purpose of this study was to investigate the biomechanical stiffening effect induced by nanoplatform-based transepithelial riboflavin/UV-A cross-linking protocol using atomic force microscopy (AFM). Methods: Twelve eye bank donor human sclerocorneal tissues were investigated using a commercial atomic force microscope operated in force spectroscopy mode. Four specimens underwent transepithelial corneal cross-linking using a hypotonic solution of 0.1% riboflavin with biodegradable polymeric nanoparticles of 2-hydroxypropyl-ß-cyclodextrin plus enhancers (trometamol and ethylenediaminetetraacetic acid) and UV-A irradiation with a 10 mW/cm2 device for 9 minutes. After treatment, the corneal epithelium was removed using the Amoils brush, and the Young's modulus of the most anterior stroma was quantified as a function of scan rate by AFM. The results were compared with those collected from four specimens that underwent conventional riboflavin/UV-A corneal cross-linking and four untreated specimens. Results: The average Young's modulus of the most anterior stroma after the nanoplatform-based transepithelial and conventional riboflavin/UV-A corneal cross-linking treatments was 2.5 times (P < 0.001) and 1.7 times (P < 0.001) greater than untreated controls respectively. The anterior stromal stiffness was significantly different between the two corneal cross-linking procedures (P < 0.001). The indentation depth decreased after corneal cross-linking treatments, ranging from an average of 2.4 ± 0.3 µm in untreated samples to an average of 1.2 ± 0.1 µm and 1.8 ± 0.1 µm after nanoplatform-based transepithelial and conventional cross-linking, respectively. Conclusions: The present nanotechnology-based transepithelial riboflavin/UV-A corneal cross-linking was effective to improve the biomechanical strength of the most anterior stroma of the human cornea.

Mitochondrial heteroplasmy profiling in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a key role in the development of a competent oocyte. Mutations of the mitochondrial genome lead to an altered energetic metabolism with negative effects on oocyte developmental competence. In this study, mtDNA heteroplasmy at an intra-oocyte level and between the different analyzed human oocytes (n = 12) was identified by a next-generation sequencing (NGS) protocol previously developed by this research group and submitted to GenBank. This method highlighted, in particular, variants in the genes involved in the respiratory chain providing a direct indication of the cell-specific damage within the mitochondrial genome as predictor of the oocyte quality.

Complete sequence of human mitochondrial DNA obtained by combining multiple displacement amplification and next-generation sequencing on a single oocyte.

Mitochondrial DNA (mtDNA) plays a key role in the development of a competent oocyte. In this study, the complete mtDNA sequence obtained for the first time by multiple displacement amplification approach in combination with next-generation sequencing from a single human oocyte is reported (GenBank accession no. KT364276). The analysis of oocyte mitochondrial mutations could provide a better understanding of the genetic variants correlated with the oocyte quality.