The Label-Free Detection and Identification of SARS-CoV-2 Using Surface-Enhanced Raman Spectroscopy and Principal Component Analysis.

The World Health Organization (WHO) declared in a May 2023 announcement that the COVID-19 illness is no longer categorized as a Public Health Emergency of International Concern (PHEIC); nevertheless, it is still considered an actual threat to world health, social welfare and economic stability. Consequently, the development of a convenient, reliable and affordable approach for detecting and identifying SARS-CoV-2 and its emerging new variants is crucial. The fingerprint and signal amplification characteristics of surface-enhanced Raman spectroscopy (SERS) could serve as an assay scheme for SARS-CoV-2. Here, we report a machine learning-based label-free SERS technique for the rapid and accurate detection and identification of SARS-CoV-2. The SERS spectra collected from samples of four types of coronaviruses on gold nanoparticles film, fabricated using a Langmuir-Blodgett self-assembly, can provide more spectroscopic signatures of the viruses and exhibit low limits of detection (<100 TCID50/mL or even <10 TCID50/mL). Furthermore, the key Raman bands of the SERS spectra were systematically captured by principal component analysis (PCA), which effectively distinguished SARS-CoV-2 and its variant from other coronaviruses. These results demonstrate that the combined use of SERS technology and PCA analysis has great potential for the rapid analysis and discrimination of multiple viruses and even newly emerging viruses without the need for a virus-specific probe.

Photomobile Polymer-Piezoelectric Composite for Enhanced Actuation and Energy Generation.

In this study, we present an innovative approach to increase the quantum yield and wavelength sensitivity of photomobile polymer (PMP) films based on azobenzene by doping the polymer matrix with noble metal nanoparticles. These doped PMP films showed faster and more significant bending under both UV as well as visible and near-infrared light regardless of whether it was coherent, incoherent, polarized, or unpolarized irradiation, expanding the potential of PMP-based actuators. To illustrate their practical implications, we created a proof-of-concept model of power generation by coupling it to flexible piezoelectric materials under simulated sunlight. This model has been tested under real operating conditions, thus demonstrating the possibility of generating electricity with variable light exposure. Additionally, our synthetic protocol is solvent-free, which is another benefit of environmental relevance. Our research lays the groundwork for the development of sunlight-sensitive devices, such as photomechanical actuators and advanced photovoltaic modules, which may break ground in the thriving field of smart materials. We are confident that the presented findings will contribute to the ongoing discourse in the field and inspire additional advances in renewable energy applications.

Development of LCEs with 100% Azobenzene Moieties: Thermo-Mechanical Phenomena and Behaviors.

Azobenzene is one of the most investigated photo-responsive liquid crystalline molecules. It can isomerize between two different isoforms, trans (E) and cis (Z) configurations, when stimulated by light. It is used as a molecular engine in photo-mobile materials (PMPs). The use of liquid crystals (LCs) as building blocks enhances the mechanical properties of the PMPs. It is not easy to obtain PMPs with monodomain configurations when the LCs are 100% azobenzene. In this work, we studied three LC mixtures, describing the thermo/mechanical phenomena that regulate the actuation of such materials. The nematic temperature of the LC elastomers was measured and the PMPs carefully characterized for their bending and speed capability. Our finding suggests that the ratio between linear and cross-linker monomer greatly influences the nematic temperature of the mixture. Furthermore, 100% azobenzene materials polymerized using dicumyl peroxide can be useful to design polarization-selective switches.

Photo-Responsivity Improvement of Photo-Mobile Polymers Actuators Based on a Novel LCs/Azobenzene Copolymer and ZnO Nanoparticles Network.

The efficiency of photomobile polymers (PMP) in the conversion of light into mechanical work plays a fundamental role in achieving cutting-edge innovation in the development of novel applications ranging from energy harvesting to sensor approaches. Because of their photochromic properties, azobenzene monomers have been shown to be an efficient material for the preparation of PMPs with appropriate photoresponsivity. Upon integration of the azobenzene molecules as moieties into a polymer, they act as an engine, allowing fast movements of up to 50 Hz. In this work we show a promising approach for integrating ZnO nanoparticles into a liquid crystalline polymer network. The addition of such nanoparticles allows the trapping of incoming light, which acts as diffusive points in the polymer matrix. We characterized the achieved nanocomposite material in terms of thermomechanical and optical properties and finally demonstrated that the doped PMP was better performing that the undoped PMP film.

LSPR immuno-sensing based on iso-Y nanopillars for highly sensitive and specific imidacloprid detection.

Imidacloprid is the most widely used insecticide in agriculture and its intensive use over the last 30 years has caused a global concern due to its potentially toxic effects on the ecosystem. Considering the recent scientific interest in novel simple methods for imidacloprid analysis, we propose a label-free sensitive and specific localised surface plasmon resonance system for the detection of the insecticide based on 2D nanostructured metasurfaces with highly performing plasmonic properties. The specificity of the sensor proposed was achieved by covalent bio-functionalization of the metasurface using a smart and easy one-step procedure mediated by carbon disulphide. The biosensor produced was tested using a set of imidacloprid standard solutions showing a competitive limit of detection, lower than 1 ng mL-1. Our novel nanosensing configuration represents a valid and reliable solution to realize low-cost portable POC tests as an alternative to the laborious and expensive methods traditionally used for insecticide detection.

The Aquatic Invertebrate Hydra vulgaris Releases Molecular Messages Through Extracellular Vesicles.

Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offers protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e., mediating the population's response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype, from structural proteins to stress induced messages promoting cell survival. Moreover, positive and negative regulators of the Wnt/ß-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to modulate both head and foot regeneration, suggesting bioactivity of the EV cargo and opening new perspectives on the mechanisms of developmental signalling. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems.

Hibernation and Radioprotection: Gene Expression in the Liver and Testicle of Rats Irradiated under Synthetic Torpor.

Hibernation has been proposed as a tool for human space travel. In recent years, a procedure to induce a metabolic state known as "synthetic torpor" in non-hibernating mammals was successfully developed. Synthetic torpor may not only be an efficient method to spare resources and reduce psychological problems in long-term exploratory-class missions, but may also represent a countermeasure against cosmic rays. Here we show the preliminary results from an experiment in rats exposed to ionizing radiation in normothermic conditions or synthetic torpor. Animals were irradiated with 3 Gy X-rays and organs were collected 4 h after exposure. Histological analysis of liver and testicle showed a reduced toxicity in animals irradiated in torpor compared to controls irradiated at normal temperature and metabolic activity. The expression of ataxia telangiectasia mutated (ATM) in the liver was significantly downregulated in the group of animal in synthetic torpor. In the testicle, more genes involved in the DNA damage signaling were downregulated during synthetic torpor. These data show for the first time that synthetic torpor is a radioprotector in non-hibernators, similarly to natural torpor in hibernating animals. Synthetic torpor can be an effective strategy to protect humans during long term space exploration of the solar system.

Combining Heavy-Ion Therapy with Immunotherapy: An Update on Recent Developments.

Clinical trials and case reports of cancer therapies combining radiation therapy with immunotherapy have at times demonstrated total reduction or elimination of metastatic disease. While virtually all trials focus on the use of immunotherapy combined with conventional photon irradiation, the dose-distributive benefits of particles, in particular the distinct biological effects of heavy ions, have unknown potential vis-a-vis systemic disease response. Here, we review recent developments and evidence with a focus on the potential for heavy-ion combination therapy.

Semiconducting polymers are light nanotransducers in eyeless animals.

Current implant technology uses electrical signals at the electrode-neural interface. This rather invasive approach presents important issues in terms of performance, tolerability, and overall safety of the implants. Inducing light sensitivity in living organisms is an alternative method that provides groundbreaking opportunities in neuroscience. Optogenetics is a spectacular demonstration of this, yet is limited by the viral transfection of exogenous genetic material. We propose a nongenetic approach toward light control of biological functions in living animals. We show that nanoparticles based on poly(3-hexylthiophene) can be internalized in eyeless freshwater polyps and are fully biocompatible. Under light, the nanoparticles modify the light response of the animals, at two different levels: (i) they enhance the contraction events of the animal body, and (ii) they change the transcriptional activation of the opsin3-like gene. This suggests the establishment of a seamless and biomimetic interface between the living organism and the polymer nanoparticles that behave as light nanotransducers, coping with or amplifying the function of primitive photoreceptors.

Endowing a plain fluidic chip with micro-optics: a holographic microscope slide.

Lab-on-a-Chip (LoC) devices are extremely promising in that they enable diagnostic functions at the point-of-care. Within this scope, an important goal is to design imaging schemes that can be used out of the laboratory. In this paper, we introduce and test a pocket holographic slide that allows digital holography microscopy to be performed without an interferometer setup. Instead, a commercial off-the-shelf plastic chip is engineered and functionalized with this aim. The microfluidic chip is endowed with micro-optics, that is, a diffraction grating and polymeric lenses, to build an interferometer directly on the chip, avoiding the need for a reference arm and external bulky optical components. Thanks to the single-beam scheme, the system is completely integrated and robust against vibrations, sharing the useful features of any common path interferometer. Hence, it becomes possible to bring holographic functionalities out of the lab, moving complexity from the external optical apparatus to the chip itself. Label-free imaging and quantitative phase contrast mapping of live samples are demonstrated, along with flexible refocusing capabilities. Thus, a liquid volume can be analyzed in one single shot with no need for mechanical scanning systems.

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion.

Control of Wnt/ß-Catenin Signaling Pathway in Vivo via Light Responsive Capsules.

The possibility to remotely manipulate intracellular pathways in single cells is among the current goals of biomedicine, demanding new strategies to control cell function and reprogramming cell fate upon external triggering. Optogenetics is one approach in this direction, allowing specific cell stimulation by external illumination. Here, we developed optical switchers of an ancient and highly conserved system controlling a variety of developmental and adult processes in all metazoans, from Hydra to mammals, the Wnt/ß-catenin signaling pathway. An intracellular modulator of the Wnt pathway was enclosed into polyelectrolyte multilayer microcapsules engineered to include self-tracking (i.e., fluorescence labeling) and light mediated heating functionalities (i.e., plasmonic nanoparticles). Capsules were delivered in vivo to Hydra and NIR triggered drug release caused forced activation of the Wnt pathway. The possibility to remotely manipulate the Wnt pathway by optical switchers may be broadly translated to achieve spatiotemporal control of cell fate for new therapeutic strategies.

Effects of Lithium Niobate Polarization on Cell Adhesion and Morphology.

Understanding how the interfacial effects influence cell adhesion and morphology is of fundamental interest for controlling function, growth, and movement of cells in vitro and in vivo. In particular, the influence of surface charges is well-known but still controversial, especially when new functional materials and methods are introduced. Here, the influence of the spontaneous polarization of ferroelectric lithium niobate (LN) on the adhesion properties of fibroblast cells is investigated. The spontaneous polarization of LN has one of the largest known magnitudes at room temperature (∼78 µC/cm(2)), and its orientation can be patterned easily by an external voltage, this motivating highly the investigation of its interaction with cells. Immunofluorescence and migration assays show strong evidence that the surface polarity regulates the adhesion functions, with enhanced spreading of the cytoskeleton on the negative face. The results suggest the potential of LN as a platform for investigating the role of charges on cellular processes, thus favoring new strategies in fabricating those biocompatible constructs used for tissue engineering. In fact, the orientation of the high-magnitude polarization can be patterned easily and, in combination with piezoelectric, pyroelectric, and photorefractive properties, may open the route to more sophisticated charge templates for modulating the cell response.

Deciphering intracellular events triggered by mild magnetic hyperthermia in vitro and in vivo.

AIM: To assess the cell response to magnetic nanoparticles under an alternating magnetic field by molecular quantification of heat responsive transcripts in two model systems. MATERIALS & METHODS: Melanoma cells and Hydra vulgaris treated with magnetic nanoparticles were subjected to an alternating magnetic field or to macroscopic heating. Effect to these treatments were assessed at animal, cellular and molecular levels. RESULTS: By comparing hsp70 expression following both treatments, thermotolerance pathways were found in both systems in absence of cell ablation or global temperature increment. CONCLUSION: Analysis of hsp70 transcriptional activation can be used as molecular thermometer to sense cells' response to magnetic hyperthermia. Similar responses were found in cells and Hydra, suggesting a general mechanism to the delivery of sublethal thermal doses.

Impact of Carbon Nano-Onions on Hydra vulgaris as a Model Organism for Nanoecotoxicology.

The toxicological effects of pristine and chemically modified carbon nano-onions (CNOs) on the development of the freshwater polyp Hydra vulgaris were investigated in order to elucidate the ecotoxicological effects of CNOs. Chemical modifications of the CNOs were accomplished by surface functionalization with benzoic acid, pyridine and pyridinium moieties. thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy confirmed the covalent surface functionalization of CNOs. Hydra specimens were exposed to the carbon nanomaterials by prolonged incubation within their medium. Uptake was monitored by optical microscopy, and the toxicological effects of the CNOs on Hydra behavior, morphology, as well as the long-term effects on the development and reproductive capability were examined. The obtained data revealed the absence of adverse effects of CNOs (in the range 0.05-0.1 mg/L) in vivo at the whole animal level. Together with previously performed in vitro toxicological analyses, our findings indicate the biosafety of CNOs and the feasibility of employing them as materials for biomedical applications.

Impact of Amorphous SiO2 Nanoparticles on a Living Organism: Morphological, Behavioral, and Molecular Biology Implications.

It is generally accepted that silica (SiO2) is not toxic. But the increasing use of silica nanoparticles (SiO2NPs) in many different industrial fields has prompted the careful investigation of their toxicity in biological systems. In this report, we describe the effects elicited by SiO2NPs on animal and cell physiology. Stable and monodisperse amorphous silica nanoparticles, 25 nM in diameter, were administered to living Hydra vulgaris (Cnidaria). The dose-related effects were defined by morphological and behavioral assays. The results revealed an all-or-nothing lethal toxicity with a rather high threshold (35 nM NPs) and a LT50 of 38 h. At sub lethal doses, the morphophysiological effects included: animal morphology alterations, paralysis of the gastric region, disorganization and depletion of tentacle specialized cells, increase of apoptotic and collapsed cells, and reduction of the epithelial cell proliferation rate. Transcriptome analysis (RNAseq) revealed 45 differentially expressed genes, mostly involved in stress response and cuticle renovation. Our results show that Hydra reacts to SiO2NPs, is able to rebalance the animal homeostasis up to a relatively high doses of SiO2NPs, and that the physiological modifications are transduced to gene expression modulation.

Gold nanoprisms for photothermal cell ablation in vivo.

AIM: To develop new methodologies for selective cell ablation in a temporally and spatially precise fashion in model organisms. MATERIALS & METHODS: living polyps (Hydra vulgaris) treated with gold nanoprisms were near-infrared (NIR) irradiated and the photothermal effects evaluated at whole-animal, cellular and molecular levels. RESULTS: Nanoprisms showed good efficiency of internalization in living specimens, with no sign of toxicity; under NIR irradiation they induced cell death and the overexpression of the hsp70 gene. CONCLUSION: gold nanoprisms could be employed as efficient heat mediators in model organisms, and NIR-triggered cell ablation may represent a new advanced tool to study cell function. Solving bioethical and economical issues, invertebrates may provide alternative models bridging the gap between cell research and preclinical studies of photothermal therapy.

Nanotoxicology using the sea anemone Nematostella vectensis: from developmental toxicity to genotoxicology.

Concomitant with the fast-growing advances in the synthesis and engineering of colloidal nanocrystals, an urgent evaluation of their toxicity on human beings and environment is strongly encouraged by public health organisations. Despite the in vitro approaches employed for toxicological screening of hazardous compounds, the use of simple and cost-effective living organisms may enormously contribute to solve unanswered questions related to embryotoxic and teratogenic effects of nanomaterials. Here, the sea anemone Nematostella vectensis (Cnidaria, Anthozoa) is presented as a novel model organism to profile bio/non-bio interactions and to show a comprehensive toxicological analysis performed on embryos, larvae and adults treated with fluorescent cadmium-based nanocrystals. Spanning from in vivo biodistribution to molecular investigations, different behaviours and effects depending on the composition and surface coatings are showed. Rod-shaped cadmium selenide/cadmium sulfide (CdSe/CdS) nanocrystals resulted in excellent imaging probes to track N. vectensis development with negligible adverse effects, while spherical CdTe nanocrystals severely impaired embryogenesis, resulting in aberrant phenotypes and deregulation of developmental genes, which raise severe worries for a safe use of this type of nanoparticles for human purposes and environmental contamination.

Imaging inward and outward trafficking of gold nanoparticles in whole animals.

Gold nanoparticles have emerged as novel safe and biocompatible tools for manifold applications, including biological imaging, clinical diagnostics, and therapeutics. The understanding of the mechanisms governing their interaction with living systems may help the design and development of new platforms for nanomedicine. Here we characterized the dynamics and kinetics of the events underlying the interaction of gold nanoparticles with a living organism, from the first interaction nanoparticle/cell membrane, to the intracellular trafficking and final extracellular clearance. By treating a simple water invertebrate (the cnidarian Hydra polyp) with functionalized gold nanoparticles, multiple inward and outward routes were imaged by ultrastructural analyses, including exosomes as novel undescribed carriers to shuttle the nanoparticles in and out the cells. From the time course imaging a highly dynamic picture emerged in which nanoparticles are rapidly internalized (from 30 min onward), recruited into vacuoles/endosome (24 h onward), which then fuse, compact and sort out the internalized material either to storage vacuoles or to late-endosome/lysosomes, determining almost complete clearance within 48 h from challenging. Beside classical routes, new portals of entry/exit were captured, including exosome-like structures as novel undescribed nanoparticle shuttles. The conservation of the endocytic/secretory machinery through evolution extends the value of our finding to mammalian systems providing dynamics and kinetics clues to take into account when designing nanomaterials to interface with biological entities.

Design of multifunctional gold nanoparticles for in vitro and in vivo gene silencing.

Over the past decade, the capability of double-stranded RNAs to interfere with gene expression has driven new therapeutic approaches. Since small interfering RNA (siRNAs, 21 base pair double-stranded RNA) was shown to be able to elicit RNA interference (RNAi), efforts were directed toward the development of efficient delivery systems to preserve siRNA bioactivity throughout the delivery route, from the administration site to the target cell. Here we provide evidence of RNAi triggering, specifically silencing c-myc protooncogene, via the synthesis of a library of novel multifunctional gold nanoparticles (AuNPs). The efficiency of the AuNPs is demonstrated using a hierarchical approach including three biological systems of increasing complexity: in vitro cultured human cells, in vivo invertebrate (freshwater polyp, Hydra ), and in vivo vertebrate (mouse) models. Our synthetic methodology involved fine-tuning of multiple structural and functional moieties. Selection of the most active functionalities was assisted step-by-step through functional testing that adopted this hierarchical strategy. Merging these chemical and biological approaches led to a safe, nonpathogenic, self-tracking, and universally valid nanocarrier that could be exploited for therapeutic RNAi.