ASFV epitope mapping by high density peptides microarrays.

African swine fever (ASF) is an acute, highly contagious and deadly infectious disease. It is a threat to animal health with major potential economic and societal impact. Despite decades of ASF vaccine research, still some gaps in knowledge are hindering the development of a functional vaccine. Worth mentioning are gaps in understanding the mechanism of ASF infection and immunity, as well as the fact that - in case of this disease - virus proteins, so-called protective antigens, responsible for inducing protective immune responses in pigs are not identified yet. In this paper we elaborate on a methodology to identify protective antigens based on epitope mapping by microarray technology. High density peptide microarrays, combined with fluorescence scanning, have been used to analyze the interaction of peptide sequences of African swine fever virus (ASFV) proteins with antibodies present in inactivated serum from infected and healthy animals. The study evidenced ASFV proteins already under the radar for vaccine development, such as p54, and identified specific sequences in those proteins that may become the focus for future vaccine candidates. Such methodology is amenable to automation and high-throughput and may help developing better targeting for next generation vaccines.

Inter-Individual Variations: A Challenge for the Standardisation of Complement Activation Assays.

Introduction: The role of the human immune system in pathologic responses to chemicals including nanomaterials was identified as a gap in current hazard assessments. However, the complexity of the human immune system as well as interspecies variations make the development of predictive toxicity tests challenging. In the present study, we have analysed to what extent fluctuations of the complement system of different individuals will have an impact on the standardisation of immunological tests. Methods: We treated commercially available pooled sera (PS) from healthy males, individual sera from healthy donors and from patients suffering from cancer, immunodeficiency and allergies with small molecules and liposomes. Changes of iC3b protein levels measured in enzyme-linked immunosorbent assays served as biomarker for complement activation. Results: The level of complement activation in PS differed significantly from responses of individual donors (p < 0.01). Only seven out of 32 investigated sera from healthy donors responded similarly to the pooled serum. This variability was even more remarkable when investigating the effect of liposomes on the complement activation in sera from donors with pre-existing pathologies. Neither the 26 sera of donors with allergies nor sera of 16 donors with immunodeficiency responded similar to the PS of healthy donors. Allergy sufferers showed an increase in iC3b levels of 4.16-fold changes when compared to PS treated with liposomes. Discussion: Our studies demonstrate that the use of pooled serum can lead to an over- or under-estimation of immunological response in particular for individuals with pre-existing pathologies. This is of high relevance when developing medical products based on nanomaterials and asks for a review of the current practice to use PS from healthy donors for the prediction of immunological effects of drugs in patients. A better understanding of individual toxicological responses to xenobiotics should be an essential part in safety assessments.

Protein-corona formation on aluminum doped zinc oxide and gallium nitride nanoparticles.

The interaction of semiconductor nanoparticles with bio-molecules attracts increasing interest of researchers, considering the reactivity of nanoparticles and the possibility to control their properties remotely giving mechanical, thermal, or electrical stimulus to the surrounding bio-environment. This work reports on a systematic comparative study of the protein-corona formation on aluminum doped zinc oxide and gallium nitride nanoparticles. Bovine serum albumin was chosen as a protein model. Dynamic light scattering, transmission electron microscopy and X-ray photoelectron spectroscopy techniques have been used to demonstrate the formation of protein-corona as well as the stability of the colloidal suspension given by BSA, which also works as a surfactant. The protein adsorption on the NPs surface studied by Bradford Assay showed the dependence on the quantity of proteins adsorbed to the available sites on the NPs surface, thus the saturation was observed at ratio higher than 5:1 (NPs:Proteins) in case of ZnO, these correlating with DLS results. Moreover, the kinetics of the proteins showed a relatively fast adsorption on the NPs surface with a saturation curve after about 25 min. GaN NPs, however, showed a very small amount of proteins adsorbed on the surface, a change in the hydrodynamic size being not observable with DLS technique or differential centrifugal sedimentation. The Circular Dichroism analysis suggests a drastic structural change in the secondary structure of the BSA after attaching on the NPs surface. The ZnO nanoparticles adsorb a protein-corona, which does not protect them against dissolution, and in consequence, the material proved to be highly toxic for Human keratinocyte cell line (HaCaT) at concentration above 25 µg/mL. In contrast, the GaN nanoparticles which do not adsorb a protein-corona, show no toxicity signs for HaCaT cells at concentration as high as 50 µg/mL, exhibiting much lower concentration of ions leakage in the culture medium as compared to ZnO nanoparticles.

Spin-Coating and Aerosol Spray Pyrolysis Processed Zn1-xMgxO Films for UV Detector Applications.

A series of Zn1-xMgxO thin films with x ranging from 0 to 0.8 were prepared by spin coating and aerosol spray pyrolysis deposition on Si and quartz substrates. The morphology, composition, nano-crystalline structure, and optical and vibration properties of the prepared films were studied using scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), X-ray diffraction (XRD), and optical and Raman scattering spectroscopy. The optimum conditions of the thermal treatment of samples prepared by spin coating were determined from the point of view of film crystallinity. The content of crystalline phases in films and values of the optical band gap of these phases were determined as a function of the chemical composition. We developed heterostructure photodetectors based on the prepared films and demonstrated their operation in the injection photodiode mode at forward biases. A device design based on two Zn1-xMgxO thin films with different x values was proposed for extending the operational forward bias range and improving its responsivity, detectivity, and selectivity to UV radiation.

Large-Sized Nanocrystalline Ultrathin ß-Ga2O3 Membranes Fabricated by Surface Charge Lithography.

Large-sized 2D semiconductor materials have gained significant attention for their fascinating properties in various applications. In this work, we demonstrate the fabrication of nanoperforated ultrathin ß-Ga2O3 membranes of a nanoscale thickness. The technological route includes the fabrication of GaN membranes using the Surface Charge Lithography (SCL) approach and subsequent thermal treatment in air at 900 °C in order to obtain ß-Ga2O3 membranes. The as-grown GaN membranes were discovered to be completely transformed into ß-Ga2O3, with the morphology evolving from a smooth topography to a nanoperforated surface consisting of nanograin structures. The oxidation mechanism of the membrane was investigated under different annealing conditions followed by XPS, AFM, Raman and TEM analyses.

A bioinspired approach to fabricate fluorescent nanotubes with strong water adhesion by soft template electropolymerization and post-grafting.

HYPOTHESIS: In this original work, we aim to control both the surface wetting and fluorescence properties of extremely ordered and porous conducting polymer nanotubes prepared by soft template electropolymerization and post-grafting. For reaching this aim, various substituents of different hydrophobicity and fluorescence were post-grafted and the post-grafting yields were evaluated by surface analyses. We show that the used polymer is already fluorescent before post-grafting while the post-grafting yield and as a consequence the surface hydrophobicity highly depend on the substituent. EXPERIMENTS: Here, we have chosen to chemically grafting various fluorinated and aromatic substituents using a post-grafting in order to keep the same surface topography. Flat conducting polymer surfaces with similar properties have been also prepared for determining the surface energy with the Owens-Wendt equation and estimating the post-grafting yield by X-ray Photoemission Spectroscopy (XPS) and Time of Flight Secondary Emission Spectrometry (ToF-SIMS). For example, using fluorinated chains of various length (C4F9, C6F13 and C8F17), it is demonstrated that the surface hydrophobicity and oleophobicity do not increase with the fluorinated chain length due to the different post-grafting yields and because of the presence of nanoroughness after post-grafting. FINDINGS: These surfaces have high apparent water contact angle up to 130.5° but also strong water adhesion, comparable to rose petal effect even if there are no nanotubes on petal surface. XPS and ToF-SIMS analyses provided a detailed characterisation of the surface chemistry with a qualitative classification of the grafted surfaces (F6 > F4 > F8). SEM analysis shows that grafting does not alter the surface morphology. Finally, fluorescence analyses show that the polymer surfaces before post-treatment are already nicely fluorescent. Although the main goal of this paper was and is to understand the role of surface chemistry in tailoring the wetting properties of these surfaces rather than provide specific application examples, we believe that the obtained results can help the development of specific nanostructured materials for potential applications in liquid transport, or in stimuli responsive antimicrobial surfaces.

New Detection Platform for Screening Bacteria in Liquid Samples.

The development of sensitive methods for the determination of potential bacterial contamination is of upmost importance for environmental monitoring and food safety. In this study, we present a new method combining a fast pre-enrichment step using a microporous cryogel and a detection and identification step using antimicrobial peptides (AMPs) and labelled antibodies, respectively. The experimental method consists of: (i) the capture of large amounts of bacteria from liquid samples by using a highly porous and functionalized cryogel; (ii) the detection and categorisation of Gram-positive and Gram-negative bacteria by determining their affinities toward a small set of AMPs; and (iii) the identification of the bacterial strain by using labelled detection antibodies. As proof of concept, the assessment of the three steps of the analysis was performed by using Escherichia coli and Bacillus sp. as models for Gram-negative and Gram-positive bacteria, respectively. The use of AMPs with broad specificity combined with labelled antibodies enabled the detection and potential categorization of a large spectrum of unknown or unexpected bacteria.

Combining microcavity size selection with Raman microscopy for the characterization of Nanoplastics in complex matrices.

Nanoplastic particulates (pNP) are widely considered as being potentially harmful to the environment and living organisms while also being technically difficult to detect and identify in the presence of biological matrices. In this study, we describe a method for the extraction and subsequent Raman analysis of pNP present in the tissues of salt-water mussels. The process combines a step of enzymatic digestion/filtering to eliminate the biological matrix with a detection/identification procedure, which uses a micro-machined surface, composed of arrays of cavities with well-defined sub-micron depths and diameters. This sensor surface, exploits capillary forces in a drying droplet of analyte solution to drive the self-assembly of suspended nanoparticles into the cavities leaving the individual particles isolated from each other over the surface. The resulting array, when analysed using confocal Raman microscopy, permits the size selective analysis of the individual sub-micron pNP trapped in the cavities structure.

Aero-Ga2O3 Nanomaterial Electromagnetically Transparent from Microwaves to Terahertz for Internet of Things Applications.

In this paper, fabrication of a new material is reported, the so-called Aero-Ga2O3 or Aerogallox, which represents an ultra-porous and ultra-lightweight three-dimensional architecture made from interconnected microtubes of gallium oxide with nanometer thin walls. The material is fabricated using epitaxial growth of an ultrathin layer of gallium nitride on zinc oxide microtetrapods followed by decomposition of sacrificial ZnO and oxidation of GaN which according to the results of X-ray diffraction (XRD) and X-ray photoemission spectroscopy (XPS) characterizations, is transformed gradually in ß-Ga2O3 with almost stoichiometric composition. The investigations show that the developed ultra-porous Aerogallox exhibits extremely low reflectivity and high transmissivity in an ultrabroadband electromagnetic spectrum ranging from X-band (8-12 GHz) to several terahertz which opens possibilities for quite new applications of gallium oxide, previously not anticipated.

Dark Field Microscopy-Based Biosensors for the Detection of E. coli in Environmental Water Samples.

Development of sensitive methods for the determination of E. coli bacteria contamination in water distribution systems is of paramount importance to ensure the microbial safety of drinking water. This work presents a new sensing platform enabling the fast detection of bacteria in field samples by using specific antibodies as the biorecognition element and dark field microscopy as the detection technique. The development of the sensing platform was performed using non-pathogenic bacteria, with the E. coli DH5α strain as the target, and Bacillus sp. 9727 as the negative control. The identification of the captured bacteria was made by analyzing the dark field microscopy images and screening the detected objects by using object circularity and size parameters. Specificity tests revealed the low unspecific attachment of either E. coli over human serum albumin antibodies (negative control for antibody specificity) and of Bacillus sp. over E. coli antibodies. The system performance was tested using field samples, collected from a wastewater treatment plant, and compared with two quantification techniques (i.e., Colilert-18 test and quantitative polymerase chain reaction (qPCR)). The results showed comparable quantification capability. Nevertheless, the present method has the advantage of being faster, is easily adaptable to in-field analysis, and can potentially be extended to the detection of other bacterial strains.

Rational design of multi-functional gold nanoparticles with controlled biomolecule adsorption: a multi-method approach for in-depth characterization.

Multi-functionalized nanoparticles are of great interest in biotechnology and biomedicine, especially for diagnostic and therapeutic purposes. However, at the moment the characterization of complex, multi-functional nanoparticles is still challenging and this hampers the development of advanced nanomaterials for biological applications. In this work, we have designed a model system consisting of gold nanoparticles functionalized with two differentially-terminated poly(ethylene oxide) ligands, providing both "stealth" properties and protein-binding capabilities to the nanoparticles. We use a combination of techniques (Centrifugal Liquid Sedimentation, Dynamic Light Scattering, Flow Field Flow Fractionation, Transmission Electron Microscopy, and Circular Dichroism) to: (i) monitor and quantify the ratios of ligand molecules per nanoparticle; (ii) determine the effect of coating density on non-specific protein adsorption; (iii) to assess the number and structure of the covalently-bound proteins. This article aims at comparing the complementary outcomes from typical and orthogonal techniques used in nanoparticle characterization by employing a versatile nanoparticle-ligands-biomolecule model system.

Label-Free Biosensor Detection of Endocrine Disrupting Compounds Using Engineered Estrogen Receptors.

Endocrine Disrupting Compounds (EDCs) are chemical substances shown to interfere with endogenous hormones affecting the endocrine, immune and nervous systems of mammals. EDCs are the causative agents of diseases including reproductive disorders and cancers. This highlights the urgency to develop fast and sensitive methods to detect EDCs, which are detrimental even at very low concentrations. In this work, we propose a label-free surface plasmon resonance (SPR) biosensor method to detect specific EDCs (17 ß-estradiol (E2), ethinyl-estradiol, 4-nonylphenol, tamoxifen) through their binding to estrogen receptor alpha (ERα). We show that the use of rationally designed ERα (as bio-recognition element) in combination with conformation-sensitive peptides (as amplification agent, resulting in increased responses) enables the detection of low parts per billion (ppb) levels of E2. As a proof of concept, this bioassay was used to detect E2 in (spiked) real water samples from fish farms, rivers and the sea at low ppb levels after concentration by solid phase extraction. In addition, the present SPR assay that combines a conformation-sensitive peptide with an array of ERα mutants is very promising for the assessment of the risk of potential estrogenic activity for chemical substances.

Characterisation of nanomaterial hydrophobicity using engineered surfaces.

Characterisation of engineered nanomaterials (NMs) is of outmost importance for the assessment of the potential risks arising from their extensive use. NMs display indeed a large variety of physico-chemical properties that drastically affect their interaction with biological systems. Among them, hydrophobicity is an important property that is nevertheless only slightly covered by the current physico-chemical characterisation techniques. In this work, we developed a method for the direct characterisation of NM hydrophobicity. The determination of the nanomaterial hydrophobic character is carried out by the direct measurement of the affinity of the NMs for different collectors. Each collector is an engineered surface designed in order to present specific surface charge and hydrophobicity degrees. Being thus characterised by a combination of surface energy components, the collectors enable the NM immobilisation with surface coverage in relation to their hydrophobicity. The experimental results are explained by using the extended DLVO theory, which takes into account the hydrophobic forces acting between NMs and collectors. Graphical abstractDetermination of hydrophobicity character of nanomaterials by measuring their affinity to engineered surfaces.

Bioinspired Rose-Petal-Like Substrates Generated by Electropolymerization on Micropatterned Gold Substrates.

Surfaces with high water-adhesion properties are promising materials for different applications in the field of water treatment and management, such as for water-harvesting systems or oil/water separation membranes. Herein, we developed rose-petal-like substrates that demonstrate interesting parahydrophobic character. This bioinspired material mimics the natural substrate thanks to a combination of two fabrication steps: (1) micropatterning to create a microstructured gold-coated substrate consisting of square pillars and (2) an electropolymerization process generating nanostructures over the micropillars. Judicious choice of the micropatterning specifications (pillar diameter and pitch), the type of electropolymerizable monomer, and the electrochemical parameters produces a material with both extremely high water contact angles (up to 160°), while retaining a remarkably high water-adhesion level. Our study suggests that a composite interface is expressed by the existence of the Wenzel state on the micropillars and the Cassie-Baxter state between the pillars ("Cassie-filled nanostructure"), as observed during our contact-angle measurements. Indeed, we show that the pitch should be small to obtain the optimal micropillar surface density. Moreover, a relatively low deposition charge of approximately 50 mC cm-2 is preferable for coating the square pillars exclusively with nanostructures.

Bioinspired Rose-Petal-Like Substrates Generated by Electropolymerization on Micropatterned Gold Substrates.

Invited for this month's cover are the collaborating groups of Dr. Thierry Darmanin at Université Côte d'Azur, France and Dr. François Rossi at JRC European Commission, Italy. The cover picture shows a novel strategy for preparing substrates having a rose-petal effect (high water adhesion). The micropatterning specifications (pillar diameter and pitch) and the electropolymerization parameters are key to obtaining both high water apparent contact angles and a high hysteresis. Read the full text of the article at 10.1002/cplu.201600387.

Gold nanoparticles increases UV and thermal stability of human serum albumin.

Ultraviolet (UV) radiation, temperature, and time can degrade proteins. Here, the authors show that gold nanoparticles significantly protect human serum albumin from denaturation when exposed to "stressing" conditions such as UV irradiation and sustained exposure in suboptimal conditions. In particular, the authors show that gold nanoparticles significantly reduce the decrease in secondary structure induced by UV irradiation or extended exposure to ambient temperature.

Multiplex cell microarrays for high-throughput screening.

Microarray technology was developed in the early 1990s to measure the transcription levels of thousands of genes in parallel. The basic premise of high-density arraying has since been expanded to create cell microarrays. Cells on chip are powerful experimental tools for high-throughput and multiplex screening of samples or cellular functions. Miniaturization increases assay throughput while reducing both reagent consumption and cell population heterogeneity effect, making these systems attractive for a wide range of assays, from drug discovery to toxicology, stem cell research and therapy. It is usual to functionalize the surface of a substrate to design cell microarrays. One form of cell microarrays, the transfected cell microarray, wherein plasmid DNA or siRNA spotted on the surface of a substrate is reverse-transfected locally into adherent cells, has become a standard tool for parallel cell-based analysis. With the advent of technology, cells can also be directly spotted onto functionalized surfaces using robotic fluid-dispensing devices or printed directly on bio-ink material. We are providing herein an overview of the latest developments in optical cell microarrays allowing high-throughput and high-content analysis.

Quantification of the cellular dose and characterization of nanoparticle transport during in vitro testing.

BACKGROUND: The constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods. RESULTS: This work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane. CONCLUSION: The fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.

Biofouling Properties of Nitroxide-Modified Amorphous Carbon Surfaces.

Amorphous carbon films exhibit attractive optical and surface properties. In this work, modified amorphous carbon films incorporating nitroxide groups (α-CNO) have been obtained by searching for a condensed analogue to classical soft antifouling materials. Thin films deposited by reactive magnetron sputtering in air discharges at varying power conditions were characterized by ellipsometry, atomic force microscopy, and water contact angle. Plasma power was observed to activate the densification and roughness of nanograined films. Most hydrophilic films deposited at 30 W exhibited the lowest refractive index, negligible optical absorption in the vis-IR, and presented a close to stoichiometric C2NO composition, as derived from X-ray photoelectron spectroscopy. Micropatterns prepared by photolithography validated the transparency-hydrophilicity of the α-CNO, as observed by water condensation contrast imaging. An albumin adsorption experiment evaluated through fluorescence revealed that α-CNO behaves as antifouling with respect to Si. Such thin antifouling films are of interest for the initiation of immobilization cascades in imaging surface plasmon resonance, where they have confirmed their antifouling contrast enhancement role. These results illustrate that the combination of a nanorough surface with nitroxide chemistry induces an antifouling behavior. In association with the optical transparency, the results invite the exploration of the bioengineering dimension of α-CNO films.